Origin and safety aspects of enterococci in Baylough, an Irish Cheddar-like cheese

Roberto Gelsomino
Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Sciences

Faculty of Sciences Deparment of Biochemistry, Physiology and Microbiology Laboratory of Microbiology

Dairy Products Research Center Teagasc, Moorepark Fermoy, Co. Cork Ireland

Origin and safety aspects of enterococci in baylough, an irish cheddar-like cheese

Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph.D) in Sciences (Biochemistry)

Roberto Gelsomino

Promoter: Prof. Dr. ir Jean Swings Co. Promoter: Prof. Dr. Tim Cogan

Front page: Floris Claesz van DIJCK (*1575, Haarlem; 1651, Haarlem) Still-life 1613 Oil on wood Frans Halsmuseum, Haarlem, The Netherlands PhD thesis, Faculty of Sciences, Ghent University, Ghent, Belgium Defence in Ghent, May 18th 2004

Examination Commission

Prof. Dr. Jos Van Beeumen (acting chairman)

Faculty of Sciences, University of Ghent

Prof. Dr. ir. Jean Swings (promoter)

Faculty of Sciences, University of Ghent

Prof. Dr. Timothy M. Cogan (co-promoter)

Visiting Professor in Microbiology, UCC and Senior Principal Research Officer, Dairy Products Research Centre, Teagasc, Fermoy, Ireland

Dr. Luc A. Devriese

Faculty of Veterinary Medicine, University of Ghent, Merelbeke

Dr. Marc Heyndrickx

Centre for Agricultural Research, Ghent

Dr. Bruno Pot

Bacteriology of Ecosystems, Institut Pasteur de Lille

Dr. Marc Vancanneyt

Faculty of Sciences, University of Ghent

Prof. Dr. Peter Vandamme

Faculty of Sciences, University of Ghent

Prof. Dr. Mario Vaneechoutte

Department of Clinical Chemistry, Microbiology & Immunology, University Hospital Ghent

DIALOGUE between Madame J. L. & her children

Children Pray dearest mother if you please Cut up your double-curded cheese, The oldest of the brotherhood. Its ripe, no doubt and nicely good! Your reputation will rise treble As we the luscious morsel nibble. Praise will flow from each partaker Both on the morsel and the maker!

Madame Your suit is vain, -upon my word, You taste not yet my double-curd; I know the hour, -the very minute In which Ill plunge my cutteau in it; Am I to learn of witless bairns How I must manage my concerns? As yet the fervid dog-star reigns And gloomy Virgo holds the reigns. Be quiet chicks, sedate and sober And house your stomachs till October; Then for a feast! Upon my word, Ill really cut my double curd.

Major Henry Livingstone Jr. (1748-1828) 7

Table of contents

Part I - Literature review

Chapter 1. General introduction about cheese.......................................................17 1.1. Cheesemaking 1.2. Biochemical pathways during cheese ripening 1.2.1. Lipolysis 1.2.2. Proteolysis 1.2.3. Citrate metabolism 1.2.4. Glycolysis 1.3. Classification of cheese 1.4. The geography of cheese production 18 21 21 22 23 23 24 26

Chapter 2. Microbiology of cheese...........................................................................30 2.1. The starter culture 2.2. Non-starter bacteria 2.3. Source of non-starter bacteria 2.4. Hygiene of milk and dairy products 30 34 36 38

Chapter 3. Baylough, an Irish Cheddar-like cheese...............................................40 3.1. Cheddar cheese 3.2. Artisanal cheeses and Baylough 40 41

Chapter 4. Enterococci..............................................................................................44 4.1. Description of the genus Enterococcus 4.1.1. Introduction 4.1.2. Taxonomy of the genus Enterococcus 4.1.3. Habitat 44 44 44 46

4.2. The occurrence of enterococci 4.2.1. Presence in cheese 4.2.2. Bacteriocin production 4.2.3. Further applications of Enterococci 4.3. Acquired and intrinsic antibiotic resistance of enterococci

47 47 50 51 52

4.3.1. Acquired antibiotic resistance and vancomycin resistance mechanism 52 4.3.2. Intrinsic antibiotic resistance 4.4. Virulence factors 4.5. The spread of VRE in Europe and the USA 4.6. Enterococci in food: yes or no? 54 54 56 59

References

61

Chapter 5. Aim of this study....................................................................................75

Part II Experimental work

Chapter 6. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory.................................................81

Gelsomino R., M. Vancanneyt, S. Condon, J. Swings, and T. M. Cogan. 2001. Enterococcal diversity in the dairy environment of an Irish cheese making factory. Int. J. Food Microbiology. 71:177-188

Chapter 7. The source of enterococci in a farmhouse raw-milk cheese..................105

Gelsomino R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings.2002. Source of enterococci in a farmhouse raw-milk cheese. Appl. Environ. Microbiol. 68:3560-3565.

10

Chapter 8. Effect of consuming a raw-milk cheese on the enterococcal flora of human feces...............................................125
Gelsomino R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings.2003. Effect of rawmilk cheese consumption on the enterococcal flora of human feces. Appl. Environ. Microbiol. 69:312-319.

Chapter 9. Antibiotic resistance and virulence traits of enterococci isolated from an Irish artisanal cheese...................................................149

Gelsomino R., G. Huys, K. DHaene, M. Vancanneyt, T. Cogan, C. M. A. P. Franz, and J. Swings. Antibiotic resistance and virulence traits of enterococci isolated from an Irish artisanal cheese. J. Food Prot. (in press)

Part III - Conclusions

Chapter 10. Conclusions and perspectives..............................................................167 10.1. Conclusions 10.2. Perspectives 167 169

Chapter 11. Summary..............................................................................................174

Chapter 12. Samenvatting.......................................................................................177 Appendix Appendix 1. List of strains isolated in this study......................................................183 Appendix 2. Curriculum vitae...................................................................................184

Acknowledgments.....................................................................................................186

11

List of abbreviations
ARE BCCM cfu DSS FAA FAO FDM FFA FHL GIT GRAS HAI HLR LAB Lb. Lc. LLR LMG MSS MNFS NSLAB PAB PCR PFGE SDS-PAGE S/M sp./spp. ssp. Sta. Str. VRE Antibiotic resistant enterococci Belgian Co-ordinated Collections of Microorganism Colony forming units Defined-strain starter Free amino acids Food and agriculture organization of the United Nations Fat in dry matter Free fatty acids Facultative heterofermentative lactobacilli Gastrointestinal tract Generally recognized as safe Hospital acquired infections High level resistance Lactic acid bacteria Lactobacillus Lactococcus Low level resistance Laboratory of Microbiology Ghent Mixed-strain starter Moisture-in-the-non-fat solids Non-starter lactic acid bacteria Propionic acid bacteria Polymerase chain reaction Pulsed-field gel electrophoresis Sodium dodecyl sulphate poly acrylamide gel electrophoresis Salt in moisture species (sing./pl.) subspecies Staphylococcus Streptococcus Vancomycin resistant enterococci

13

Part I Literature review

Chapter 1. General INTRODUCTION about cheese

The production of cheese from milk is an ancient process, as is shown by prehistoric findings. The oldest of these findings about cheesemaking is the so-called dairy frieze from the third millennium BC (Fig. 1). This frieze shows Sumeric priests (the ancient cheese experts) in the various stages of cheese production (http://www.formaggio.it/storia1.htm). Also in the Bible in the book of Samuel (which is believed to have been written around 1100 BC) we read under Samuel 17,29: And honey, and butter, and sheep, and cheese of kine, for David, and for the people that [were] with him, to eat. Throughout centuries testimonies over cheesemaking can be found from the Romans to the Middle Ages. Also Homer (~800 BC), in book 9 of his Ulysses, describes Polyphem, a one-eyed giant who makes cheese from the milk he gets from his sheep and goats: while he into a cave > Of huge receipt his high-fed cattle drave, >All that he milk'd; > could not stir it there. > Thus making sure, he kneel'd and milk'd his ewes, > And braying goats, with all a milker's dues; > Then let in all their young. Then quick did dress > His half milk up for cheese, and in a press > Of wicker press'd it This is probably the first written recipe of cheesemaking (http://www.bartleby.com/111/chapman22.html, http://www.formaggio.it/storia1.htm;). It is generally believed that cheese must have resulted from the simple observation that milk left in a ruminants stomach ends up by coagulating. This might be considered the first technological cheesemaking discovery (Johnson, 1989; http://www.formaggio.it/storia1.htm). Cheesemaking then developed from the need to preserve the constituents of milk.

17

Figure 1. One of the oldest findings about cheesemaking is the Sumeric frieze (obove a detail), which has been dated to the 3rd millennium BC. Below the one-eyed giant polyphem threatening Ulysses and his friends (Annibale Carraci, 1595-1605, Palazzo Farnese, Roma)

1.1. Cheesemaking There is no such thing as a standard cheesemaking recipe as every cheese varies in one or other manufacturing step. The only common step in all cheesemaking processes is the transformation of a liquid into a solid, or, defined differently, a dehydration process in which the fat and casein in milk are

18

concentrated between 6- and 12-fold, depending on the variety (Fox, 1993). There are, nonetheless, basic steps common to a lot of cheese varieties: Acidification. Until the end of the 19th century the indigenous microflora of milk was responsible for acidification. Due to unpredictable acid production rates starter cultures were then introduced. Basically this was milk, which had been incubated until it coagulated. It was then used as starter and if it produced good quality cheese, it was sub-cultured. These starters were originally available as liquid cultures but they were subsequently made available as vacuum-dried cultures, then freeze-dried and more recently as either frozen or freeze-dried, concentrated cultures, which were suitable for direct inoculation of milk in the cheese vat. It is now the practice to add a starter culture of selected lactic acid-producing bacteria to achieve a uniform acid production. These are called starter cultures and will be discussed in detail later (section 2.1) (Cogan, 1996; Stanley, 2003). Pasteurisation: The milk is generally pasteurised at 72oC for 15 sec for cheese making, but about 10% of all the cheese made in Europe is made from raw milk. Pasteurisation inactivates all pathogens, which might be present in the raw milk (Mycobacterium tuberculosis, Brucella abortus, Staphylococcus aureus, Listeria monocytogenes) and significantly reduces other adventitious bacteria present. To-day pasteurised milk is virtually sterile, and counts of <5x103 cfu/ml are not uncommon (Manners and Craven, 2003a; Manners and Craven, 2003b)

Coagulation. This is the essential characteristic step in the manufacture of all cheese varieties and is achieved by (i) addition of rennin (chymosin) or a mixture of pepsin and chymosin proteinases. Chymosin is an extract from the bovine abomasum, and is added to the milk, which is normally pre-warmed to ~30oC, after the starter culture. The curd which is formed is then cut with knives into small particles, the size of which depends on the cheese being made. (ii) by starter acidification to pH ~4.6 and (iii) a combination of 1 and 2 and heat (Fox, 1993; Hill, 2000).

19

Dehydration. Once the rennet gel is cut or broken, syneresis, i.e. contraction of the curd and expulsion of the whey, occurs rapidly. Acidification by starter cultures through lactose fermentation results in a decrease in pH, which helps to expel whey from the curd. This, in turn, reduces the moisture level and aids in preservation of the milk as cheese (Cogan, 1996). Cooking, also referred to as scalding (although boiling temperatures are never reached), sometimes occurs and further helps expulsion of the whey. The cooking temperature varies between 35C in the case of soft cheeses to 55C in the case of some hard Italian cheeses. The rate and extent of syneresis are influenced by pH of the whey, cooking temperature and rate and time of stirring. The whey is then drained, i.e., the whey and curd are separated from each other, and the curd is salted. This type of heating must be distinguished from that given to the so called pasta filata (or plastic curd) cheeses like Mozzarella, where the curd is heated with hot water (up to 67C) to plasticize it and produce a more fibrous cheese (http://www.caseificiocorvino.com/schedamoz.htm). Most cheeses are then brined in 20-23% (saturated) NaCl once they are removed from the moulds. Cheddar cheese is an exception. Its curd is cut into small particles before it is moulded and it is then dry salted, moulded and pressed overnight. Brining can last for a few hours (small cheeses) to several days (large cheeses). Salt plays an important role in cheesemaking. It slows down acidification by inhibiting the starter and decreases enzymatic activity. If the curd is brined it is usually placed in moulds first and pressed; however, dry salting can occur before moulding (Cheddar) or after moulding (Gruyre) (Cogan, 1996; Banks and Horne, 2003). Ripening. High moisture cheeses are ripened at relatively high temperatures of ~15oC for short periods, perhaps 4-8 weeks, while low moisture cheeses like Cheddar are ripened at 7-9oC for 6-12 months. During this time extremely complex biochemical changes occur through the action of (i) coagulant, (ii) indigenous milk enzymes, (iii) starter bacteria and their enzymes and (iv) the secondary microflora and their enzymes. It is due to the action of these four factors that every cheese develops its unique characteristics (Hill, 2000; Farkye, 2003). The major biochemical changes during cheese ripening are discussed in the following section. Most if not all cheesemaking steps change according to the type of cheese made. Other factors influence the final product including the use of raw or

20

pasteurised milk, the use of different starter cultures and the adjustment of milk components such as milk fat or calcium content. In addition, the duration and temperature of cooking, the amount of whey drained, the duration of moulding, the amount of salt added and the duration and temperature of ripening are some of the factors that change the water activity, pH and salt content, which, in turn, influence the moisture content, acidity, firmness and texture of the cheese. The effect of these factors may not be great individually but the interaction of all of them leads to the diversity of cheese, which now exists in hundreds of different varieties (Johnson, 1998; Fox, 1993; Olson, 2003).

1.2. Biochemical pathways during cheese ripening The extremely complex set of biochemical changes which occur during ripening include lipolysis, proteolysis, glycolysis, deamination, decarboxylation, desulphurisation of amino acids, -oxidation of fatty acids, esterification etc. It is not my purpose to review this part of cheese development. Instead, a short overview of the principal ripening reactions is presented although much of the biochemical changes are due to microbial activity and are important for the formation of flavour compounds.

1.2.1. Lipolysis Satisfactory flavour development is one of the biggest problems encountered in the manufacture of low-fat products implying that lipolysis is essential for flavour development. Milk fat contains short- and intermediate-chain fatty acids (butanoic, hexanoic, octanoic and decanoic acid), which contribute directly to cheese flavour (Ha and Lindsay, 1993). This is achieved by enzymatic hydrolysis of triglycerides to fatty acids and glycerol, mono- or diglycerides. Lipolysis is very high in hard Italian cheeses and blue mould cheeses. High lipolysis is undesirable in cheeses like Edam, Mozzarella, Camembert or Cheddar, where free fatty acids (FFA) may range

21

between 300 and 1500 mg/kg but it is highly desirable in cheeses like Parmesan or Roquefort, where FFA reach ~30000 mg/kg. Lipases responsible for lipolysis originate from different sources like milk, rennet, starter etc. (McSweeney and Sousa, 2000). These lipases can lead to off flavours such as goaty, muttony and sweatlike which derive from the volatile fatty acids 4-ethyloctanoic acid, 4methyloctanoic acid and 3-methylbutanoic acid, respectively (Ha and Lindsay, 1993; Farkye, 2003).

1.2.2. Proteolysis Proteolysis is probably the most important biochemical event, taking place during cheese manufacture. Proteolysis begins during cooking, and hence contributes to textural changes. But it is also important in contributing to flavour and off-flavour development, liberating free amino acids (FAA), which contribute to the background flavour of most cheese varieties (e.g., alanine, serine, and threonine are responsible for sweetness, valine, isoleucine and tyrosine for bitter). The FAA are further catabolized through enzymatic reactions involving deamination, decarboxylation, desulphurization etc. to products which are chemically transformed into the compounds responsible for flavour development (Farkye, 2003). Initial hydrolysis of casein is caused by coagulation of the milk. The two major enzymes involved are chymosin and pepsin, which hydrolyse casein to large peptides. Most of the coagulant is lost in the whey. The non-starter microflora may play a significant role in proteolysis as they rapidly reach high numbers, especially in cheeses made from raw milk while starter bacteria are only weakly proteolytic. In many cases a secondary starter is added to the cheese in order to accelerate ripening. Such secondary starters comprise Penicillium roqueforti (for blue cheese), Penicillium camembertii (Camembert and Brie), Propionibacterium freudenreichii (Emmentaler) or Brevibacterium linens (smear- or surface-ripened cheeses). The final products of proteolysis are FAA and medium and small peptides. Further catabolism of FAA results in ammonia, amines, phenols, methanthiol and alcohols, all of which contribute to flavour (McSweeney and Sousa, 2000; Farkye, 2003).

22

1.2.3. Citrate metabolism Most of the citrate contained in milk is lost in the whey during cheesemaking. Nevertheless, the low concentration in the cheese mass is of considerable importance since it is metabolised to flavour compounds by mesophilic, citrate utilizing (Cit+) starter bacteria. These bacteria (i.e. the flavour producers, usually Leuconostoc lactis, Leuconostoc mesenteroides, and Cit+ Lactococcus lactis ssp. lactis) co-metabolize citrate and lactose rather than using citrate as an energy source. The major flavour compounds in citrate metabolism are acetate and diacetyl. Citrate metabolism is especially important in Dutch cheeses, where the CO2 formation is responsible for eye formation (Hugenholtz, 1993).

1.2.4. Glycolysis Lactose is metabolised to L- or D- lactate or both isomers by the starter and non-starter lactic acid bacteria (NSLAB). The NSLAB of some cheeses (Cheddar and some Dutch varieties) isomerize L-lactate to D-lactate. Ca-D-lactate is less soluble than Ca-L-lactate and results in crystallization and the formation of white visible specks (e.g. in Parmigiano Reggiano). Some of these specks are also composed of calcium phosphate or tyrosine (Bottazzi, 2003). In Emmental L-lactate is metabolised to propionic acid, acetate and CO2 by the propionic acid bacteria (Steffen et al., 1993). Acetate is an important flavour component and can be formed also from citrate or from amino acids. (Farkye, 2003). Late gas and off-flavour (mainly due to the production of butyric acid) production may result from metabolism of lactate by Clostridium spp. (Thomas and Crow, 1983).

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1.3. Classification of cheese As seen in section 1.1 changes in the manufacturing steps result in different products and it is the fact that cheese exists in so many varieties that makes it so difficult to classify. One of the most common methods is to classify on the basis of moisture content into hard (<40% moisture), semi-hard (40-45%) and soft cheeses (>45%). Several other classification schemes exist but most of them have deficiencies. A classification in fat content results in some cheeses being assigned to two or even three categories, for example Brie as Cheese, soft > 60% fat, Cheese, soft 46 - 60% fat and Cheese, soft 30 - 45% fat (for further details see also http://www.ianunwin.demon.co.uk/eurocode/dnotes /n006.htm#CheeseType). Attempts have been made to classify cheeses according to criteria which include method of production, level of fat, fat in dry matter, moisture in fat-free substance, texture, method of coagulation of proteins, the microorganisms used during manufacture and their influence during maturation and preservation techniques. In Table 1 the classification is modified from Scott (Fox, 1993), who classified cheeses primarily on the basis of moisture content and sub-divided these groups on the basis of cooking temperature and/or secondary microflora.

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Table 1: Classification of cheese according to moisture content, cooking temperature and microflora. Modified from Scott (Fox, 1993) *Scalding is the heating of the curd to usually 35C Hard cheese (moisture content 20-42%)
Normal lactic starter as only microflora Normal lactic starter as only microflora or Propionic acid bacteria

Low scald* Edam Fontina Cheshire

Medium scald* Cheddar Derby Svecia

High scald* Parmiggiano Emmental Beaufort

Plastic curds (pasta filata) Scamorza Provolone Mozzarella Cheddar Mozzarella

Semi-hard cheeses (moisture content 44-55%) low scald

Normal lactic starter as only microflora Smear coat Blue veined internal mould

St. Paulin Lancashire Trappiste

Herve Limburger Tilsit

Roquefort Gorgonzola Stilton Livarot

Acid coagulated, Normally unripened, fresh cheese

Soft cheeses (moisture content >55%) very low or no scald

Smear coat or Surface mould Surface mould Normal lactic starter as only microflora

Brie Bel Paese Maroilles

Camembert Neufchatel Carre d'est

Colwich Lactic Bondon

Ricotta Cottage Quarg Roquefort

Ricotta

1.4. The geography of cheese production Cheese has long been recognised as one of the international dairy industrys long-term growth sectors. The market continues to exhibit growth, spurred in particular by new product development, segmentation, and increased efforts to brand what has usually been a commodity sector. In 2000, cheese production in the European Union rose to over 6.5 million tonnes. This represented an increase of 5.4% compared with 1997 and 8.2% compared with 1995. With the exception of the Netherlands, all the major producer states recorded increases of the order of 6 %. Italy, Portugal and Greece saw the sharpest increases (ranging from 12% to 23%) (http://www.eu-datashop.de/download/EN/sta_kurz/thema5/nn.02_17.pdf). A glance at the production data within the EU reveals that France, Germany, Italy and The Netherlands are the greatest cheese producers. Together they produced over 5 000 000 tonnes in the year 2001, compared to the ~7 000 000 tonnes of the EU (FAOSTAT, 2001). But this has not always been so. In his history of Scotland, Smith (1995) narrates that cheese production declined from the early 30s to the late 40s decreasing from 9 800 tonnes to 3 200 tonnes. It is interesting to note that farmhouse cheese, which in 1937 had been some 4 400 tonnes, reached an absolute minimum in 1947 of 600 tonnes. But things were going better from then on for the creameryproduced cheeses thanks to mechanisation: In the 50s and 60s cheese production in the UK doubled. Cheese production slowed down but increased steadily afterwards: from 1970 to 1998 the average annual growth rate of cheese consumption in the USA was 4.5% (World Dairy, 1999; Smith, 1995). Farmhouse cheese-production on the other hand, still decreased (Smith, 1995). In the sixties and seventies, with the introduction of supermarkets, small businesses were displaced, especially independently owned small businesses that contributed significantly to local civic life (Shils, 1997). It is only in the last number of years that farmhouse cheeseproduction is rising (Lassaut, 2001; ONeill, 2000; Bord Bia, 2003). In the USA, Canada, New Zealand, South Africa and Australia the favourite cheeses are Cheddar, Mozzarella and processed cheeses. Together they dominate the market in the countries mentioned. Specialty cheeses like blue cheeses, Gouda, Edam, Camembert follow the hit list. (US Dairy Export Council, 2003) The varieties

26

produced in these countries are of European origin. Countries in Asia, Africa and Latin America produce little cheese compared with European or North American standards. However, even if on a very small scale, cheese is produced in these regions, frequently by nomads or shepherds. Very little scientific information is available on most of them. Some are produced by rennet or acid coagulation, but in many cases defined starter cultures are not used so that acid production is very variable (Phelan and Renaud, 1993). In some countries quite interesting techniques for different steps of cheese manufacturing are being used: in India concentration by boiling, sometimes with the addition of sugar, is widespread; in Indonesia coagulation happens by means of bromelain obtained from pineapples, while in Benin they add crushed stems of the plant Bryophylum sp. In Cyprus, Halloumi is made by heating the whey to 80-90C to further precipitate and remove the proteins in it and by submerging pieces of curd into the hot whey; Tulum Peyneri in Turkey is ripened for 3 to 4 months in a goat skin or, in some cases, in the rumen of a sheep or goat. In Tanzania some families filter the whey from soured milk in a piece of cloth, which is then hung over the fire place in the kitchen for about one week before being consumed. For further information on non-European cheeses see Phelan and Renaud (1993) or the booklet issued by the FAO (1990). In addition, Table 2 gives an overview of the quantities of the most important cheese varieties produced around the world in 2001. The table was created by merging information from FAO (1990), the statistical database of the FAO (FAOSTAT, 2001), Phelan and Renaud (1993) and various information pages from the internet (US Dairy Export Council, 2003).

27

Table 2. Cheese production (x1000 tonnes) in the world and some representative cheese varieties (US Dairy Export Council; FAO, 1990; FAOSTAT, 2001; Phelan and Renaud, 1993).
Country
Africa Egypt Sudan South Africa Niger Tunisia Marocco Nigeria Ethiopia Botswana Algeria Angola Mauritania Tanzania Zimbabwe Zambia Benin Chad Kenia Madagaskar Mali Zaire North and Central America USA Canada Mexico Nicaragua Cuba Guatemala Costa Rica Honduras Panama Dominican Rep. El Salvador South America Argentina Venezuela Chile Colombia Brasil Uruguay Peru Ecuador Bolivia Asia Iran China Japan Turkey Israel Myanmar Lebanon Uzbekistan Yemen Azerbaijan Kazakhstan Tajikistan Cyprus

quantity cheese varieties (e.g.)

733 480 152 35 15 14 8 8 6 4 2 2 2 2 2 1 (a) (a) (a) (a) (a) (a) 4594 4024 344 154 17 14 11 9 9 8 2 2 717 420 97 57 52 38 29 9 8 7 913 224 217 123 123 102 32 17 17 12 11 7 7 6 Daani, Ras, Karish Bibbna, Mudafera, Tchoukou

Wara, Chukumara Ayib Aoules, Takamart

Woagachi, Wahashi Pont Belile Mboreki Ya Iria Fromage, Fromage Blanc Wagashi Mashanza

Cotija, Oaxaca, Chihuaua Patagras Palmito Quesillo de Honduras Queso de Freir

Goya, Tafi De Mano Chanco Queso Blanco Queijo de Coalho, Queiso de Manteiga Colonia Queso Andino Queso Andino Quesillo, Queso Benianco

Liqvan Hurood, Aarul, Biaslag Cheddar, Edam, Gouda, Mozzarella Beyaz Peynir, Mihalic Peynir; Teleme Lebbene Akawieh, Karichee Taizz cheese

Halloumi

28

Table 2. continued
Kyrgyzstan Armenia Jordan Mongolia Turkmenistan Bangladesh Afghanistan Buthan India Indonesia Iraq Nepal Pakistan The Philippines Qatar Saudi Arabia Syria Europe France Germany Italy Netherlands Poland Russian Federation UK Denmark Greece Spain Switzerland Austria Czech Rep. Sweden Ireland Ukraine Hungary Finland Norway Portugal Belarus Belgium/Luxemburg Lithuania Slovakia Bulgaria Romania Croatia Slovenia Estonia Serbia-Montenegro Latvia Albenia Bosnia Herzg. Iceland Moldava Rep. Macedonia Oceania Australia New Zealand 4 3 3 2 2 1 (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) (a) 9000 1770 1743 1087 662 510 433 385 318 240 181 174 163 140 130 127 117 108 98 82 77 71 62 58 58 47 39 24 22 17 14 13 10 9 5 5 1 665 376 289 Armenian String Naboulsi, Djamid

Chhana, Ponir Karut, Kimish Panier Chhana, Churtsi Channa, Panir Tahu Susu Atau Dadih Awshari Chhana, Chhurpi, Shosim Panir, Peshawari Kesong Puti White cheese Ekt Mesanarah, Medaffarah

Beaufort, Brie, Camembert, Livarot Quark, Klosterkse, Bierkse Gorgonzola, Pecorino, Taleggio Gouda, Leerdamer, Edam Oszczypek, Podhalanski Daralagjazsky, Gornyaltajski Cheddar, Stilton, Leicester Esrom, Havarti, Maribo Feta, Kaseri Cabrales, Manchego, Roncal Emmental, Gruyere, Appenzell Graukse, Mondseer Abertam Graddost, Herrgardsost, Prastost Cashel Blue, Gubbeen Balaton, Lajta, Liptauer Juustoleipa, Lappi, Turunmaa Gammelost, Geitost, Gjetost Evora de l'Alentejo, Rabacal Herve, Passendale, Oud Brugge Bryndza, Oschtjepka Sirene Brinza, Telemea Peg Posavec

Avaxtskyr, Braudostur

Hipi Iti

(a)

Although FAOSTAT does not list any cheese production in this country, Phelan and

Renaud (1993) and FAO (1990) mention some cheese varieties typical for this country.

29

Chapter 2. Microbiology of cheese

2.1. The starter culture One of the most important processes in cheese manufacture is the acidification of the milk to the desired pH and this is the primary function of the starter bacteria. Starter cultures can be divided into three types, mesophilic, thermophilic and artisanal starters. Mesophilic starters always contain acid producing and flavour producing strains. The acid producing strains are unable to metabolise citrate (Cit-) and are Lactococcus lactis ssp. cremoris (mainly) and Lactococcus lactis ssp. lactis). These are the dominant organisms in mixed cultures and comprise 90-99% of the microflora. The flavour producing strains in a mesophilic starter are citrate utilizing (Cit+) strains of Leuconostoc lactis, Leuconostoc mesenteroides, and/or Lactococcus lactis ssp. lactis. The terms citrate utilizing and flavour producing are used interchangeably since citrate metabolism results in the production of compounds like acetate and diacetyl (see section 1.2.3) which impart flavour to the unripe or green curd (McSweeney and Sousa, 2000; Stanley, 2003). Depending on the presence or absence of flavour producer, mesophilic cultures are divided into (i) D-types containing Cit+ Lactococcus lactis ssp. lactis, (ii) L-types containing Leuconostoc spp., (iii) DL-types containing both species and (iv) O-types containing no flavour producer (Cogan, 1996). Table 3 gives an overview of the most important cheese starters and their desired functions. Thermophilic starters contain both coccal and rod-shaped bacteria. The coccus is invariably Streptococcus thermophilus whereas the rod is Lactobacillus helveticus, Lactobacillus delbrueckii ssp. lactis or Lactobacillus delbrueckii ssp. bulgaricus. Str. thermophilus, Lactobacillus delbrueckii ssp. lactis and Lactobacillus

30

delbrueckii ssp. bulgaricus only ferment the glucose portion of lactose. Lactobacillus helveticus can use galactose as energy source and is therefore used with Str. thermophilus because they have the ability to metabolize galactose. A symbiotic relationship exists between Str. thermophilus and the lactobacilli with the latter organisms producing amino acids, especially glycine, valine and histidine, as a result of proteolytic activity on the casein, which stimulates the growth of Str. thermophilus. The latter organism, in turn, produces formic acid and CO2 from lactose, which stimulate the lactobacilli (Stanley, 2003). Some strains of Lactobacillus delbrueckii ssp. lactis are able to use galactose and these are now more commonly used than Lb. helveticus in thermophilic starters for cheese manufacture. Thermophilic cultures have an optimum growth temperature of 42- 45C, compared to ~27oC for mesophilic cultures (Limsowtin et al., 1996). A third type of starter culture is the artisanal starter culture. This is derived from the practice of using part of a previous batch of a fermented product to inoculate a new or fresh batch. As they are produced with every cheese batch and rely on selective pressure (depending on the cheese preparation), their composition is variable and poorly defined. They can contain both mesophilic and thermophilic strains (Frank and Hassan, 1998). Starter cultures are usually deliberately added to the milk for cheese making but in some artisanal cheeses, particularly raw milk cheeses produced in Spain, no starter cultures are added. Instead the cheesemaker relies on adventitious, contaminant lactic acid bacteria present in the milk to produce the necessary acid in the cheese (Nuez and Medina, 1979; Del Pozo et al., 1988).

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Table 3: Cheese starters and their main functions (modified from Cogan, 1996).

Important Function

Starter Micro-organism Str. thermophilus Lb. helveticus Lb. delbrueckii ssp. lactis Lb. casei Cit- Lactococcus lactis ssp. lactis Lactococcus lactis ssp. cremoris Cit+ Lactococcus lactis subsp. lactis Leuconostoc mesenteroides ssp. cremoris

Lactic Diacetyl Lipolysis Proteolysis CO2 Acetoin Product acid + + Hard cheese; pasta filata; e.g., Swiss type cheeses (with Propionibacterium spp.) + + + + + + + + + + + + + Hard cheese, high scald, e.g.,Parmigiano Regiano Hard cheese, low scald, e.g., Cheddar Soft cheese ripened and unripened; semihard cheese; mould-ripened cheese; hard cheeses, low and medium scald, e.g., Cheddar, Gouda, Camembert Ripened soft cheese; semihard cheese; hard cheeses, low and medium scalde.g., Gouda, Camembert, Brie Ripened and unripened soft cheeses, e.g.,Gouda, Edam

+ +

+ +

+ +

+ +

+ +

+ +

Thermophilic and mesophilic cultures are commercially available in two types. The first are mixed-strain starters (MSS). These starter cultures derive from artisanal cultures and have been propagated under controlled conditions for several years. They are thought to contain several strains but experimental proof of this is lacking (Cogan, personal communication). However, continuous sub-culturing can lead to variability in strain and species composition. In contrast to MSS, the exact strain and species composition of defined-strain starters (DSS) is known. DSS are usually blends of two or more strains (Cogan, 1996; Limsowtin et al., 1996). During cheese manufacture, the starter cells in the curd increase from an initial number of 106 cfu/ml of milk to 109 cfu/g of cheese and, in the process produce lactic acid from lactose, which causes the pH of the cheese curd to decrease to 4.6-5,3 depending on the cheese. Non-viral agents may inhibit the growth of the starter (e.g. residual antibiotics from treatment of cows for mastitis) but bacteriophages (or phage) are still the most prevalent cause of decreased starter activity in cheese manufacture. The decreased starter activity results in fermentations in which acid production is noticeably reduced or, in extreme cases, fails completely. The phage tail is initially attached to the cell wall of the host cell and then injects its DNA into the bacterial cell. The phage DNA takes over the biosynthetic machinery of the host cell to produce more phage DNA and, as a result, more phage particles are produced in a short time, perhaps 100 particles in 60 min and the cell lyses. The new phages in turn attack new cells. The net result is that very quickly the entire bacterial culture is lysed. Phage have a very narrow host range and one of the advantages of MSS is that the phage only attack closely related strains and the other strains are able to continue to grow and produce acid (Sandine, 1996; Frank and Hassan, 1998). At the beginning of ripening, starter organisms dominate the microflora of cheese (~109 cfu/g) and during ripening they loose viability, lyse and release their intracellular enzymes in a process referred to as autolysis. Interest in autolytic strains is receiving considerable attention since it was demonstrated that cheese manufactured with autolytic strains was less likely to suffer from bitter flavour defects (Beresford and Williams, 2003). Starters vary in their ability to lyse and faster lysing organisms are also thought to produce a better flavour in the cheese (Fox et al., 2000).

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2.2. Non-starter bacteria During ripening, non-starter lactic acid bacteria (NSLAB) grow from relatively small numbers of 102-103 cfu/g at the beginning to >107 cfu/g after several weeks. The majority of the NSLAB are lactobacilli, but, in some cheese, pediococci and enterococci are also found. Other bacteria, which are deliberately added to some cheeses, e.g., propionibacteria to Swiss-type cheese, and coryneforms to red smearor surface-ripened cheeses also grow (Beresford and Williams, 2003). Non-starter lactobacilli: This group constitutes the majority of the nonstarter bacteria in probably all cheese varieties during ripening. The dominant species found in cheese are facultatively heterofermentative lactobacilli (FHL) particularly Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. The source of NSLAB is the raw milk or factory environment. Many of them survive pasteurisation and cooking temperatures. In contrast to the starter cells, the non-starter lactobacilli grow relatively quickly from initial levels of 102 to 103 cfu/ml to 108 cfu/ml and die off very slowly. Despite extensive studies, the role of non-starter lactobacilli in the development of cheese flavour is still not clear (Fox et al., 2000). Some strains of Lactobacillus plantarum and Lactobacillus brevis are known to produce various off flavours, e.g., gassy, putrid, fruity (van den Berg, 2003). Enterococci. Enterococci are an essential part of the natural microflora in many dairy products of the Mediterranean countries and they dominate over lactobacilli and lactococci in some cheeses, especially artisanal cheeses. The enterococcal population ranges from 105107 cfu g-1 in cheeses from Italy, France, Spain and Greece. The species most commonly isolated in cheese are Enterococcus faecium and Enterococcus faecalis (Beresford and Williams, 2003; Delgado and Bayo, 2004). Pediococci. Pediococci are found only occasionally as a minor part of the non-starter bacteria in some hard cheeses. Their influence on the production of cheese flavour is not clear as most pediococci do not ferment lactose and therefore do not grow very well in cheese (Litopoulou-Tzanetaki et al., 1989; Fox et al., 2000).

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Leuconostocs. Although some Leuconostoc species are considered as spoilage bacteria, certain strains produce acetate and diacetyl from citrate (see above) and they are widely used as starter in L and DL cultures. However, their occurrence is not restricted to cheeses with Leuconostoc-containing starters. Leuconostoc spp. have been isolated from artisanal cheeses produced from raw milk in Greece, Italy and France (Frank and Hassan, 1998; Beresford and Williams, 2003) Micrococci. Micrococci are obligate aerobes. They have been isolated in low numbers from a variety of cheeses including Cheddar and Iberian cheeses. The lack of oxygen inside most cheeses inhibits their growth and this explains their low numbers. Micrococcus spp. are believed to contribute positively to the maturation of different cheeses (Bhowmilk and Marth, 1990; Beresford and Williams, 2003). Staphylococci. Staphylococci are salt-tolerant, facultative anaerobes which also grow quite well in cheese. The pathogenic Staphylococcus aureus is found quite often at the initial stages of ripening but dies off relatively quickly. Staphylococci also occur on the surface of smear-ripened cheeses. The species most commonly found are Staphylococcus xylosus and Staphylococcus epidermidis (Beresford and Williams, 2003). Coryneforms. They are very salt tolerant, generally withstanding 10% NaCl, and grow on the surface of surface ripened cheeses such as Livarot, Tilsiter or Gubbeen. Yeasts on the surface of the cheese, originating from raw milk and air, metabolize the lactate causing a change in pH of the cheese surface towards neutrality. This favours the growth of coryneforms. The term coryneform has no taxonomic status but generally includes Arthrobacter, Brevibacterium, Brachybacterium, Corynebacterium and Microbacterium spp., all of which have been isolated from surface ripened cheesees. The only species, which is deliberately inoculated onto the cheese surface is Brevibacterium linens. During ripening the microflora of the surface changes into a not yet fully characterised one where Arthrobacter spp., Corynebacterium spp., Rhodococcus spp., other coryneform bacteria and yeasts like Debaryomyces hansenii and Geotrichum candidum grow (Brennan et al., 2002; Beresford and Williams, 2003). Propionic acid bacteria. Propionic acid bacteria (PAB) metabolise lactate to propionate, acetate, CO2 and H2O and are found in Swiss-type cheeses. The CO2 is responsible for the eye formation while acetic acid and propionate contribute to

35

flavour development. They survive the high cooking temperature required for these cheeses (50-52C) and their growth is stimulated by increasing the ripening temperature from 12 to 22C. PAB are found at levels of 108-109 cfu g-1 after a few weeks of ripening. Then the temperature is decreased to limit further growth (Cogan, 1996; Frank and Hassan; 1998). Coliforms. Coliforms are mostly present in raw milk cheeses. According to Bramley and McKinnon (1990), the presence of coliforms in raw milk is not evidence of direct faecal contamination as they can build up in milk residues and milking equipment or be the cause of mastitis. Coliforms are present in low levels in cheese and usually disappear or decrease after the first few weeks of ripening (Litopoulou-Tzanetaki and Tzanetakis, 1992; Macedo et al., 1995). The presence of coliforms can be the cause of blowing defect in white-brined cheeses (Abd El-Salam, 2003).

2.3. Source of non-starter bacteria In the EU, over 120 million tons of milk were produced in the year 2000 (http://europa.eu.int/comm/agriculture/publi/agrep2000/2000_it.pdf). Different housing conditions, different milking equipment and different storage conditions are found on farms. Once collected, the milk is cooled and the number and types of microorganisms present depend on the temperature and duration of storage (Bramley and McKinnon, 1990; Slaghuis et al., 1991). Milk is a highly nutritious food containing valuable sources of sugar (lactose), proteins (casein and whey proteins), vitamins and minerals. It is clear that milk can be contaminated in different ways and at different times before being cooled and processed. If it is not cooled promptly the bacteria will grow and cause it to spoil (Manners and Craven, 2003a). Unless cows suffer from mastitis, milk is sterile just before exiting the cows teats. The bacteria most commonly implicated in mastitis are Streptococcus agalactiae and Staphylococcus aureus, while Streptococcus uberis and Escherichia coli play a minor role. If contamination does not start from within the teats, milk becomes contaminated with microorganisms from the exterior of the udder. This

36

happens when cows teats become soiled with dung, mud, straw, sand etc. If not removed, this dirt is washed into the milk together with bacteria associated with it. These are mainly micrococci, staphylococci, streptococci, enterococci and bacilli (Bramley and McKinnon, 1990; Slaghuis et al., 1991). Air is not an important source of microorganisms in milk (bacterial loads of air seldom exceed 200 cfu/l) although small numbers may fall into the milk. Mainly micrococci but also coryneforms, Bacillus spores, streptococci and Gram-negative rods are responsible for contamination from the air (Bramley and McKinnon, 1990). Many water supplies from farms come from wells, lakes, springs. Some of them may be contaminated, e.g. with coliforms, streptococci and clostridia, if they are not properly protected from rodents, birds, insects and dust. For this reason hypochlorite is frequently added to the water if this water is used to wash and rinse equipment which may come in contact with milk, e.g. milking machines and pasteurizers. Improperly cleaned milking machines and milk storage tanks are the major sources of microorganisms in raw milk (Garg and Mital, 1991). Because of the complexity of the milking machines and their components, milk residues and microorganisms can remain trapped in crevices, joints, dead ends, ridges. A study by Mettler and Carpentier (1997) showed how easily gaskets of pasteurizers can wear and become contaminated with Bacillus, Microbacterium, Micrococcus, and Lactococcus spp., which predominate on milking equipment surfaces. Milkers and farmers are important not only for the dirt or dust they can dislodge but also for the organisms they carry on their clothes and hands. Waes (1973) showed that bacteria on hands are not very numerous but from the ones found almost 90% belong to Enterococcus faecalis. Enterococci may gain entrance to the milk primarily via contaminated water supplies, dung, equipment, insects and, as mentioned, handlers. They are generally found in raw milk in the range of 103 to 105cfu/ml raw milk. The most commonly found Enterococcus species are E. faecalis and E. faecium, which can also be the source of environmental mastitis (Jones, 1998).

37

2.4. Hygiene of milk and dairy products With a product like cheese, subject to risk of microbial contamination, awareness of health hazards arising from the consumption of contaminated food has grown in the last decades. Milk is a source of nutrition and must therefore be protected from contamination or deterioration as it can lead to infections, economic loss and maybe death (http://nano.med.umich.edu/food_bacteria_background.htm). A wide variety of factors can influence the microbial safety of cheese. Staphylococcus aureus, Salmonella spp. and Listeria monocytogenes are of concern in cheese. In the European council directive 92/46/EEC (Anonymous, 1992) Listeria monocytogenes and Salmonella spp. are categorized as pathogenic microorganisms, Staphylococcus spp. and Escherichia coli are categorized as organisms indicating poor hygiene while coliforms and the total count are guideline organisms which should help the producers ensuring proper operation of their establishments and in implementing the system and the procedure for carrying out their own checks on their production (Anonymous, 1992). A statistically based sampling plan and the decision criteria to be applied to a particular batch, based on examination of a prescribed number of sample units by definite methods has been defined by the EU (Table 4). The widely accepted sampling plan is the three-class plan as defined by the International Commission on Microbiological Specifications for Foods (Opinion of the Scientific Committee on Veterinary Measures Relating to Public Health, 1999). In Table 4 the 92/46/EEC directives are described for raw milk and cheese made from raw and thermized (not pasteurised) milk. Every country belonging to the European union has to follow the above-mentioned directive (Anonymous, 1992).

38

Table 4. Microbiological criteria for milk and cheese made from raw and thermized milk in cfu g-1 or ml-1 (92/46/EEC directive, Anonymous, 1992)
Products Raw cows' milk (1) pathogenic bacteria (m=M=c=0) organisms indicating poor hygiene (n = 5, c = 2) E. coli Listeria monocytogenes (3) Salmonella (4) Staphyloccus aureus n n m M m M M n 1 X 105 1 1 5 500 2000 Cheese made from raw milk and from thermized milk (2) 5 5 1000 1 X 104 1 X 104 1 X 105

indicator organisms (5)

Total counts

n number of sample units examined from a lot m the threshold value for the number of bacteria (the result is considered satisfactory if the number of bacteria in all sample units does not exceed m M maximally allowed value for the number of bacteria; the result is considered unsatisfactory if the number of bacteria in one or more sample units is M or more c number of sample units where the bacteria count may be between m and M the sampling being considered acceptable if the bacteria count of the other sample units is m or less. (1) Raw cows milk intended for direct human consumption. (2) Thermization is the heating of raw milk for at least 15 seconds at a temperature between 57C and 68C (Anonymous, 1992; 92/46/EEC, Chapter 1, Article 2, 6). (3) Absent in 1g (n = 1, c = 0) (4) Absent in 25g (n = 5, c = 0). The 25g sample has to consist of 5 specimens of 5g taken from different parts of the same food. (5) There are no directives for total coliforms for raw cows milk and cheese made from raw milk and from thermized milk.

39

Chapter 3. Baylough, an Irish cheddar-like cheese

3.1. Cheddar cheese As the cheese in this study is a Cheddar-like cheese, I will discuss the major characteristics of Cheddar cheese. Cheddar is one of the most widely produced and eaten cheeses in the world. Cheddar cheeses were originally made in England; today it is manufactured in many countries, particularly English speaking ones, all over the world. It is only made from raw or pasteurised cows milk. The milk is inoculated with starter strains (Lactococcus lactis ssp. lactis or Lactococcus lactis ssp. cremoris). After acidification to pH ~6.4 at 32C, rennet is added to form a coagulum. The curd is then cut and cooked at 38C. After the whey is drained the curd is cheddared. This means piling and re-piling blocks of warm curd on top of each other in the cheese vat for a period of time. These are worked and turned to further expel whey and also produce the characteristic fibrous texture of Cheddar cheese. During the cheddaring process, the curd also becomes further acidified (to pH 5.2-5.5) by the continuing action of the starter culture on residual lactose in the curd. After cheddaring the curd is milled and dry-salted. As salting slows down acidification, the stage at which it occurs is important in determining the fate of lactose. Cheddar cheese is dry-salted and this means that lactose is subsequently metabolised much slower than in brinesalted cheeses. This results in the presence of ~10 g lactose/kg cheese after moulding and overnight pressing. The lactose is metabolised completely within the first two weeks of ripening while, simultaneously, non-starter lactic acid bacteria, particulary facultatively heterofermentative lactobacilli (FHL), grow.

40

Fully cured Cheddar (curing occurs at 7-9C for 2 month to 2 years) is a semi-hard or hard cheese shaped like a drum. Normally, the colour of Cheddar ranges from white to pale yellow. Some Cheddars, however, have a colour added, e.g. annatto, which is extracted from the Brazilian plant, Bixa orellana, giving the cheese a yellow-orange colour (Cogan, 1996; www.foodscience.cornell.edu/fs406/FS406-L07.ppt). At the end of ripening, a typical Cheddar should have a pH of ~5.3. After salting the potential for further decrease in pH depends on the residual lactose level however, generally the high buffering capacity of the cheese curd prevents any further decrease in pH. The salt in moisture (S:M) ratio determines the actual pH by controlling the activity of the starter after salting. In Cheddar the S:M should lie between 4.0 and 6.0%. Other two parameters usually found to describe cheese are the fat in dry matter (FDM) and the moisture-in-the-non-fat solids (MNFS). As many cheeses now are found as half fat, reduced fat and full fat, the FDM value can change between ~28 and ~52 while the MNFS in Cheddar is usually ~54 +/-0.5 (Fenlon et al., 2000)

3.2. Farmhouse cheeses and Baylough In the 60s and 70s supermarket shopping became a trend. The pressure from large chain stores to supply cheaper, mass-produced cheese to meet the trend towards this supermarket shopping meant that farmhouse cheesemakers went out of business. Shelf life, ease of packaging and quantity came before taste and texture (Shils, 1997). However, in the last years, farmhouse cheese-production is rising (Lassaut, 2001; ONeill, 2000; Bord Bia, 2003). Farmhouse cheese is defined as cheese made from the milk of a single farm (Pujol, 1997) and is usually made with milk from the cheesemakers own herd. The self-sufficient cheesemaker, assured of the high quality of their own raw materials, is given a head start over larger creameries, which blend the milk of several different herds and then pasteurise it to a bland uniformity. Regarding the issue of premises located within farmyards, the issue is the proximity of the food premises, be it meat

41

or cheese, livestock, manure storage or animal feed storage. In fact, a good example is the farmhouse cheese sector where a production facility is located within the farmyard but appropriate separation ensures that there is no risk to the finished product (O`Mahony, 2002). Attributes such as taste, traditional and artisanal quality and healthiness and safety of the product have become primary factors motivating consumers to buy farmhouse products (Revell and Franois, 1997). In Ireland these features helped the consumption of farmhouse cheese rise from 500 tonnes in 1996 to 1000 tonnes in 2000 (ONeill, 2000). One of these Irish farmhouse cheeses is the Cheddar-like Baylough cheese. Although the chemical composition of Baylough fits quite well with the Cheddar characteristics, the cheese does not get a lot of cheddaring and ripening, which is why it is referred to as Cheddar-like. The farmhouse is located near Clogheen, Co. Tipperary. On the internet we read A mixed herd of Fresians and distinctive, Red and White Dutch cows provides the full-cream milk for Baylough, a hard-pressed, waxed cheese which can mature for many months. There are several varieties of this cheese, including Oak-smoked Garlic and Herbs Fresh Garlic) and they are sold locally in different stores around Ireland Baylough, (http://www.cheese.com/Description.asp?Name= http://www.cookingisfun.ie /letters/2002/Culinaryed18mar02.htm). Baylough is a raw-milk Cheddar-like cheese, made at farmhouse level, from the milk of a herd of 27 cows, by the herdowners wife. The milking parlour is located about 1 mile from the cheese making plant to which the milk is transported in a stainless steel tank before cheesemaking. The cheese is manufactured using a proprietary method (the manufacture steps are summarized in Figure 2) and a DL mixed-strain starter culture (Flora Danica, Chr. Hansens Laboratory), which contains 62% Lactococcus lactis ssp. cremoris, 19% Lactococcus lactis ssp. lactis biovar diacetylactis, 2% Lactococcus lactis ssp. lactis, 16% Leuconostoc cremoris and 1% unknown species (Lodics and Steenson, 1990) and was ripened for several months. Each cheese lot undergoes a microbiological control in a local laboratory. As with all raw milk cheeses, it was suspected to contain strains of the genus Enterococcus as part of the non-starter bacterial flora.

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Figure 2. Production of the Cheddar-like cheese Baylough. Raw Milk heated to 31C addition of a DL mixed strain starter culture* acidified milk (~pH 6.4, after ~30 min.) addition of rennet curd formation (after ~45 min.) cutting of the curd cooking at 38C (~30 min.) draining of the whey curd after drainage cheddaring, salting and milling curd in moulds waxing step ripening at 12C for ~8 weeks

Baylough cheese
*the starter culture contains 62% Lc. lactis ssp. cremoris, 19% Lc. lactis ssp. lactis biovar. diacetylactis, 2% Lc. lactis ssp. lactis, 16% Leuconostoc cremoris and 1% unknown species (Lodics and Steenson, 1990).

Chapter 4. enterococci

4.1. Description of the genus Enterococcus 4.1.1. Introduction Enterococci occur widely in the environment but are mainly associated with the gastrointestinal tract and, because of this, their presence in food is seen as an indicator of poor hygiene. They are an essential part of the natural microflora in many dairy products especially in Southern European countries. The source of enterococci in cheese is not clear. However, their presence in cheese is a matter of debate since they have recently emerged as nosocomial pathogens. On the other hand, although no consensus has been reached on whether enterococci should be considered as GRAS (generally recognized as safe), enterococci from dairy products show higher sensitivity to antibiotics and they have got a long history of safe use (Giraffa et al., 1997; Franz et al., 1999; Hardie and Whiley, 1997; Beresford and Williams, 2003)

4.1.2. Taxonomy of the genus Enterococcus The genus Enterococcus consists of Gram-positive, oxidase negative, catalase negative, non-spore forming, ovoid cells arranged in single cells, pairs or chains (Fig. 3). Enterococci are facultative anaerobes with an optimum growth temperature of 35C. In 1937 they were described by Sherman as organisms that grow from 10 to 45C, in 6.5% NaCl and at a pH of 9.6, survive heating at 60C for 30 min., and react

44

with Lancefield group D antisera (Stiles and Holzapfel, 1997). Since then things have changed and not all species meet all of these criteria. Exceptions include Enterococcus dispar (Collins et al., 1991), Enterococcus sulfureus (Martinez-Murcia and Collins, 1991) and Enterococcus malodoratus (Collins et al., 1984), which do not grow at 45C and Enterococcus cecorum and Enterococcus columbae, which do not grow at 10C (Devriese et al., 1993). Most enterococcal species are capable of growing in 6.5% NaCl. However, according to Devriese et al. (1993), E. cecorum, E. columbae and Enterococcus avium grow poorly and slowly or not at all in 6.5% salt. E. sulfureus, Enterococcus casseliflavus and Enterococcus mundtii are yellowpigmented (Martinez-Murcia and Collins, 1991). The motility seems to create some discordance; e.g. according to Devriese et al. (1993) only Enterococcus gallinarum is motile while Schleifer and Kilpper-Blz (1987) ascribe motility to E. gallinarum and E. casseliflavus and Morrison et al. (1997) to E. casseliflavus and Enterococcus flavescens. However, Collins et al. (1984) describe E. casseliflavus and E. gallinarum as non-motile. Haemolytic reactions may vary in accordance with the blood used (e.g. horse, sheep, calf) (Facklam and Teixeira, 1998).

Figure 3. Electronic microscope image of enterococci. Enterococci belong to the loosely defined group of lactic acid bacteria (LAB). One of the earliest definitions of LAB was given in 1919 by Orla-Jensen as Gram-

45

positive, non-motile, non-spore forming, rod- and coccus-shaped organisms that ferment carbohydrates and higher alcohols to form chiefly lactic acid. Current genera include Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, Pediococcus and Tetragenococcus among others (Stiles and Holzapfel, 1997). The name enterocoque was first used in a paper in 1899. The name was proposed to emphasize the intestinal origin of this strain. Several enterococcal species, like E. faecalis and E. faecium, originally belonged to the genus Streptococcus, a term coined in 1906 (Muray, 1990; Devriese et al., 1993; Deasy, 2000). It was only in 1984 when Schleifer and Kilpper-Blz showed by DNA-DNA and DNA-rRNA hybridisation that some species were so distantly related to streptococci that they should be transferred to another genus (Schleifer and KilpperBlz, 1984). At present, 27 different Enterococcus species are recorded on the list of valid names. Recently newly described strains comprise Enterococcus canis (De Graef et al. 2003), Enterococcus gilvus, Enterococcus pallens (Tyrrell et al. 2002), Enterococcus haemoperoxidus, Enterococcus moraviensis (Svec et al. 2001), Enterococcus phoeniculicola (Law-Brown and Meyers 2003), Enterococcus ratti (Teixeira et al. 2001), and Enterococcus villorum (Vancanneyt et al. 2001). Further reclassification may occur in the future. E. casseliflavus and E. flavescens for instance, can barely be distinguished (Descheemaeker et al. 1997) and Enterococcus solitarius is more closely related to Tetragenococcus than to Enterococcus (Williams et al., 1991).

4.1.3. Habitat The habitat of enterococci is very diverse as the nature of these bacteria allows them to survive and grow also in harsh environments. The main habitat is the gastrointestinal tract (GIT) of mammals (Franz et al., 1999). Enterococci are the predominant, Gram-positive cocci in human stools with 105-108 cfu/g faeces (Jett et al., 1994; Kleessen et al., 2000). E. faecalis is often the dominant species found in human faeces (Murray, 1990; Facklam and Teixeira, 1998; Franz et al., 1999). E. faecium, Enterococcus hirae, E. avium and Enterococcus durans have also been found (Murray, 1990; Franz et al., 1999; Khn et al., 2003). Some authors (Murray,

46

1990; Franz et al., 1999; Khn et al., 2003) suggest that the presence of E. faecium and E. faecalis in humans is dependent on the geographical location. Some enterococci are also found in the upper GIT, the lower and upper urogenital tracts and in the oral cavity (Murray, 1990; Morrison et al., 1997). Although enterococci are prevalent in the human intestinal tract, they are also frequently isolated from the animal intestinal tract. E. faecium is the most frequently occurring Enterococcus species in dairy cows (Latham et al., 1978; Devriese et al., 1992. Leclerc et al., 1996) but E. faecalis, E. hirae and E. casseliflavus are also found (Devriese et al., 1987; Devriese et al., 1992). In addition, enterococci are found in water, soil, plants, vegetables, birds and insects (Martin and Mundt, 1972; Devriese et al., 1992; Godfree et al., 1997; Facklam and Teixeira, 1998; Franz et al., 1999).

4.2. The occurrence of enterococci 4.2.1. Presence in cheese The two most important food products, which contain enterococci, are meat and cheese. The presence of enterococci in the GIT of animals implies a high potential of contamination of the meat at the time of slaughter. This can cause spoilage in raw meat like salami but also in cooked and processed meat, as enterococci are able to survive heat processing (Franz et al. 1999, Giraffa, 2002). In contrast, the presence of enterococci in cheese may be desirable and is, in some cases, seen as an essential part of the microflora. Enterococci are commonly found in high numbers in cheeses produced in Italy, France, Portugal, Spain and Greece from raw or pasteurized goats, ewes, water buffaloes or cows milk (Cogan et al., 1997) (Table 5). Different species of enterococci are found in cheese but E. faecalis and E. faecium are the species most commonly isolated (Neviani et al., 1982; Trovatelli et al., 1987; Fontecha et al., 1990; Litopoulou-Tzanetaki and Tzanetakis, 1992; Devriese et al., 1995; Arizcun et al., 1997; Cogan et al., 1997; Suzzi et al., 2000). The numbers of enterococci in cheese vary between 104 and 108 cfu/g cheese (Carrasco de Mendoza

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et al., 1992; Arizcun et al., 1997; Franz et al., 1999). In a survey of 48 samples of fresh, soft and ripened semi-hard cheeses, enterococci were found in more that 96% of them (Giraffa et al., 1997). Some workers have reported that they cause deterioration in flavour (Lopez-Diaz et al., 1995; Thompson and Marth, 1986), while others report enterococci to play a major role in improving flavour development and cheese quality (Dahlberg and Kosikowsky, 1948; Clark and Reinbold, 1966; Kurman, 1968; Neviani et al., 1982; Litopoulou-Tzanetaki et al., 1993). This is achieved by the production of flavour components such as acetaldehyde, acetoin and diacetyl (Trovatelli et al., 1987) and through proteolysis, lipolysis and citrate metabolism. These pathways were already discussed and only the contribution of enterococci will be mentioned here. Lipolysis and proteolysis are seen as the principal reactions responsible for the flavour development in Cheddar cheese (Dovat et al., 1970; Jensen et al., 1975a). Generally enterococci show very weak proteolytic activity, E. faecalis being the most proteolytic species (Jensen et al., 1975a; Arizcun et al., 1997; Suzzi et al., 2000). High proteolytic activities by enterococci have been reported in such different cheeses as Arza (Centeno et al., 1995), Cebreiro (Centeno et al., 1999) and Feta (Sarantinopoulos et al., 2002). Fayed et al. (1989) produced a yogurt-like product with Enterococcus spp. and found an overall high quality of the product as well as high proteolytic values. The lipolytic activity, i.e. the ability to hydrolyse milk triglycerides, is essential in bacteria used in cheese as it provides aroma due to liberation of volatile fatty acids. The lipolytic activity in enterococci is very variable; only E. faecalis showing a degree of lipolysis worthy of incorporating into the cheese (Carrasco de Mendoza et al., 1992). An increase in fatty acids has been observed in Cheddar cheese (Jensen et al., 1975b), Feta cheese (Sarantinopoulos et al., 2002), Picante cheese (Freitas et al., 1999) and Cebreiro cheese (Centeno et al., 1999) while none of the strains isolated by Suzzi et al. (2000) from Semicotto Caprino showed lipolytic activity.

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Table 5. European cheeses in which enterococci have been found. Country France Cheese Comt Beaufort Mont d'Or Roquefort Pichtogalo Chanion Feta/Teleme Orinotyri Cheddar-like Fiore Sardo Casu Axedu Fontina Toma Mozzarella Caciotta Semicotto caprino Pecorino Sardo Picante da Beira Baixa Serra da Estrela Manchego Arza Cabrales La Serena Majorero Milk Cow Cow Cow Ewe Ewe/Goat Ewe Ewe Cow Sheep Goat Cow Cow Cow Cow-Sheep Goat Ewe Goat+Ewe Ewe Cow Cow Cow + Ewe/Goat Ewe Goat Dominant Enterococcus spp. Enterococcus spp. Enterococcus spp. Enterococcus spp. E. faecalis Enterococcus spp. Enterococcus spp. E. faecalis E. casseliflavus/E. faecalis E. faecium E. faecium E. faecium E. faecium Enterococcus spp. Enterococcus spp. E. faecium/E. faecalis Enterococcus spp. E. faecium/E. faecalis E. faecium E. faecium E. faecalis E. faecium E. faecium E. faecalis Reference Bouton et al., 1998 Cogan and Rea, 1996 Sozzi and Maret, 1973 Devoyod, 1969 Papageorgiou et al., 1998 Tzanetakis and Litopoulou-Tzanetaki, 1992 Prodromou et al., 2001 present study Ledda et al., 1978 Cogan and Rea, 1996 Cogan and Rea, 1996 Caserio et al., 1976 Morea et al., 1999 Cogan and Rea, 1996 Suzzi et al., 2000 Mannu et al., 1999 Freitas et al., 1999 Macedo et al. 1995 Martinez-Moreno, 1976 Centeno et al., 1995 Nuez and Medina, 1979 Del Pozo et al., 1988 Fontecha et al., 1990

Greece

Ireland Italy

Portugal Spain

Little is known about the ability of enterococci to metabolize citrate. Early reports (Campbell and Gunsalus, 1944; Coventry, 1978) showed that enterococci metabolized citrate to acetate and formate. There appear to be no further detailed investigations of the metabolism of citrate by enterococci. Rea and Cogan (2003) found that enterococci metabolise citrate only when glucose has been exhausted.

4.2.2. Bacteriocin production Bacteriocins, called enterocins in the case of enterococci, are, according to Klaenhammer (1993), small proteins produced by microorganisms that can have narrow inhibit or broad-host ranges (McAuliffe et al., 2001). Enterocins belong to classes I, IIa, and IIc. These are described as follows: Class I: small peptides (<5 kD) containing the unusual amino acids, lanthionine or/and -methyl lanthionine (cyl LL and cyl LS), Class IIa: heat stable, non-lanthionine containing, Listeria-active peptides (<10 kD; Enterocin A, Enterocin CRL 35, Enterocin 31, Enterocin P, Enterocin SE-K4, Mundticine, Bacteriocin RC714), and Class IIc: heat stable, non-lanthionine containing, thiol-activated peptides requiring reduced cystein residues for activity (<10 kD; Enterocin B, Enterocin AS-48, Enterocin L50A, Enterocin L50B, Enterocin EJ97, Enterocin Q, Enterocin 1071A, Enterocin 1071B) (Foulquie-Moreno, 2002). Several authors have successfully used Enterococcus spp. (usually as cocultures) or the isolated enterocins in food production to reduce the occurrence of Listeria monocytogenes in cheese and meat (Giraffa et al., 1995; Nuez et al., 1997; Farias et al., 1999; Aymerich et al., 2000a; Aymerich et al., 2000b; Callewaert et al., 2000; Laukov and Czikkov, 2001; Rodriguez et al., 2001). Other authors have observed that enterocins lyse starter cells and free intracellular enzymes and thus accelerate the cheese ripening and increase flavour production (Garde et al., 1997; Oumer et al., 2001). Enterococci seem to produce enterocins also against Staphylococcus aureus, Clostridium spp. and Vibrio cholerae (Giraffa, 2003).

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4.2.3. Further applications of enterococci Enterococci have also been considered as indicators of faecal contamination of water and food (Kenner et al., 1961; Lopez et al., 1995, Audicana et al., 1995). Godfree et al. (1997) advise every microbiologist to decide which indicator system is more appropriate for the particular problem under consideration as several groups of microbes have been employed as indicator of faecal pollution. In the UK, enterococci are used as indicators of secondary faecal contamination in samples where E. coli are absent (Anonymous, 2002). Enterococci have also been suggested as starter adjuncts for their flavour and texture improving activity and have successfully been used as part of starter cultures in a variety of cheeses (Franz et al., 1999; Sarantinopoulos et al., 2002) and a yoghurt-like product (Fayed et al., 1989). Enterococci are usually part of a starter (as so called starter-adjuncts) as they have a low acidifying activity (Carrasco de Mendoza et al., 1992; Giraffa, 2003). Seale (1986) reported the use of enterococci as silage inoculants. Several authors describe enterococci as organisms with properties that allow them to be used as probiotics (Fuller, 1989; Dunne et al., 2001; Franz et al. 2001). The reduction of cholesterol levels was studied in vitro by Rossi et al. (1994, 1999) with E. faecium CRL 183. They showed a reduction of over 50% in the level of cholesterol. Another strain, which has also been thoroughly studied, is E. faecium SF68, which reduces the risk of developing antibiotic-associated diarrhoea or acute diarrhoea (Hgenauer et al., 1998, Vanderhoof and Young, 1998) and hepatic encephalopathy (Loguercio et al., 1995). A study by Benyacoub et al. (2003) proves a higher immune response in dogs after they received E. faecium SF68. Klein et al. (1998) state that E. faecium has mainly been used as an animal and human probiotic and E. faecalis primarily as a human probiotic. However, the FAO/WHO report (2001) recommends that enterococci not be used as a probiotics for humans due to their high level resistance to vancomycin and their association with nosocomial infections.

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4.3. Acquired and intrinsic antibiotic resistance of enterococci Antimicrobial resistance can be divided into two types: acquired and intrinsic resistance. Acquired resistance results from either a mutation in the host DNA or DNA acquired from an external source. Intrinsic (or inherent) is a species-specific resistance, in which the responsible genes are believed to reside in the chromosome (Murray, 1990).

4.3.1. Acquired antibiotic resistance and vancomycin resistance mechanism If acquired resistance does not occur by mutation, it occurs by acquisition of new DNA either by transduction (the transfer of genetic information between cells through the mediation of a virus [phage] particle), transformation (the transfer of genetic information from a donor to a recipient using naked DNA, a process not known to occur in enterococci), and conjugation (the unidirectional transfer of genetic information between cells by cell-to-cell contact; Murray, 1998). Murray (1990) quotes various papers where up to 80% of the enterococci have shown resistance to tetracycline. Several genes that encode antibiotic resistance have been found in Mycoplasma spp., Campylobacter spp., Neisseria gonorrhoeae and Clostridium spp., among others. Resistance to chloramphenicol and erythromycin and high-level resistance (HLR) to clindamycin have been reported (Morrison et al., 1997; Murray, 1998). HLR to glycopeptides such as vancomycin has been of particular concern as it confers resistance to an antibiotic, which has proven bactericidal against enterococci. An even bigger problem is represented by the threat of vancomycin resistant enterococci (VRE) which spread their resistant genes to more dangerous organisms such as methicillin-resistant Sta. aureus (MRSA) (Morrison et al., 1997). Noble et al. (1992) demonstrated that the vancomycin resistance from E. faecalis NCTC 12201 could be transferred by conjugation to Sta. aureus B111.

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Five patterns of vancomycin resistance are known: VanA, VanB, VanC, VanD and VanE. VanC and VanD are intrinsic and will be discussed in the following section (4.3.2.). VanA shows inducible high level resistance to the glycopeptides vancomycin (MIC >512g/ml) and teicoplanin (MIC >64g/ml, both are glycopeptides) and VanB, produces moderate to high level resistance to vancomycin and low-level resistance (LLR) to teicoplanin (0.5g/ml). VanA and VanB are most commonly found in E. faecium and E. faecalis, they are acquired and inducible and have also been found in lactococci, Oerskovia, arcanobacteria and Streptococcus bovis (Murray, 1998). Vancomycin, a highly efficient antibiotic against the majority of Grampositive bacteria, is a glycopeptide, which inhibits cell-wall synthesis. A peptidoglycan precursor in enterococci (as in most Gram postive bacteria) has a terminal D-alanyl-D-alanine group (D-Ala-D-Ala) to which vancomycin binds inhibiting cell-wall synthesis by interfering with the attachment of further building blocks to the cell wall (Gholizadeh and Courvalin, 2000). In vancomycin resistant enterococci vanA, which is a complex of genes (vanH, vanR, vanS, and vanX), produces, in the presence of vancomycin, a substrate with a terminal D-Ala-D-X, where X is usually lactate. This leads to a 1 000-fold drop in binding affinity of vancomycin to the D-Ala-D-Lac terminus than to the D-Ala-D-Ala terminus and is thus not able to block the building of the cell wall. At the molecular level, VanA and VanB resistance result from the same substitution at the termini of peptidoglycan chains involved in cell wall synthesis. They are acquired resistances and are transferable via transposons belonging to the Tn3 family (VanA) and to transposon Tn 1547 (VanB) (Gholizadeh and Courvalin, 2000). The vanD gene, which is related to vanA and vanB, also encodes D-Ala-D-Lac and is expressed constitutively. It is characterized by moderate levels of vancomycin and LLR to teicoplanin in E. faecalis and E. faecium (Park et al., 1997; Perichon et al., 1997). vanE, which is related to vanC, synthesizes D-Ala-D-Ser peptidoglycan precursors (Fines et al., 1999; Gholizadeh and Courvalin, 2000; Park et al., 1997; Perichon et al., 1997). Fraimow et al. (1994) reported the findings of vancomycin dependent enterococci (VDE).

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4.3.2. Intrinsic antibiotic resistance Enterococci are naturally resistant to a series of -lactams like penicillin, ampicillin, semisynthetic penicillins, and cephalosporins (MICs 2 to 32 g/ml, 1-4 g/ml, 8-50 g/ml, 6.3->100 g/ml, respectively). The resistance to clindamycin, and its related lincomycin, is another feature of enterococci with low-level resistance MICs ranging between 12.5 to 100 g/ml. Another important group of antibiotics comprises the aminoglycosides to which E. faecalis has low-level resistance e.g. kanamycin, streptomycin (250 g/ml), and gentamicin (8-64 g/ml) (Murray, 1990). The constitutive, non-transmissible VanC resistance is responsible for lowlevel resistance to vancomycin and teicoplanin. VanC occurs naturally in E. gallinarum (vanC-1 gene), E. casseliflavus (vanC-2 gene), and E. flavescens (vanC-3 gene). VanC resistance involves a switch to a D-Ala-D-ser terminus rather than a DAla-D-Lac terminus (Patel, 1999).

4.4. Virulence factors Enterococci are believed to be able to colonise the GIT as they are commensals of the human intestine. Colonization is not seen as a virulence factor but it may increase the potential pathogenicity of other strains. Eaton and Gasson (2001) and Franz et al. (2001) found more virulence traits in E. faecalis than in other Enterococcus species. Adherence to host tissue is considered crucial for infection and a first step towards colonization. Potential recipient strains (e.g., plasmid-free strains of E. faecalis) secrete small peptide signal molecules known as sex pheromones. These pheromones induce, in strains carrying a particular plasmid (donor cells), genes leading to the production of aggregation substance (Agg) that facilitates formation of mating aggregates with nearby recipients. An example of this system is the highly transmissible, pheromone responsive plasmid pAD1, which also carries the cytolysin determinants (Gilmore et al., 1994). Once a copy of the plasmid has been acquired, the recipient shuts down production of that pheromone but continues to secrete

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pheromones specific for other plasmids (Clewell, 1993). Without adhesion substances like Agg and surface carbohydrates, enterococci would be swept away by gastrointestinal motility. Agg may mediate adherence to urinary tract epithelial cells, resulting in urinary tract infection (UTI), and may promote adherence to endocardial tissue, resulting in endocarditis. Enterococci are believed to originate in the intestinal tract. They exit the epithelial cells spreading to distant sites (Jett et al., 1994). Agg mediates contact to other cells to facilitate plasmid transfer. It has also been shown that in some cases Agg promotes greater increases in enterococcal adherence to macrophages and phagocytosis. Once phagocytosed, enterococci promote intracellular survival due to inhibition of the oxidative burst (production by the phagocyte of large quantities of reactive oxygen species which are released into the phagocytic vesicle eliciting microbicidal results): an adhesin and invasin at the same time (Franz et al., 1999; Rakita et al., 1999; Smuth et al., 2000). Agg was found in E. faecalis strains in both food and clinical isolates (Eaton and Gasson, 2001; Franz et al., 2001; Semedo et al., 2003). Esp and Ace are two adhesins similar to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), which are protein adhesins that mediate adherence. Esp (enterococcal surface protein) is a chromosomally encoded enterococcal adhesin. The incidence of Esp was shown to be higher among clinical strains (i.e. from patients with bacteremia or endocardatis) of E. faecalis than isolates from healthy individuals (i.e. individuals without any history of hospitalization). Esp may help enterococci to evade detection by the immune system (Shankar et al., 1999). Franz et al. (2003) suggest that Esp promotes colonization also due to its similarity with MSCRAMM. Another putative virulent agent may be the Enterococcus faecalis adhesin Ace (adhesin of collagen from E. faecalis). Nallapareddy et al. (2000) showed that Ace was expressed by enterococci during human infections and that E. faecalis can adhere to different types of collagen and laminin (which are found in the extracellular matrix and used by microbes for colonization) but additional studies are needed to evaluate the contribution of Ace to the virulence of E. faecalis. Very little is also known about adhesin-like antigens (EfaAfs and EfaAfm of E. faecalis and E. faecium antigen A). So far EfaAfs is the only one of the two

55

antigens showing pathogenicity in animal models and is suggested to play a role in endocarditis (Singh et al., 1998). In some cases EfaA is the only virulence determinant present in food strains (Eaton and Gasson, 2001; Mannu et al., 2003). At present, there is no evidence of whether virulence determinants other than Agg play a role in invasion of translocation of enterococci (Franz et al., 2003; Temedo et al., 2003). Cytolysin (haemolysin) is a toxin, which enhances virulence and is expressed by E. faecalis strains associated with disease. It causes rupture of a variety of target membranes, including bacterial cells, erythrocytes, and other mammalian cells. Its activity is observed as a haemolytic zone on blood agar (Huycke et al, 1998). Its operon is transmissible by plasmids. Cytolysin is located on the highly transmissible pheromone-responsive conjugative plasmid, pAD1 (Jett et al., 1994). Cytolysin is involved in the majority of infections, it is also found in enterococci isolated from healthy humans but can also be absent from enterococci causing disease. Cytolysin is therefore not a prerequisite for virulence (Johnson, 1994). Gelatinase (GelE) and serine protease (SprE) are two proteases, which are important in the pathogenicity in animal models. Pillai et al. (2002) studied the presence of fsr, the genes that regulate the expression of gelE and sprE. The fsr locus is present in 70% of E. faecalis clinical isolates and is, according to Pillai et al. (2002), an indication for a virulent sub-population within enterococci, which generally have a low pathogenic potential. No studies have yet been carried out on the association of the hyaluronidase of enterococci and infection (Jett et al., 1994; Franz et al., 1999; Mundy et al., 2000).

4.5. The spread of VRE in Europe and the USA Over the last two decades enterococci have gained importance as pathogens for nosocomial infections (hospital acquired infections or HAI). They are the cause of a variety of infections including endocarditis and neonatal, central nervous system or respiratory tract infections (Hardie and Whiley, 1997). They may also infect the abdomen, biliary tract, burn wounds, soft tissues, paranasal sinuses, ear, eye, and

56

periodontal tissue (Holdeman et al., 1976). Of major concern are the sources of hospital-acquired infections. Many of these have been identified but a high percentage remains obscure in origin. Some presumably originate from the GIT. It is believed that enterococci translocate across an intact intestinal tract and spread hematogenously to distant sites (Graninger and Ragette, 1992; Jett et al., 1994; Adams, 1999). Enterococci have become the focus of attention also due to their increasing resistance to antibiotics, especially vancomycin. In Europe the first VRE were reported in 1986 and in the USA in 1988 (Morrison et al., 1997). In 1993, VRE were the third leading cause of nosocomial infections in the USA (Aguirre and Collins, 1993), second in 1995 (Morris et al, 1995), and first in 1998 (Huycke et al, 1998), accounting for ~10% of such cases (Witte, 1997; Franz et al., 1999; Kim, 1999). In Europe their incidence is much lower. In 1995 VRE accounted for <1% for the infections in Belgium and Italy, 1-5% in Germany, The Netherlands, Portugal, Spain and Switzerland, with only France showing high levels (9.1%) (Witte, 1997). A more recent study by Goossens et al. (2003) shows that the UK and Italy were the countries with the highest rate of VRE with 10.4% and 19.6%, respectively. VRE are mostly endemic and monoclonal, favouring the suggestion of a common source or cross-contamination (e.g., the use of rectal thermometers). From 1990 to 1992 a clone of VanB phenotype E. faecium was isolated from nine patients at three hospitals in the USA proving evidence of an inter-hospital clonal transmission (Kim, 1999). Although the degree to which VRE contribute to mortality has proven difficult to estimate because of co-morbidities often present simultaneously (Chavers et al., 2003), about 85-90% of the enterococcal infections are thought to be caused by E. faecalis while 5-10% are caused by E. faecium (Patel, 1999, Adams, 1999). According to Coque et al. (1996), in the USA VRE are characterized by nosocomial outbreaks, while in Europe, the isolation of VRE from healthy humans, animals and environmental sources is considered to indicate that these organisms are part of the normal flora and suggest that the food chain may be the origin. Since the first outbreak of VRE the use of vancomycin has constantly risen. In 1996, 11 200 kg of vancomycin were used in the US, where it was introduced in the 1950s, while only a quarter of that amount was used in France, Germany, Italy, UK and the Netherlands combined (Kirst et al., 1998). Due to the minor use of

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antibiotics in Europe the problem of VRE has not grown to the same proportions as in the US (Wegener et al., 1999). In Europe and Australia antibiotics such as avoparcin (avoparcin and vancomycin belong to the same class of glycopeptides) are used to a great extent as growth promoters for animals. In the USA avoparcin has not been approved for use in animals but the human use of vancomycin is far greater than in Europe (Collignon, 1999). Evidence that feeding antibiotics to chickens induces selection of antibiotic resistant enterococci (ARE), is provided by the study where the antibiotic resistance levels were high when antibiotics were used (up to 100%), while the resistance level reaches 0% when the antibiotics were not been used (Collignon, 1999; Teuber et al., 1999). Bager et al. (1997) report that there is a link between the use of avoparcin and VRE in animals. From the animals, ARE are transmitted to food as proven by numerous studies where ARE, resistant to a whole range of antibiotics (Teuber et al., 1999), are found in dairy products (Teuber, 1996 and 1999; Giraffa et al., 2000), meat products (Wegener et al., 1997; Quednau et al., 1998; Son et al., 1999) and even vegetables such as salads, tomatoes and peppers (Corpet, 1998). But ARE are not only isolated from food but also from livestock, animal carcasses, hospitalised patients and non-hospitalized patients (McDonald et al., 1997; Witte, 1997). McDonald et al. (1997) report a higher prevalence of VRE among people working on animal farms or meat processing plants than among urban residents in The Netherlands. This study, together with those of van den Braak et al. (1998) and Collignon (1999), suggests that VRE enter the human GIT by working with or ingesting contaminated food. In Europe several antibiotics used as growth promoters have been banned: avoparcin in January 1997, ardacin in January 1998 and bacitracin zinc, virginiamycin, tylosin phosphate and spiramycin in December 1998. After that ban a sharp drop of VRE in food has occurred in Europe (Teuber et al., 1999). There are only four substances still authorized as growth promoting agents and none of them belong to classes used in human or veterinary medicine (EUcommunication, 2001). The four substances are monensin sodium, salinomycin sodium, flavophospholipol and avilamycin (EU-Council regulation, 1998) but there are plans to ban also these four growth promoters from 1 January 2006 (EU-common position, 2003).

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4.6. Enterococci in food: yes or no? LAB are ubiquitous in fermented foods and are common components of the human GIT. For hundreds of years humans have eaten food containing LAB and this has led to the general conclusion that LAB are safe. There have been a number of reports of human infection caused by LAB. Not only enterococci, but also lactobacilli, leuconostocs, pediococci, lactococci and aerococci have been isolated from humans and animals and considered as pathogens (Rogasi et al., 1998; Scano et al., 1999; Barton et al., 2001; Ebnother et al., 2002; Gillespie et al., 2002; Cheng et al., 2003; Schmidtke and Carson, 2003). As has been observed in the previous sections, enterococci occupy a special position among LAB. Virulence factors, which seem to be strain specific (Franz et al., 2001), have often been found in enterococci isolated from different sources, also from food (Eaton and Gasson, 2001; Franz et al., 2001). Franz et al. (2003) argue that strains with multiple virulence factors pose a higher risk than strains with a single one but that clear data are lacking on this issue. They raise the question whether food strains possess an intrinsic lower pathogenic potential or not. A view also shared by Semedo et al. (2003) who argue that a more permissive attitude regarding their use in artisanal cheeses may be pursued as only a low risk is involved. As it is, knowledge on virulence factors is not satisfactory, and comparative studies between food and clinical isolates are limited. For Huycke et al. (1998) the problem lies in the limited knowledge concerning colonization . But while some time ago enterococci were put forward as co-cultures in food, now scientists are aware of the possible health hazard enterococci can pose. Can cause human disease and might be a hazard to workers is cited from the last directive from the European Parliament, which puts Enterococcus spp. into risk group 2 (EU-directive, 2000). Enterococci are still considered for use as probiotics or starter cultures, but with reservations: strains should carefully be evaluated for the presence of all known virulence traits, suggest Franz et al. (2003) and Giraffa (2002). Both scientists, however, are aware that enterococci may readily acquire virulence determinants due to their gene transfer mechanism, even in meat and cheese, as shown by Cocconcelli et al (2003). Teuber et al. (1996, 2003) find it irresponsible to feed ARE to people, knowing that they possess gene transfer mechanisms able to transmit resistances to

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other microorganisms. They suggest that cheese should be produced only with pasteurized or microfiltered milk. The European Union looks at this problem on a higher scale creating awareness on the use and misuse of antimicrobial agents (EUcommunication, 2001) as there is evidence that enterococci become vancomycin resistant. This view is shared also by other scientists (Bager et al., 1997; McDonald et al., 1997; Collignon, 1999; Teuber et al., 1999). Morbidity of enterococcal infections is described differently. According to Franz et al. (2003) and Garbutt et al. (2000) morbidity is low, although Jett et al. (1994) and Graninger and Ragette (1992) report mortality rates of 31% and 43%, respectively, to enterococcal bacteremia. Peters et al. (2003) isolated enterococci from food in Germany and found both enterococci that were sensitive to antibiotics used to treat enterococcal bacteremia and enterococci that were resistant towards tetracyclin, which the authors regard as a cause of concern. Several aspects still remain to be elucidated: which and how virulence factors contribute to a disease and whether virulence traits-containing enterococci can be harmless. The path of transmission in the environment, the strategy of enterococci to cause diseases and whether enterococci from food possess an intrinsically lower pathogenicity are still not clear. Another important question is whether VRE acquisition increases a patients severity of illness or whether a patients severity of illness increases the likelihood of VRE acquisition (Chavers et al., 2003). Only after answering these questions, can a risk assessment be made and control measures taken (Huycke et al., 1998). Cimons (2001) has already an apocalyptic view (Imagine a world without fine, aged cheeses made from raw milk no Gruyre, no Parmigiano Reggiano, no Roquefort, no Vermont Shepherd) having in mind a world, where milk unavoidably has to be pasteurized or even, more dramatically, where these cheeses have to be removed from the shops altogether.

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References

Abd El-Salam, M. H. 2003. White brined varieties. p. 1098-1103, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Adams, M. R. 1999. Safety of industry lactic acid bacteria. J. Biotechnol. 68:171-178. Aguirre, M., and M. D. Collins. 1993. Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75:95-107. Anonymous. 1992. 92/46/EEC. Council Directive of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heat-treated milk and milk-based products. Official Journal of the European Communities L 268, 14/9/1992. Anonymous. 2002. The microbiology of drinking water (2002) Methods for the Examination of waters and associated materials. Part 5 A method for the isolation and enumeration of enterococci by membrane filtration. http://www.dwi.gov.uk/regs/pdf/part5.pdf, Drinking water inspectorate, London, UK Arizcun, C., Y. Barcina, and P. Torre. 1997. Identification and characterization of proteolytic activity of Enterococcus spp. isolated from milk and Roncal and Idiazabal cheese. Int. J. Food Microbiol. 38:17-24. Audicana, A., I. Perales, and J. J. Borrego. 1995. Modification of kanamycin-esculin-azide agar to improve selectivity in the enumeration of fecal streptococci from water samples. Appl. and Environ. Microbiol. 61:4178-4183. Aymerich, T., M. G. Artigas, M. Garriga, J. M. Monfort, and M. Hugas. 2000a. Effect of sausage ingredients and additives on the production of enterocins A and B by Enterococcus faecium CTC 492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages. J. Appl. Microbiol. 88:686-694. Aymerich, T., M. Garriga, J. Ylla, J. Vallier, J. M. Monfort, and M. Hugas. 2000b. Application of enterocins as biopreservatives against Listeria innocua in meat products. J. Food Prot. 63:721-726. Bager, F., M. Madsen, J. Christensen, and F. M. Aarestrup. 1997. Avoparcin used as a growth promoter is associated with the occurrence of vancomycin-resistant Enterococcus faecium on Danish poultry and pig farms. Prev. Vet. Med. 31:95-112. Banks, J. M., and D. S. Horne. 2003. Chemistry of gel formation. p. 1056-1062, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Barton, L. L., E. D. Rider, and R. W. Coen. 2001. Bacteremic infection with Pediococcus: vancomycin-resistant opportunist. Pediatrics. 4:775-776. Benyacoub, J., G. L. Czarnecki-Maulden, C. Cavadini, T. Sauthier, R. E. Anderson, E. J. Schiffrin, T. von der Weid. 2003. Supplementation of food with Enterococcus faecium (SF68) stimulates immune functions in young dogs. J. Nutr. 133:1158-62. Beresford, T., A. Williams. 2003. Microbiological changes during ripening. In Cheese: Chemistry, Physics, Microbiology, P. F. Fox, P. McSweeney, T. Guinee and T. M. Cogan (eds.), 3rd edition, Academic Press. London (in press).

61

Bhowmik, T., and E. H. Marth. 1990 Role of Micrococcus and Pediococcus species in cheese ripening: a review. J. Dairy Sci. 73:859-866. Bord Ba. 2003. Bord Bia Annual Review 2002. Irish Food Board, Dublin, Ireland. Bottazzi, V. 2003. Manufacture of extra hard cheeses. p. 1067-1073, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Bouton, Y., P. Guyot, and R. Grappin. 1998. Preliminary characterization of microflora of Comt cheese. J. Appl. Microbiol. 85:123-131. Bramley, A. J., and C. H. McKinnon. 1990. The microbiology of raw milk, p. 163-208, In The microbiology of the milk, R. K. Robinson (ed.), 2nd ed. Elsevier Applied Science, London, UK. Brennan, N. M., A. C. Ward, T. P. Beresford, P. F. Fox, M. Goodfellow, and T. M. Cogan. 2002. Biodiversity of the bacterial flora on the surface of a smear cheese. Appl. Environ. Microbiol. 68:820-830. Callewaert, R., M. Hugas, and L. De Vuyst. 2000. Competitiveness and bacteriocin production of Enterococci in the production of Spanish-style dry fermented sausages. Int. J. Food Microbiol. 57:33-42. Carrasco de Mendoza, M., H. E. Scarinci, M. del Huerto Garat, and A. C. Simonetta. 1992. Technological properties of enterococci in lactic starters: acidifying and lipolytic activities. Microbiologie-Aliments-Nutrition. 10:289-293. Caserio, G., M. A. Bianchi, G. Beretta, C. Cantoni, and F. Ardizzone. 1976. Ricerche preliminari sulla maturazione della Toma vercellese. Il latte, 2:67-80. Centeno, J. A., A. Cepeda, and J. L. Rodrquez-Otero. 1995. Identification and preliminary characterization of strains of enterococci and micrococci isolated from Arza raw cows-milk cheese. Die Nahrung. 39:55-62. Centeno, J. A., S. Menendez, Ma. Hermida, and J. L. Rodrguez-Otero. 1999. Effects of the addition of Enterococcus faecalis in Cebreiro cheese manufacture. Int. J. Food Microbiol. 48:97-111. Chavers, L. S., S. A. Moser, W. H. Benjamin Jr., S. E. Banks, J. R. Steinhauer, A. M. Smith, C. N. Johnson, E. Funkhouser, L. P. Chavers, A. M. Stamm, and K. B. Waites. 2003. Vancomycin-resistant enterococci: 15 years and counting. J. Hosp. Inf. 53:159-171. Cheng, W., C. H. Wang, and J. C. Chen. 2003. Effect of benzalkonium chloride stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii. Dis. Aquat. Organ. 53:223-229. Cimons, M. 2001. Food safety concerns drive FDA review of fine cheeses. ASM News. 67:85-89. Clark, W. S., and G. W. Reinbold. 1966. Enterococci in young Cheddar cheese. J. Dairy Science. 49:1214-1218. Clewell, D. B. 1993. Bacterial sex pheromone-induced plasmid transfer. Cell. 73:9-12. Cocconcelli, P. S., D. Cattivelli, and S. Gazzola. 2003. Gene transfer of vancomycin and tetracycline resistance among Enterococcus faecalis during cheese and sausage fermentations. Int. J. Food Microbiol. 88:315-323.

62

Cogan, T. M. 1996. History and taxonomy of starter cultures, p. 1-22, In Dairy Starter Cultures, T. M. Cogan and J.-P. Accolas (ed.). VCH Publishers, New York, USA. Cogan, T. M., M. Barbosa, E. Beuvier, B. Bianchi-Salvadori, P. S. Cocconcelli, I. Fernandes, J. Gomez, R. Gomez, G. Kalantzopoulos, A. Ledda, M. Medina, M. C. Rea, and E. Rodriguez. 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64:409-421. Cogan, T. M., and M. C. Rea. 1996. Artisanal European Cheeses. Published by the European Commission, Directorate Generale XII, Science, Research and Development, Office for Official Publications of the European Communities, 1996, Luxembourg. Collignon, P. J. 1999. Vancomycin-resistant enterococci and use of avoparcin in animal feed: is there a link? Med. J. Aust. 171:144-146. Collins, M. D., D. Jones, J. E. A. Farrow, R. Kilpper-Blz, and K. H. Schleifer. 1984. Enterococcus avium nom. rev., com. nov.; E. casseliflavus nom. rev., com. nov.; E. durans nom. rev., com. nov.; E. gallinarum com. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol. 34:220-223. Collins, M. D., U. M. Rodrigues, N. E. Pigott, and R. R. Facklam. 1991. Enterococcus dispar sp. nov. a new Enterococcus species from human sources. Lett. Appl. Microbiol. 12:95-98. Coque, T. M., J. F. Tomayko, S. C. Ricke, P. C. Okhyusen, and B. E. Murray. 1996. Vancomycin-resistant enterococci from nosocomial community and animal sources in the United States. Antimicrob. Agents Chemother. 40:2605-2609. Corpet, D. E. 1998. Antibiotic resistant bacteria in human food. Revue Md. Vt. 149:819-822. Dahlberg, A. C., and F. V. Kosikowsky. 1948. The development of flavor in American Cheddar cheese made from pasteurized milk with Streptococcus faecalis starter. J. Dairy Sci. 31:275284. Deasy, B. M. 2000. Design of 16S rRNA-targeted primers for identification of species of Pediococcus, p. 159-176. In Method for the rapid identification of dairy lactic acid bacteria. Ph.D. thesis at DPRC Moorepark, Fermoy and Department of Microbiology, University College Cork, Ireland. De Graef E. M., L. A. Devriese, M. Vancanneyt, M. Baele, M. D. Collins, K. Lefebvre, J. Swings, and F. Haesebrouck. 2003. Description of Enterococcus canis sp. nov. from dogs and reclassification of Enterococcus porcinus Teixeira et al. 2001 as a junior synonym of Enterococcus villorum Vancanneyt et al. 2001. Int. J. Syst. Evol. Microbiol. 53:1069-1074. Delgado, S., and B. Mayo. 2004. Phenotypic and genetic diversity of Lactococcus lactis and Enterococcus spp. strains isolated from Northern Spain starter-free farmhouse cheese. Int. J. Food Microbiol. 90:309-319. Del Pozo, B. F., P. Gaya, M. Medina, M. A. Rodriguez-Marin and M. Nuez. 1988. Changes in the microflora of La Serena ewes milk cheese during ripening. J. Dairy Res. 55:449-455. Descheemaeker, P., C. Lammens, B. Pot, P. Vandamme, and H. Goossens. 1997. Evaluation of arbitrarily primed PCR analysis and Pulsed-Field Gel Electrophoresis of large genomic DNA fragments for identification of enterococci important in human medicine. Int. J. Syst. Bacteriol. 47: 555-561. Devoyod, J. J. 1969. La flore microbienne du fromage de Roquefort, IV. Les entrocoques. Lait. 49:637-650.

63

Devriese, L. A., A. Van de Kerckhove., R.Kilpper-Blz, and K. H. Schleifer. 1987. Characterization and identification of Enterococcus species isolated from the intestines of animals. Int. J. System. Bacteriol. 37:257-259. Devriese L. A., L. Laurier, P. De Herdt, and F. Haesebrouck. 1992. Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows. J. Appl. Bacteriol. 72:29-31. Devriese, L. A., B. Pot, and M. D. Collins. 1993. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75:399-408. Devriese, L. A., B. Pot, L. Van Damme, K. Kersters, and F. Haesebrouck. 1995. Identification of Enterococcus species isolated from foods of animal origin. Int. J. Food Microbiol. 26:187197. Dovat, A. M., G. W. Reinbold, E. G. Hammond, and E. R. Vedamuthu. 1970. Lipolytic and proteolytic activity of enterococci and lactic group streptococci isolated from young Cheddar cheese. J. Milk Food Tech. 33:365-372. Dunne, C., L. Murphy, S. Flynn, L. OMahony, S. OHalloran, M. Feeney, D. Morrisey, G. Thornton, G. Fitzgerald, C. Daly, B. Kiely, E. M. M. Quigley, G. C. OSullivan, F. Shanahan, and J. K. Collins. 1999. Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical traits. Antonie van Leeuwenhoek. 76:279-292. Eaton, T. J., and M. J. Gasson. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67:1628-1635. Ebnother, C., M. Altwegg, J. Gottschalk, J. D. Seebach, and A. Kronenberg. 2002. Aerococcus urinae endocarditis: case report and review of the literature. Infection. 30:310-313. EU-Common position. 2003. Common Position (EC) No 20/2003 adopted by the Council on 17 March 2003 with a view to adopting Regulation (EC) No/2003 of the European Parliament and of the Council of on additives for use in animal nutrition. Official Journal of the European Union C 113 E/1. 13.5.2003. (The title of the common position is not complete as it is yet a draft for proposal.) EU-communication. 2001. Communication from the commission on a community strategy against antimicrobial resistance. Commision of the European community. Volume I, COM(2001) 333 final, Brussels, 20.06.2001. http://europa.eu.int/comm/health/ph/others/antimicrobresist/am_02_en.pdf EU-Council regulation. 1998. Council regulation (EC) No 2821/98 of 17 December 1998 amending, as regards withdrawal of the authorisation of certain antibiotics, Directive 70/524/EEC concerning additives in feedingstuffs. Official Journal of the European Communities. L 351/4. 29.12.1998. EU-directive. 2000. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work. Official Journal of the European Communities. L 262/21. 17.10.2000. Facklam, R. R., and L. M. Teixeira. 1998. Enterococcus. p. 669-682. In Topley & Wilsonss Microbiology and microbial infections, A. Balows and B. I. Duerden (eds.), vol. II (systematic bacteriology), 9th, Arnold, London, UK FAO. 1990. The technology of traditional milk products in developing countries. Anim. Production and Health Paper No. 85. (http://www.fao.org/docrep/003/t0251e/t0251e00.htm)

64

FAO/WHO report. 2001. Health and Nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Report of a Joint FAO/WHO expert consultation. Cordoba, Argentina. 1-4 October 2001. http://www.fao.org/es/ESN/Probio/probio.htm FAOSTAT. 2001. Food Balance Sheets of 2001 of the Statistical Database of the FAO. http://apps.fao.org/lim500/wrap.pl?FoodBalanceSheet&Domain=FoodBalanceSheet&Langua ge=english Farias, M. E., M. Nuez de Kairuz, F. Sesma, J. Palacios, A. P. de Ruiz Holgado, and G. Oliver. 1999. Inhibition of Listeria monocytogenes by the bacteriocin enterocin CRL35 during goat cheese making. Milchwiss. 54:30-32. Farkye, N. Y. 2003. Chemistry and microbiology of maturation. p. 1062-1066, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Fayed, E. O., A. E. A. Hagrass, A. A. Aly, and Y. A. El-Samragy. 1989. Use of enterococci starter culture in the manufacture of a yogurt-like product. Cult. Dairy Prod. J. February:16-23. Fenlon, M. A., T. P. Guinee, C. Delahunty, J. Murray, and F. Crowe. 2000. Composition and sensory attributes of retail Cheddar cheese with different fat contents. J. Food Comp. Anal. 13:13-26 Fines, M., B. Perichon, P. Reynolds, D. F. Sahm, and P. Courvalin. VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405. Antimicrob. Agents Chemother. 43:2161-2164. Fontecha, J., C. Pelaez, M. Juarez, T. Requena, and C. Gomez. 1990. Biochemical and microbiological characteristics of artisanal hard goats cheese. J. Dairy Sci. 73:1150-1157. Foulqui-Moreno, M. R. 2002. Microbiological and technological characterization of bacteriocinproducing enterococci. Ph.D. thesis at VUB, Faculty of Sciences, Department of Applied and Biological Sciences, Brussels, Belgium. Fox., P. F. 1993. Cheese: An overview. p. 1-35, In Cheese: Chemistry, Physics & Microbiology, P. F. Fox (ed.), Vol. 1- General Aspects, 2nd Edition, Academic Press, London, UK. Fox, P. F., T. P. Guinee, T. M. Cogan, P. L. H. McSweeney. 2000. Microbiology of cheese ripening. p. 206-235, In Fundamentals of cheese science, (no eds.) Aspen Publishers, USA. Fox, P. F., and McSweeney, P. L. H. 1993. Proteolysis in cheese during ripening. Food Rev. Int. 12:457-509. Fraimow, H. S., D. L. Jungkind, D. W. Lander, D. R. Delso, and J. L. Dean. 1994. Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth. Ann. Intern. Med. 121:22-26. Frank, J. F., and A. N. Hassan. 1998. Starter Cultures and their use. p. 213-249, In Applied dairy microbiology, E. M. Marth, and J. M. Steele (eds.). Marcel Dekker, Inc., New York, USA. Franz, M. A. P. C., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47:1-24 Franz, M. A. P. C., A. B. Muscholl-Silberhorn, N. M. K. Yousif, M. Vancanneyt, J. Swings, and W. H. Holzapfel. 2001. Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Appl. Environ. Microbiol. 67:4385-4389.

65

Franz, M. A. P. C., M. E. Stiles, K. H. Schleifer, and W. H. Holzapfel. 2003. Enterococci in foods - a conundrum for food safety. Int. J. Food Microbiol. 88:105-122. Freitas, A. C., A. E. Pintado, M. E. Pintado, and F. X. Malcata. 1999. Role of dominant microflora of Picante cheese on proteolysis and lipolysis. Int. Dairy J. 9:593-603. Fuller, R. 1989. Probiotics in man and animals; a review. J. Appl. Bacteriol. 66:365-378. Garbutt, J. M., M. Ventrapragada, B. Littenberg, and L. M. Mundy. 2000. Association between resistance to vancomycin and death in cases of Enterococcus faecium bacteremia. Clin. Inf. Dis. 30:466-472. Garde, S., P. Gaya, M. Medina, and M. Nuez. 1997. Acceleration of flavour formation in cheese by a bacteriocin-producing adjunct lactic culture. Biotechn. Lett. 19:1011-1014. Garg, S. K., and B. K. Mital. 1991. Enterococi in milk and milk products. Crit. Rev. Microbiol. 18:15-45. Gholizadeh, Y., and P. Courvalin. 2000. Acquired and intrinsic glycopeptide resistance in enterococci. Int. J. Antimicrob. Agents. 16:S11-S17. Gilmore, M. S., R. A. Segarra, M. C. Booth, C. P. Bogie, L. R. Hall, and D. B. Clewell. 1994. Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants. J. Bacteriol. 176:7335-7344. Giraffa, G., N. Picchioni, E. Neviani, and D. Carminiati. 1995. Production and stability of an Enterococcus faecium bacteriocin during Taleggio cheesemaking and ripening. Food Microbiol. 12:301-307. Giraffa, G., D. Carminati, and E. Neviani. 1997. Enterococci isolated from dairy products: A review of risks and potential technological use. J. Food Protection. 60:732-738. Giraffa, G., A. M. Olivari, and E. Neviani. 2000. Isolation of vancomycin-resistant Enterococcus faecium from Italian cheeses. Food Microbiol. 17:671-677. Giraffa, G. 2002. Enterococci from foods. FEMS Microbiol. Lett. 26:163-171. Giraffa, G. 2003. Functionality of enterococci in dairy products. Int. J. Food Microbiol. 88:215-222. Godfree, A. F., D. Kay, and M. D. Wyer. 1997. Faecal streptococci as indicators of faecal contamination in water. J. Appl. Microbiol. Symposium Supplement. 83:110S-119S. Goossens, H., D. Jabes, R. Rossi, C. Lammens, G. Privitera, and P. Courvalin. 2003. European survey of vancomycin-resistant enterococci in at-risk hospital wards and in vitro susceptibility testing of ramoplanin against these isolates. J. Antimicrob. Chem. 51:Suppl. S3, iii 5-iii 12. Graninger, W., and R. Ragette. 1992. Nosocomial bacteremia due to Enterococcus faecalis without endocardatis. Clin. Inf. Dis. 15:49-57. Ha, J. K., and R. C. Lindsay. 1993. Release of volatile branched-chain and other fatty acids from ruminant milk fats by various lipases. J. Dairy Sci. 76:677-690. Hardie, J. M., and R. A. Whiley. 1997. Classification and overview of the genera Streptococcus and Enterococcus. J. Appl. Microbiol. Symposium Supplement. 83:1S-11S. Hill, A. R. 2000. Welcome to our cheese site. Edited by the University of Guelph, Ontario, Canada. Downloaded from http://www.foodsci.uoguelph.ca/cheese.

66

Hgenauer, C., H. F. Hammer, G. J. Krejs, and E. C. Reisinger. 1998. Mechanisms and management of antibiotic-associated diarrhea. Clin. Inf. Dis. 27:702-710. Holdeman, L. V., I. J. Good, and W. E. C. Moore. 1976. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31:359-375. Hugenholtz, J. 1993. Citrate metabolism in lactic acid bacteria. FEMS Microbiol. Rev. 12:165-168. Huycke, M. M., D. F. Sahm, and M. S. Gilmore. 1998. Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg. Inf. Dis. 4:239-249. Jensen, J. P., G. W. Reinbold, C. J. Washam, and E. R. Vedamuthu. 1975a. Role of enterococci in Cheddar cheese: proteolytic activity and lactic acid development. J. Milk Food Technol. 38:3-7. Jensen, J. P., G. W. Reinbold, C. J. Washam, and E. R. Vedamuthu. 1975b. Role of enterococci in Cheddar cheese: organoleptic considerations. J. Milk Food Technol. 38:142-145. Jett, B. D., M. M. Huycke, and M. S. Gilmore. 1994. Virulence of enterococci. Clin. Microbiol. Rev. 7:462-478. Johnson, A.P. 1994. The pathogenicity of enterococci. J. Antimicrob. Chemother. 33:1083-1089. Johnson, M. E. 1998. Cheese products. p. 213-249, In Applied dairy microbiology. E. M. Marth, and J. M. Steele (eds.). Marcel Dekker, Inc., New York, USA. Jones, T. O. 1998. Bacteriology of clinical mastitis in England and Wales historical perspective In: Proceedings XX World Buiatrics Congress , Sydney 6-10 july 1998, Vol I, Poster presentations: 544. Kenner, B. A., H. F. Clark, and P. W. Kabler. 1961. Cultivation and enumeration of streptococci in surface waters. J. Appl. Microbiol. 9:15-20. Kim, W-J. 1999. Molecular epidemiology of glycopeptide-resistant enterococci. The 2nd international symposium on antimicrobial resistance. May 7, 1999. Samsung Medical Center, Seoul, Korea. Kirst, H. A., D. G. Thompson, and T. I. Nicas. 1998. Historical yearly usage of vancomycin. Antimicrob. Agents Ch. 42:1303-1304. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12:39-86. Kleessen, B., E. Bezirtzoglou, and J. Mtt. 2000. Culture-based knowledge on biodiversity, development and stability of human gastrointestinal microflora. Microb. Ecol. Health Dis. Suppl. 2:53-63. Klein, G., A. Pack, C. Bonaparte, and G. Reuter. 1998. Taxonomy and physiology of probiotic lactic acid bacteria. Int. J. Food. Microbiol. 41:103-125. Khn, I., A. Iversen, L. G. Burman, B. Olsson-Lijequist, A. Franklin, M. Finn, F. Aarestrup, A. M. Seyfarth, A. R. Blanch, X. Vilanova, H. Taylor, J. Caplin, M. A. Moreno, L. Dominguez, I. A. Herrero, and R. Mllby. 2003. Comparison of enterococcal populations in animals, humans, and the environment a European study. Int. J. Food Microbiol. 88:133145.

67

Kurmann, J. A. 1968. ber die Ursachen der qualittsfrdernden Wirkung auf Emmentaler und Greyerzerkse bei einer Einimpfung von Streptokokkus faecalis in keimarmer Rohmilch. Milchwiss. 23:193-198. Lassaut, B. 2001. Presentation of the Origin Labeled Productss sectors, DOLPHINS: Development of origin labelled products; humanity, innovation and sustainability, November 2001, downloaded from www.origin-food.org/pdf/olp/olp-fr.pdf Latham, M. J., and D. J. Jayne-Williams. 1978. Streptococci in the alimentary tract of ruminants. p. 207-243. In Streptococci. Skinner, F.A. & Quesnel, L.B. (eds.). Academic press, London, UK. Laukov, A., and S. Czikkov. 2001. Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on bryndza, a traditional Slovak dairy product from sheep milk. Microbiol. Res. 156:31-34. Leclerc, H., L. A. Devriese, and D. A. A. Mossel. 1996. Taxonomical changes in intestinal (faecal) enterococci and streptococci: consequences on their use as indicators of faecal contamination in drinking water. J. Appl. Bacteriol. 81:459-466. Ledda, A., A. Murgia, and S. Arizza. 1978. Indagine sulla tecnologia di fabbricazione del Fiore Sardo. Scienza e tecnica lattiero-casearia. 29:145-159. Limsowtin, G. K. Y., I. B. Powell, and E. Parente. 1996. Types of starters, p. 102-129. In Dairy Starter Cultures, T. M. Cogan and J.-P. Accolas (ed.). VCH Publishers, New York, USA. Litopoulou-Tzanetaki, E., D. C. Graham, and Y. Beyatli. 1989. Detection of Pediococci and other nonstarter organisms in American Cheddar cheese. J. Dairy Sci. 72:854-858. Litopoulou-Tzanetaki, E., and N. Tzanetakis. 1992. Microbiological study of white-brined cheese made from raw goat milk. Food Microbiol. 9:13-19. Litopoulou-Tzanetaki, E., N. Tzanetakis, and A. Vafopoulou-Mastrojiannaki. 1993. Effect of the type of lactic starter on microbiological chemical and sensory characteristics of Feta cheese. Food Microbiol. 10:31-41. Lodics, T. A., and L. R. Steenson. 1990. Characterization of bacteriophages and bacteria indigenous to a mixed-strain cheese starter. J. Dairy Sci. 73:2685-2696. Loguercio, C., R. Abbiati, M. Rinaldi, A. Romano, C. del Vecchio Blanco, and M. Coltorti. 1995. Long-term effects of Enterococcus faecium SF68 versus lactulose in the treatment of patients with cirrhosis and grade 1-2 hepatic encephalopathy. J. Hepatol. 23:39-46. Lopez-Diaz, T. M., J. A. Santos, C. J. Gonzles, B. Moreno, and M. L. Garca. 1995. Bacteriological quality of a traditional Spanish blue cheese. Milchwiss. 50: 503-504. Low-Brown, J., and P. R. Meyers. 2003. Enterococcus phoeniculicola sp. nov., a novel member of the enterococci isolated from the uropygial gland of the Red-billed Woodhoopoe, Phoeniculus purpureus. Int. J. System. Evol. Microbiol. 53:683-685. Macedo, A. C., F. X. Malcata, and T. A. Hogg. 1995. Microbiological profile in Serra ewes cheese during ripening. J. Appl. Bacteriol. 79:1-11. Manners, J., and H. Craven. 2003a. Liquid milk for the consumer p. 3947-3951, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK.

68

Manners, J., and H. Craven. 2003b. Processing of liquid milk. p. 3951-3957, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Mannu, L., A. Paba, M. Pes, R. Floris, M. F. Scintu, and L. Morelli. 1999. Strain typing among enterococci isolated from home-made Pecorino Sardo cheese. FEMS Microbiol. Lett. 170:2530. Mannu, L., A. Paba, E. Daga, R. Comunian, S. Zanetti, I. Dupr, aldn L. A. Sechi. 2003. Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin. Int. J. Food Microbiol. 88:291-304. Martin, J. D., and J. O. Mundt. 1972. Enterococci in insects. Appl. Microbiol. 24:575-580. Martinez-Moreno, J. L. 1976. Flora microbiana del queso Manchego. III. Estreptococcos. Anales del Instituto Nacional de Investigaciones Agrarias, Serie General 4: 93-102. Martinez-Murcia, A. J., and M. D. Collins. 1991. Enterococcus sulfureus, a new yellow-pigmented Enterococcus species. FEMS Microbiol. Lett. 80:69-74. McAuliffe, O., R. P. Ross, and C. Hill. 2001. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol. Rev. 25:285-308. McDonald, L. C., M. J. Kuehnert, F. C. Tenover, W. R. Jarvis. 1997. Vancomycin-resistant enterococci outside the health-care setting: prevalence, sources, and public health implications. Emerg. Inf. Dis. 3:311-317. McSweeney, P. L. H., and M. J. Sousa. 2000. Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review. Lait. 80:293-324. Mettler, E., and B., Carpentier. 1997. Localisation, dnombrement et identification de la contamination microbienne aprs nettoyage de joints en EPDM dun circuit de pasteruisation de lindustrie laitire. Lait. 77:489-503. Morea, M., F. Baruzzi, and P. S. Cocconcelli. 1999. Molecular and physiological characterization of dominant bacterial populations in traditional Mozzarella cheese processing. J. Appl. Microbiol. 87:574-582. Morris Jr. J. G., D. K. Shay, J. N. Hebden, R. J. McCarter, B. E. Perdue, W. Jarvis, J. A. Johnson, T. C. Dowling, L. B. Polish, and R. S. Schwalbe. 1995. Enterococci resistant to multiple antimicrobial agents, including vancomycin. Ann. Intern. Med. 123:250-259. Morrison, D., N. Woodford, and B. Cookson. 1997. Enterococci as emerging pathogens of humans. J. Appl. Microbiol. Symposium Supplement. 83:89S-99S. Mundy, L. M., D. F. Sahm, and M. Gilmore. 2000. Relationship between enterococcal virulence and antimicrobial resistance. Clin. Microbiol. Rev. 13:513-522. Murray, B. E. 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3:46-65. Murray, B. E. 1998. Diversity among multidrug-resistant enterococci. Emerg. Inf. Dis. 4:37-47. Nallapareddy, S. R., X. Qin, G. M. Weinstock, M. Hk, and B. E. Murray. 2000. Enterococcus faecalis adhesin, Ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Inf. Immun. 68:5218-5224. Neve, H. 1996. Bacteriophage, p157-189. In Dairy Starter Cultures, T. M. Cogan and J.-P. Accolas (ed.). VCH Publishers, New York, USA.

69

Neviani, E., G. Mucchetti, G. Contarini, and S. Carini. 1982. Ruolo degli enterococchi nei formaggi italiani I. Il latte. 7:722-728. Noble, W. C., Z. Virani, and R. G. A. Cree. 1992. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol. Lett. 93:195-198. Nuez, M., and M. Medina. 1979. La flora lactique du fromage bleu de Cabrales. Lait. 59:497-513. Nuez, M., J. L. Rodrguez, E. Garca, P. Gaya, and M. Medina. 1997. Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese. J. Appl. Microbiol. 83:671-677. Olson, N. F. 2003. Types of cheese. p. 1045-1051, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. OMahony, P. 2003. Article in The Irish Times from 2/03/03. Pat OMahony, Director of Consumer Protection, Food Safety Authority of Ireland (FSAI), downloaded from http://www.angelfire.com/ia/japan/bonina.html. ONeill, E. 2000. Production of farmhouse cheese. Factsheets of Teagasc, by J. Whiriskey and L. Connolly; Rural Development Centre, Athenry, Co. Galway. Factsheet No. 8, January 2000 Opinion of the Scientific Commettee on Veterinary Measures Relating to Public Health. 1999. The evaluation of microbiological criteria for food products on animal origin for human consumption. 23 September 1999. European Commission. Health and Consumer Protection Directorate-Generale. http://europa.eu.int/comm/food/fs/sc/scv/out26_en.html Oumer, A., P. Gaya, E. Fernandez-Garcia, R. Mariaca, S. Garde, M. Medina, and M. Nuez. 2001. Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture. J. Dairy Res. 68:117-129. Papageorgiou, D. K., A. Abrahim, M. Bori, and S. Doundounakis. 1998. Chemical and bacteriological characteristics of Pichtogalo Chanion cheese and mesophilic starter cultures for its production. J. Food Prot. 61:688-692. Park, I.-S., C.-H. Lin, and C. T. Walsh. 1997. Bacterial resistance to vancomycin: overproduction, purification and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-DSer ligase. Proc. Natl. Acad. Sci. 94:10040-10044. Patel, R. 1999. Glycopeptide-reistant enterococci: clinical perspectives. The 2nd International Symposium on Antimicrobial Resistance. May 7, 1999. Samsung Medical Center, Seoul, Korea. See also: http://www.ansorp.org/isaar/1999/symposium/3_Patel1.pdf Perichon, B., P. Reynolds, and P. Courvalin. 1997. VanD-type glycopeptide-resistant Enterococcus faecium BM4339. Antimicrob. Agents Chemoter. 41:2016-2018. Gillespie, R. S., J. M. Symons, and R. A. McDonald. 2002. Peritonitis due to Leuconostoc species in a child receiving peritoneal dialysis. Pediat. Nephrol. 17:966-968. Peters, J., K. Mac, H. Wichmann-Schauer, G. Klein, and L. Ellerbroek. 2003. Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany. Int. J. Food Microbiol. 88:311-314. Phelan, J. A., and J. Renaud. 1993. Some non-European cheese varieties. p. 421-465. In Cheese: Chemistry, Physics and Microbiology, P. F. Fox (ed.), 2nd Edition, Academic Press, London, UK.

70

Pillai, S. K., G. Sakoulas, H. S. Gold, C. Wennersten, G. M. Eliopoulos, R. C. Moellering, Jr., and R. T. Inouye. 2002. Prevalence of the fsr locus in Enterococcus faecalis infections. J. Clin. Microbiol. 40:2651-2652. Prodromou, K., P. Thasitou, E. Haritonidou, N. Tzanetakis, and E. Litopoulou-Tzanetaki. 2001. Microbiology of Orynotyri, a ewes milk cheese from the Greek mountains. Food Microbiol. 18:319-328. Pujol, D. 1997. The collective organisation of a sector for the local valorisation of agricultural resources: the example of cheese processing. Leader European Observatory, Dossier N 1, 1997. http://europa.eu.int/comm/archives/leader2/rural-en/biblio/produ/produ_2.pdf Quednau, M., S. Ahrn, A. C. Petersson, and G. Molin. 1998. Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork. J. Appl. Microbiol. 84:1163-1170. Rakita, R. M., N. N. Vanek, K. Jacques-Palaz, M. Mee, M. M. Mariscalco, G. M. Dunny, M. Snuggs, W. B. Van Winkle, and S. I. Simon. 1999. Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation. Inf. Imm. 67:6067-6075. Rea, M. C., and T. M. Cogan. 2003. Glucose prevents citrate metabolism by enterococci. Int. J. Food Microbiol. 88:201-206. Revell, B. J., and M. Franois. 1997. On-farm processing of the products of livestock systems. Proceedings of the conference Livestock systems in European rural development, Nafplio, Greece, 23-25 Janury 1997. Rodriguez, E., J. L. Arqus, P. Gaya, M. Nuez, and M. Medina. 2001. Control of Listeria monocytogenes and monitoring of bacteriocin-producing lactic acid bacteria by colony hybridization in semi-hard raw milk cheese. J. Dairy Res. 68:131-137. Rogasi, P. G., S. Vigano, P. Pecile, and F. Leoncini. 1998. Lactobacillus casei pneumonia and sepsis in a patient with AIDS. Case report and review of the literature. Ann. Ital. Med. Int. 13:180-182. Rossi, E. A., G. S. de Giori, A. P. de Ruiz Holgado, and G. F. de Valdez. 1994. In vitro effect of Enterococcus faecium and Lactobacillus acidophilus on cholesterol. Microbiol. Alim. Nutr. 12:267-270. Rossi, E. A., R. C. Vendramini, I. Z. Carlos, Y. C. Pei, and G. F. de Valdez. 1999. Development of a novel fermented soymilk product with potential probiotic properties. Eur. Food Res. Technol. 209:305-307. Sarantinopoulos P., G. Kalantzopoulos, and E. Tsakalidou. 2001. Citrate metabolism by Enterococcus faecalis FAIR-E 229. Appl. Environ. Microbiol. 67:5482-5487. Sarantinopoulos P., G. Kalantzopoulos, and E. Tsakalidou. 2002. Effect of Enterococcus faecium on microbiological, physiochemical and sensory characteristics of Greek Feta cheese. Int. J. Food. Microbiol. 76:93-105. Scano, F. L. Rossi, A. Cattelan, G. Garretta, F. Meneghetti, P. Cadrobbi, and D. Sgarabotto. 1999. Leuconostoc species: a case-cluster hospital infection. Scand. J. Infect. Dis. 31 :371373. Schleifer, K. H., and R. Kilpper-Blz. 1984. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. System. Bacteriol. 34:31-34.

71

Schleifer, K. H., and R. Kilpper-Blz. 1987. Molecular and chemotaxonomic approaches to the classification of streptococci, enterococci and lactococci: a review. System. Appl. Microbiol. 10:1-9. Schmidtke, L. M., and J. Carson. 2002. Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited conditions. Vet. Microbiol. 93:63-71. Seale, D. R. 1986. Bacterial inoculants as silage additives. J. Appl. Bact. 61 (suppl): 9S-26S. Semedo, T., M. Almeida Santos, M. F. Silva Lopes, J. J. Figueiredo Marques, M. T. Barreto Crespo, and R. Tenreiro. 2003. Virulence factors in food, clinical and reference enterococci: a common trait in the genus? Syst. Appl. Microbiol. 26:13-22. Shankar, V., A. S. Baghdayan, M. M. Huycke, G. Lindhal, and M. S. Gilmore. 1999. Infectionderived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Inf. Immun. 67:193-200. Shils, E. B. 1997. Who are the mega-retail discount chains - how did they evolve? What ar the latest developments and expected trends? Chapter II from the Shils Report: Measuring the economic and sociological impact of the mega-retail discount chains on small enterprises in urban, suburban and rural communities. Downloaded from www.lwamall.com/rpa/rpashils.htm Singh, K. V., T. M. Coque, G. M. Weinstock, B. E. Murray. 1998. In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification. FEMS Immunol. Med. Microbiol. 21:323-331. Slaghuis, B. A., T. De Vries, and J. G. P. Verheij. 1991. Bacterial load of different materials which can contaminate milk during production. Milchwiss. 46:574-577. Smith, J. 1995. Creamery Days - The start (1920-1950), p. 69-89. In Cheesemaking in Scotland a history, The Scottish Dairy Association, Paisley, Scotland, UK. Son, R., F. Nimita, G. Rusul, E. Nasreldin, L. Samuel, and M. Nishibuchi. 1999. Isolation and molecular characterization of vancomycin-resistant Enterococcus faecium in Malaysia. Lett. Appl. Microbiol. 29:118-122. Sozzi, T., and R. Maret. 1973. Etude sur la microflore lactique du fromage Vacherine Mont-dOr, Lait. 53:280-294. Stanley, G. 2003. Starter cultures employed in cheese-making. p. 1051-1056, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Steffen, C., P. Eberhard, J. O. Bosset, and M. Regg. 1993. Swiss-type varieties. p. 83-110. In Cheese: chemistry, physics and microbiology, P. F. Fox (ed.). Chapman & Hall, London, UK. Stiles, E. M., and W. H. Holzapfel. 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36:1-29. Smuth, S. D., A. Muscholl-Silberhorn, R. Wirth, M. Susa, R. Marre, and E. Rozdzinski. 2000. Aggregation substance promotes adherence, phagocytosis and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst. Inf. Imm. 68:4900-4906.

72

Suzzi, G., M. Caruso, F. Gardini, A. Lombardi, L. Vannini, M. E. Guerzoni, C. Andrighetto, and M. T. Lanorte. 2000. A survey of the enterococci isolated from an artisanal Italian goats cheese (semicotto caprino). J. Appl. Microbiol. 89:267-274. vec, P., L. A. Devriese, I. Sedlek, M. Baele, M. Vancanneyt, F. Haesebrouck, J. Swings, and J. Doka. 2001. Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water. Int. J. Syst. Evol. Microbiol. 51:1567-1574. Teixeira, L. M., M. G. S. Carvalho, M. M. B. Espinola, A. G. Steigerwalt, M. P. Douglas, D. J. Brenner, and R. R. Facklam. 2001. Enterococcus porcinus sp. nov. and Enterococcus ratti sp. nov. associated with enteric disorders in animals. Int. J. System. Evol. Microbiol. 51:1737-1743. Temedo, T., M. Almeida Santos, M. F. Silva Lopes, J. J. Figueiredo Marques, M. T. Barreto Crespo, and R. Tenreiro. 2003. Virulence factors in food, clinical and reference enterococci: a common trait in the genus? System. Appl. Microbiol. 26:13-22. Teuber, M., V. Perreten, and F. Wirsching. 1996. Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiologie. Lebensm. Technol. 29:182-198. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek. 76:115-137. Teuber, M., F. Schwarz, and V. Perreten. 2003. Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a rawfermented sausage. Int. J. Food Microbiol. 88:325-329. Thomas, T. D., and V. L Crow. 1983. Mechanism of D(-)lactic acid formation in Cheddar cheese. N. Z. J. Dairy Sci. Technol. 18:117-124. Thompson, T. L., and E. H. Marth. 1986. Changes in Parmesan cheese during ripening: microflora coliforms, enterococci, anaerobes, propionibacteria and staphylococci. Milchwiss. 41:201205. Trovatelli, L.D., and A. Schiesser. 1987. Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwiss. 42:717-718. Tyrrell, G. J., L. A. Turnbull, L. M. Teixeira, J. Lefebvre, M. G. S. Carvalho, R. R. Facklam, and M. Lovgren. 2002. Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. isolated from human clinical specimens. J. Clin. Microbiol. 40:1140-1145. Tzanetakis, N., and E. Litopoulou-Tzanetaki. 1992. Changes in numbers and kinds of lactic acid bacteria in Feta and Teleme, two Greek cheeses from Ewes milk. J. Dairy Sci. 75:13891393. US Dairy Export Council. 2003. http://usdec.org/products/cheeseoverview.htm and other various Internet addresses where valuable information could be obtained. Canadian Dairy industry profile, and cheese sites from different countries: www.dairyinfo.gc.ca/dairyprofile.pdf, (http://www.sirena.hr/nagrade.asp?lang=eng, http://www.the-vu.com/English_Cheeses.htm, http://www.newzealandmilk.com/cheese/body.html, http://www.ljmlek.si/eng/products/products07.htm, http://www.bongrain.com/gb/voyage/info_b.php3?num=1, http://www.theworldwidegourmet.com/cheeses/uk/cheddar.htm, http://greekproducts.com/greekproducts/cheese/ Vancanneyt, M., C. Snauwaert, I. Cleenwerck, M. Baele, P. Descheemaeker, H. Goossens, B. Pot, P. Vandamme, J. Swings, F. Haesebrouck, and L. A. Devriese. 2001. Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets. Int. J. System. Evol. Microbiol. 51:393-400.

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Van den Berg, G. 2003. Dutch-type cheeses. p. 1129-1135, In Encyclopedia of food sciences and nutrition, Caballero, B., Trugo, L. C., and P. M. Finglas (eds.), 2nd ed. Academic Press, Oxford, UK. Van den Braak, N., A. van Belkum, M. van Keulen, J. Vliegenthart, H. A. Verbrugh, and H. P. Endtz. 1998. Molecular characterization of vancomycin-resistant enterococci from hospitalized patients and poultry products in The Netherlands. J. Clin. Microbiol. 36:19271932. Vanderhoof, J. A., and R. J. Young. 1998. Use of probiotics in childhood gastrointestinal disorders. J. Ped. Gastroent. Nutr. 27:323-332. Wegener, H. C., M. Madsen, N. Nielsen, and F. M. Aarestrup. 1997. Isolation of vancomycin resistant Enterococcus faecium from food. Int. J. Food. Microbiol. 35:57-66. Wegener, H. C., F. M. Aarestrup, L. B. Jensen, A. M. Hammerum, and F. Bager. 1999. Use of antimicrobial growth promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe. Emerg. Inf. Dis. 5:329-335. Williams, A. M., U. M. Rodrigues, and M. D. Collins. 1991. Intragenic relationships of enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA. Res. Microbiol. 142:67-74. Witte, W. 1997. Wie gross ist das Gefhrdungspotential durch Vancomycin-resistente Enterokokken? Dtsch. med. Wschr. 122:1161-1163. World Dairy. 1999. The world market for cheese. World Dairy, Vol. 4, No. 5

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Chapter 5. Aim of this study

Cheese has existed for thousands of years. Its consumption in developed nations has been steadily increasing over the past 20 years. In the US, average annual consumption per head is 12,7 kilograms a year, while in Europe it is 17 kilograms. The cheese microflora differs from cheese to cheese but can comprise starter cultures, non-starter-lactic acid bacteria, staphylococci, and coliforms. A taxon frequently found in cheese, especially in raw-milk cheeses, is Enterococcus. The source of enterococci in raw milk cheese, however, is not clear. Enterococci are ubiquitous and therefore found quite easily in the GIT of mammals, in the farming environment and in raw milk cheese. In recent years, attention has focused on enterococci, as they have become the cause of a variety of infections due to their increasing resistance to antibiotics. They might be able to colonize the human GIT or spread their genes to bacteria that normally reside in the GIT. The overall objective of this work was to produce knowledge on the origin and safety of enterococci found on an Irish farm and in the cheese produced on that farm. The first step is to determine the diversity of enterococci in an Irish farmhouse raw-milk Cheddar-type cheese by identifying enterococci isolated from the raw-milk, the cheese, the cheese-making environment and the faeces of the cows and humans associated with the cheesemaking. At the same time valuable information will be obtained about the additional microflora of this particular cheese. The three trials will be undertaken in July 1998, in September 1998 and in October 1999. Enterococci will be isolated from KAA agar and identified using RAPD and SDS-PAGE. Furthermore the origin of enterococci in this cheese will be investigated. Strains found in cheese will be typed and compared with isolates from the human

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faecal samples, the milking equipment and the environment using Pulsed Field Gel Electrophoresis (PFGE). This method has shown to be highly discriminatory and is generally accepted as the method of choice for typing strains of enterococci. There is evidence suggesting that enterococci colonize the GIT only transiently as they are unable to compete with the indigenous flora but this conclusion is not well documented. Studies exist about the passage of lactobacilli and bifidobacteria but not about enterococci. To determine whether enterococci can endure the passage through the human GIT, the effect that consumption of cheese containing enterococci has on the enterococcal flora will also be studied. The faeces of three healthy humans will be investigated before, during and after consumption of cheese. Enterococci obtained from the isolation will be characterised and compared using PFGE. Finally the safety aspects of the representative enterococcal strains isolated from cheese will be studied. Resistances to the most common antibiotics will be tested phenotypically. The presence of resistance genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) will be investigated. In addition, all strains will be phenotypically and genotypically tested for the presence of (potential) virulence factors (Agg, Gel, Cyl, Esp, Ace, EfaAfs and EfaAfm). This study should allow us to get an insight into the ecology of enterococci present on this particular farm and the source of enterococci in the cheese. It is intended to learn something about enterococcal levels of the GIT of the persons living on the farm and involved with the cheesemaking, where they spread, and how safe enterococci in cheese are.

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Chapter 8. Effect of cheese consumption

Baylough, an Irish Cheddar- type cheese

?
cheese consumption 3 subjects Chapter 6. Enterococcal diversity on a cheesemaking factory Irish Farm Milk/cheese/environment farmers faeces cows faeces
clones originating from cheese: 10 9 D1 Ds1 8 7 6 5 4 Fm3 Fm3 3 2 Fm2 Fm2 Fm4 Fm10 1 Fm1 0 1 2 3 4 5 6 7 8 9 10 weeks Fm9 Fm13 Fm15 F13 Fs13 Fm7 Fm6 isolates Fs7 F7 Fm5 Fm11 F2 Fs2 F2 Fs2 F2 Fs2 Fs8 F8 Fm8 C4 Cs4 Fm12 C1 Cs1 C2 Cs2 C2 Cs2 H1 He1 F12 Fs12 F11 Fs11 F10 Fs10 Fm14 Fm17 F17 Fs17

1 control (faeces)

Fs9 F9

Fm16

F16 Fs16 F15 Fs15 F14 Fs14

Isolation of 1449 strains from KAA identification by RAPD

Fm18 Fm18

cheese consumption

species E. casseliflavus E. casseliflavus E. casseliflavus E. durans E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis

LMG number LMG 19844 LMG 19845 LMG 20235 LMG 20231 LMG 19846 LMG 20228 LMG 20229 LMG 20230 LMG 20234

original number Cs1 Cs2 Cs3 D Fs1 Fs2 Fs3 Fs4 Fs6

Strain E. casseliflavus E. casseliflavus E. casseliflavus E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis

LMG number LMG 19844 LMG 19845 LMG 20235 LMG 19846 LMG 20228 LMG 20229 LMG 20230 LMG 20234

Phenotypic resistance St St, Tc St St, Tc St, Tc, Ka, Cm St, Tc, Ka, Cm St, Tc, Ka, Cm, Er St, Ka

tet genes nd tet(M) nd tet(M) tet(M), (L) tet(M), (L) tet(M) nd

Tn916 + + + + -

erm(B) + -

gel + + + + + + +

Chapter 7. Source of enterococci in cheese

Chapter 9. Antibiotic resistance and virulence traits

Part II Experimental work

Chapter 6. Enterococcal diversity in the environment of an Irish cheddar-type cheesemaking factory

Gelsomino R.a, b, M. Vancanneytb, S. Condonc, J. Swingsb, and T. M. Cogan.a*

a b c

Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland BCCM/LMG Culture Collection, University of Gent, Ledeganckstraat 35, Belgium Department of Microbiology, University College Cork, Cork, Ireland

International Journal of Food Microbiology (2001) Vol. 71, p. 177-188.

Summary Enterococci are natural residents of human and animal intestinal tracts and grow to high levels in a variety of artisanal cheeses. The aim of this study was to determine the diversity of enterococci in a farmhouse raw-milk cheese production unit. Putative enterococci were isolated from the faeces of all the cows and all the people associated with the cheesemaking, from the milk and cheese during manufacture and ripening and from the environment in three separate trials. Almost 1400 isolates were screened using a genus specific primer. The results indicated that all human, milk, curd and cheese isolates but only 33.7%, 6.7% and 4.4% of the bovine isolates from the 3 trials, respectively, were members of the genus Enterococcus. RAPD-PCR was used to type the enterococcal isolates. In general, only E. faecium was found in the bovine faecal samples while E. casseliflavus dominated the human faeces, milk and cheese followed by lower numbers of E. faecalis. Environmental sampling of the water in the milking parlour and rinses of the cows teats, the bulk-milk storage tank and the milking machine corroborated these results as E. casseliflavus and E. faecalis were the only Enterococcus species found in these samples. The putative vancomycin-resistant enterococci (VRE), isolated in Trial 1, were shown to be Pediococcus ssp. by genotypic and phenotypic analysis.

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Introduction Enterococci are ubiquitous, Gram positive, catalase-negative cocci that occur and grow in a variety of dairy and other food products. They have been used as silage inoculants (Seal, 1986), dairy starter cultures (Giraffa et al., 1997) and probiotics (Fuller, 1989) and although they do not grow well in milk (Cogan et al., 1997) they seem to play a major role in improving flavour development and quality of cheese (Dahlberg et al., 1948; Walter et al., 1956; Clark et al., 1966; Kurmann, 1968; Neviani et al., 1982a). Nevertheless, they are not considered GRAS (generally recognized as safe) organisms (Giraffa et al., 1997). Enterococci are commonly found in high levels in a variety of cheeses produced in Italy, Portugal, Spain and Greece from raw or pasteurized goats, ewes, water buffaloes or cows milk (Cogan et al., 1997). Although they can be found on insects, in soil and on plants, their natural habitat is the intestinal tract of humans and animals (Garg et al., 1991). Previous studies have shown that Enterococcus faecalis is the most commonly found Enterococcus species in human faeces (Cooper et al., 1955; Saramago Stern et al., 1994; Leclerc et al., 1996; Facklam et al., 1998) while E. faecium is the most common in the intestinal tract of dairy cattle (Leclerc et al., 1996). Enterococci are less prevalent in dairy cows faeces but there have been reports of finding E. faecium, Enterococcus hirae and E. faecalis (Medrek et al., 1962; Mieth, 1962; Latham et al., 1978; Devriese et al., 1992); these workers also report that Streptococcus bovis is the most frequently occurring organism in cows faeces. Enterococci are used as indicators of faecal contamination (Kenner et al., 1961; Lopez et al., 1995) and they are known to cause food spoilage, urinary tract infections, endocarditis and neonatal, central nervous system and respiratory tract infections. The ability of enterococci to exchange extrachromosomal elements which encode antibiotic resistance genes, particularly vancomycin, have been of particular concern during the last 10 years (Hardie et al., 1997). Vancomycin Resistant Enterococci (VRE) and a case of a Vancomycin Dependant Enterococcus (VDE) have been reported (Fraimow et al., 1994). These organisms may enter the milk either directly from human or animal faeces or indirectly from contaminated water sources, exterior of the animal and/or

83

from the milking equipment and bulk-milk storage tank. Different species of enterococci are found in cheese but E. faecalis and E. faecium are the species most commonly isolated (Franklin et al., 1963; Fryer, 1969; Neviani et al., 1982a; Trovatelli et al., 1987; Fontecha et al., 1990; Devriese et al., 1995; Arizcun et al., 1997; Cogan et al., 1997; Suzzi et al., 2000). The aim of this study was to determine the diversity of enterococci in an Irish raw-milk, farmhouse Cheddar-type cheese by typing enterococci isolated from the raw-milk, the cheese and the cheese-making environment and the faeces of the cows and humans associated with the cheesemaking.

84

Material and methods Cheese production: Bay Lough cheese was used. This is a raw milk Cheddar-type cheese, made at farmhouse level, from the milk of a herd of 27 cows, by the herdowners wife. The milking parlour is located about 1 mile from the cheese making plant to which the milk was transported in a stainless steel tank before cheese-making. The cheese was manufactured using a proprietary method with a DL mixed-strain starter culture (Flora Danica, Chr. Hansens Laboratory) which contains 62% Lactococcus lactis ssp. cremoris, 19% Lactococcus lactis ssp. lactis biovar. diacetylactis, 2% Lactococcus lactis ssp. lactis, 16% Leuconostoc cremoris and 1% unknown species. (Lodics et al., 1990) and was ripened for several months. Sample collection: The milk in the bulk tank, the milk in the cheese vat, the starter, the curd after cooking and at moulding and the cheese the following day were sampled. The cheese was also sampled after 1, 2, 3, 4, 6 and 8 weeks of ripening. Three trials were undertaken: the first in July 1998, the second in September 1998 and the third in October 1999. Bovine faecal samples of all the 27 dairy cows on the farm were obtained by manual rectal retrieval and transferred to sterile containers. The family involved in cheesemaking (2 adults and 2 teenagers) was also asked to collect their faecal samples in sterile containers as close as possible to the time the cows and milk were sampled. All samples were chilled and transported to the laboratory. During the third trial the following environmental samples were also taken: A) Five hundred milliliters of the tap water in the milking parlour was membrane filtered. B) The milking machine was rinsed with ~50 l of water and sampled, it was then washed with water containing sodium hypochlorite (12ml sodium hypochlorite in 50 l water), sampled again, and then with 50 l of water containing 0.002% sodium thiosulfate to inactivate residual chlorine and sampled again. Five hundred milliliters of the chlorinated water were membrane filtered. C) The bulk tank was rinsed with 5 l of Maximum Recovery Diluent (MRD, Oxoid) and sampled. D) Milk samples were also taken at different stages from the bulk tank: after the first filling in the morning, before the second filling in the evening, before the third filling in the morning, after the third filling and shortly before the milk was put into the cheese vat (2 h later). E)

85

The four teats of each cow were rinsed with 80 ml MRD and all teat-rinses were mixed together. The environmental samples were diluted and plated on KF agar. Bacteriological analysis: Curd and cheese were emulsified 1:10 in 2% (w/v) trisodium citrate, pH 8.75, and plated, with the milk samples, on the following media: coliforms on Violet Red Bile Agar (VRB, Merck) incubated for 18h at 30C (IDF 73B, 1998); non starter lactic acid bacteria (NSLAB) on Lactobacillus selective agar (LBS, Becton Dickinson Microbiology Systems, USA) incubated for 5 days at 30C; starter cells on LM17 agar (Difco) incubated for 3 days at 30C; staphylococci on Baird Parker-Agar (Merck) incubated at 37 for 3 days; enterococci on Kanamycin Aesculin Azide Agar (KAA) incubated at 37 for 24h; total counts on Plate Count Agar (PCA, Merck) incubated at 30C for 72h. Faecal samples were emulsified 1:10 in sterile MRD and plated on KAA, KFStreptococcusagar (Difco) supplemented with 2,3,5-triphenyl-tetrazolium chloride (TTC, final concentration 100 g/ml, BDH) and vancomycin (final concentration 6g/ml, Sigma) was used to determine vancomycin resistant enterococci (VRE). VRE were determined only during Trial 1. An additional test to check for VRE consisted of adding 0.1ml of a 1:10 dilution of all samples to 5ml Buffered Peptone Water containing 6g Vancomycin/ml. After incubation for 18h at 37C, a loopful was streaked on KF-plates containing Vancomycin. KAA and KF plates were incubated at 37C for one and two days respectively. KF agar was used instead of KAA to isolate VRE to avoid any interaction that might occur between the two antibiotics, vancomycin and kanamycin. In Trial 3, KF-Streptococcus agar was used instead of KAA for the detection of enterococci in all samples and was incubated for 2 days at 37C Isolation of strains: Twenty colonies were picked from the highest dilution of each sample taken from the bulk tank, the cheese vat, the curd before moulding and from the cheese the following day and at 2, 4, 6 and 8 weeks of ripening. Ten colonies were picked from the highest dilution of each faecal sample. The colonies were purified twice on KAA and grown overnight in Basal Medium (BM) which contained 2% Tryptose (Oxoid), 0.5% NaCl (AnalaR), 0.5% Yeast extract (Merck), 0.5% Glucose (AnalaR), pH 6.85. Cultures were maintained at -80C in a 1:2 glycerol-BM mixture. Isolates from stock were streaked on BM plates and a single colony was transferred to broth.

86

Phenotypic

characterisation:

All

1449

isolates

were

examined

microscopically and checked for catalase production using 3% (v/v) H2O2 on single colonies grown overnight on BM agar at 37C. Growth of catalase negative cocci was measured in BM broth containing 6.5% salt after 7days, in 10% sterile (121C, 5 min) reconstituted skim milk (RSM) after 6h, 16h and 7 days, in BM broth at 10C after 2, 5 and 7 days and at 45C after 2 days. Growth was determined by measuring the pH. A pH difference of at least 0.5 between the uninoculated control and the culture was considered to indicate growth. Columbia blood agar plates containing 0.1g/100ml aesculin and 7ml/100ml calf blood were used to record haemolytic reactions on strains identified as enterococci from the first trial. Compositional analyses: Standard procedures were used to analyse the content of fat (IDF 5B, 1986), NaCl (IDF 88A, 1988), protein (IDF 20B, 1993) and moisture (IDF 4A, 1982) in the cheese after 8 weeks of ripening. The pH was measured on a cheese slurry prepared from 20g of cheese and 12g water (British Standard 770, 1976). PCR with genus-specific primer: Genomic DNA from all strains was extracted from overnight cultures using the procedure described of Coakley et al. (1996). DNA amplification was performed using the procedures of Deasy et al. (2000) for enterococci and Deasy (2000) for pediococci. RAPD typing: DNA was prepared using the rapid procedure described by Pitcher et al. (1989). Random primer D11344 was used to type enterococcal isolates to species level. DNA amplification was performed using the procedure described by Descheemaker et al. (1997). Numerical analysis was performed using the Pearson coefficient and the UPGMA (unweighted pair group method) with the GelCompar software, version 4.2 (Applied Maths, Kortrijk, Belgium). Fatty Acid Analysis: Cells were grown twice for 24 h on Trypticase Soy agar (BBL, Becton Dickinson Microbiology Systems, USA) supplemented with 15g Bacto Agar (Difco) at 28C. Fatty acids were extracted, separated and analyzed using the procedure described by Vancanneyt et al. (1996). Profiles were identified using the commercial TSBA 40 library (MIDI Inc. Newark, Delaware). Identification by SDS-PAGE: Cells were grown for 24 hours on BM-agar at 37C. Whole cell protein extracts were prepared with a slight modification as described by Pot et al. (1994): after the washing step the cells were sonicated for 2

87

minutes, with an output control of 3 and a duty cycle of 50% using a Branson Sonifier 250 (Branson Ultrasonics, Danbury, USA). Conversion, normalization and further analysis were performed using the Pearson coefficient and the UPGMA with the GelCompar software, version 4.2 (Applied Maths, Kortrijk, Belgium).

88

Results Development of the flora in the cheese. The total counts and the counts of the different groups of bacteria in the raw milk in the bulk tank and in the cheese vat (Table 1) showed that the raw milk in each of the 3 trials was of satisfactory quality. There was little increase in numbers between milking and placing the milk in the cheese vat ~18h later. No enterococci were found in the starter culture. The enterococci, lactobacilli, coliforms and staphylococci all grew during manufacture (data not shown). Their behavior in ripening is shown in Fig. 1. The lactobacilli were the only group of organisms to show an increase in numbers. The enterococci remained more or less constant while the numbers of coliforms, staphylococci and starters all decreased. Table 1. Count (cfu/ml) of different groups of organisms in the milk from the bulk tank and the cheese vat in each trial. Trial 1 Trial 2 Trial 3 Bulk tank Cheese vat Bulk tank Cheese vat Bulk tank Cheese vat Total viable count Enterococci Lactobacilli Staphylococci Coliforms 18400 500 100 40 80 18100 500 300 50 110 16800 130 60 20 80 20100 160 40 10 500 3000 230 60 160 101 3700 260 50 230 400

There was considerable variation in the number of the different bacterial groups between each trial and some variation in the enterococci within a trial (Figure 1). However, the trends in each trial were similar. While enterococci were present in low numbers in the raw milk (~102/ml) (Table 1), their number increased during cheesemaking to final numbers of 1.4 X 104, 2.6 X 104 and 5.8 X 103 cfu/g in Trials 1, 2 and 3 respectively (Fig. 1)

89

Figure 1. Development of enterococci, Non Starter Lactic Acid Bacteria (NSLAB), coliforms, Staphylococcus aureus and starters in each trial during ripening.

Enterococci
1,0E+05 1,0E+04 cfu/g cfu/g 1,0E+03 1,0E+02 0 10 20 30 Ripening T ime, d 40 50 60 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 0 10

NSLAB

20

30 Ripening T ime, d

40

50

60

Coliforms
1,0E+04 1,0E+03 cfu/g 1,0E+02 1,0E+01 1,0E+00 0 10 20 30 Ripening T ime, d 40 50 60 1,0E+02 0 10 cfu/g 1,0E+03 1,0E+04

S. aureus

20

30 Ripening T ime, d

40

50

60

Starter
1,0E+09 1,0E+08 cfu/g 1,0E+07 1,0E+06 0 10 20 30 Ripening T ime, d 40 50 60

Compositional profile of the cheese. The results of the compositional analyses are shown in Table 2 and did not show significant differences between the 3 trials. The salt in moisture (S/M), the pH and the ratio of moisture to nonfat substance (MNFS) were all within the range of a First grade Cheddar cheese while

90

the fat in dry matter (PDM) was within 50-52 which is considered to be the range for a Premium grade Cheddar cheese (Lawrence et al., 1984).

Table 2. Compositional analysis of the cheeses in the three trials at 8 weeks of ripening. parameters pH MNFSa (%) FDMb (%) S/M (%)
a b c c

range 4.84 - 4.95 54 57 50 52 3.6 - 4.1

MNFS, ratio of moisture to nonfat substance, moisture/(100-fat) x 100. FDM, fat in dry matter, fat/(100-moisture) x 100. S/M, ratio of salt to moisture, salt/moisture x 100.

Selective isolation of the strains. In each trial, 160 strains were isolated from the milk, the curd and the cheese and 40 strains were isolated from the human faeces. Two hundred and seventy isolates from KAA or KF were obtained from the cows faeces in each of Trials 1 and 2 and 250 in Trial 3, as there were 2 fewer cows in the latter trial (Table 3). In Trial 3 ~2 X 102 cfu/ml were found in the milk sampled from the bulk tank at different fillings. Five colonies were isolated from each sampling point. Five colonies were found, and subsequently isolated, on the KF plates from the rinse of the bulk-milk storage tank. Prior to chlorination the milking machine contained ~2.9 X 103 enterococci per ml of rinse; no enterococci were found in the chlorinated rinse while after deactivation of the chlorine the number of enterococci found was 10 per ml. Five colonies were isolated from these samples before and after

91

Table 3. Total numbers of strains isolated per trial and the distribution of Enterococcus ssp.
Trial 1 Sample total E. E. E. E. total E. Trial 2 E. E. total E. Trial 3 E. E. E.

isolates casseliflavus faecalis faecium hirae isolates casseliflavus faecalis faecium isolates casseliflavus faecalis faecium durans Milk from the bulk tank Milk from the cheese vat Cheese before moulding Cheese at day 1 Cheese at week 2 Cheese at week 4 Cheese at week 6 Cheese at week 8 Cows faeces1 Faeces human 1 (adult) Faeces human 2 (adult) Faeces human 3 (teenager) Faeces human 4 (teenager)2 water3 Rinsed bulk tank Milking machine Cows' teats rinses4
1 2

20 20 20 20 20 20 20 20 270 10 10 10 10

14 17 17 14 15 18 20 20

6 3 3 6 5 2

20 20 20 20 20 20 20 20 90 1 270 10 10 10 10

20 20 20 20 20 20 20 20 10 10 10 10 10 8

40 20 20 20 20 20 20 20 250 10 10 10 10 6 5 10 8

35 14 14 11 16 7 17 15 10 4 4 6

5 6 6 9 4 13 3 5 11 6 6 3 2 1 2

9 3 6 9

1 7 4 1

4 8

All the non-enterococci, catalase negative organisms screened with SDS-PAGE (136 strains) were Str. bovis. One strain in Trial 3 was S. saprophyticus. 3 Of the 4 non-enterococcal strains 2 were S. saprophyticus, 1 was B. cereus and one could not be identified. 4 6 were S. saprophyticus and 2 could not be identified.

chlorination. Twenty-six colonies from the cows teat rinses were found on the 2.5 ml-filter. Only 8 were cocci. Six colonies were found on the 500ml-filter from the tap water from the milking parlour. Vancomycin resistant colonies were only examined in Trial 1. None were found in any of the cows faeces in the 1:10 dilution and only 5, 3 and 2 colonies in the milk in the vat, the cheese the following day and the cheese ripened for 11 days, respectively. Phenotypic characterisation. In total, 1449 strains were isolated i.e. 470, 470 and 450 strains from the cheese, the humans and cows faeces from Trial 1, 2 and 3 respectively, 6 from the water in the milking parlour, 10 each from the milking machine rinses before and after chlorination, 8 from the cows teats, 25 from the bulk-milk storage tank at different stages during milking and 10 from KF plates containing vancomycin. All strains were catalase-negative cocci, except 53 bovine isolates and one human faeces isolate from Trial 3, which were catalase positive. This may have been due to the use of KF rather than KAA in this trial. The catalase negative cocci from the milk, the curd, the cheese samples and the human faeces grew at 10C and 45C, in broth containing 6.5% salt and in RSM after 7 days. Some of the isolates from the cows faeces were also able to grow at these conditions. However, numerous isolates from the latter did not grow at 10C or in 6.5% salt. None of the enterococci showed apparent hemolytic activity. The vancomycin resistant strains were able to grow at 45C and in broth containing 6.5% salt but not at 10C. Of the 8 organisms found on the cows teats, 2 were catalase negative and 6 were catalase positive cocci while of the 6 cocci found in the water 3 were catalase positive and 3 catalase negative. Two of the latter strains grew at 10C and 45C and in 6.5% salt. The third one and the 2 catalase negative strains found in the teat rinses grew in broth containing 6.5% salt but not at 10C or 45C. Genotypic analysis. All catalase negative cocci were analyzed with the genus specific primer developed by Deasy et al. (2000). Only isolates that grew at 10C and 45C and in 6.5% salt reacted positively with the genus specific primer for enterococci (Figure 2) while all the strains that did not grow at 10C and/or in 6.5% salt failed to give a band on the agarose gel. This analysis confirmed that all the isolates from the milk, the cheese and the human isolates were enterococci but

93

1 2

3 4 5 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Figure 2. Agarose gel showing PCR products generated with the specific enterococcal primer developed by Deasy (2000). Mol. wt. ladders (lanes 1, 12, 23), 3 human isolates, Trial 1 (lanes 2, 3, 4), milk from the bulk tank, Trial 1 (lane 5), curd before moulding, Trial 1 (lane 6), cheese ripened for 2 weeks, Trial 1 (lane 7), human isolates, Trial 2 (lanes 8, 9, 10), milk from the bulk tank, Trial 2 (lane 11), curd before moulding, Trial 2 (lane 13), cheese ripened for 2 and 4 weeks, Trial 2 (lane 14, 15), human isolates, Trial 3 (lanes 16, 17, 18), milk from the bulk tank, Trial 3 (lane 19), curd before moulding, Trial 3 (lane 20), cheese ripened for 4 and 6 weeks, Trial 1 (lane 21, 22).

only 91, 18 and 11 out of 270, 270 and 250 isolates respectively from the animal faeces were enterococci. The vancomycin resistant isolates from Trial 1 reacted positively with the pediococcal primer and not with the enterococcal primer. RAPD based typing. RAPD analysis was used to type the species of enterococci in all samples. The milk and cheese samples from the different stages of cheese ripening and the human faeces were dominated by E. casseliflavus, followed by lower numbers of E. faecalis. In Trial 2 only E. faecalis was isolated from three of the human samples and E. durans was isolated from two samples in Trial 3. Two catalase negative isolates from the water sample were identified by RAPD as E. faecalis. The cows faeces were dominated by E. faecium in all trials (Table 3) but, in Trial 2, 10 of the 18 isolates were E. faecalis. One isolate of E. hirae was found. Figure 3 shows a selection of RAPD band

94

patterns of the most common Enterococcus species from different sources in different trials

30 40 40 50 60 60 70 70 80 80 90 90 100

Trial 1 cow nr. 18 Trial 1 cow nr. 9 E. faecium Trial 1 cow nr. 3 Trial 2 cow nr. 4 Trial 3 cow nr. 19 Trial 1 milk bulk tank Trial 1 human (teenager) Trial 3 curd before moulding E. faecalis Trial 2 human (adult) Trial 3 human (teenager) Trial 3 cheese at 2 weeks Trial 1human (adult) Trial 1 milk cheese vat Trial 2 cheese at 8 weeks E. casseliflavus Trial 3 milk bulk tank Trial 2 milk bulk tank Trial 2 cheese at 6 weeks Trial 3 human (adult) Trial 1 milk bulk tank Trial 3 cheese at 4 weeks Figure 3. RAPD band patterns and corresponding dendrogram of some of the E. faecium, E. faecalis and E. casseliflavus strains found during the 3 trials. Most of the strains belong to one of the band patterns shown below. Numerical analysis was performed using the Pearson coefficient and the UPGMA (unweighted pair group method) using the GelCompar software, version 4.2 (Applied Maths, Kortrijk, Belgium)

Almost all band patterns of E. faecium, E. casseliflavus and E. faecalis found belong to one of the band patterns shown in the figure. All three clusters divide into two subclusters but this can be seen visually only for E. casseliflavus. SDS-PAGE and Fatty Acid Analysis. Fatty Acid Profiling and SDS-PAGE were used to identify the non-enterococci. The vancomycin resistant, catalase negative

95

strains found to be pediococci by the genus specific primer were confirmed to be Pediococcus pentosaceus by SDS-PAGE. Six colonies were isolated from the filtered tap water in the milking parlour. Three were catalase-negative and three catalase-positive. Two of the three catalasepositive isolates were shown to be Sta. saprophyticus and the third Bacillus cereus by fatty acid analysis. Two of the catalase-negative strains were E. faecalis as shown by phenotypic analysis and RAPD typing while one strain, which grew in broth containing 6.5% salt but not at 10C could not be identified. Six of the eight colonies isolated from the cows teat rinses were catalasepositive. Fatty acid analysis showed that they all were Sta. saprophyticus. The two catalase-negative strains grew in 6.5% salt but not at 10C and did not react with the enterococcus probe; they could not be identified. Fifty-three catalase-positive strains were found in some of the cows faeces, in one human faeces and in some cheese samples. Fatty acid analysis showed them to be Sta. saprophyticus. From all isolates found in the cows faeces that did not grow under the conditions typical for enterococci, i.e. growth at 10C and 45C and in broth containing 6.5% salt, 136 isolates were chosen (2 from each cow). These were all found to be Str. bovis after examination of whole cell proteins by SDS-PAGE. One catalase negative coccus found in the water and 2 catalase negative cocci found in the teat rinses could not be identified.

96

Discussion In this study, the milk and cheese, samples from the environment and the faeces of every cow and human associated with a small artisanal cheesemaking operation were sampled for enterococci in three independent trials within a period of 15 months. Both phenotypic and genotypic analyses were used for identification of the isolates. E. casseliflavus and E. faecalis were the most frequently isolated species in both the milk, the curd and the cheese samples and in the human faeces. This result was surprising but was confirmed in all trials. The results for the humans largely disagree with the findings of Saramago-Stern et al. (1994), Facklam and Texeira. (1989) and Leclerc et al. (1996) who stated that E. faecalis was the dominant enterococcal species in humans. In our study the E. casseliflavus population dominated not only in the humans intestine but also in the milk, the curd and the cheese, which also disagrees with the earlier reports of Neviani et al., 1982a; Trovatelli and Schiesser, 1987; Fontecha et al., 1990; Devriese et al., 1995; Arizcun et al., 1997; Cogan et al., 1997; Suzzi et al., 2000 for cheese. We do not have an adequate explanation for the analogous flora in both niches. The cheese used in the present study was a raw milk cheese and it is possible that E. casseliflavus does not withstand pasteurisation of milk and hence would not be found in pasteurised milk cheese. Human contamination of the milk may have occurred. However, the cheese is consumed by the cheesemaking family and this may have been the source of enterococci in the humans. A feeding trial would have to be undertaken to resolve this issue. Very few enterococci were found in the cows faeces; they were isolated from 10, 3 and 2 cows of the 27, 27 and 25 cows in Trials 1, 2 and 3, respectively. The recovery of enterococci from the bovine faecal samples ranged from 1 to 10 isolates in those samples who contained them. In each of the 3 trials enterococci were isolated from different cows except for one cow from which 10 and 2 E. faecium strains were isolated in Trials 1 and 2, respectively. This poor recovery confirms the results of Devriese et al. (1992). Almost all enterococci found were E. faecium, which is one of the most commonly found species in the intestine of cows (Medrek and Barnes, 1962; Mieth, 1962; Latham and Jayne-Williams, 1978; Devriese et al., 1992). The non-enterococcal catalase-negative strains found in the cows faeces were also typed phenotypically. They did not give the typical response for Enterococcus ssp.

97

but grew at 45C. Two isolates from each cows faeces were shown to be Str. bovis by SDS-PAGE corroborating the findings of Medrek and Barnes (1962), Mieth (1962), Latham and Jayne-Williams (1978) and Devriese et al. (1992) that Str. bovis is the most common organism in the cows intestine. In the three trials, the dominant species of enterococci found in the cows faeces, was E. faecium, whereas E. faecalis and E. casseliflavus dominated the milk and cheese. The fact that very few enterococci were isolated from the cows faeces is a complicating factor in the experiments, as it is possible that E. casseliflavus and E. faecalis may have been present in low numbers in the cows faeces but were missed because the cows faeces were dominated by Str. bovis. No Str. bovis was found in the milk, the curd or the cheese although it was the dominant organism occurring in the cows faeces. This suggests that the cows were not the source of enterococci in the milk and cheese. A more selective medium in which enterococci can grow and Str. bovis cannot is currently being evaluated. In Trial 3, KF agar was used instead of KAA agar because of the poor recovery of enterococci by KAA in the animal faeces. However, no difference was noted between both agars in the recovery of enterococci except that some catalase positive strains were isolated from the cows and humans faeces from KF plates. The vancomycin resistant organisms isolated from the milk and cheese KF agar containing vancomycin were shown to be Pediococcus pentosaceus by phenotypic and genotypic analyses and SDS-PAGE. This confirms the findings of Litopoulou-Tzaneki et al. (1989) and Bhowmik and Marth (1990) that Pediococcus, which is intrinsically resistant to vancomycin (Mackey et al., 1993), is an organism commonly found in cheese. The milk and the cheese were of satisfactory initial microbiological quality (Table 1 and Fig. 1). Numbers of enterococci as well as staphylococci, coliforms and lactobacilli increased during manufacture. The difference in total viable counts was probably due to psychrotrophic organisms. Only lactobacilli grew subsequently during ripening. The behavior of the bacteria present in the cheese compares well with previous results. Mesophilic lactobacilli increase in numbers in all cheeses that have been examined while starter bacteria dominate the microflora of cheese at the beginning of the ripening period but their number decrease during ripening (Fox et al., 2000). Both staphylococci and coliforms decrease during ripening as reported by Fontecha et al.

98

(1990) and Macedo et al. (1995). Enterococci were present in small but stable numbers throughout the ripening period. Although previous studies report a decrease during curing (Clark and Reinbold, 1966; Fontecha et al., 1990) or a die-out after 30 days (Neviani et al., 1982b) the present results support those of Jensen et al. (1973) and Neviani et al. (1982a) who showed that enterococci were able to grow and survive during the ripening process. In this study the faeces of every animal and human in contact with the cheesemaking was sampled in a small artisanal cheese factory. We are not aware of any other such studies in the literature with which to compare our results. Whether these findings can be extrapolated to other artisanal cheese producers is not clear but should be investigated. The relationship between the isolates of E. casseliflavus and E. faecalis in the human faeces and milk and cheese are not clear as RAPD typing could not give sufficient information about the relationship at the strain level. As can be seen in Fig. 3, the same band pattern occurs in almost all the strains of the same species not only from different sources but also in the 3 different trials. A study of the clonal relationships between the isolates needs to be undertaken and this is currently being examined.

99

Acknowledgments This work was funded by a Teagasc Walsh Fellowship. The authors wish to thank Dick and Anne Keating for facilitating the study of their cheese and Tom Condon for his help in sampling the cows faeces. Marc Vancanneyt, Jean Swings and Tim Cogan acknowledge the European Communitys project 'Enterococci in food fermentations: Functional and safety aspects' (FAIR program FAIR-CT97-3078).

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References

Arizcun, C., Barcina, Y., Torre, P. 1997. Identification and characterization of proteolytic activity of Enterococcus ssp. isolated from milk and Roncal and Idiazabal cheese. Int. J. Food Microbiol. 38, 17-24. Bhowmik, T., Marth, E. H. 1990. Role of micrococcus and pediococcus species in cheese ripening: A Review. J. Dairy Sci. 73, 859-866. British Standard Methods. 1976. Chemical analysis of cheese. Part 5. Determination of pH values. BS 770. Clark, W. S., Reinbold, G. W. 1966. Enterococci in young Cheddar cheese. J. Dairy Science. 49, 12141218. Coakley, M., Ross, R. P., Donnelly, D. 1996. Application of the polymerase chain reaction to the rapid analysis of brewery yeast strains. J. Inst. Brew. 102, 349-354. Cogan, T. M., Barbosa, M., Beuvier, E., Bianchi-Salvadoris, B., Cocconcelli, P. S., Fernandes, I., Gomez, J., Gomez, R., Kalantzopoulos, G., Ledda, A., Medina, M., Rea, M. C., Rodriguez, E. 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64, 409-421. Deasy, B. M., Rea, M. C., Fitzgerald, G. F., Cogan, T. M., Beresford, T. P. 2000. A rapid PCR based method to distinguish between Lactococcus and Enterococcus. Syst. Appl. Microbiol. 23. Deasy, B. M. 2000. Design of 16S rRNA-targeted primers for identification of species of Pediococcus. in: Methods for the rapid identification of dairy lactic acid bacteria. Ph.D. thesis, University College Cork. 159-176. Descheemaeker, P., Lammens, C., Pot, B., Vandamme, P., Goosens, H. 1997. Evaluation of arbitrarily primed PCR analysis and Pulsed-Field Gel Electrophoresis of large genomic DNA fragments for identification of enterococci important in human medicine. Int. J. Syst. Bacteriol. 47 (2), 555561. Devriese, L. A., Van de Kerckhove, A., Kilpper-Blz, R., Schleifer, K. H. 1987. Characterization and identification of Enterococcus species isolated from the intestines of animals. Int. J. System. Bacteriol. 37 (3), 257-259. Devriese, L. A., Laurier, L., De Herdt, P., Haesebrouck, F. 1992. Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows. J. of Appl. Bacteriol. 72, 2931. Devriese, L. A., Pot, B., Van Damme, L., Kersters, K., Haesebrouck, F. 1995. Identification of Enterococcus species isolated from foods of animal origin. Int. J. of Food Microbiol. 26, 187197. Facklam, R. R., Teixeira, L. M. 1998. Enterococcus. In A. Balows and B. I. Duerden (Ed.), Topley & Wilsonss Microbiology and microbial infections, vol. 2., 9th ed., Arnold, London. pp. 669-682. Fontecha, J., Pelaez, C., Juarez, M., Requena, T., Gomez, C. 1990. Biochemical and microbiological characteristics of artisanal hard goats cheese. J. Dairy Sci. 73, 1150-1157. Fox, P. F., Guinee, T. P., Cogan, T. M., McSweeney, P. L. H. 2000. Microbiology of cheese ripening. In Fundamentals of cheese science, Aspen Publishers, USA. pp. 206-235.

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Fraimow, H. S., Jungkind, D. L., Lander, D. W., Delso, D. R., Dean, J. L. 1994. Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth. Ann Intern. Med. 121, 22-26. Franz, C. M. A. P., Holzapfel, W. H., Stiles, M. E. 1999. Enterococci at the crossroads of food safety? Int. J. Food. Microbiol. 47, 1-24. Fuller, R. 1989. Probiotics in man and animals, A Review. J. Appl. Bacteriol. 66, 365-378. Garg, S. K., Mital, B. K. 1991. Enterococci in milk and milk products. Crit. Rew. Microbiol. 18 (1), 1545. Giraffa, G., Domenico, C., Neviani, E. 1997. Enterococci isolated from dairy products: A review of risks and potential technological use. J. of Food Protection. 60 (6), 732-738. Hardie, J. M., Whiley, R. A. 1997. Classification and overview of the genera Streptococcus and Enterococcus. J. of Appl Microbiol. Symposium Supplement. 83, 1S-11S. IDF Standard 4A, 1982. Determination of the total solid contents. International dairy federation. Bruxelles. IDF Standard 5B, 1986. Determination of fat content. Gravimetric method. International dairy federation. Bruxelles. IDF Standard 20B, 1993. Determination of nitrogen content. International dairy federation. Bruxelles. IDF Standard 88A, 1988. Determination of chloride content. International dairy federation. Bruxelles. Jensen, J. P., Reinbold, G. W., Washam, C. J., Vedamuthu E. R. 1973. Role of enterococci in Cheddar cheese: Enterococci during manufacture and curing. J. Milk Food Technol. 36 (12), 613-618. Kurmann, J. A. 1968. ber die Ursachen der qualittsfrdernde Wirkung auf Emmentaler und Greyerzerkse bei einer Einimpfung von Streptococcus faecalis in keimarmer Rohmilch. Milchwissenschaft. 23, 193-197. Latham, M. J., Jayne-Williams, D. J. 1978. Streptococci in the alimentary tract of ruminants. In Skinner, F.A. & Quesnel (Ed.), Streptococci. L.B. Academic press, London. pp. 207-243. Lawrence, R. C., Heap, H. A., Gilles, J. 1984. A controlled approach to cheese technology. J. Dairy Sci. 67, 1632-1645. Leclerc, H., Devriese, L. A., Mossel, D. A. A. 1996. Taxonomical changes in intestinal (faecal) enterococci and streptococci: consequences on their use as indicators of faecal contamination in drinking water. J. Appl. Bacteriol. 81, 459-466. Litopoulou-Tzanetaki, E., Graham, D. C., Beyatli, Y. 1989. Detection of pediococci and other nonstarter organisms in American Cheddar cheese. J. Dairy Sci. 72, 854-858. Lodics, T. A., Steenson, L. R. 1990. Characterization of bacteriophages and bacteria indigenous to a mixed-strain cheese starter. J. Dairy Sci. 73, 2685-2696. Lopez-Diaz, T. M., Santos, J. A., Gonzales, C. J., Moreno, B., Garcia, M. L. 1995. Bacteriological quality of a traditional Spanish blue cheese. Milchwissenschaft. 50 (9), 503-504. Macedo, A. C., Malcata, F. X., Hogg, T. A. 1995. Microbiological profile in Serra ewes cheese during ripening. J. Appl. Bacteriol. 79, 1-11. Mackey, T., Lejeune, V., Janssens, M., Wauters, G. 1993. Identification of vancomycin-resistant lactic acid bacteria isolated from humans. J. Clin. Microbiol. 31, 2499-2501.

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Medrek, T. F., Barnes, E. M. 1962. The distribution of group D streptococci in cattle and sheep. J. Appl. Bacteriol. 25, 159-168. Mieth, H. 1962. Untersuchung ber das Vorkommen von Enterokokken bei Tieren und Menschen, III. Mitteilung: Die Enterokokkenflora in den Faeces von Rindern. Zentralblatt fr Bakteriologie, Parasitenkunde. Inf.-krankheiten/ Abt. I. 185, 47-52. Neviani, E., Mucchetti, G., Contarini, G., Carini, S. 1982a. Ruolo degli enterococci nei formaggi italiani I. Il latte. 7, 722-728. Neviani, E., Mucchetti, G., Contarini, G., Carini, S. 1982b. Ruolo degli enterococci nei formaggi italiani III. Il latte. 7, 902-907. Pitcher, D. G., Saunders, N. A., Owen, R. J. 1989. Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. Letters in Appl. Microbiol. 8, 151-156. Pot, B., Vandamme, P., Kersters, K. 1994. Analysis of electrophoretic whole-organism protein fingerprints. In: Goodfellow, M., and ODonnel, A. G. (Ed.), Chemical methods in procaryotic systematics. John Wiley and sons. USA, pp. 493-521. Saramago Stern, C., Siqueira Carvalho, M. G., Martins Teixeira, L. 1994. Characterization of enterococci isolated from human and nonhuman sources in Brazil. Diagn. Microbiol. Infect. Dis. 20, 61-67. Seale, D. R. 1986. Bacterial inoculants as silage additives. J. Appl. Bacteriol. 61 (suppl), 9S-26S. Suzzi, G., Caruso, M., Gardini, F., Lombardi, A., Vannini, L., Guerzoni, M. E., Andrighetto, C., Lanorte, M. T. 2000. A survey of the enterococci isolated from an artisanal Italian goats cheese (semicotto caprino). J. Appl. Microbiol. 89, 267-274. Trovatelli, L. D., Schiesser, A. 1987. Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwissenschaft. 42 (11), 717-718. Vancanneyt, M., Witt, S., Abraham, W. R., Kersters, K., Fredrickson, H. L. 1996. Fatty acid content in whole-cell hydrolysates and phospholipid fractions of Pseudomonas: a taxonomic evaluation. System. Appl. Microbiol. 19, 528-540.

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Chapter 7. the source of enterococci in a farmhouse raw-milk cheese

Gelsomino R.a, b, M. Vancanneytb, T. M. Cogan.a, S. Condonc, and J. Swingsb
a b c

National Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland BCCM/LMG Bacteria Culture Collection, University of Gent, Ledeganckstraat 35, Gent, Belgium Department of Microbiology, University College Cork, Cork, Ireland

Applied and Environmental Microbiology (2002) Vol. 68, p. 3560-3565

Summary Enterococci are widely distributed in raw milk cheeses and are generally thought to positively affect flavour development. Their natural habitats are the human and animal intestinal tracts but they are also found in soil, on plants, in the intestine of insects and birds. The source of enterococci in raw milk cheese is unknown. In the present study, an epidemiological approach, using Pulsed-Field Gel Electrophoresis (PFGE), was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the faeces of cows and humans, associated with the cheesemaking unit and the environment, including the milking equipment, the water used on the farm and the cows teats. Nine different PFGE patterns, 3 of E. casseliflavus, 5 of E. faecalis and 1 of E. durans were found. The same 3 clones, 1 of E. faecalis and 2 of E. casseliflavus, dominated almost all milk, cheese and human faecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination suggesting that a niche where enterococci could grow was present, and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human faeces are due to consumption of the cheese. Cows faeces were not considered to be the source of enterococci in the cheese as E. faecium and Str. bovis, which largely dominated the cows intestinal tract, were not found in either the milk or the cheese.

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Introduction Enterococci are found in a variety of artisanal cheeses made from raw or pasteurized goats, sheep, water-buffaloes or cows milk (Cogan et al, 1997; Franz et al., 1999). Their role in flavour development in cheese is not clear; some workers have reported that they cause deterioration in the flavour (Thompson and Marth, 1986; Lopez et al., 1995;), while others believe they play a major role in improving flavour development and cheese quality (Dahlberg and Kosikowsky, 1948; Walter et al., 1956; Clark et al., 1966, Kurman, 1968; Neviani et al., 1982). The most common enterococci in cheese are Enterococcus faecalis and Enterococcus faecium (Dave et al., 1980; Centeno et al., 1995; Arizcun et al., 1997; Cogan et al., 1997, Stiles and Holzapfel, 1997; Suzzi et al., 2000). Enterococci have become important over the past decade as they are the most frequently encountered nosocomial pathogens and appear to have increasing antimicrobial resistance (Noskin, 1997). An even greater threat is the transfer of vancomycin resistance from vancomycin resistant enterococci (VRE) to methicilinresistant Staphylococcus aureus (Michel and Gutman, 1997). Enterococci in food usually indicate poor bacteriological quality and poor hygiene during manufacture (Lopez et al., 1995). Therefore, there is a need to determine their source in food. The natural habitat of enterococci is the mammalian intestinal tract (Franz et al, 1999). E. faecalis is often the dominant species found in human faeces (Murray, 1990; Facklam and Teixeira, 1998; Franz et al, 1999); however, E. faecium, Enterococcus hirae, Enterococcus avium and Enterococcus durans have also been found (Murray, 1990; Godfree et al., 1997; Franz et al, 1999). Some enterococci are also found in the lower and upper urogenital tracts and in the oral cavity (Murray, 1990; Morrison et al., 1997). Although enterococci are prevalent in the human intestinal tract, they are less frequently isolated from the animal intestinal tract. E. faecium is the most frequently occurring species in dairy cows (Medrek and Barnes, 1962; Mieth, 1962; Martin et al., 1972; Latham et al., 1978; Devriese et al., 1992) but E. faecalis, E. hirae and Enterococcus casseliflavus are also found (Devriese et al., 1987; Devriese et al., 1992). In addition, enterococci are found in water, soil,

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plants, vegetables, birds and insects (Martin et al., 1972; Godfree et al., 1997; Facklam and Teixeira, 1998; Franz et al, 1999). In a previous study (Gelsomino et al, 2001), enterococci were isolated from a Cheddar type cheese during manufacture and ripening, from the milk it was made from, from the faeces of the personnel involved in cheesemaking and from the faeces of the dairy cows present on the farm. In addition, strains were isolated from the environment and the milking equipment, the tap water, the milking machine, and the cows teats. All these isolates were identified to species level by RAPD (random amplified polymorphic DNA). The dominant species in milk, cheese and human faeces was E. casseliflavus followed by lower numbers of E. faecalis. The cows faeces contained mainly E. faecium. The enterococci isolated from the environmental sources were mainly E. casseliflavus and E. faecalis. As the RAPD technique was not sensitive enough to distinguish between strains, no conclusion could be drawn about the clonal relationships of the enterococci in the cheese and their putative source. Pulsed Field Gel Electrophoresis (PFGE) has proven to be highly discriminatory and is generally accepted as the method of choice for typing strains of enterococci (Struelens et al., 1992; Gordillo et al., 1993; Maslow et al., 1993; Van Belkum et al., 1998). The aim of this study was to determine the sources of enterococci in the Cheddar cheese by comparing the strains isolated from the rawmilk and the cheese samples with the isolates from the human faecal samples and the isolates from the milking equipment and the environment using PFGE. As a result of this study valuable information has been obtained about the biodiversity of enterococci in a ripening cheese and the personnel associated with one cheese over a period of 15 months.

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Materials and Methods Isolation of strains. Six-hundred and forty-six enterococcal strains, isolated from the 3 trials described by Gelsomino et al. (Gelsomino et al, 2001) were used. These included 120 E. casseliflavus and 20 E. faecalis isolated from the milk, 304 E. casseliflavus and 56 E. faecalis isolated from the curd and the cheese, 61 E. casseliflavus, 49 E. faecalis and 9 E. durans isolated from the human faecal samples, 10 E. faecalis isolated from cows faeces, and 12 E. casseliflavus and 5 E. faecalis isolated from the milking equipment and the environment. KAA containing 6.5% salt (KAAS [wt/vol]) was tested as a selective medium for enterococci. Growth of type strains of enterococci (E. avium, Enterococcus cecorum, E. casseliflavus, Enterococcus columbae, Enterococcus dispar, E. durans, E. faecalis, E. faecium, Enterococcus flavescens, Enterococcus gallinarum, E. hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus saccharolyticus, Enterococcus solitarius, Enterococcus sulfurous) (BCCM/LMG) and Streptococcus bovis (R-16101 and R-16102, Research Collection of the Laboratory of Microbiology, University of Gent, Belgium) were tested on both KAA and KAAS. Two additional experiments were undertaken during the present study. The first consisted in mixing the first few strands of milk from each cows teat, which are normally disposed of, together, and plating them on KAA and KAAS. The second additional experiment consisted of collecting faecal samples by manual rectal retrieval and plating them on KAAS as well as on KAA. Where possible 10 putative enterococci were isolated per cow. In both of these experiments, when all 27 cows in the herd were milked, samples were taken from the bulk milk and the cheese at day 1 after manufacture. Samples were emulsified and plated and individual colonies isolated as described previously (Gelsomino et al, 2001). In this way, 131 isolates were obtained from the milk (n=30), the cheese (n=30) and the cows faeces, (n=71) from both KAA and KAAS plates.

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RAPD typing. DNA was prepared using the rapid procedure described by Pitcher et al. (Pitcher et al., 1989). Random primer D11344 was used to identify the new isolates from the milk, the cheese, the cows faeces and the strands of milk to species level in the 2 additional experiments as described previously (Gelsomino et al, 2001). DNA amplification was performed using the procedure described by Descheemaeker et al. (1997). Pulsed Field Gel Electrophoresis (PFGE). The strains were grown overnight at 37C on BM (2% Tryptose [Oxoid, Basingstoke, England], 0.5% NaCl [Merck, Darmstadt, Germany], 0.5% Yeast Extract [Merck, Darmstadt, Germany], 0.5% Glucose [Merck, Darmstadt, Germany], pH 6.85) agar. All reagents were obtained from Sigma (St. Louis, USA) unless otherwise stated. One loopful of cells from an overnight culture was washed 3 times in 1 ml EET buffer (100 mM EDTA, 10 mM EGTA, 10 mM Tris-HCl [pH 8.0]). After centrifugation the pellet was resuspended in EC buffer (6 mM Tris-HCl [pH 7.6], 1 M NaCl (Merck, Darmstadt, Germany), 100 mM EDTA [pH 8.0], 0.5% Polyoxyethylene 20 cetyl ether [Brij 58], 0.2% deoxycholate, 0.5% N-laurylsarcosyl) and mixed with an equal volume of 1.6% (wt/vol) low melting-point agarose (Bio-Rad, Richmond, USA) in EC buffer and pipetted into plug molds. The solidified plugs were incubated overnight at 37C in 1 ml EC buffer-lysozyme solution (2.88 mg lysozyme per ml of EC buffer). The lysis buffer was replaced with 1 ml of protein digestion solution (3.3 mg pronase E in 1 ml EET buffer containing 1.6% [wt/vol] SDS) and the plugs were incubated again overnight at 37C. The agarose plugs were washed 3 times for 1 h in EET buffer, twice for 1 h in Milli-Q water and once for 1 h in the appropriate restriction buffer (Buffer Y+/Tango, MBI Fermentas, St.Leon-Rot, Germany) at room temperature. The restriction was carried out overnight at 27.5C in 300l restriction buffer containing 30 units Sma I (MBI Fermentas). The digestion was stopped by adding 0.5 ml of 0.5 M EDTA (pH 8.0) and the plugs were stored at 4C. The restriction fragments were separated by PFGE in a contour clamped homogeneous electric field MAPPER system (Bio-Rad) by loading pieces of the plugs in 1% (wt/vol) PulsedField Certified Agarose (Bio-Rad) gel prepared with a 0.5X TBE buffer (45 mM

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Tris-HCl, 45 mM boric acid, 1 mM EDTA). Electrophoresis of E. casseliflavus was performed in 2 l 0.5X TBE at 14C for 22 h at 6 V/cm, an angle of 120 with pulse times ramping linearly from 0.41 s to 15.11 s. For all the other Enterococcus strains pulse times ramping linearly from 5 s to 30 s were chosen. A Staphylococcus aureus strain (R-6314, Department for Medical Microbiology, University of Antwerp) was used as a molecular weight marker. The genome was prepared as described above with the exception that 500 U of mutanolysin were added to the lysozyme solution. The gels were stained with ethidium bromide. Conversion, normalisation and analysis of the band patterns were performed using GelCompar software (Applied Maths, Belgium). Correlation coefficients and levels of similarity were calculated using the Dice coefficient and cluster analysis using the unweigthed pair group method (UPGMA). Growth of Str. bovis in milk. The two strains of Str. bovis were grown overnight in BM broth and in BM broth containing lactose instead of glucose. One ml of each culture was centrifuged and resuspended in 1 ml of Buffered Peptone Water (Oxoid). After washing, the cells were diluted in order to obtain an initial number of cells between 103 and 104 per ml of milk. Fifty ml of fresh raw milk and 50 ml of heat-treated milk (20 min at 95C) were inoculated and incubated on a stirring plate at 4C, 15C and 37C. Samples taken periodically during incubation for 3 days were plated on KAA and incubated for 24 h at 37C. Control samples were also plated on Plate Count Agar (PCA, Oxoid), which was incubated at 30C for 3 days.

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Results Selectivity of the isolation medium. The selectivity of KAA for the isolation of enterococci from cows faeces is very low as the vast majority of isolates from the medium were found to be Str. bovis (Gelsomino et al, 2001). The ability of 2 Str. bovis and 17 BCCM/LMG Enterococcus type strains to grow on KAA and KAAS was determined. E. avium, E. cecorum, E. columbae, E. pseudoavium, E. malodoratus did not grow or grew very poorly on KAAS (data not shown). All the other type strains grew as well on KAAS as on KAA while the 2 Str. bovis strains did not grow at all on KAAS. Seventy-one enterococci were isolated from the faeces of 12 of the 27 cows present on the farm on KAAS at dilutions 10-1 to 10-3. No enterococci were found in the faeces of the other 15 cows. In contrast, all samples plated on KAA showed growth up to dilution 10-4, indicating the presence of Str. bovis. No enterococci were found in the first strands of milk from all the teats of the cows either on KAA or KAAS. This corroborates the results of the previous study (Gelsomino et al, 2001). An average of 7.3 X 103 enterococci/ml was found in the bulk milk ~3 h after the cows' teats and faeces were sampled on both the KAA and KAAS while an average of 2 X 105 enterococci/ml was found in the cheese on both KAA and KAAS. Species identification. In the 2 additional experiments, the isolates on both KAA and KAAS from the milk (n=30) and the cheese (n=30), which was made shortly after the cows were milked, were shown to be E. casseliflavus (50%) and E. faecalis (50%) by RAPD. All isolates from the cows faeces (n=71) were shown to be E. faecium by RAPD and were not categorised any further. Strain typing. PFGE was performed on the 646 isolates of E. casseliflavus, E. faecalis and E. durans previously isolated (Gelsomino et al, 2001). All 646 PFGEband patterns were clustered and compared visually and with the GelCompar software. Only 9 different clones were found in the 646 isolates tested. An example of each of these is shown on Figure 1. In this figure E. casseliflavus patterns are marked with Cs, E. faecalis patterns with an Fs and E. durans patterns with a

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Ds. The patterns marked Fs1a/Fs1b/Fs1c and Cs1a/Cs1b differed by only one band and were, in accordance with the definition of Tenover et al. (Tenover et al., 1995), considered to represent the same clone. Table 1 shows the distribution of all the clones found in the milk, cheese, faeces and the environment and Fig. 2 shows the percentage of the most common clones in each of the 3 trials. Three of the 9 clones, Cs1, Cs2 and Fs1, largely dominate the milk, the cheese and the human faeces. These 3 clones, 2 E. casseliflavus and 1 E. faecalis, were found throughout the 3 trials, and 2 of them, E. casseliflavus Cs1 and Cs2, were also present in the environmental samples, i.e. the bulk tank and the milking machine rinses. E. casseliflavus Cs3 appeared less abundantly in the cheeses and in adult 1 but was not evenly distributed over the three trials. Other clones were occasionally found. Four E. faecalis isolates of clone Fs2 were found in the milk from the bulk tank and 1, 4 and 1 isolates of this clone were found in adults 1 and 2 and teenager 2, respectively, in Trial 1. Ten isolates in the cows faeces during Trial 2 and one isolate from the rinse of the bulk tank were found to be clone Fs2. Only 1 isolate of clone Fs3 (E. faecalis) was found in the cheese of Trial 1, ripened for 11 days. In addition, 6 and 3 isolates of E. durans were found in the faeces of adult 2 and teenager 2, respectively, and they all show the same band pattern (Ds). The two Fs4 clones were found in the tap water while the two Fs6 clones were found in the milking machine (Figure 2 and Table 1). A representative isolate of each clone has been deposited in the BCCM/LMG Bacteria Collection with the following numbers: LMG 19846 (Fs1), LMG 20228 (Fs2), LMG 20229 (Fs3), LMG 20230 (Fs4), LMG 20234 (Fs6), LMG 20231 (Ds), LMG 19844 (Cs1), LMG 19845 (Cs2), and LMG 20235 (Cs3). Growth of Streptococcus bovis in milk. Large numbers of Str. bovis were found in cows faeces when plated on KAA but no Str. bovis isolates were found in any milk sample (Gelsomino et al, 2001). This raised the question whether Str. bovis grows in milk.

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Sc

Cs1a Cs1b Cs2 Cs3

Sf

Fs1a Fs1b Fs1c Fs2

Fs3

Fs4

Fs6

Ds

Figure 1: Agarose gel showing the PFGE-band patterns of the 9 clones found and digested with Sma I. A Sta. aureus strain was used as molecular weight marker; Sc and Sf are Sta. aureus strains used as a molecular weight marker run with the PFGE parameters for E. casseliflavus and E. faecalis, respectively. Cs, Fs and Ds signify E. casseliflavus, E. faecalis and E. durans, respectively, while Cs1a and Cs1b and Fs1a, Fs1b and Fs1c are considered to be the same clones.

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Table 1. Strain distribution of the isolates found in milk, cheese, faeces and the environment. E. casseliflavus Cs1 sample Milk bulk tank cheese vat Cheese before moulding at 1 day at 11 days at 25 days at 39 days at 53 days Faeces adult 1 adult 2 teenager 1 teenager 2 cow 27 trial 2 Environment water rinse emty bulk tank rinse milking machine before chlorination rinse milking machine after chlorination Total 46 32 29 16 35 19 35 22 8 15 5 6 Cs2 23 19 22 29 14 23 20 30 7 2 5 9 Cs3 Fs 1 7 9 9 15 8 15 3 5 10 3 20 10 1 4 1 10 2 2 2 5 3 1 2 Clones Fs 2 4 E. faecalis Fs 3 Fs 4 Fs 6 E. durans Ds Total 80 60 60 60 60 60 60 60 30 30 30 29 10 2 5 5 5 646

2 3 2 3 4

6 3

100%

80%

60%

others Fs 1 Cs1 Cs2 Cs3

40%

20%

0% Trial 1 [40] Trial 2 [40] Milk Trial 3 [60] Trial 1 [120] Trial 2 [120] Cheese Trial 3 [120] Trial 1 [40] Trial 2 [40] Human Faeces Trial 3 [39] Trial 1 [0] Trial 2 [0] Environment Trial 3 [17]

Figure 2: Distribution of the clones found in each of the 3 trials (July 1998, September 1998 and October 1999). Numbers in brackets give the total number of isolates in that particular trial. Cs1, Cs2 and Cs3 are different clones of E. casseliflavus and Fs1 a clone of E. faecalis.

Both glucose-grown and lactose grown cells of the 2 strains of Str. bovis survived for at least 3 days at 4oC and grew at 15C and at 37oC from 1,5 X 103 cfu/ml to 9.6 X 107 cfu/ml in 2 days in the raw and heat-treated milk indicating that the natural inhibitors present in the raw milk did not prevent the growth of Str. bovis. In 3 days, the total bacterial counts on Plate Count Agar increased from 4.6 X 104 to 7.0 X 106 cfu/ml in the raw milk at 4oC and from 3.2 X 104 to 3.5 X 108 cfu/ml in the raw milk at 37oC (data not shown); bacteria with which Str. bovis was able to compete. No colonies were detected on KAA or PCA in the uninoculated heat-treated milk indicating that the heat treatment was sufficient to inactivate any contaminating bacteria present.

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Discussion In this study, an epidemiological approach was used to identify the sources of enterococci in cheese using PFGE. Nine different PFGE-band patterns, which were considered to be distinct clones, were found among 646 isolates from the milk, the cheese, the human faeces, the equipment and the environment (Fig. 1). Of these, 5 were produced by E. faecalis (Fs1-Fs4, F6), 1 by E. durans (Ds) and 3 by E. casseliflavus (Cs1-Css3). The dominant species found in the cheese and the milk was E. casseliflavus (Gelsomino et al, 2001), although E. faecalis and E. faecium are reported to be the most common species of Enterococcus in cheese (Trovatelli and Schiesser, 1987; Devriese et al, 1995; Arizcun et al., 1997; Cogan et al., 1997; Suzzi et al., 2000) whereas E. casseliflavus is more often associated with plants (Devriese et al., 1992; Leclerc et al., 1996). Two E. casseliflavus clones, Cs1 and Cs2, accounted for 47% and 36% of the 500 isolates from the milk and cheese respectively. E. faecalis clone Fs1 was present in lower numbers (14%). Except for the milk and the cheese in Trial 2, where clone Fs1 was not present (Fig. 2), these 3 clones were found in all samples of the milk and the cheese throughout the 3 trials (Table 1) and constituted 97% of all isolates found in the milk and the cheese. E. casseliflavus clone Cs3 was found quite often in the ripening cheese but not in the milk. Of the other E. faecalis isolates, only 4 isolates of clone Fs2 were found in the bulk tank milk while clone Fs3 was found only once, in the cheese ripened for 11 days. The dominant species in the human faeces was E. casseliflavus followed by lower numbers of E. faecalis (Gelsomino et al, 2001). This result disagrees with previous studies which showed that E. faecalis is the most common species in the human intestine (Leclerc et al., 1996; Saramago et al., 1994; Facklam and Teixeira, 1998). Strain identification with PFGE showed that the same clones occurred in the milk, the cheese and the human faeces. Within the human faeces E. faecalis clone Fs1 was found in high numbers (36% of isolates), followed by E. casseliflavus clones Cs1 (28.% of isolates) and Cs2 (19% of isolates). Clone Cs2 was only found in Trial 1 while clones Fs1 and Cs1 occurred in all trials (Fig. 2). E. casseliflavus clone Cs3 was found in adult 1 in Trial 3 while E. faecalis clone Fs2 was distributed over 3 of

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the 4 members of the family. The 9 isolates of E. durans gave identical patterns and were found in adult 2 and teenager 2 in Trial 3 (Ds). As both the milk and cheese samples and the human samples contained the same Enterococcus species and, more importantly, the same 3 dominant clones, human contamination could be regarded as the possible source of enterococci in cheese. Another possibility is that the enterococci in the human faeces are due to ingestion of the milk and the cheese. The latter is more likely as the family eat their own cheese. In the previous study (Gelsomino et al, 2001) only a few enterococci were found in the cows faeces. All of them were E. faecium except 10 isolates from cow No. 27 in Trial 2. A more selective medium (KAAS) was used in the present study to isolate putative enterococci from the bovine faeces and E. faecium was still the only species found, and again in very low numbers; some sample of cows faeces showed no colonies even at dilution 10-1. In contrast, growth on KAA without NaCl occurred up to dilution 10-4, indicating the presence of Str. bovis (Gelsomino et al, 2001). As indicated, E. faecalis was isolated only from cow No. 27 in Trial 2 (This cow was present in the herd of Trials 1 and 2 but was culled in Trial 3) and all isolates belonged to clone Fs2. The same clone was found in the milk in Trial 3 and also in human faeces in Trial 1. We have no adequate explanation for this result but we believe that cows faeces could not have contaminated the milk, as Fs2 was already present in the human faeces in Trial 1. Enterococci were not found on the teats (Gelsomino et al, 2001) or in the udder (the present study). In addition, no clones of E. casseliflavus or E. faecalis found in the milk or the cheese, could be traced back to cows, except for the one already discussed. Furthermore, the only Enterococcus species found in cows faeces, except for the one cow mentioned above, was E. faecium, which was also not found in the milk or cheese. Therefore, we conclude that the cows faeces were not the source of enterococcal contamination of the cheese and milk. This is corroborated by the findings that Str. bovis, which is the most dominant species in cows faeces and grows quite well on KAA, can grow in milk but was not found in the milk or the cheese. Both the milking machine and bulk tank are normally sterilized with HydrosanT before chlorination. Both were rinsed with sterile water and the rinses examined for enterococci. Ten isolates of E. casseliflavus clone Cs2 were found in

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the rinses of the milking machine and bulk tank and 2 isolates of clone Cs1 were found in the rinse of the bulk tank. Clone Fs2 was found in the rinse of the bulk tank and in the milk of the bulk tank. E. faecalis clones Fs4 and Fs6 were also found in the tap water and in the milking machine. The fact that the dominant clones in the milk, the cheese and the humans are found in the milking equipment strongly suggests that enterococcal contamination starts there. Although the milking machine has been rinsed and sterilized with a 0.024% sodium hypochlorite solution (Gelsomino et al, 2001) in order to maintain good hygiene in the milking equipment, disinfection might not be fully effective, so that milk residues and bacteria are not completely eliminated from the equipment. Crevices, joints, dead ends and fittings are danger points where bacteria tend to accumulate and grow, infecting the milk directly (Bramley et al., 1990; Slaghuis et al., 1991). The possibility of a biofilm also being present (Ven, 1999) cannot be ruled out. This might explain why enterococci were found even after chlorination. It is also possible that E. casseliflavus survives better in the milking equipment than E. faecalis. In conclusion, data from the present study demonstrate that enterococci survive and grow in the hidden corners of the milking machine and the bulk tank thus infecting the milk directly. From the milk the enterococci are transferred into the cheese and from there probably into the human intestinal tract by ingestion. But whether these strains were brought into the equipment via the air or poor hygienic housing conditions is not obvious. The original source of the enterococci in the milking machine is not clear.

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Acknowledgments This work was funded by a Teagasc Walsh Fellowship. The authors wish to thank Dick and Anne Keating for facilitating the study of their cheese and Tom Condon for his help in sampling the cows faeces. Marc Vancanneyt, Jean Swings and Tim Cogan acknowledge the European Communitys project 'Enterococci in food fermentations: Functional and safety aspects' (FAIR program FAIR-CT97-3078).

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References

Arizcun, C., Y. Barcina, and P. Torre. 1997. Identification and characterization of proteolytic activity of Enterococcus ssp. isolated from milk and Roncal and Idiazabal cheese. Int. J. Food Microbiol. 38:17-24. Ayers, S. H., and C. S. Mudge. 1923. Streptococci of feces and mouth of cows. J. Inf. Dis. 33:155160. Bramley, A. J., and C. H. McKinnon. 1990. The microbiology of raw milk. p. 163-208. In R. K. Robinson (ed.) The microbiology of the milk, 2nd ed. Elsevier Applied Science, London. Centeno, J. A., A. Cepeda, and J. L. Rodriquez-Otero. 1995. Identification and preliminary characterization of strains of enterococci and micrococci isolated from Arza raw cows-milk cheese. Die Nahrung. 1:55-62. Clark, W. S., and G. W. Reinbold. 1966. Enterococci in young cheddar cheese. J. Dairy Sci. 49:1214-1218. Cogan, T. M., M. Barbosa, E. Beuvier, B. Bianchi-Salvadoris, P. S. Cocconcelli, I. Fernandes, J. Gomez, R. Gomez, G. Kalantzopoulos, A. Ledda, M. Medina, M. C. Rea, and E. Rodriguez. 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64:409-421. Dahlberg, A. C., and F. V. Kosikowsky. 1948. The development of flavor in American Cheddar cheese made from pasteurized milk with Streptococcus faecalis starter. J. Dairy Sci. 31:275284. Dave, J. M., M. V. Desai, K. G. Upadhayay, and P. N. Thakar. 1980. Incidence of enterococci in buffalo milk Cheddar cheese. GAU Res. J. 5:44-47. Descheemaeker, P., C. Lammens, B. Pot, P. Vandamme, and H. Goosens. 1997. Evaluation of arbitrarily primed PCR analysis and Pulsed-Field Gel Electrophoresis of large genomic DNA fragments for identification of enterococci important in human medicine. Int. J. Syst. Bacteriol. 47: 555-561. Devriese, L. A., A. Van de Kerckhove., R. Kilpper-Blz, and K. H. Schleifer. 1987. Characterization and identification of Enterococcus species isolated from the intestines of animals. Int. J. System. Bacteriol. 37:257-259. Devriese, L. A., B. Pot, L. van Damme, K. Kersters, and F. Haesebrouck. 1995. Identification of Enterococcus species isolated from foods of animal origin. Int. J. Food Microbiol. 26:187197. Devriese, L. A., L. Laurier, P. De Herdt, and F. Haesebrouck. 1992. Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows. J. Appl. Bacteriol. 72:29-31. Facklam, R. R., and L. M. Teixeira. 1998. Enterococcus. p. 669-682. In A. Balows and B. I. Duerden (ed.), vol. II (systematic bacteriology) of Topley & Wilsonss Microbiology and microbial infections, 9th ed. Arnold, London. Franz, C. M. A. P., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47:1-24

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Gelsomino, R., M. Vancanneyt, S. Condon, J. Swings, and T. M. Cogan. 2001. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory. Int. J. Food Microbiol. 71:177-188. Godfree, A. F., D. Kay, and M. D. Wyer. 1997. Faecal streptococci as indicators of faecal contamination in water. J. Appl. Microbiol. Symposium Supplement. 83:110S-119S. Gordillo, M. E., K. V. Singh, and B. E. Murray. 1993. Comparison of ribotyping and Pulsed-Field Gel Electrophoresis for subspecies differentiation of strains of Enterococcus faecalis. J. Clin. Microbiol. 31:1570-1574. Kurmann, J. A. 1968. ber die Ursachen der qualittsfrdernde Wirkung auf Emmentaler und Greyerzerkse bei einer Einimpfung von Streptokokkus faecalis in keimarmer Rohmilch. Milchwiss. 23:193-197. Latham, M. J., and D. J. Jayne-Williams. 1978. Streptococci in the alimentary tract of ruminants. p. 207-243. In Streptococci ed. Skinner, F.A. & Quesnel, L.B. Academic press, London. Leclerc, H., L. A. Devriese, and D. A. A. Mossel. 1996. Taxonomical changes in intestinal (faecal) enterococci and streptococci: consequences on their use as indicators of faecal contamination in drinking water. J. Appl. Bacteriol. 81:459-466. Lopez-Diaz, T. M., J. A. Santos, C. J. Gonzales, B. Moreno, and M. L. Garcia. 1995. Bacteriological quality of a traditional Spanish blue cheese. Milchwiss. 50: 503-504. Martin, J. D., and J. O. Mundt. 1972. Enterococci in insects. Appl. Microbiol. 24:575-580. Maslow, J. N., M. E. Mulligan, and R. D. Arbeit. 1993. Molecular Epidemiology: Application of contemporary techniques to the typing of microorganisms. Clin. Inf. Dis. 17:153-164. Medrek, T. F., and E. M. Barnes. 1962. The distribution of group D streptococci in cattle and sheep. J. Appl. Bact. 25:159-168. Michel, M., and L. Gutmann. 1997. Methicilin-resistant Staphylococcus aureus and vancomycinresistant enterococci: therapeutic realities and possibilities. Lancet. 349:1901-1906. Mieth, H. 1962. Untersuchung ber das Vorkommen von Enterokokken bei Tieren und Menschen, III. Mitteilung: Die Enterokokkenflora in den Faeces von Rindern. Zentralbl. Bakteriol. Parasitenk. Inf.-krankh./ Abt. I. 185:47-52. Morrison, D., N. Woodford, and B. Cookson. 1997. Enterococci as emerging pathogens of humans. J. Applied Microbiol Symposium Supplement. 83:89S-99S. Murray, B. E. 1990. The life and times of the enterococcus. Clin. Microbiol. Rev. 3:46-65. Neviani, E., G. Mucchetti, G. Contarini, and S. Carini. 1982. Ruolo degli enterococci nei formaggi italiani I. Il latte. 7:722-728. Noskin, G. A. 1997. Vancomycin-resistant enterococci: Clinical, microbiologic and epidemiologic features. J. Lab. Clin. Med. 130:14-20. Pitcher, D. G., N. A. Saunders, and R. J. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidinium thiocyanate. Letters in Appl. Microbiol. 8: 151-156. Saramago Stern, C. M. G. Squeira Carvalho, L. Martins Teixeira. 1994. Characterization of enterococci isolated from human and nonhuman sources in Brazil. Diagn. Microbiol. Infect. Dis. 20:61-67.

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Slaghuis, B. A., T. De Vries, and J. G. P. Verheij. 1991. Bacterial load of different materials which can contaminate milk during production. Milchwiss. 46:574-577. Stiles, E. M., and Holzapfel W. H. 1997. Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36:1-29 Struelens, M. J., A. Deplano, C. Godard, N. Maes, and E. Serruys. 1992. Epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using Pulsed-Field Gel Electrophoresis. J. Clin. Microbiol. 30:2599-2605. Suzzi, G., M. Caruso, F. Gardini, A. Lombardi, L. Vannini, M. E. Guerzoni, C. Andrighetto, and M. T. Lanorte. 2000. A survey of the enterococci isolated from an artisanal Italian goats cheese (semicotto caprino). J. Appl. Microbiol. 89:267-274. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by Pulsed-Field Gel Electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239. Thompson, T. L., and E. H. Marth. 1986. Changes in parmesan cheese during ripening: microflora coliforms, enterococci, anaerobes, propionibacteria and staphylococci. Milchwiss. 41:201205. Trovatelli, L.D., and A. Schiesser. 1987. Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwiss. 42:717-718. Van Belkum, A., W. Van Leeuwen, M. E. Kaufmann, B. Cookson, F. Forey, J. Etienne, R. Goering, F. Tenover, C. Steward, F. OBrien, W. Grubb, P. Tassios, N. Legakis, A. Morvan, N. El Solh, R. De Ryck, M. Struelens, S. Salmenlinna, J. Vuopio-Varkila, M. Kooistra, A. Talens, W. Witte, and H. Verbrugh. 1998. Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of Sma I macrorestriction fragments: a multicenter study. J. Clin. Microbiol. 36:1653-1659. Ven Poulsen, Lena. 1999. Microbial biofilm in food processing. Lebensm.-Wiss. u.-Technol. 32:321-326. Walter, H. E., A. M. Sadler, J. P. Malkames, and C. D. Mitchell. 1956. A simplified short-time method for making Cheddar cheese from pasteurized milk. J. Dairy Sci. 39:917 (Abstr.).

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Chapter 8. Effect of consuming a raw milk cheese on the enterococcal flora of human feces

Roberto Gelsominoa, Marc Vancanneyta, Timothy M. Cogan.b, and Jean Swingsa

a b

BCCM/LMG Bacteria Culture Collection, University of Gent, Ledeganckstraat 35, Gent, Belgium National Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland

Applied and Environmental Microbiology (2003) Vol. 69, p. 312-319.

Summary Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract (GIT). In the present study the composition of the enterococcal fecal flora in three healthy humans was analysed before, during and after the daily consumption of ~125g of a raw-milk Cheddar-type cheese containing 3.2 X 104 enterococci/g cheese. Enterococcal counts ranged between 1.4 X 102 and 2.5 X 108 cfu/g of feces and varied within subjects and from week to week. The cheese contained mainly Enterococcus casseliflavus and a small population of Enterococcus faecalis. Clonal relationships were determined by PFGE. Before and after consumption of the cheese, human samples contained mainly Enterococcus faecium with some of the clones being resident. During consumption of the cheese one particular transient clone of E. faecalis, clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of E. casseliflavus from the cheese were also found in the feces of one of the subjects during cheese consumption. These result suggest that a clone need not to be present in a food in high numbers to establish itself in the intestine.

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Introduction The human gastrointestinal tract (GIT) harbours a complex bacterial ecosystem. Up to 1014 bacteria may be present (Adlerberth et al., 2000; Fons et al., 2000) comprising 400-500 species although generally only a few predominate (Kleessen et al., 2000). The major bacterial groups are normally stable (Holdeman et al., 1976). Studies in which known strains were ingested showed that certain strains are detected in faeces constantly and over a long period, while others are found only occasionally (Adlerberth et al., 2000; Wallick and Stuart, 1943). Sears et al. (1949) called the persistent strains resident and the strains found occasionally transient. The human intestinal flora is a complex network of mutual or/and antagonistic interactions. To establish in the intestine, bacteria must either adhere to the mucosa in order not to be swept away by peristalsis (Adlerberth et al., 2000) or multiply at a rate exceeding their rate of elimination (Kleessen et al., 2000); bacteria must also compete for nutrients, growth factors, binding sites and colonisation resistance towards already established bacteria (Adlerberth et al., 2000) which generate an environment that is inhibitory towards potential competitors. This can be accomplished by changes in pH and oxidation-reduction potential and by the production of H2S and volatile fatty acids (8). Recent studies have monitored the passage of lactobacilli and bifidobacteria through the GIT (Bunte et al., 2000; Kimura et al., 1997; Srensen et al., 2001). Enterococci are the predominant, Gram-positive cocci in human stools with 105-108 cfu/g feces (Jett et al., 1994; Kleessen et al., 2000) and Enterococcus faecalis is the commonest Enterococcus species found (Facklam and Teixeira, 1998; Leclerc et al., 1966; Saramago Stern et al., 1994). Franz et al. (1999) and Murray (1990) suggest that the presence of Enterococcus faecium and E. faecalis in humans is dependent on the geographical location. Enterococci are the cause of a variety of infections including endocarditis and neonatal, central nervous system or respiratory tract infections (Hardie and Whiley, 1997). They may also infect the abdomen biliary tract, burn wounds, soft tissues, paranasal sinuses, ear, eye and peridontal tissue (Jett et al., 1994). Despite these involvements, the study of these organisms at a strain level is limited. Of major concern are the sources of nosocomial infections. Many of these have been

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identified, but a high percentage remains obscure in origin and some presumably originate from the GIT. It is believed that enterococci exit the epithelial cells or migrate in phagocytes and spread hematogenously to distant sites (Jett et al., 1994). Enterococci have become the focus of attention also due to their increasing resistance to antibiotics. Not only are they resistant to vancomycin, they are also resistant to teicoplanin, penicillins and aminoglycosides. In addition, vancomycin dependent enterococci have also been reported (Fraimow et al., 1994). The presence of vancomycin resistant enterococci (VRE) in hospitals is met with considerable apprehension (Franz et al., 1999; Gardiner et al., 2000; Morris et al., 1995; Morrison et al., 1997). A major issue of concern is the transfer of antibiotic resistance from enterococci to more virulent pathogens such as multiple-drug resistant staphylococci (Morrison et al., 1997). VRE have also been isolated from food (Franz et al., 1999; Giraffa et al., 1997; Gardiner et al., 2000; Perreten et al., 1997). From this point of view the statement by Garg and Mital (1991), a food should be free not only from disease-producing organisms, but also from those that have the potential of causing disease seems legitimate. Nevertheless enterococci are used as silage inoculants (Seale, 1986), starter cultures (Giraffa et al., 1997) and probiotics (Franz et al., 1999; Fuller, 1989), possess antilisterial activities (Elotmani et al., 2002; Nunez et al., 1997) produce various metabolic compounds that can interfere with the growth of undesirable bacteria (Fonds et al., 2000) and have a beneficial role in ripening and flavor development of cheese (Clark and Reinbold, 1966; Kurmann, 1986; Neviani, et al., 1982). Finally, enterococci from dairy products show higher sensitivity to antibiotics and they had a long history of safe use (Giraffa et al., 1997). In a previous study (Gelsomino et al., 2002) the enterococcal flora of a rawmilk, farmhouse cheese was compared with the microflora of human and bovine feces. The cheese and the human feces contained two dominant strains of Enterococcus casseliflavus and one of E. faecalis followed by lower numbers of other clones from the two species. The cows were not the source of enterococci in the cheese but the presence of both E. casseliflavus clones in the milking equipment suggested that contamination of the milk starts there. The presence of identical clones of enterococci in human feces and cheese was explained by consumption of milk and cheese by the humans, but this was not proven.

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In the present paper we describe the results of a study in which 3 healthy human subjects consumed cheese containing enterococci (Gelsomino et al., 2001; Gelsomino et al., 2002). The purpose of our investigation was to determine the impact that consumption of cheese containing enterococci had on the enterococcal flora of the feces. Valuable information was also obtained about the regular enterococcal flora.

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Materials and methods Collection of samples: Four healthy Belgian subjects (2 males and 2 females) who were between 26 and 52 years old participated in this study. Fecal samples were collected weekly, in sterile containers, by the volunteers, over a 10-12 week period, except in week 4. The first 3 weeks was the control period after which three of the four volunteers began to consume between 100 and 150 g of an Irish farmhouse raw milk Cheddar-type cheese (Gelsomino et al., 2001) daily, giving a daily dose of 3.2 X 106 4.8 X 106 enterococci for 4 weeks. The feces were not sampled during the first week of cheese consumption. Weekly fecal samples were also collected for 3 weeks after cheese consumption had stopped. The 4th volunteer was used as a control. During this period the volunteers maintained their lifestyles and usual diet, including, in some cases, other cheeses. None of the subjects was administered antibiotics during the study period. If, for any reason, a weekly fecal sample was not obtained, the experiment was prolonged by one week. Isolation of strains: The cheese was emulsified 1:10 in a 2% (wt/vol) trisodium citrate solution, pH 8.75, homogenized with the aid of a Stomacher and plated on Kanamycin Esculin Azide (KAA, Merck) agar. Fecal samples (3-6g) were immediately diluted 1:10 in sterile peptone saline solution (PSS, [0.1% Peptone, Oxoid; 8.5% NaCl, Merck]), emulsified in a Stomacher, diluted in PSS and plated on KAA agar. All plates were incubated overnight at 37C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2). Four hundred and eighty colonies were picked from plates of the cheese showing over 100 colonies. Ten colonies were randomly picked from the highest dilution of each fecal sample. All colonies were purified twice on KAA and once on BM agar (2% Tryptose [Oxoid], 0.5% NaCl [Merck], 0.5% Yeast extract [Merck], 0.5% Glucose [Merck], pH 6.85). Cultures were maintained at -20C in a 1:2 glycerol-BM broth mixture. Isolates from stock were streaked on BM plates. Strain typing by Pulsed Field Gel Electrophoresis (PFGE). As the running conditions for PFGE are different for the yellow-pigmented E. casseliflavus than for other enterococci, all isolates were first distinguished by colony-color when grown on BM agar. The yellow colonies were assumed to be E. casseliflavus as shown in previous studies (Gelsomino et al., 2001; Gelsomino et al., 2002) while the white

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colonies represented E. faecalis, E. faecium, Enterococcus hirae and other enterococci, commonly present in feces. The strains were grown overnight at 37C on BM (2% Tryptose [Oxoid, Basingstoke, England], 0.5% NaCl [Merck, Darmstadt, Germany], 0.5% Yeast Extract [Merck, Darmstadt, Germany], 0.5% Glucose [Merck, Darmstadt, Germany], pH 6.85) agar. All reagents were obtained from Sigma (St. Louis, USA) unless otherwise stated. One loopful of cells from an overnight culture was washed 3 times in 1 ml EET buffer (100 mM EDTA, 10 mM EGTA, 10 mM Tris-HCl [pH 8.0]). After centrifugation the pellet was resuspended in EC buffer (6 mM Tris-HCl [pH 7.6], 1 M NaCl (Merck, Darmstadt, Germany), 100 mM EDTA [pH 8.0], 0.5% Polyoxyethylene 20 cetyl ether [Brij 58], 0.2% deoxycholate, 0.5% N-laurylsarcosyl) and mixed with an equal volume of 1.6% (wt/vol) low melting-point agarose (BioRad, Richmond, USA) in EC buffer and pipetted into plug molds. The solidified plugs were incubated overnight at 37C in 1 ml EC buffer-lysozyme solution (2.88 mg lysozyme per ml of EC buffer). The lysis buffer was replaced with 1 ml of protein digestion solution (3.3 mg pronase E in 1 ml EET buffer containing 1.6% [wt/vol] SDS) and the plugs were incubated again overnight at 37C. The agarose plugs were washed 3 times for 1 h in EET buffer, twice for 1 h in Milli-Q water and once for 1 h in the appropriate restriction buffer (Buffer Y+/Tango, MBI Fermentas, St.Leon-Rot, Germany) at room temperature. The restriction was carried out overnight at 27.5C in 300l restriction buffer containing 30 units Sma I (MBI Fermentas). The digestion was stopped by adding 0.5 ml of 0.5 M EDTA (pH 8.0) and the plugs were stored at 4C. The restriction fragments were separated by PFGE in a contour clamped homogeneous electric field MAPPER system (Bio-Rad) by loading pieces of the plugs in 1% (wt/vol) Pulsed-Field Certified Agarose (Bio-Rad) gel prepared with a 0.5X TBE buffer (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA). Electrophoresis of E. casseliflavus was performed in 2 l 0.5X TBE at 14C for 22 h at 6 V/cm, an angle of 120 with pulse times ramping linearly from 0.41 s to 15.11 s. For all the other Enterococcus strains pulse times ramping linearly from 5 s to 30 s were chosen. A Staphylococcus aureus strain (R-6314, Department for Medical Microbiology, University of Antwerp) was used as a molecular weight marker. The genome was prepared as described above with the exception that 500 U of

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mutanolysin were added to the lysozyme solution. The gels were stained with ethidium bromide. Species identification by SDS-PAGE. Cells were grown for 24 hours on BM-agar at 37C under microaerophilic conditions. Whole cell protein extracts and protein separation was carried out as described by Gelsomino et al. (Gelsomino et al., 2002) on 1 to 3 isolates from each clone clustered by PFGE.

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Results Total counts of enterococci in cheese and human feces. The cheese used contained an average of 3.2 X 104 enterococci/g (sd = 5.6 X 103; data not shown). Figure 1 shows the number of enterococci in the fecal samples as well as the clonal diversity at each sampling point. A black bar on the x-axis illustrates the period of cheese consumption. The subjects started eating the cheese in week 4 during which no samples were taken.

Figure 1. Diagrams showing the numbers of enterococci and the species and strains recovered from the feces of single subjects before, during and after cheese consumption. (Fs = E. faecalis, Fm = E. faecium, Cs = E. casseliflavus, Ds = E. durans, He = E. hirae). Numbers refer to different clones. The black bars show the period of cheese consumption. Column-colors in the legend refer to the strains that were recovered from both humans and cheese.
Subject A
10
Ds1 Fs8 Fm8 Cs4 Fm 12 Cs1 Cs2 Cs2

clones originating from the cheese E. faecalis strain Fs2 E. casseliflavus strain Cs1 E. casseliflavus strain Cs2 species abbreviations DsEnterococcus durans FsEnterococcus faecalis FmEnterococcus faecium HeEnterococcus hirae

He1 Fs 12 Fs 11 Fs 10

Fm 17 Fm 16

Fs 17 Fs 16 Fs 15 Fs 14

8
Fm7 Fm6

Fs9

6 isolates

Fs7 Fm5 Fm 11 Fs2 Fs2 Fs2

Fm 14

Fm3

Fm 13 Fm4 Fm 10 Fm9

Fm 15

Fs 13

Fm2

Fm1

0 1 2 3 4 5 6 7 8 9

Fm 18

10 week

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Fig. 1. continued

Subject B
10
Cs5

Fm 26 Fm 25 Fm 24

Fs 19 Fm 31 Fm 30 Fm 29

Fs 20 Fm 32

Fm 33

Fm 35

6 isolates

Fs 18

Fm 23 Fm 22

4
Fm 21 Fm 28

Fs2

Fs2

Fs2

Fs 21 Fs2 Fm 34

2
Fm 19 Fm 20 Fm 27

0 1 2

10 week

Subject C
10

Fm 43 Fm 42 Fm 41 Fm 40 Fm 37 Fm 38

Fs 23

He 2 Fs 24 Fs 26

Fs 22

6 isolates

Fs 20

Fm 38

Fs2

Fs 25 Fm 38

Fm 44

4
Fm 39 Fs2

2
Fm 36

Fm 39

10

11 week

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Fig. 1. continued

Control
10
He3 Fm 53

Fs 28

Fm 49 Fm 48 Fm 47

Fs 27 Fm 59 Fm 58 Fm 57

Fm 60 Fm 58

He4 Fm 61 Fs 29 Fs 27 Fm 66 Fm 65 Fm 53 Fm 64

He5 Fs 30 Fm 68 Fm 67 Fm 65 Fm 63

He4 Fs 32 Fs 31 Fm 69 Fm 68

Fm 52 Fm 51 Fm 50

Fs 27

6 isolates

Fm 46

4
Fm 46

Fm 55

Fm 56

2
Fm 45

Fm 45

Fm 54

Fm 54

Fm 45 Fm 45

Fm 62 Fm 63 Fm 62 Fm 62

0 1 2 3 4 5 6 7 8 9

10

11

12 week

Recovery of enterococci in the feces varied from subject to subject and from week to week. In subject A, the level of enterococci remained stable during the first 6 weeks at 1.9 X 103 cfu/g after which it increased to 1.6 X 106 cfu/g. Subject B had stable numbers of enterococci in the first 3 weeks (8.7 X 104 cfu/g) and in the last 3 weeks (1.8 X 105 cfu/g) but the level peaked at 5.7 X 107 cfu/g during cheese consumption. Subject C had numbers of ~1 X 108 cfu/g during the first five weeks and a lower number during the last 3 weeks (9.1 X 105 cfu/g). The number of enterococci in the control subject increased during the first 3 weeks from 4.1 X 105 cfu/g to 3.4 X 107 cfu/g, remained stable around 7.3 X 105 cfu/g between weeks 6-8 and decreased from 5.8 X 106 cfu/g to 1.3 X 104 cfu/g in the last three weeks (Figure 2). Species identification by SDS-PAGE. Four hundred and eighty colonies were isolated from plates of the cheese showing >102 colonies. These were divided into yellow-pigmented, sticky colonies (n = 443) and white, thick-creamy colonies (n = 37) and counted. Forty randomly selected yellow-pigmented colonies were identified as E. casseliflavus and the 37 white colonies as E. faecalis by SDS-PAGE.

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1,0E+10

1,0E+08 cfu enterococci/g faeces A B C ctrl

1,0E+06

1,0E+04

1,0E+02

1,0E+00 0 2 4 6 8 10 12 week

Figure 2. Total counts of enterococci found in subjects A, B, C and the control person.

After PFGE had been performed on the fecal isolates and the band patterns had been clustered, 1 to 3 isolates from each clone were also identified by SDS-PAGE as E. faecium, E. faecalis, E. casseliflavus, E. durans or E. hirae. During the pre-consumption period (weeks 1-3) subjects A, B and C showed the presence of high numbers of E. faecium in their feces (73%, 70% and 100% respectively). Other species were detected in lower numbers: E. faecalis (16.5%), Enterococcus durans (6.5%) and E. casseliflavus (3.5%) in subject A, and E. faecalis (26.5%) and E. casseliflavus (3.5%) in subject B. In the control subject the distribution of enterococcal species remained fairly stable during the entire experiment. E. faecium (84.5%) was the most frequently encountered species with much lower numbers of E. faecalis (11.0%) and E. hirae (4.5%). During cheese consumption the detection frequency of E. faecalis was much higher in subjects A, B and C. Subject A showed high numbers of E. faecalis (76.5% of isolates) followed by lower numbers of E. casseliflavus (23.5% of isolates) while, in subject B, E. faecalis was the only species detected. In subject C, 33% of the

136

isolates were still E. faecium and they were all found in week 6. E. faecalis was found in weeks 5 and 7 (67% of isolates). In the post-consumption period E. faecium appeared again in subjects A, B and C (56.5%, 46.5% and 56.5% of isolates, respectively) while E. faecalis was present at 40%, 53.5% and 40% of isolates, respectively. E. hirae was present in small numbers in subjects A (3.5%) and C (3.5%). Strain typing by PFGE. A total of 437 isolates were typed by PFGE (360 human isolates, i.e. 10 isolates picked from 4 human fecal samples over 9 sampling points and 77 isolates from the cheese, i.e. 40 yellow and 37 white colonies). In the present study we labelled the E. faecalis clones Fs1-Fs32, the E. faecium clones Fm1-Fm69, the E. casseliflavus clones Cs1-Cs5, the E. durans clone Ds1 and the E. hirae clones He1-He5. Thirty-nine E. casseliflavus isolates were identical to clone Cs2 and 1 to clone Cs1 of E. casseliflavus from the previous study (Gelsomino et al., 2002), while 33 E. faecalis isolates were identical to clone Fs1 and 4 to clone Fs2. It was presumed that the other 403 yellow isolates from the cheese were either clone Cs1 or Cs2. All these clones, except Fs2, were also found in the cheese in the previous study proving that the flora of this particular cheese remains constant over a period of at least 3 years. Clone Fs2 was found in the milk and the human feces in the previous study. Taking into consideration that the average enterococcal content in the cheese was 3.2 X 104 cfu/g cheese, we conclude that each gram of cheese contained at least 2.7 X 102 cfu of clone Fs2, 7.4 X 102 cfu/g cheese of clone Cs1, 2.2 X 103 cfu of clone Fs1 and ~2.9 X 104 cfu of clone Cs2. None of the clones detected in the cheese were found in the human feces in the 3 week pre-consumption period. The clones found in this period, especially in the control and in subjects A and B, were very diverse. Subject C showed different dominant clones in each week, Fm37 in week 1, Fm38 in week 2 and Fm39 in week 3 (Fig. 1). During consumption of the cheese, E. faecalis clone Fs2, which was a minor component of the cheese, dominated the feces of all cheese-consuming subjects, especially subjects A (73.5% of isolates) and B (96.5% of isolates). In addition, the feces of subject A contained E. casseliflavus clones Cs1 (16.5%) and Cs2 (6.5%), both of which were also found in the cheese. The latter was the dominant clone in the

137

cheese. Subject Cs feces contained clone Fs2 but only in weeks 5 and 7; in week 6, E. faecium clone Fm38, which had also been found in the pre-consumption period, was the only clone detected (Fig. 1). During the first week of cheese consumption, a number of other E. faecalis clones were isolated one of which (Fs20) was isolated from the feces of two different subjects (B and C). In the post-consumption period, E. faecalis clone Fs2 disappeared from the feces of all subjects, except subject B, where it was detectable for one more week. New clones of E. faecalis appeared in all subjects and E. faecium appeared again in subjects A, B and C. However, all these clones were different from the ones recovered in the cheese. As in the pre-consumption period, the feces of subjects A, B and C showed dominant clones, e.g. Fm44 and Fs25 in weeks 10 and 11 in subject C. All the clones found in the feces of subject C are shown in Fig. 3. Clone Fm38 was recovered in the pre- and post-consumption periods as well as during the cheese consumption period. Comparison of the PFGE band patterns of all fecal isolates was made with GelCompar and visually. All clones, except clone Fs20, present in the feces of one subject were unique to that subject and were not present in another subject (data not shown).

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Fm37

Fm38

Fm36

Fm39

Fm43

Fm40

Fm38

Fm38

Fm41

Fm42

Fm39

Fm44

Fs20

Fs22

Fs23

Fs25

Fs24

Fs26

Fs2

He2

SA

pre-consumption

consumption

post-consumption

Figure 3. PFGE agarose gel showing the strains recovered from subject C. A Sta. aureus strain was used as molecular weight marker (SA). Clone Fm38 was found before, during and after the cheese consumption period.

SA

139

Discussion The aim of this study was to determine the common clones of enterococci in human feces and the effect that daily consumption of a raw milk cheese, containing known clones of enterococci, has on the fecal enterococcal microflora. We assumed, like Tannock et al. (Tannock et al., 2000), that the bacteria in feces reflect the bacteria in the distal large bowel although a study by Savage (1997) claims that fecal samples alone cannot reveal the composition and localization of bacterial communities in the colonic lumen. However, it is the common method used to study the flora of the GIT (Bunte et al., 2000; Kimura et al., 1997; Sears et al., 1949; Srensen et al., 2001). The common enterococcal flora in humans was studied during three weeks (pre-consumption period) in subjects A to C and during 12 weeks in the control subject. Only the clones isolated from the feces during the first three weeks were considered to reflect the regular flora as the effect of cheese consumption could last through the post-consumption period. Total enterococcal counts varied in each of the subjects from week to week. In the pre-consumption period, E. faecium was the dominant organism in all subjects (70-100%) followed by E. faecalis. These findings confirm the statements of Devriese et al. (1995) and Murray (1990) that E. faecalis and E. faecium are the dominant species in the human intestine. Generally, each sample of feces contained 2-7 different clones. Very often the subjects showed dominant clones or clones that are recovered over several weeks like Fm38 in subject C or Fm45 in the control (Figs. 1 and 3). According to the definition of Sears et al. (1949) these clones are resident, whereas the clones that appear only once are transient. In the only cheese-consuming subject where a resident clone could be detected (Fm 38 in subject C), it is precisely this clone that comes back first after the cheese consumption. No resident clone was found in subjects A and B. This may be due to the low numbers of isolates taken per sample. E. hirae clone He4 in the control subject seems to be resident too, although it was found only twice. According to Sears et al. (1949) though, resident strains eventually disappear to be replaced by other resident strains. During cheese consumption, the subjects showed a drastic change in their fecal flora. The cheese contained an average enterococcal count of 3.2 X 104 cfu/g,

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which is within the range (103-107 cfu/g cheese) of enterococci found in cheese by Fryer (1969). Four clones were detected in the cheese (Fs1, Fs2, Cs1 and Cs2). Three of them (Fs1, Cs1, Cs2) were the same as those recovered from the same cheese in the previous study (Gelsomino et al., 2002). The incidence of these clones differs when compared to the previous trials. Also during cheese consumption no correlation seemed to exist between the amount of enterococci/g ingested and the number of enterococci/g in the feces as decreasing and increasing of numbers of enterococci occurred all the time. The majority of the strains isolated from the feces during the consumption period belonged to the same clones as those detected in the cheese. Although the cheese contained primarily E. casseliflavus clone Cs2, almost all clones detected in the feces of subjects A, B and C belonged to E. faecalis clone Fs2 (Fig. 1). Why the finding of clone Fs2 was interrupted by clone Fm38 in subject C is not clear but the reasons might be found in dietary habits or in the low number of isolates taken. Clone Fs2, although a minor component of the enterococcal clones in the cheese, apparently thrived best in the human intestine as soon as cheese consumption began, and disappeared as soon as the cheese consumption came to an end. This clone most likely found optimal conditions in the bowel and proliferated as long as there was a continuous supplementation of that particular clone. It is possible that other enterococcal strains were brought into the human GIT by consuming the cheese but were missed because they were in low numbers. An example of this happening is that E. faecalis clone Fs20 was found twice in subject C and once in subject B. Srensen et al. (38) proved, in a similar experiment, that enterococci in a suspension, pass the GIT, but in contrast, in this study we could prove the effect of the food, in this case cheese, as a carrier. Similar studies with other organisms (Lactobacillus rhamnosus, Lactobacillus paracasei and Bifidobacterium lactis) confirm the present finding (Bunte et al., 2000; Matsumoto et al., 2000; 38). A similar result was found in a previous study (Gelsomino et al., 2002) where isolates from the raw-milk, the cheese and the milking equipment were compared with isolates from the four cheesemakers fecal samples. In that study three clones, 2 of E. casseliflavus (Cs1 and Cs2) and 1 of E. faecalis (Fs1), largely dominated the milk, cheese and human fecal samples. Clone Fs2 was never isolated from the cheese in that study; however, it was found in the raw milk and in the human feces. In the

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present study, this clone was present in very low numbers in the cheese but was the dominant clone isolated from the feces during the cheese consumption period. Why different clones dominate the feces of the Irish cheesemakers and the Belgian consumers is not known. This is due to the two groups living in different geographical areas, as stated by Franz et al. and Murray (Franz et al., 1999; Murray, 1990) or, that the dominant clones of the cheese also dominate the GIT of the cheesemaking family due to consumption over several years. These findings support the idea that the fecal flora of the cheesemaking family reflects consumption of the cheese rather than human fecal contamination of the milk (Gelsomino et al., 2002). According to Franz et al. (1999), enterococci are able to colonize the GI tract as they are part of the normal intestinal flora. The allochtonous clone Fs2 (found in a place other than where it originated from) is, however, not able to colonize the intestine as it disappears quickly after cheese consumption ends. In subjects A and C, clone Fs2 disappears when cheese consumption stops while, in subject B, it remains for another week (Fig. 1). This may be subject dependent. Tannock et al. (2000) obtained similar results with a strain of Lactobacillus rhamnosus. The clones probably undergo a reduction in numbers due to the variable conditions in the stomach, the changing nature of the intestinal contents, the rate of movement, the competition for nutrients or binding cites etc. There may also be an antagonistic effect between the enterococci or other bacteria in the GIT and the new enterococci from the cheese such as clone Fs2 which can only be detected in the feces when relatively large amounts of it are consumed. One of the first reports of a similar experiment was that of Sears et al. (1949). E. coli strains were swallowed deliberately in large numbers but they were recovered for limited periods only and were not established as residents. The present findings corroborate these results. Enterococci do not seem to adhere to the intestinal mucosa. Although adhesion is regarded by numerous authors as an essential feature for probiotics (Dunne et al., 2001; Morelli, 2001; Tannock et al., 2000) there is concern over the use of probiotic bacteria that contain antibiotic resistance genes as these could be transmissible to other bacteria especially if adhering to the mucosa. Another recommendation regards the level of viable cells. According to Ziemer and Gibson (1998) probiotics should establish and flourish in the intestine while others (Garg and Mital, 1991; Reid, 2001) recommend a daily intake of at least 107 cfu/g or ml. E.

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faecalis clone Fs1 and E. casseliflavus clone Cs2 were present in the cheese in relatively high numbers, 2.2 X 103 and 3.0 X 104 cfu/g, respectively. But it is E. faecalis clone Fs2 which is present in the cheese at only 267 cfu/g (giving a daily consumption of 2.6 X 104 to 4.0 X 104 cfu) which proliferates and colonizes the intestine during cheese consumption. These results prove that a clone present in very low numbers in cheese can establish itself in the intestine without having to be present in the food in high numbers.

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Acknowledgments This work was funded by a Walsh Fellowship from Teagasc, Dublin. The authors wish to thank Dick and Anne Keating for supplying the cheese and the 4 volunteers who eat the cheese.

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References

Adlerberth, I., M. Cerquetti, I. Poilane, A. Wold, and A. Collignon. 2000. Mechanisms of colonisation and colonization resistance of the digestive tract. Microb. Ecol. Health Dis. Suppl. 2:223-239. Bunte, C., C. Hertel, and W. P. Hammes. 2000. Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract. System. Appl. Microbiol. 23:260-266. Clark, W. S., and G. W. Reinbold. 1966. Enterococci in young cheddar cheese. J. Dairy Science. 49:1214-1218. Devriese, L. A., B. Pot, L. van Damme, K. Kersters, and F. Haesebrouck. 1995. Identification of Enterococcus species isolated from foods of animal origin. Int. J. Food Microbiol. 26:187197. Dunne, C., L. OMahony, L. Murphy, G. Thornton, D. Morrisey, S. OHalloran, M. Feeney, S. Flynn, G. Fitzgerald, C. Daly, B. Kiely, G. C. OSullivan, F. Shanahan, and J. K. Collins. 2001. In vitro selection criteria for probiotics bacteria of human origin: correlation with in vivo findings. Am. J. Clin. Nutr. 73 (suppl):386S-92S. Elotmani, F., A.M. Revol-Junelles, O. Assobhei, and J. B. Millire. 2002. Characterization of antiListeria monocytogenes bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis strains isolated from Rab, a Maroccan traditional fermented milk. Curr. Microbiol. 44:10-17. Facklam, R. R., and L. M. Teixeira. year. Enterococcus. p. 669-682. In A. Balows and B. I. Duerden (ed.), vol. II (systematic bacteriology) of Topley & Wilsonss Microbiology and microbial infections, 9th ed. Arnold, London. Fons, M., A. Gomez, and T. Karjalainen. 2000. Mechanisms of colonisation and colonisation resistance of the digestive tract. Microb. Ecol. Health Dis. Suppl. 2:240-246. Fraimow, H. S., D. L. Jungkind, D. W. Lander, D. R. Delso, and J. L. Dean. 1994. Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth. Ann Intern. Med. 121:22-26. Franz, C. M. A. P., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47:1-24 Fryer, T. F. 1969. Microflora of cheddar cheese and its influence on cheese flavour. Dairy Sci. Abstr. 31:471-490. Fuller, R. 1989. Probiotics in man and animals; A Review. J. Appl. Bacteriol. 66:365-378. Garg, S. K., and B. K. Mital. 1991. Enterococi in milk and milk products. Crit. Rev. Microbiol. 18:15-45. Gardiner, G. E., E. OSullivan, J. Kelly, M. A. E. Auty, G. F. Fitzgerald, J. K. Collins, R. P. Ross, and C. Stanton. 2000. Comparative survival rates of human-derived probiotics Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying. Appl. Environ. Microbiol. 66:2605-2612. Gelsomino, R., M. Vancanneyt, S. Condon, J. Swings, and T. Cogan. 2001. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory. Int. J. Food. Microbiol. 71:177-188.

145

Gelsomino, R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings. 2002. The source of enterococci in a farmhouse raw-milk cheese. Appl. Environ. Microbiol. 68:3560-3565. Giraffa, G., C. Domenico, and E. Neviani. 1997. Enterococci isolated from dairy products: A review of risks and potential technological use. J. of Food Protection. 60:732-738. Hardie, J. M., and R. A. Whiley. 1997. Classification and overview of the genera Streptococcus and Enterococcus. J. of Appl Microbiol. Symposium Supplement. 83:1S-11S. Holdeman, L. V., I. J. Good, and W. E. C. Moore. 1976. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31:359-375. Jett, B. D., M. M. Huycke, and M. S. Gilmore. 1994. Virulence of enterococci. Clin. Microbiol. Rev. 7:462-478. Kimura, K., A. L. McCartney, M. A. McConnell, and G. W. Tannock. 1997. Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains. Appl. Environ. Microbiol. 63:3394-3398. Kleessen, B., E. Bezirtzoglou, and J. Mtt. 2000. Culture-based knowledge on biodiversity, development and stability of human gastrointestinal microflora. Microb. Ecol. Health Dis. Suppl. 2:53-63. Kurmann, J. A. 1968. Ueber die Ursachen der qualittsfrdernde Wirkung auf Emmentaler und Greyerzerkse bei einer Einimpfung von Streptokokkus faecalis in keimarmer Rohmilch. Milchwissenschaft. 23:193-197. Leclerc, H., L. A. Devriese, and D. A. A. Mossel. 1996. Taxonomical changes in intestinal (faecal) enterococci and streptococci: consequences on their use as indicators of faecal contamination in drinking water. J. Appl. Bacteriol. 81:459-466. Matsumoto, M., T. Tadenuma, K. Nakamura, H. Kume, T. Imai, R. Kihara, M. Watanabe, and Y. Benno. 2000. Effect of Bifidobacterium lactis LKM 512 yogurt on fecal microflora in middle to old aged persons. Microbial Ecol. Health Dis. 12:77-80. Morelli, L. 2001. Taxonomy and physiology of lactic acid bacteria, effects and functions on nutrition. Report of a Joint FAO/WHO expert consultation on Evaluation on health and nutritional properties of probiotics in food including powder milk with lactic acid bacteria. Cordoba, Argentina. 1-4 October 2001. http://www.fao.org/es/ESN/Probio/probio.htm Morris Jr. J. G., D. K. Shay, J. N. Hebden, R. J. McCarter Jr., B. E. Perdue, W. Jarvis, J. A. Johnson, T. C. Dowling, L. B. Polish, and R. S. Schwalbe. 1995. Enterococci resistant to multiple antimicrobial agents, including vancomycin. Ann. Intern. Med. 123:250-259. Morrison, D., N. Woodford, and B. Cookson. 1997. Enterococci as emerging pathogens of humans. J. Applied Microbiol Symposium Supplement. 83:89S-99S. Murray, B. E. 1990. The life and times of the enterococcus. Clin. Microbiol. Rev. 3:46-65. Neviani, E., G. Mucchetti, G. Contarini, and S. Carini. 1982. Ruolo degli enterococci nei formaggi italiani I. Il latte. 7:722-728. Nuez, M., J. L. Rodrguez, E. Garcia, P. Gaya, and M. Medina. 1997. Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese. J. Appl. Microbiol. 83:671-677.

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Perreten, V., B. Kollffel, and M. Teuber. 1997. Conjugal transfer of the Tn916-like transposon TnFO1 from Enterococcus faecalis isolated from cheese to other Gram-positive bacteria. Syst. Appl. Microbiol. 20:27-38. Reid, G. 2001. Regulatory and clinical aspects of dairy probiotics. Report of a Joint FAO/WHO expert consultation on Evaluation on health and nutritional properties of probiotics in food including powder milk with lactic acid bacteria. Cordoba, Argentina. 1-4 October 2001. http://www.fao.org/es/ESN/Probio/probio.htm Saramago Stern, C. M. G. Squeira Carvalho, L. Martins Teixeira. 1994. Characterization of enterococci isolated from human and nonhuman sources in Brazil. Diagn. Microbiol. Infect. Dis. 20:61-67. Savage, D. C. 1977. Microbial ecology of the gastrointestinal tract. Ann. Rev. Microbiol. 31:107-133. Seale, D. R. 1986. Bacterial inoculants as silage additives. J. Appl. Bact. 61 (suppl): 9S-26S. Sears, H. J., I. Brownlee, and J. K. Uchiyama. 1949. Persistance of individual strains of Escherichia coli in the intestinal tract of man. J. Bacteriol. 59:293-301. Srensen, T. L., M. D. M. Blom, D. L. Monnet, N. Frimodt-Mller, R. L. Poulsen, and F. Espersen. 2001. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. N. Engl. J. Med. 345:1161-1166. Tannock, G. W., K. Munro, H. J. M. Harmsen, G. W. Welling, J. Smart, and P. K. Gopal. 2000. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl. Environ. Microbiol. 66:2578-2588. Tuomola, E., R. Crittenden, M. Playne, E. Isolauri, and S. Salminen. 2001. Quality assurance criteria for probiotics bacteria. Am. J. Clin. Nutr. 73 (suppl):393S-8S. Wallick, H, and C. A. Stuart. 1943. Antigenic relationships of Escherichia coli isolated from one individual. J. Bacteriol. 45:121-126. Ziemer, C. J., and G. R. Gibson. 1998. An overview of probiotics, probiotics and synbiotics in the functional food concept: perspectives and future strategies. Int. Dairy J. 8:473-479.

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Chapter 9. antibiotic resistance and virulence traits of enterococci isolated from an irish artisanal cheese

Roberto Gelsomino1, Geert Huys1, Klaas DHaene1, Marc Vancanneyt2, Timothy M. Cogan3, Charles M. A. P. Franz4, and Jean Swings1, 2

Laboratory of Microbiology,

BCCM/LMG Bacteria Culture Collection, Ghent University,

K. L. Ledeganckstraat 35, Gent, Belgium

3 4

National Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland Federal Research Centre for Nutrition, Institute of Biotechnology and Molecular Biology,

Haid-und-Neu-Strasse 9, D-76131 Karlsruhe, Germany

Submitted to the Journal of Food Protection

Abstract A representative selection of 8 Enterococcus strains from a collection of over 600, previously isolated from an Irish artisanal cheese, was subjected to phenotypic and genotypic analysis of their antibiotic resistance and virulence properties. Resistance genes encoding resistance to tetracycline (tet(M) and tet(L)) and/or erythromycin (erm(B)) were detected in five strains. In addition, all strains were found to contain two or more virulence genes of those tested (agg, gel, cyl, esp, ace, efaAfs and efaAfm). Further investigation into the transferability and environmental dissemination of these resistance and virulence traits will allow risk assessment and safety evaluation of the consumption of artisanal cheeses.

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Introduction Enterococci can be found in a variety of cheeses made from raw or pasteurised milk (Cogan et al., 1997). Some authors have reported beneficial roles for them in developing the sensory properties of cheese while others have attributed probiotic characteristics to them (Franz et al., 1999). Their application in food preservation results from their antilisterial activity based on bacteriocin production (Franz et al., 1999, Giraffa et al., 1995; Nuez et al., 1997). Enterococci are frequently encountered nosocomial pathogens (Noskin, 1997) and cause infections like bacteraemia, endocarditis, neonatal central nervous system and urinary tract infections (Hardie and Whiley, 1997). In addition to their intrinsic antibiotic resistance to cephalosporins and low level resistance to clindamycin and aminoglycosides, an increasing number of food enterococci have developed atypical resistances against various therapeutic antibacterial agents including vancomycin (Giraffa et al., 2002; Robredo et al., 2000; Wegener et al., 1997), tetracyclines (Teuber et al., 1999; Huys et al. 2004), gentamicin (Donabedian et al., 2003) and streptogramin (Simjee et al., 2002). The increase of vancomycin-resistant enterococci is of particular importance, as in many cases vancomycin is considered the last resort for treatment of multiple-resistant Enterococcus infection (Murray, 1990). A number of virulence factors have been linked to the pathogenicity of Enterococcus faecalis including cytolysin (CylA), enterococcal surface protein (Esp), aggregation substance (Agg), and gelatinase (GelE). The cytolysin CylA causes rupture of a variety of membranes, including those of bacterial cells, erythrocytes, and other mammalian cells (Huycke et al, 1998). The Esp determinant can cause disease in mouse and may assist enterococci to evade detection by the immune system (Shankar et al., 1999). The aggregation substance is believed to facilitate formation of mating aggregates with nearby recipients as well as facilitate binding to eucaryotic cells, which may be crucial steps in colonization and infection (Franz et al., 2003). Other enterococcal adhesins include the adhesin of collagen in E. faecalis (Ace) (Nallapareddy et al., 2000) and the adhesin-like E. faecalis and E. faecium antigen A (EfaAfs and EfaAfm, respectively) (Franz et al., 2003). Gelatinase is a protease that hydrolyses bioactive peptides. Gelatinase producing E. faecalis

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were commonly found in intensive care units in Germany and in patients with endocarditis and other nosocomial infections (Khnen et al., 1988; Coque et al., 1995). Franz et al. (2001) reported that 10.4% and 78.7% of E. faecium and E. faecalis strains, respectively, isolated from cheese, were positive for one or more of these virulence factors. Current evidence suggests that virulence and antibiotic resistance properties in enterococci are often strain-specific (Huys et al., 2004; Eaton and Gasson, 2001; Franz et al., 2001). This finding may have important implications for the safety evaluation of industrial Enterococcus strains used in food manufacturing and of food products that contain enterococci as natural constituents. The objective of the present study was to determine the presence of antibiotic resistance and virulence traits among a set of eight enterococcal strains. These strains were shown to be the representative strains of the dominant enterococcal flora in the microbial ecosystem of Baylough, an Irish artisanal cheese.

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Material and Methods Bacterial strains. The strains used in this study were isolated from Baylough, an Irish artisanal cheese, and were identified and typed in the course of previous studies (Gelsomino et al., 2001; 2002). The 8 strains in question represent unique strain types observed among over 600 isolates obtained from the cheese mentioned above. Three of them, LMG 19844, LMG 19845 and LMG 20235 (original isolate nr. Cs1, Cs2 and Cs3 respectively) belong to the species E. casseliflavus, and 5 strains, LMG 19846, LMG 20228, LMG 20229, LMG 20230 and LMG 20234 (Fs1, Fs2, Fs3, Fs4 and Fs6, respectively) belong to E. faecalis (Table 1). All strains have been deposited in the BCCM/LMG Bacteria Collection, Gent University, Gent, Belgium (http://www.belspo.be/bccm/lmg.htm#main). Phenotypic and genotypic assessment of antibiotic resistance traits and detection of int genes. Strains were screened for phenotypic resistance to 12 antibiotics using the disc diffusion method and Mueller-Hinton agar (Oxoid). The following antibiotic discs (Oxoid) were used: tetracycline (30 g), doxycycline (30 g), minocycline (30 g), ampicillin (10 g), streptomycin (10 g), kanamycin (30 g), rifampicin (30 g), vancomycin (30 g), erythromycin (15 g), gentamicin (10 g), trimethoprim (1.25 g), and chloramphenicol (30 g). When available, inhibition zones were interpreted following the guideline tables of the National Committee for Clinical Laboratory Standards (1999). All isolates were subjected to MIC testing in the range of 4-512 g/ml tetracycline based on the micro-broth dilution method using cation-adjusted Mueller-Hinton II broth (Beckton-Dickinson 212322, Maryland) as recommended by NCCLS (1999). Following a 24 h incubation, the MIC value was visually read from a microtiter plate. In cases of doubt, the MIC was read as the concentration at which >80% inhibition of growth occurred. Each batch of MIC determinations included the control strain E. faecalis LMG 8222 (= ATCC 29212). Total genomic DNA was prepared using a protocol based on the method of Pitcher et al. (1989). For all detection assays, a common PCR core mix (total volume, 50 l) was used consisting of 1PCR buffer (Applied Biosystems, Warrington, United Kingdom), deoxynucleoside triphosphates (Applied Biosystems) at a concentration of 200 M each, 1 U of AmpliTaq DNA polymerase (Applied

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Biosystems), and 20 pmol of each primer (Sigma-Genosys Ltd., Cambridgeshire, UK). A 50 ng portion of intact total DNA was used as PCR template. The presence of efflux genes tet(K) and tet(L) was determined as previously described (Gevers et al., 2003). The tet genes encoding ribosomal protection (RP) proteins were detected using the RP-group specific primer pairs DI and DII (Gevers et al., 2003) and Ribo2FW and Ribo2-RV (Aminov et al., 2001). Isolates harbouring an RP-type tet gene were further subjected to PCR detection of tet(M), tet(O), and tet(S) as previously described (Gevers et al., 2003). In addition to tet gene detection, the presence of erythromycin resistance gene erm(B) (Gevers et al., 2003) and of the vancomycin resistance genes vanA, vanB, vanC1 and vanC2 (Dutka-Malen et al., 1995) was investigated using previously described PCR primers. The occurrence of conjugative transposons of the Tn916-Tn1545 family was determined using the primers Int-FW and Int-RV targeting the transposon integrase (int) gene (Doherty et al., 2000; Gevers et al., 2003). All PCR reactions were performed in a GeneAmp 9600 PCR system (Perkin-Elmer) using previously described amplicon conditions for each primer set (Gevers et al., 2003). PCR amplicons were subjected to electrophoresis on 1% agarose gels and visualised using ethidium bromide fluorescence. Phenotypic and genotypic assessment of virulence traits. Genomic DNA was prepared as described previously (Lewington et al., 1987). PCR assays were performed using pairs of previously reported primers for the detection of the following genes (positive control strains are indicated between brackets): agg (E. faecalis LMG 20947 [FAIR-E 404] and plasmid pAD1), gel (E. faecalis LMG 20869 [FAIR-E 313] and E. faecalis LMG 20823 [FAIR-E 265]) efaAfs, efaAfm (E. faecalis MMH594 and E. faecium BFE 2262, respectively) (Eaton and Gasson, 2001), esp (E. faecalis MMH594) (Shankar et al., 1999), ace (E. faecalis V583) and cylA (E. faecalis LMG 20681 [FAIR-E 77]) (Rich et al., 1999). Production of gelatinase on gelatin agar was performed as previously described (Franz et al., 2001). For the detection of haemolysin/cytolysin the strains were streaked on commercially obtained horse blood agar plates (Columbia Agar with 5% horse blood, Becton Dickinson, Heidelberg, Germany), incubated at 37C for 3 days and examined daily for production of clear zones of beta haemolysis. All virulence traits studied have previously been found in human isolates.

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Results and discussion

During previous studies (Gelsomino et al., 2001; 2002) over 600 isolates were obtained from Baylough, an Irish artisanal cheese, over a period of 15 months. All isolates were typed with Pulsed-field gel electrophoresis from which only 8 unique strain types were found. One representative strain of each of these 8 types was included in this study and considered to represent the total enterococcal flora of the Baylough cheese over a period of 15 months. Results from disc diffusion testing indicated that all strains, regardless of their species designation, were susceptible to ampicillin, vancomycin, gentamicin, rifampicin, and trimethoprim (the last two are based on inhibition zones of >21 and >23 mm, respectively, as no NCCLS guidelines are available) and showed uniform resistance to streptomycin when using the 10 g disc. However, it should be highlighted that a disc with a higher content of streptomycin (300g) is recommended to record high-level resistance in enterococci. The vancomycin-susceptible phenotype was confirmed by the finding that none of the strains contained the vanA, vanB, and vanC1 resistance genes (data not shown). The three E. casseliflavus strains contained the vanC2 gene, which is in agreement with the results of Dutka-Malen et al. (1995) showing that this species is intrinsically resistant to low levels of vancomycin. The fact that these three strains did not show a phenotypic vancomycin resistance could be explained by the inability of the disc diffusion method to recognize low-level resistances to vancomycin. Strain-specific resistance traits were observed for the tetracyclines (5 of 8 strains), kanamycin (4 of 8 strains), chloramphenicol (3 of 8 strains), and erythromycin (1 of 8 strains). Except for E. casseliflavus strain LMG 19845, which displayed tetracycline resistance (TcR), all observed resistances were restricted to E. faecalis strains (Table 1). E. faecalis strain LMG 20230 displayed multiple resistance to all four antibiotics. Teuber et al. (1999) have reported that multiple resistance to tetracycline, erythromycin, chloramphenicol, and gentamicin can occur among Enterococcus isolates from various types of cheese. Resistance to tetracyclines, erythromycin, and chloramphenicol are considered atypical and thus possibly acquired in enterococci (Teuber et al., 1999). In the present study, we further investigated the molecular basis of the first two mentioned resistance phenotypes. As shown in Table 1, resistance to tetracycline in four E.

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faecalis isolates and in E. casseliflavus LMG 19845 was linked to the presence of the ribosomal protection (RP) gene tet(M) sometimes in combination with the efflux gene tet(L) (Table 1). To our knowledge, this is the first report of the tet(M) gene occurring in members of the species E. casseliflavus. The single strain displaying erythromycin resistance, E. faecalis LMG 20230, contained the macrolide resistance erm(B) gene which is widespread among food enterococci (Teuber et al., 1999). The observation that all TcR isolates carried a representative of the RP tet gene group complies with the minocycline resistance phenotype found in disc diffusion testing, which is mediated only by RP mechanisms in enterococci (Chopra and Roberts, 2001). The two E. faecalis strains that showed the highest MIC value for tetracycline (256 g/ml) harboured both tet(M) and tet(L), whereas the remaining strains which displayed lower MIC values in the range of 32128 g/ml, contained only tet(M) (Table 1). This reinforcement effect has recently also been reported by Huys et al. (2004) for food enterococci and seems to indicate that strains containing both an active efflux and a RP mechanism can achieve a higher level of Tc resistance. As expected, no tet genes were detected in the strains showing the lowest MIC (E. casseliflavus LMG 19844 and LMG 20235 and E. faecalis LMG 20234). In the majority of enterococci and streptococci originating from clinical or food sources, Tc resistance genes are more frequently located on conjugative transposons than on plasmids (Teuber et al., 1999). From the five tet(M)-carrying strains included in this study, four contained the integrase element int, indicating that they harbour a member of the broad host range Tn916-Tn1545 conjugative transposon family (Table 1). Members of this family carry the tet(M) gene, which is sometimes joined by genetic elements encoding resistance to additional antibiotics such as erythromycin and kanamycin in the case of Tn1545 (Clewell et al., 1995). Conjugal transfer of Tn916-Tn1545-associated tet(M) and of tet(L) genes by food enterococci has previously been reported for isolates from cheese and meat products (Teuber et al., 1999; Huys et al., 2004). In order to assess the potential risk involved with the presence of resistance gene-carrying enterococci in the cheese investigated in this study, detailed analyses of the potential mobility of the detected tet or erm genes are required.

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Table 1. Phenotypic and genotypic virulence traits and antibiotic resistance properties of Enterococcus isolates from Baylough cheese.
Strain E. casseliflavus E. casseliflavus E. casseliflavus E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis LMG number code Gela gel Haem cyl agg ace efaAfm efaAfs LMG 19844 LMG 19845 LMG 20235 LMG 19846 LMG 20228 LMG 20229 LMG 20230 LMG 20234 Cs1 Cs2 Cs3 Fs1 Fs2 Fs3 Fs4 Fs6 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Van VanC2 Phenotypic esp genesb gene resistancec + - St + + St, Tc + - St + St, Tc - St, Tc, Ka, Cm + St, Tc, Ka, Cm + St, Tc, Ka, Cm, Er - St, Ka MIC (Tc) (mg/ml) 4 64 4 32 256 256 128 4 tet genes Tn916 erm(B) tet(M) tet(M) tet(M), (L) tet(M), (L) tet(M) + + + + + -

a b c

Phenotypic assessment vanA, vanB, and vanC1

Phenotypic resistances determined from disc diffusion susceptibility testing. Cm, chloramphenicol; Er, erythromycin; Ka, kanamycin; St,

streptomycin; Tc, tetracycline.

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All strains included in this study contained one or more virulence genes that have previously been found in human isolates. Except for E. faecalis strain LMG 20229 all strains carried the gelE gene, although phenotypic gelatinase activity could only be detected in two E. faecalis strains (LMG 19846 and LMG 20230). Similar results were obtained for haemolysis testing. All three E. casseliflavus strains and E. faecalis strain LMG 20230 showed haemolytic activity and carried the cylA gene, whereas two other E. faecalis strains (LMG 20228 and LMG 20234) displayed no haemolytic activity although they both carried the cylA gene. Collectively, these results seem to indicate that several of the investigated strains contained silent gelE and cylA genes. According to Eaton and Gasson (2001), there are several factors such as environmental or temporal parameters that may account for the apparent lack of phenotypic expression of enterococcal virulence genes. The same authors also reported up to 50% difference between gelatinase and haemolytic activity and the presence of GelE and CylA determinants in enterococci isolated from food. In a previous study of Franz et al. (2001), 48.9% of the investigated E. faecalis strains isolated from different cheeses displayed gelatinase activity. Other authors (Khnen et al., 1988; Coque et al., 1995) have reported that 54-64% of enterococci isolated from nosocomial infections had a gelatinase positive phenotype. Haemolysin plays an important role in enterococcal virulence, and although this virulence property is believed to increase the severity of infection (Franz et al., 2001), it has also been found in faecal isolates from healthy individuals (Franz et al., 2003). Similar to the case of gelatinase, the presence of the cylA gene is not always linked to the phenotypic expression of haemolysin activity. It has been speculated that this phenomenon may be due to low-level gene expression or to the presence of an inactive gene product (Eaton and Gasson, 2001). All strains contained the agg gene responsible for the clumping factor aggregation substance, which is often linked to haemolysin production encoded by the pheromone-responsive plasmids (Franz et al., 2001). The Agg factor has also previously been found in food and clinical isolates of E. faecalis (Eaton and Gasson, 2001; Franz et al., 2001). Smuth et al. (2002) have shown that the Agg factor promotes a greater increase in enterococcal adherence to macrophages and phagocytosis. Once phagocytosed, enterococci promote intracellular survival probably due to inhibition of the respiratory burst (Smuth et al., 2000). In addition

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to AS, some of the strains in this study also possessed the ace gene (4 of 8 strains) or the adhesin-like antibodies EfaAfm (2 of 8 strains) and EfaAfs (7 of 8 strains) (Table 1). It has been suggested that the EfaAfm plays a role in endocarditis (Lowe et al., 1995). Four strains (E. faecalis strains LMG 19846, LMG 20229, and LMG 20230 and E. casseliflavus strain LMG 19845) were found to contain the esp gene. The incidence of Esp+ enterococci was shown to be higher among clinical strains of E. faecalis than isolates from healthy individuals (Shankar et al., 1999). This was confirmed by Eaton and Gasson (2001) where the esp gene was detected in more than half of the clinical isolates but only rarely occurred in food enterococci. Collectively, the current study has demonstrated that the enterococcal subpopulation present in artisanal cheese types can harbour various antibiotic resistance and virulence traits. All virulence traits found in our strains have also been found in human clinical isolates. However, the current data set does not allow speculation on the safety evaluation of the cheese itself. Risk assessment requires research on the potential source and the in-vitro and in-situ transferability of these resistance and virulence properties.

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Acknowledgments This study was made possible thanks to a Walsh Fellowship from Teagasc, Dublin, Ireland. Part of this work was supported by the Fund for Scientific Research - Flanders (Belgium) (F.W.O.-Vlaanderen) (contract G.0309.01). G. Huys is a postdoctoral fellow of the Fund for Scientific Research - Flanders (Belgium) (F.W.O.-Vlaanderen).

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References

Aminov, R.I., N. Garrigues-Jeanjean, and R.I. Mackie. 2001. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl. Environ. Microbiol. 67:22-32. Chopra, I., and M. Roberts. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 65:232-260. Clewell, D.B., S.E. Flannagan, and D.D. Jaworski. 1995. Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol. 3:229-236. Cogan, T. M., M. Barbosa, E. Beuvier, B. Bianchi-Salvadoris, P. S. Cocconcelli, I. Fernandes, J. Gomez, R. Gomez, G. Kalantzopoulos, A. Ledda, M. Medina, M. C. Rea, and E. Rodriguez. 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64:409-421. Coque, T.M., J. E. Patterson, J. M. Steckelberg, and B. E. Murray, B.E. 1995. Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from the feces of hospitalized and communitybased persons. J. Infect. Dis. 171:1223-1229. Doherty, N., K. Trzcinski, P. Pickerill, P. Zawadzki, and C. Dowson. 2000. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:2979-2984. Donabedian, S.M., L.A. Thal, E. Hershberger, M.B. Perri, J.W. Chow, P. Bartlett, R. Jones, K. Joyce, S. Rossiter, K. Gay, J. Johnson, C. Mackinson, E. Debess, J. Madden, F. Angulo, and M.J. Zervos. 2003. Molecular characterization of gentamicin-resistant enterococci in the United States: evidence of spread from animals to humans through food. J. Clin. Microbiol. 41:1109-1113. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33:24-27. Eaton, T. J., and M. J. Gasson. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67:1628-1635. Franz, M. A. P. C., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47:1-24 Franz, C. M. A. P., A. B. Muscholl-Silberhorn, N. M. K. Yousif, M. Vancanneyt, J. Swings, and W. H. Holzapfel. 2001. Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Appl. Environ. Microbiol. 67:4385-4389. Franz, C. M. A. P., M. E. Stiles, K. H. Schleifer, and W. H. Holzapfel. 2003. Enterococci in foods a conundrum for food safety. Int. J. Food Microbiol. 88:105-122 Gevers, D., M. Danielsen, G. Huys, and J. Swings. 2003. Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl. Environ. Microbiol. 69:1270-1275.

161

Gelsomino, R., M. Vancanneyt, S. Condon, J. Swings, and T. M. Cogan. 2001. Enterococcal diversity in the environment of an Irish Cheddar-type cheesemaking factory. Int. J. Food Microbiol. 71:177-188. Gelsomino, R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings. 2002. The source of enterococci in a farmhouse raw-milk cheese. Appl. Environ. Microbiol. 68:3560-3565. Giraffa, G., N. Picchioni, E. Neviani, and D. Carminiati. 1995. Production and stability of an Enterococcus faecium bacteriocin during Taleggio cheesemaking and ripening. Food Microbiol. 12:301-307. Giraffa, G. 2002. Enterococci from foods. FEMS Microbiol. Lett. 26:163-171. Hardie, J. M., and R. A. Whiley. 1997. Classification and overview of the genera Streptococcus and Enterococcus. J. of Appl Microbiol. Symposium Supplement. 83:1S-11S. Huycke, M. M., Sahm, D. F., and Gilmore, M. S. 1998. Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg. Inf. Dis. 4:239-249. Huys, G., K. DHaene, J.-M. Collard, and J. Swings. 2004. Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ Microbiol. 70:1555-1562. Jett, B. D., M. M. Huycke, and M. S. Gilmore. 1994. Virulence of enterococci. Clin. Microbiol. Rev. 7:462-478. Khnen, E., F. Richter, K. Richter, and L. Andries. 1988. Establishment of a typing system for group D streptococci. Zentralbl. Bakteriol Hyg A 267:322-330. Lewington, J., S. D. Greenaway, and B. J. Spillane. 1987. Rapid small scale preparation of bacterial genomic DNA suitable for cloning and hybridization analysis. Lett. Appl. Microbiol. 5:5153. Lowe, A.M., P. A. Lambert, and A. W. Smith. 1995. Cloning of an Enterococcus faecalis endocarditis antigen: Homology with adhesins from some oral streptococci. Infect. Immun. 63:703-706. Murray, B. E. 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3:46-65. Nallapareddy, S. R., X. Qin, G. M. Weinstock, M. Hk, and B. E. Murray. 2000 Enterococcus faecalis adhesin, Ace, mediates attachment to extracellular matrix proteins collagen type IV and laminin as well as collagen type I. Infect. Immun. 68:5218-5224. National Committee for Clinical Laboratory Standards (NCCLS). 1999. Performance standards for antimicrobial susceptibility testing. Ninth informational supplement. M100-S9, vol. 19, no. 1. Noskin, G. A. 1997. Vancomycin-resistant enterococci: Clinical, microbiologic and epidemiologic features. J. Lab. Clin. Med. 130:14-20. Nuez, M., J. L. Rodrguez, E. Garcia, P. Gaya, and M. Medina. 1997. Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture and ripening of Manchego cheese. J. Appl. Microbiol. 83:671-677. Pitcher, D.G., N.A. Saunders, and R.J. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8:151-156.

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Rich, R.L., Kreikemeyer, B., Owens, R.T., LaBrenz, S., Narayana, S.V.L., Weinstock, G.M., Murray, B.E., and Hk, M. 1999. Ace is a collagen-binding MSCRAMM from Enterococcus faecalis. J. Biol. Chem. 274, 26939-26945. Robredo, B., K.V. Singh, F. Baquero, B. Murray, and C. Torres. 2000. Vancomycin-resistant enterococci isolated from animals and food. Int. J. Food Microbiol. 54:197-204. Shankar, V., A. S. Baghdayan, M. M. Huycke, G. Lindhal, and M. S. Gilmore. 1999. Infectionderived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein. Infect. Immun. 67:193-200. Simjee, S., D.G. White, J. Meng, D.D. Wagner, S. Qaiyumi, S. Zhao, J.R. Hayes, and P.F. Mc Dermott. 2002. Prevalence of streptogramin resistance genes among Enterococcus isolates recovered from retail meats in the Greater Washington DC area. J. Antimicrob. Chemother. 50:877-882. Smuth, S. D., A. Muscholl-Silberhorn, R. Wirth, M. Susa, R. Marre, and E. Rozdzinski. 2000. Aggregation substance promotes adherence, phagocytosis and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst. Infect. Immun. 68:4900-4906. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek 76:115-137. Wegener, H.C., M. Madsen, N. Nielsen, and F.M. Aarestrup. 1997. Isolation of vancomycin resistant Enterococcus faecium from food. Int. J. Food Microbiol. 35:57-66.

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Part III Conclusions

Chapter 10. Conclusions and perspectives

10.1 Conclusions Enterococci are present in the GIT of both humans and animals and in the farm environment. In addition, the nature of these bacteria, which allows them to survive and grow in harsh environments, makes them ideal to study the source of enterococci in raw milk cheeses. The diversity of enterococci in an Irish farmhouse cheese and the farms environment were studied in order to characterize the enterococcal microflora present in the human and animal intestinal tract, the milk, the cheese and the manufacturing equipment. A further aim consisted of tracing and locating the enterococci back to their source. The following conclusions were drawn: The source of contamination were neither the cows nor the humans but the milking equipment. This was not expected but was not surprising either, as it is known that enterococci can accumulate in joints and fittings of milking machines infecting the milk directly (Slaghuis et al., 1991). In addition, E. faecium is the most frequently occurring Enterococus species in dairy cows (Devriese et al., 1992) but none were found in the cheese. This was also corroborated by the abundant findings of Streptococcus bovis in the cows faeces while none were found in the cheese. The same few enterococcal clones repeatedly survive chlorine sterilization and infect the milk each time anew. We are not aware of any other study where such an epidemiological approach was used. The enterococcal population of the milk and cheese was constant over time as shown by the results of the three trials. The same strains present in milk and cheese dominate the enterococcal flora in the human GIT of the cheese-

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making family. Three strains, two E. casseliflavus and one E. faecalis, were the most abundant. From the literature we know that E. faecalis is the dominant species in the human intestine and that E. faecalis and E. faecium are very commonly isolated from cheese (Murray, 1990; Cogan et al., 1997; Franz et al., 1999). A surprising finding was that E. casseliflavus, although never found in such large numbers, was present in cheese and in human faeces. This finding was confirmed in all trials. It was assumed that an analogous flora in the farmhouse family was due to cheese consumption. We performed a cheese consumption study to determine the common enterococcal clones in human faeces and cheese, in which three persons consumed Baylough cheese on a daily basis for three weeks. This led to the following conclusions: Enterococci ingested with food survive trans-intestinal passage. Our result confirms a study by Srensen et al. (2001) although they did not use food as a carrier, which might influence the passage through the GIT. Similar studies, but with other organisms, were performed by Bunte et al. (2000) and Matsumoto et al. (2000) with the same outcome. Enterococci present in very small numbers in cheese (2.7 x 102 cfu/g) can survive and proliferate in the human GIT. It was surprising that this strain could establish itself so well and grow to such large numbers in the GIT. The presence of this strain, however, stopped when cheese consumption came to an end. Adherence and persistence in the GIT causes some dispute. Some authors regard it as an essential feature (Ziemer and Gibson, 1998; Dunne et al., 2001) while others are concerned over the use of probiotic bacteria that contain antibiotic resistance genes and recommend that strains, which do not establish in the GIT be used (Garg and Mital, 1991; Reid, 2001). In the last phase of our study we determined antibiotic resistance (genes) and potential virulence (genes) of the enterococci from Baylough cheese and we concluded that: In several strains we detected resistance genes to tetracycline and erythromycin. All strains contained two or more virulence genes. Various

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studies report the finding of enterococci harbouring antibiotic resistances and potential virulence traits from food in general, among which also cheese (Eaton and Gasson, 2001; Franz et al., 2001). Knowledge on the spread of antibiotic resistance and virulence genes is still very limited. As suggested by Perreten et al. (1997) epidemiological studies are necessary to clarify the contribution of antibiotic resistant microrganisms in food to the general phenomenon of the antibiotic resistance, which, in clinically important and pathogenic species, has become an almost worldwide occurrence. To our knowledge this is the first study concerning the source of enterococci in a raw milk cheese combined with the determination of their antibiotic resistance and virulence factors. This study shows that potentially pathogenic enterococci may be found in food. We realise that the extent of risk has still to be established. Of course it has to be considered that this is a case study with one type of cheese (Baylough) from one particular farm in Ireland, and it is premature to draw conclusions about cheeses made in other farms.

10.2 Perspectives Before resistance genes and virulence traits were discovered in them, enterococci were considered relatively innocuous lactic acid bacteria and regarded as GRAS (generally recognized as safe). The emergence of VRE has begun to worry the disease community for mainly two reasons. First of all, VRE have shown in vitro to be able to transfer their vancomycin resistance gene to Staphylococcus aureus. Secondly, enterococci resistant to vancomycin leave few other possibilities as therapeutic alternative. The spread of VRE was different in USA and Europe. While in the USA a careless and imprudent use of antibiotics in medical surroundings seemed to be the key factor (McDonald et al., 1997), in Europe the use of avoparcin as growth promoter seems to be the largest contributor to vancomycin resistance (Collignon, 1999; Teuber et al., 1999). Next to the antibiotic resistance there are

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virulence traits such as cytolytic toxin, gelatinase and aggregation substance, which are believed to enhance the ability to cause disease. Several questions remain unanswered. The knowledge on the consequences of the known virulence factors is not satisfactory. It is also not yet clear whether enterococci, which possess several virulence traits, pose a higher risk than enterococci with only one virulence trait. Surveillance, prevention, control and research are becoming increasingly important in food safety issues. Surveillance can be done in hospitals and health care settings when specimens taken from diseased patients are routinely analysed for antibiotic resistance and virulence genes. It is important to monitor antibiotic use in human and veterinary medicine and in agriculture and link it to clinical information. Prevention and control, in the day-to-day life, is also important in health care settings. Concern and worry was created by the study of Badri et al. (1998) who showed not only contaminated hands of gloved and ungloved health care workers but also the presence of VRE on their hands after glove removal. As shown by the Centers for Disease Control and Prevention, improved hand hygiene is able to terminate outbreaks in health care facilities, to reduce transmission of antimicrobial resistant organisms and reduce overall infection rates (http://www.cdc.gov/handhygiene/; Weinstein, 2001). Promotion of an appropriate use of antibiotics can help us to decrease the incidence of antibiotic resistant strains. In Europe this is especially important in agriculture. Several antibiotics used as growth promoters were banned between 1997 and 1998, including avoparcin, ardacin, bacitracin zinc, virginiamycin, tylosin phosphate and spiramycin. After that ban a sharp drop of VRE has occurred in Europe (Teuber et al., 1999). Some substances are still authorized as growth promoting agents but there are plans to ban also these four growth promoters (monensin sodium, salinomycin sodium, flavophospholipol and avilamycin) from 1 January 2006 (EU-common position, 2003). Another form of prevention is pasteurisation, which can reduce the number of enterococci in cheese although this will not please lovers of raw milk products. Also ultrafiltration (UF) is increasingly used in the industry. It is mainly used for milk standardisation but Teuber et al. (1996) recommend it to eliminate dangerous

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bacteria, among them enterococci. One of the major problems with UF is fouling of the filtration membrane and this still requires further research (Razavi et al. 2004). Detecting and researching enterococci has become important more recently since the discovery of virulence traits and antibiotic resistance genes. But enterococci have always been extensively used for their functional and safety properties. Research should provide genome sequences and other powerful tools to identify target genes such as antibiotic resistance and virulence trait genes. A comparison of genome sequences will help us to better understand the technological and health benefits of enterococci such as lactic acid production, proteolysis and lipolysis, production of antimicrobials and, from a medical point of view, such undesired genes as potential virulence genes, antibiotic resistance genes and biogenic amines (Franz et al., 1999; Sarantinopoulos et al., 2001). Another useful tool is DNA-arrays, which, in combination with whole genome sequences, allow sequence comparisons of a large number of strains and provide evidence of unwanted genes. The next aim for research should be to evaluate rapid, affordable and accurate diagnostic methods for human and veterinary uses, which can easily be implemented in routine settings. This way new strategies can be developed which prevent colonization and infection with enterococci containing undesirable genes. A topic, which deserves some attention, is how the source of virulence factors and antibiotic resistances in enterococci is linked to the whole bacterial population from the milk, the cheesemakers, the herd and the milking equipment and whether the virulence factors have their origin in organisms other than enterococci. This could not only reveal possible solutions in breaking the transfer chain but also draw our attention to genera that in our eyes are still innocuous, like enterococci were 20 years ago.

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References
Badri, S., N. Sahgal, A. Tenorio, K. Law, B. Hota, M. Matushek, M. Hayden, G. Trenholme, and R. Weinstein. 1998. Effectiveness of gloves in preventing the transmission of vancomycinresistant Enterococcus (VRE) during patient care activities. In: Program of the 36th Annual Meeting of the Infectious Diseases Society of America, November 11-14, 1998, Denver, USA. Bunte, C., C. Hertel, and W. P. Hammes. 2000. Monitoring and survival of Lactobacillus paracasei LTH 2579 in food and the human intestinal tract. System. Appl. Microbiol. 23:260-266. Cogan, T. M., M. Barbosa, E. Beuvier, B. Bianchi-Salvadori, P. S. Cocconcelli, I. Fernandes, J. Gomez, R. Gomez, G. Kalantzopoulos, A. Ledda, M. Medina, M. C. Rea, and E. Rodriguez. 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64:409-421. Collignon, P. J. 1999. Vancomycin-resistant enterococci and use of avoparcin in animal feed: is there a link? Med. J. Aust. 171:144-146. Devriese, L. A., L. Laurier, P. De Herdt, and F. Haesebrouck. 1992. Enterococcal and streptococcal species isolated from faeces of calves, young cattle and dairy cows. J. Appl. Bacteriol. 72:29-31. Dunne, C., L. OMahony, L. Murphy, G. Thornton, D. Morrisey, S. OHalloran, M. Feeney, S. Flynn, G. Fitzgerald, C. Daly, B. Kiely, G. C. OSullivan, F. Shanahan, and J. K. Collins. 2001. In vitro selection criteria for probiotics bacteria of human origin: correlation with in vivo findings. Am. J. Clin. Nutr. 73 (suppl):386S-92S. Eaton, T. J., and M. J. Gasson. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl. Environ. Microbiol. 67:1628-1635. EU-Common position. 2003. Common Position (EC) No 20/2003 adopted by the Council on 17 March 2003 with a view to adopting Regulation (EC) No/2003 of the European Parliament and of the Council of on additives for use in animal nutrition. Official Journal of the European Union C 113 E/1. 13.5.2003. (The title of the common position is not complete as it is yet a draft for proposal.) Franz, C. M. A. P., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? Int. J. Food Microbiol. 47:1-24 Franz, M. A. P. C., A. B. Muscholl-Silberhorn, N. M. K. Yousif, M. Vancanneyt, J. Swings, and W. H. Holzapfel. 2001. Incidence of virulence factors and antibiotic resistance among enterococci isolated from food. Appl. Environ. Microbiol. 67:4385-4389. Garg, S. K., and B. K. Mital. 1991. Enterococi in milk and milk products. Crit. Rev. Microbiol. 18:15-45. Matsumoto, M., T. Tadenuma, K. Nakamura, H. Kume, T. Imai, R. Kihara, M. Watanabe, and Y. Benno. 2000. Effect of Bifidobacterium lactis LKM 512 yogurt on fecal microflora in middle to old aged persons. Microbial Ecol. Health Dis. 12:77-80. McDonald, L., M. Kuehnert, F. Tenover, W. Jarvis, and Centers for Disease Control and Prevention. 1997. Vancomycin-resistant enterococci outside the healthcare setting: prevalence, sources, and public health implications. Emerg. Infect. Dis. 3:311-317.

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Murray, B. E. 1990. The life and times of the enterococcus. Clin. Microbiol. Rev. 3:46-65. Perreten, V., B. Kollffel, and M. Teuber. 1997. Conjugal transfer of the Tn916-like transposon TnFO1 from Enterococcus faecalis isolated from cheese to other gram-positive bacteria. Syst. Appl. Microbiol. 20:27-38 Razavi, M. A., A. Mortazavi, and M. Mousavi. 2004. Application of neural networks for crossflow milk ultrafiltration simulation. Int. Dairy J. 14:69-80. Reid, G. 2001. Regulatory and clinical aspects of dairy probiotics. Report of a Joint FAO/WHO expert consultation on Evaluation on health and nutritional properties of probiotics in food including powder milk with lactic acid bacteria. Cordoba, Argentina. 1-4 October 2001. http://www.fao.org/es/ESN/Probio/probio.htm Sarantinopoulos, P., C. Andrighetto, M. D. Georgalaki, M. C. Rea, A. Lombardi, T. M. Cogan, G. Kalantzopoulos and E. Tsakalidou. 2001. Biochemical properties of enterococci relevant to their technological performance. Int. Dairy J. 11:621-647. Srensen, T. L., M. D. M. Blom, D. L. Monnet, N. Frimodt-Mller, R. L. Poulsen, and F. Espersen. 2001. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. N. Engl. J. Med. 345:1161-1166. Slaghuis, B. A., T. De Vries, and J. G. P. Verheij. 1991. Bacterial load of different materials which can contaminate milk during production. Milchwiss. 46:574-577. Teuber, M., V. Perreten, and F. Wirsching. 1996. Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiologie. Lebensm. Technol. 29:182-198. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek. 76:115-137. Weinstein, R. A. 2001. Controlling antimicrobial resistance in hospitals: Infection control and use of antibiotics. Emer. Infect. Dis. 7:188-192. Ziemer, C. J., and G. R. Gibson. 1998. An overview of probiotics, probiotics and synbiotics in the functional food concept: perspectives and future strategies. Int. Dairy J. 8:473-479.

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Chapter 11. Summary

Enterococci are natural residents of human and animal intestinal tracts and grow to high levels in a variety of artisanal cheeses. In the first part of this study we analysed the typical flora of Baylough cheese and determined the diversity of enterococci on a farm. For this purpose putative enterococci were isolated from the faeces of all the cows and all the people associated with the cheesemaking, from the milk and cheese during manufacture and ripening, and from the environment in three separate trials. In a first step almost 1800 putative enterococci were screened using a genus specific primer. The results indicated that all human, milk, curd and cheese isolates were members of the genus Enterococcus. RAPD-PCR was used to identify the enterococcal isolates. While the bovine faecal samples were dominated by E. faecium, the non-enterococcal strains of the cows were Streptococcus bovis. In the human faeces, milk and cheese samples E. casseliflavus were dominant, followed by lower numbers of E. faecalis. The same two species could be found in the environmental samples, the water in the milking parlour, rinses of the cows teats, the milking machine and the bulk-milk storage tank. In the following part of this study PFGE (restriction enzyme SmaI) was used to type the enterococcal isolates to a clonal level. All strains that were isolated in the first part could be represented by nine strains: three E. casseliflavus (LMG 19844, LMG 19845, LMG 20235), five E. faecalis (LMG 19846, LMG 20228, LMG 20229, LMG 20230, LMG 20234) and one E. durans (LMG 20231). The same 3 clones, 1 of E. faecalis (LMG 19846) and 2 of E. casseliflavus (LMG 19844 and LMG 1945), dominated almost all milk, cheese and human faecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination. This suggests the presence of a niche where enterococci could grow and contaminate the milk.

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The question at this point arose whether the enterococci in humans and faeces were the same due to consumption of the cheese and whether enterococci would survive the passage through the GIT. In the study that followed the composition of the enterococcal faecal flora in three healthy humans was analysed before, during and after the daily consumption of cheese made in the same plant under the same conditions. PFGE was used to determine the clonal relationship. Before and after consumption of the cheese, human samples contained mainly E. faecium. During consumption of the cheese one particular transient clone of E. faecalis, clone LMG 20228, which was present in small numbers in the cheese (2.7 x 102 cfu/g), largely dominated the faeces. E. casseliflavus LMG 18944 and LMG 18945, dominant in the cheese (7.4 x 102 en 2.9 x 104 cfu/g cheese) were also found in the faeces of one of the subjects during cheese consumption. The same nine clones recovered from cheese and the environment were subjected to phenotypic and genotypic analysis of their antibiotic resistances and virulence properties. All strains showed phenotypic resistance to streptomycin. In those five strains which showed phenotypic resistance to tetracycline, genes encoding resistance to tetracycline (tet(M) and tet(L)) were detected. In the only strain which showed an erythromycin resistant phenotype, the erm(B) gene were detected. Additionally, all strains were found to contain two or more virulence genes of those tested (agg, gel, cyl, esp, ace, efaAfs and efaAfm) resulting in some reservations about the safety of these strains. An important conclusion in the present study is that cows faeces were not considered to be the source of enterococci in the cheese. E. faecium and Str. bovis largely dominated the cows intestinal tract but were not found in either the milk or the cheese. Furthermore we deduce that the homologies in the enterococcal population between cheese and farmers are found in the cheese consumption and that the cheese was contaminated by enterococci residing in the milking machine. A significant conclusion is also that a clone needs not to be present in a food in high numbers to establish itself in the intestine. Additionally, we conclude that cheese can contain enterococci with virulence traits and resistances to clinically important antibiotics.

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It is and remains a case study, so conclusions for other farms should not be drawn. Additional studies about the putative source and safety aspects of enterococci are advised. DGGE would be very helpful in establishing the similarity between samples, which could have been resulted in less sampling. Nevertheless, the methods chosen for this study served its purpose very well.

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Chapter 12 Samenvatting

Enterokokken zijn aanwezig in het menselijk en dierlijk darmkanaal en komen voor in verschillende ambachtelijke kazen. De enterokokken flora van Baylough, een Cheddar-achtige kaas die geproduceerd wordt op een welbepaalde Ierse boerderij, werd bestudeerd met als doel de origine van deze organismen op te sporen. Hierbij werd de diversiteit van enterokokken van de gezinsleden, melkkoeien en omgevingsstalen op de boerderij bepaald. Enterokokken werden selectief gesoleerd uit fecale stalen van zijn vier bewoners en 27 melkkoeien, uit de melk en de kaas (tijdens het productie- en rijpingsproces) en uit de omgeving. Dit gebeurde tijdens drie afzonderlijke staalnames over een periode van 15 maanden. Ongeveer 1800 isolaten werden uitgezuiverd, en gescreend met een genusspecifieke primer. Alle isolaten uit de mensen, de melk, de wrongel en de kaas waren vertegenwoordigers van het genus Enterococcus terwijl nagenoeg geen isolaten van de koeien positief reageerden met de genusspecifieke primer. RAPD-PCR werd gebruikt om de isolaten te identificeren. De weinige enterokokken uit de fecale stalen van de koeien waren E. faecium. De in hoofdzaak niet-enterokokken isolaten van de melkkoeien behoorden tot het species Streptococcus bovis. In de menselijke fecale flora, in de kaas en in de melk overheersten E. casseliflavus en E. faecalis. Beide laatstgenoemde species werden tevens ook in de volgende omgevingsstalen gevonden: het water in de stal, het water voor de spoelingen van de spenen, de melkmachine en de koeltank voor de melk. In een verdere studie werd PFGE (SmaI restrictie enzyme) gebruikt om de enterokokken isolaten tot op klonaal niveau te typeren. Alle verzamelde isolaten behoorden tot slechts 9 stammen, drie E. casseliflavus stammen (LMG 19844, LMG 19845, LMG 20235), vijf E. faecalis stammen (LMG 19846, LMG 20228, LMG 20229, LMG 20230, LMG 20234) en n E. durans stam (LMG 20231). Drie van deze stammen, E. faecalis LMG 19846 en E. casseliflavus LMG 19844 en LMG

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19845, overheersten bijna alle stalen van melk, kaas en menselijk fecaal materiaal. De twee E. casseliflavus stammen werden echter ook na reiniging en chlorinatie in de tank van kaasbereiding en in de melkmachine gevonden. Dit suggereert de aanwezigheid van een niche waar enterokokken blijvend aanwezig zijn en telkens opnieuw de melk besmetten. In een daaropvolgende studie waren twee belangrijke vragen aan de orde: (i) Is een analoge enterokokkenpopulatie in het darmkanaal van de kaasproducenten en in de kaas toe te schrijven aan de consumptie van de kaas en (ii) kunnen enterokokken de passage door het gastrointestinaal kanaal overleven? Hiertoe werd de enterokokken flora in drie gezonde mensen voor, tijdens en na een dagelijkse consumptie van Baylough kaas geanalyseerd. Isolaten uit fecaal materiaal werden gedentificeerd en getypeerd. PFGE werd gebruikt om de clonale verwantschap te bepalen. Voor en na de consumptie van kaas was in elk van de proefpersonen E. faecium dominant. Tijdens de consumptie van kaas overheerste hoofdzakelijk de van de kaas afkomstige E. faecalis stam LMG 20228. Deze kloon was slechts in kleine hoeveelheden in de kaas te vinden (2.7 x 102 cfu/g) en verdween uit het darmkanaal na consumptie. In een van de proefpersonen werden tijdens de kaasconsumptie ook de stammen E. casseliflavus LMG 19844 en LMG 19845, dominant aanwezig in de kaas (7.4 x 102 en 2.9 x 104 cfu/g kaas), gevonden. Vervolgens werden de veiligheidsaspecten van de negen stammen uit de kaas en de omgevingsstalen bepaald. Met een fenotypische en genotypische benadering werd de aanwezigheid van antibiotica resistenties en virulentiefactoren nagegaan. Alle stammen waren resistent tegen streptomycine. In vijf stammen, die resistent waren tegen tetracycline, werd het tet(M) en tet(L) gen gedetecteerd. De stam waar het erm(B) gen aanwezig was toonde ook een fenotypische resistentie tegen erythromycine. In alle stammen werden bovendien twee of meer virulentiegenen gedetecteerd (agg, gel, cyl, esp, ace, efaAfs and efaAfm) en heeft tot besluit dat de veiligheid van deze stammen in vraag kan gesteld worden. De belangrijkste besluiten van dit doctoraat zijn dat koeien niet de primaire bron van enterokokken besmetting in Baylough kaas zijn vermits E. faecium en Str. bovis, die met grote hoeveelheden in het darmkanaal van koeien aanwezig zijn, noch in de melk noch in de kaas gevonden werden. De onderzochte kaas werd telkens

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opnieuw door dezelfde enterokokken gecontamineerd, aanwezig in de melkmachine en melktank, waar ze de reiniging overleven. Verder besluiten we dat een zelfde enterokokken populatie in de kaas en in het gastrointestinaal kanaal van de mensen toe te schrijven is aan de consumptie van kaas. Onze resultaten tonen aan, dat een Enterococcus stam in lage hoeveelheden aanwezig in voeding zich in het darmkanaal dominant kan manifesteren en groeien. Onze studies tonen aan dat de enterokokken uit Baylough kaas virulentiefactoren en resistenties tegen klinisch belangrijke antibiotica bevatten, wat hun veiligheid in vraag stelt. We moeten ervan uit gaan dat conclusies niet veralgemeend kunnen worden naar alle kaasboerderijen. Bijkomende analoge studies omtrent de mogelijke bron en veiligheidsaspecten van enterokokken in andere ambachtelijke kazen zijn aangewezen. Om de werklast te verminderen en een snellere screening naar variaties in enterokokken populaties toe te laten zou DGGE een waardig alternatief kunnen vormen voor de uitzuivering van honderden isolaten. Niettemin hebben de gekozen methoden in deze studie de vooropgestelde vragen beantwoord.

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Appendix 1

List of strains isolated in this study

original number Cs1 Cs2 Cs3 D Fs1 Fs2 Fs3 Fs4 Fs6 year of isolation 1998 1998 1998 1998 1998 1998 1998 1999 1999

Species E. casseliflavus E. casseliflavus E. casseliflavus E. durans E. faecalis E. faecalis E. faecalis E. faecalis E. faecalis

LMG number LMG 19844 LMG 19845 LMG 20235 LMG 20231 LMG 19846 LMG 20228 LMG 20229 LMG 20230 LMG 20234

origin milk bulk tank milk bulk tank cheese human faeces milk bulk tank milk bulk tank cheese water milking machine

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appendix 2 Curriculum vitae Name Date of Birth Place of birth Gelsomino, Roberto 6th March 1972 Merano (Italy)

Education 1986-1991 1991-1995 1995-1998 secondary school A. Einstein with scientific background in Merano (Italy) Leopold Franzens Universitt in Innsbruck (Austria) thesis at the Institut fr alpenlndische Milchwirtschaft in Rotholz (Austria), Magister der Naturwissenschaft (MSc degree in Microbiology; Hygiene and Medical Microbiology as option). Graduated in April 1998. 1998-1999 Beginning of the PhD at Teagasc, Moorepark, Dairy Products Research Centre, Fermoy, Ireland since September 1999 PhD at the University Gent-Laboratorium voor Mikrobiologie since September 2001: EU-project: Biodiversity and anti-listerial activity of surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit and Gubbeen cheese Activities -1996/1997: Chemistry teacher at the Technical School G. Matteotti in Merano (Italy) -Since October 2002 some one-hour lectures and assistance in the practical courses of microbiology -Supervision of a Master student (Hinde Benbrahim): Karakterisering van de microflora van roodsmeerkazen

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Research papers:
Sebastiani, H., R. Gelsomino and H. Walser. 1997. Cultures for the Improvement of Texture in Quarg. Int. Dairy Fed: Texture of Fermented Milk Products & Dairy Research. 9802:78-92. Gelsomino R., M. Vancanneyt, S. Condon, J. Swings, and T. M. Cogan. 2001. Enterococcal diversity in the dairy environment of an Irish cheese making factory. Int. J. Food Microbiol. 71:177-188 Gelsomino R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings. 2002. Source of enterococci in a farmhouse raw-milk cheese. Appl. Environ. Microbiol. 68:3560-3565. Gelsomino R., M. Vancanneyt, T. M. Cogan, S. Condon, and J. Swings. 2003. Effect of raw-milk cheese consumption on the enterococcal flora of human feces. Appl. Environ. Microbiol. 69:312-319. Gelsomino R., M. Vancanneyt, and J. Swings. 2004. Reclassification of Brevibacterium liquefaciens Okabayashi & Masuo 1960 as Arthrobacter nicotianae Giovannozzi - Sermanni 1959. Int. J. Syst. Evol. Microbiol. 54:615-616. Gelsomino R., M. Vancanneyt, T. T. M. Vandekerckhove, and J. Swings. Development of a 16S rRNA primer for the detection of Brevibacterium spp. Lett. Appl. Microbiol. (in print) Gelsomino R., M. Vancanneyt, and J. Swings. Reclassification of Enterococcus solitarius as Tetragenococcus solitarius, comb. nov. and emended description of the genus Tetragenococcus. Int. J. Syst. Evol. Microbiol (in preparation) Gelsomino R., G. Huys, K. DHaene, M. Vancanneyt, T. Cogan, C. M. A. P. Franz, and J. Swings. Antibiotic resistance and virulence traits of enterococci isolated from an Irish artisanal cheese. J. Food Prot. (submitted)

Oral presentations:
Gelsomino, R., T. M. Cogan, S. Condon and M. Vancanneyt. 1999. The Source of enterococci in an Irish Farmhouse Raw-Milk Cheese. 29th Annual University College Cork Food Science and Technology Research Conference, 16-17 September 1999, Cork, Ireland. R. Gelsomino, M. Vancanneyt, T. M. Cogan, and J. Swings. 2002. The effect of raw-milk cheese consumption on the enterococcal faecal flora. Enterococci in Foods; Functional and Safety Aspects. 30-31 May 2002, Berlin Germany

Other:
R. Gelsomino. 1997. Milchsurekulturen zur Verbesserung der Textur in Quark. (Master Thesis). Institut fr alpenlndische Milchwirtschaft in Rotholz (Austria).

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Acknowldgments Ringraziamenti

When I decided to begin my PhD I knew I would have to live most of the time in Fermoy, Co. Cork and a few months in Gent. I had never heard of either of them but my adventurous soul made me accept. For personal reasons it turned out to be exactly the opposite but I have learned to appreaciate the bad and good sides of both. The person who made this possible was the man who I felt looked at me not only as a PhD student but also as a person: Prof. Dr. Tim Cogan. Tim, it is not only your expertise but also your understanding, patience, kindness, helpfulness and big humanity that added considerably to my graduate experience. I doubt that I will ever be able to convey my appreciation fully, but I owe you my eternal gratitude. I would like to acknowledge Teagasc for Walsh Fellowship funding and the opportunity to carry out this research. I would like to express my gratitude to Prof. Dr. ir. Jean Swings for inviting me in the Laboratium voor Microbiologie in Gent. Thanks to you I was able to continue my studies and this means a lot to me. Furthermore you gave me the possibility to learn more about academic life, thank you very much! I appreciate also the help of the Keiting family. Ann and Dick, thank you for all your help, assistance, and support. This study was only possible thanks to you. A very big thanks goes to Dr. Marc Vancanneyt, to his patience, his explanations, his big help. Marc, this work would not have been possible without you. Another big thanks goes to the people in the lab In Ireland, Mary and Finbarr, thanks for helping me with all the plates in triplicates, and we are talking triple digits here! To the good friends I have made there Brid D., Brid L., Noelle, Nora thanks for

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the laughs. In Belgium thanks to Cindy and Karen, you were a precious help in the beginning and throughout the PhD, thanks also for my first words in Dutch ever (een bruin broodje met kaas en hesp). Thanks to everybody who helped me in learning everything that I know now, and for the laughs we had in the lab, Dirk D., Margo, Maggy, Liesbeth, Ben, Geert, Moudjahid, Leen, Ann, Katleen, Nele, Katrien, Bart, and Renata. Paul, thanks a lot to you too. You were the gateway to cultural Vlaanderen in particular and Belgium in general, you taught me my first steps in appreciating all the old stuff I am stacking at home. Its catching dust, but it is oh so beautiful! Mamma, pap, e Omi, difficile rinraziarvi doverosamente in poche righe. Voglio solo dirvi grazie per avere sempre mostrato, per quello che faccio, una fiducia cieca e priva di incertezze, spronandomi sempre ad andare avanti per la mia strada. Grazie per il vostro appoggio, che mi avete dato, soprattutto per telefono, perch vi ho privato troppo spesso della mia presenza a casa. Amici, italiani, conosciuti in Belgio: Savina, Cristina, Francesco e Alberto. Un grazie affettuoso anche a voi, per avere condiviso momenti di ansia ma anche tante situazioni divertenti e gratificanti. Un grazie di cuore anche mia sorella Antonella. Non solo per laiuto e lappoggio che mi hai dato ma anche per lallegria che sprigioni e le risate che ci facciamo insieme. Thank you too, Pery, I should have taught you how to cook Italian, but the arroz a la Cubana was just too yummy. Thanks also to Silvie, who helped me in keeping weight. Thanks for cooking French for me and for not bypassing the chocolate isle at the supermarket. A very big hug goes to Wolfgang, Andreas, Petra, Jochen, Martin and Heidi (Waikiki-Koko). With you I shared very important and intensive moments during my PhD and you were always there; in hard times, when I was really down and you didnt

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fail to support me, and in happy and funny moments, were we shared so many laughs. Thank you so much!! Het laatste dankwoordje aan mijn vrienden in Belgie: Greta, Wilfried en Geert voor de lachertjes, de vakanties (met de slang), de etentjes. Jurgen, Gregory en Peter voor de kookinspiraties, Daniel en Bart voor de etentjes, de babbeltjes en de competitieve squashavonden (stond ik in de weg?). Een grote dank ook aan Marino voor de steun, het gezelschap en al de hulp die je me gaf. En zeker voor de wetenschappelijke informatie in Melle: ik hoop de camionbestuurder, die jouw autodeur weggezogen heeft, het weet te waarderen. Ik doe dat in iedere geval.

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