Klebsiella pneumoniae as a Spoilage Organism

in Mozzarella Cheese
SALVATORE MASSA, FAUSTO GARDINI, MILENA SINIGAGLIA, and MARIA ELlSABElTA GUERZONI Dipartimento di Protezione e Valotizzazione Agroalimentare Universita' degli Studi d Bologna Via San Giacomo. 7-1-40126 Bologna, Italy ABSTRACT

A high concentration of total and fecal coliforms (5 x 108 and 3 x lo7 cfu/g, respectively) was observed in samples representative of two production lots of Mozzarella cheese from a local dairy. Contamination was manifested by the swelling of plastic pouches and the formation of gas holes in Mozzarella cheese. Aerobic and anaerobic sporeformers, heterofermentative lactic acid bacteria, propionibacteria, and yeasts were absent. Of the 41 isolates identified, there were 37 Klebsiella pneumoniae, 2 Klebsiella oxytoca, 1 Enterobacter aerogenes, and 1 Escherichia coli. Exposure at 63'C for 15 min caused the death of all total and fecal coliforms. (Key words: deterioration, gas, Mozzarella cheese, Klebsiella pneumniae)
INTRODUCTION

duction in fresh cheese (9, 15), reports are lacking on this type of deterioration specifically in Mozzarella cheese. Only recently has a case been described regarding abnormal gas production in American-type Mozzarella cheese f r o m Propionibacterium freundenreichii (6). A case of spoilage, characterized by swelling of plastic pouches and formation of gas holes in cheese, occurred in two production lots, corresponding to 2 different d, at a local dairy. These two lots were removed from the market, resulting in serious economic loss. In this present communication, we report the results of a microbiological study conducted to determine the agent responsible for gas production in Mozzarella cheese samples.
MATERIALS AND METHODS Sarnpllng and Mlcrobiologlcal Analysls

Mozzarella cheese is a soft, plastic cheese typical of Italy and of notable economic relevance because of the steady rise in its production and consumption. It is a particularly perishable cheese, having a brief commercial shelf-life (20 to 30 d) that is dependent on the extent and degree of refrigerated storage. Other authors have described diverse cases of spoilage: extremely soft consistency (13), appearance of anomalous pigmented spots on the surface (11, 17), development of bitter taste (5), production of slime, and surface flaking (26). Although yeasts, coliforms, heterofermentative lactic acid bacteria, clostridia, and Bacillus spp. have often been associated with gas pro-

Received September 16, 1991. Accepted February 24, 1992.

Fifteen swollen polyethylene pouches of Mozzarella cheese, representative of two production lots, were randomly selected and sampled. The samples showed a gas defect a few days after production; these samples were transported on ice to the laboratory and analyzed within l h of their receipt. Typically, Italian Mozzarella cheese is 58 to 62% water, 16 to 18% protein, 17 to 19% fat, and 3.4% ash. The cheeses were examined microbiologically for total and fecal coliform counts, respectively, at 37 and 4432 on violet red bile agar (Diico, Detroit, MI) and confirmed in 2% brilliant green bile broth (Difco) fermentation tubes. Aerobic and anaerobic sporeformers were enumerated on plate count agar (Difco) following treatment at 75C for 15 min; heterofermentative lactobacilli and propionibacteria were estimated using the selective media proposed by McDonald et al. (20) and Peberdy and Fryer (24), respectively. Yeasts

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TABLE 1. Microbiological analysis of 15 samples of Mozzarella cheese i n expanded plastic pouches. Percentage of contaminated sam~les
~~~

Microorganism Total coliforms' Fecal coliforms2 Spore-forming bacteria3 Heterofementative lactobacilli Propionibacteria YeaSts

Lot A (n = 8)

Lot B (n = 7)
100

la0 la0
0 0 0 0

100 0 0 0 0

'Average densities expressed as per gram: 4.8 x 10' for lot A and *Average densities expressed as per gram: 2.5 x lo7 for lot A and 3Aerobic and anaerobic.

colony-forming units 5.3 x lo8 for lot B. colony-forming units 3.4 x lo7 for lot B.

were enumerated on malt extract agar (Oxoid, Basingstoke, Engl.).

Characterlzatlon of isolates

Because coliform bacteria were isolated in large numbers, these organisms were selected for further analyses. The brilliant green bile broth tubes with gas were streaked onto Endo agar (Difco) plates and incubated at 37'C for 24 h. Forty-one colonies, randomly chosen and purified in tryptic soy agar (Difco), were initially characterized as to their Gram reaction l l isolates were tested in and morphology. A OF (oxidative fermentative) basal medium Determination of pH m c o ) containing 1% glucose to determine pH was determined directly by immersing their mode of carbohydrate utilization. Identifi- the electrode of a pH meter (Beckman Instrucation was performed using conventional ments, Fullerton, CA) into the cheese; the pH phenotypic traits, according to the methods of meter was calibrated at 22'C.

Edwards and Ewing (8). Moreover, the same strains were identified with API 20 E test strips (BioMerieux, CharbonniBres-les-Bas, France), according to the manufacturer's instructions. Two cell suspensions of Klebsiella pneumoniae (strains prevalently isolated from spoiled cheese of lots A and B; API profile 5215773) were prepared from pure culture in tryptic soy agar. For thermoresistance, an appropriate quantity of cell suspension was added to 250-ml Erlenmeyer flasks containing preheated sterile skim milk (Difco) prepared in 10% solution; find c e l l concentration was approximately 107 cfU/ml. Quadruplicate sets of inoculated skim mi& were incubated in a water bath without shaking at 60 and 6332. After 15 min, 1 ml of skim milk was rapidly withdrawn and diluted in chilled peptone-water. Klebsiella pneumoniae counts were determined by using tryptic soy agar. In order to verify the isolates of K.pneumoniae as the causative organism of gas production, sealed vials containing cubes of about 1 g of fresh Mozzarella cheese and 1 ml of whey were inoculated with 106 ceUs/g. The evolution of C02 over time was evaluated by means of the gas chromatographic analysis of the headspace. The gas chromatographic conditions were those described by Guerzoni et al. (12). Uninoculated samples were prepared as a control.

TABLE 2. Identification of 41 isolates from violet red bile agar (VRBA) plates incubated at 37 and 44T. 37'c
No.
W C

Species Klebsiella pnewnonioe Klebsiella oxytoco Enterobocter oerogenes K . pneumonioe K . oxytoco K . pneumonioe K . oxytoco E. oeropenes

No.

Species K . pneumonioe Escherichia coli K. pneumonioe

K . pneumonioe

Lot A

8
1 1

9
1

Lot B

Total

10 1 18 2
1

10

19
1

E. coli

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TABLE 3. Percentage of survival of two isOlates following

heating (15 min) in skim milk. Isolates
Initial Tempconcentration W C 63'C

(cfu/g) Klebsiella pneumoniae T1 1.3 x lo7 1.9 x 106 K . nneumoniae ~2

~~

- 1.15
62

0 0

lStmin from violet red bile agar incubated at 37'C. 2S~ah from violet red bile agar incubated at WC.

RESULTS AND DISCUSSION

The results of the microbiological analysis of the 15 samples of Mozzarella cheese are reported in Tables 1 and 2. Aerobic and anaerobic sporeformers, heterofermentative lactic acid bacteria, propionibacteria, and yeasts were absent. The spoilage was not accompanied by significant variations in pH, ranging between 5.2 to 5.4; pH was considered to be normal for this type of product. The gas production observed can be atuibuted essentially to extensive contamination by coliforms. The density of indicator bacteria was roughly 5 x 108 and 3 x lo7 cfdg for total and fecal coliforms, respectively. Of the 41 isolates identified, there were 37 (90.2%) K. pneumoniae, 2 (4.9%) Klebsiella oxytoca, 1 (2.4%)Enterobacter aerogenes, and 1 (2.4%) Escherichia coli. Exposure at 63'C for 15 min caused the death of the K. pneumoniae strains taken from the violet red bile agar plates incubated at 37 and 44T (Table 3). A l l isolated strains were able to produce gas from lactose (confiied test in brilliant green bile broth fermentation tubes within 24 h). Moreover, 91% of the 37 isolates of K. pneumoniae were able to produce more than 70% of gas in the headspace of the sealed vials containing Mozzarella cheese and stored at 10C for 48 h. In the control samples, the gas value was always lower than 20%. Klebsiella pneumoniae is widely recognrzed as an opportunistic pathogen and as an agent of pneumonia, u r i n a r y tract infection, and gastroenteritis in stressed patients (16). At the same time, K . pneumoniae has been isolated on numerous occasions from nonclinical environments as a major component of the microbial flora (4, 14, 19, 23). The bacterium has

been recovered from acid foods having a high sugar content (7, 21). Klebsiella pneumoniae is also associated with outbreaks of mastitis in cattle and is the cause of a variety of infections in other domestic and wild animals (3, 27). The surfaces o f cow teats can be colonized by Klebsiella organisms present in high numbers in sawdust bedding used for dairy herds (25). Bedding materials can be an important reservoir for this agent of coliform mastitis (22). Thus, it is not surprising that K . pneumoniae was the most frequently isolated species and most probably the cause of the observed spoilage. It is diffkult to pinpoint the sources of the microorganism because the possible origins of contamination are numerous. However, strains of K. pneumoniae that are positive for fecal coliforms and able to grow at low temperatures (10"C), although rare, have been reported by others (1, 2, 10). Given the lack of thermoresistance of K . pneumoniae the most probable contamination source is likely to have occurred after pasteurization. However, stretching under hot water, which in this particular type of cheese increases the temperature of the body up to 57C (18). is unable to eliminate high concentrations of coliforms, which eventually recontaminated the milk after pasteurization. Further studies will strive to determine the actual origin of these contaminants, because they represent a serious danger both to human health and to the economy of the dairy industry.
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