FOODBORNE PATHOGENS AND DISEASE Volume 10, Number 8, 2013 Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2012.

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Transmission and Persistence of Methicillin-Resistant Staphylococcus aureus in Milk, Environment, and Workers in Dairy Cattle Farms
1 1 1 1 1 Suk-Kyung Lim, Hyang-Mi Nam, Gum-Chan Jang, Hee-Soo Lee, Suk-Chan Jung, and Tae-Sun Kim 2

Abstract

The aim of this study was to determine the presence and persistence of methicillin-resistant Staphylococcus aureus (MRSA) in milk, farm environment, and farmers on 22 dairy cattle farms in Korea during 20082009. Genetic relatedness among the MRSA isolates was also investigated. Of 1146 samples examined, 35 of 559 (6.3%) quarter milk samples from 371 cows, four of 86 (4.7%) hand and nose samples from 43 farmers, and 6 of 501 (1.2%) farm environment samples were MRSA positive. Except for three isolates, all MRSA were classied into ST72-spa t324SCCmec IV with PVL negative, the most predominant clonal type among community-associated MRSA in South Korea. All 35 MRSA-positive milk samples from 19 cows were obtained from a single farm (Farm G) out of 22 (4.5%) farms tested. The farm G was revisited 1 year later and milk samples were collected for examination of MRSA again. Two of six previous MRSA-positive cattle that had been kept on the farm still harbored MRSA genetically identical to MRSA strains, which were isolated from the same farm a year ago. The results of this study provide the evidence of transmission of MRSA among cattle, farm environment, and farmers and also long-term persistence of MRSA in animals.

Introduction

taphylococcus aureus (S. aureus) is one of the most important pathogens causing mastitis in dairy cattle (Moon et al., 2007; Nam et al., 2011). Although methicillin is not generally used for treatment of mastitis, methicillinresistant Staphylococcus aureus (MRSA) has been recovered sz-Kaszanyitzky et al., 2007; Moon from dairy cattle ( Juha rkyilmaz et al., 2010; Vanderhaeghen et al., 2010; et al., 2007; Tu Nam et al., 2011). Furthermore, horizontal transmission of MRSA between dairy cattle and farm workers has been sz-Kaszanyitzky et al., 2007), suggesting that reported ( Juha human contact with MRSA-positive animals or vice versa could provide opportunity for transmission of MRSA between them. Presence of MRSA in the environment might also be one of the sources of MRSA infection in animals, where it may survive for several months. Detection of MRSA from various environmental samples associated with animals including dust, farm rat, and environmental wipes has been previously reported (Weese et al., 2004; Friese et al., 2012). So far, studies on MRSA have been focused primarily on rkyilmaz the occurrence of MRSA in animals (Lee 2006; Tu

et al., 2010; Nam et al., 2011; Lim et al., 2012). However, in order to develop strategies on the prevention and control of MRSA on dairy farms, rst it is necessary to identify the source of MRSA on the farms. The aims of this study were, therefore, to examine the presence of MRSA in milk, farm environment, and farmers on dairy farms in Korea and to determine genetic relatedness of MRSA strains isolated using molecular genetic methods. In addition, the persistence of MRSA strains in MRSA-positive dairy cattle was also investigated 1 year after the rst examination was completed. Materials and Methods Sample collection A total of 1146 samples (559 quarter milk samples from 371 cows, 501 environmental samples, and 86 swab samples of 43 farmers: one hand sample and one nasal swab each) were collected from 22 different dairy cattle farms between February and September 2008. Twenty-two farms were selected for this study based on the likelihood of mastitis, the farmers interest, and compliance. These farms were located in ve different provinces ( Jeonnam, n = 3; Jeonbuk, n = 8; Chungnam, n = 4; Gangwon, n = 3; Gyeonggi, n = 4) in Korea.

1 2

Animal, Plant, and Fisheries Quarantine and Inspection Agency, Gyeonggi-do, Republic of Korea. Public Health and Environment Institute of Gwangju, Gwangju, Republic of Korea.

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732 The quarter milk samples were taken aseptically from 371 lactating cows directly after milking from 10 to 70 samples on each farm. Milk samples with somatic cell counts greater than 200,000 cells/mL were subjected to MRSA and methicillinresistant coagulase-negative staphylococci (MRCNS) isolation. For farmers nasal sampling, a cotton-tipped swab with Stuarts medium (Becton Dickinson, Sparks, MD) and for the farmers hand sampling, four gauze pads (10 cm 10 cm) wetted with 1% sterile skim milk were used, respectively. A total of 501 environmental samples: 102 milk cups, 210 teat swabs from 210 cows, 43 oor swabs, 44 fence swabs, 42 ventilation fan swabs, 30 water and 30 feed samples were collected. Environment samples were also collected using four gauze pads wetted with 1% of sterile skim milk, and the samples were placed in a sterile plastic bag. Drinking water and feed samples were collected in buckets and placed into sterile 50-mL containers. The samples were immediately transported to the laboratory in ice-cooled containers and analyzed within 24 h of collection. In order to examine the persistence of MRSA in MRSApositive cattle and environment 1 year later, the only farm (farm G) from which MRSA were isolated from milk samples among 22 farms studied previously was revisited in March 2009. A total of 28 samples (22 milk samples from six remaining MRSA-positive cows and ve newly introduced cows, four environmental samples, and two samples of one MRSA-positive farmers hands and nasal cavities) were collected from farm G in the same manner as mentioned above. Isolation and identication of MRSA and MRCNS All swab samples of environmental and farmers hands were individually placed in a sterile stomacher bag containing 50 mL of 1% sterile skim milk. Each swab sample was homogenized for 30 s. Nasal swabs, 1 mL of milk, and 1 mL of homogenized gauze swab samples were inoculated into 9 mL Mueller Hinton broth (Becton Dickinson) containing 6.5% NaCl. After incubation at 37C for overnight, 1 mL of this preenrichment broth was transferred into 9 mL of Tryptone Soy Broth containing cefoxitin (3.5 mg/L) and incubated for a further 1620 h at 37C. Then one loopful of this broth was spread onto a chromogenic MRSA agar (Oxoid, Hampshire, UK) selective for MRSA and incubated for 2448 h at 37C. Based on colony morphology and color, three suspected colonies were subcultured on blood agar. Presumptive MRSA and MRCNS colonies were identied by detecting the Staphylococcus 16S rRNA, clfA, and mecA gene by polymerase chain reaction (PCR) (Mason et al., 2001). Antimicrobial susceptibility Antimicrobial susceptibility was determined by the disk diffusion method on Mueller Hinton agar according to the Clinical and Laboratory Standard Institute (CLSI, 2008) guidelines using commercial antimicrobial-containing discs obtained from Becton Dickinson (BBL Sensi-Disk). The antimicrobial agents tested were cefoxitin (30 lg), penicillin (10 units), vancomycin (30 lg), erythromycin (15 lg), clindamycin (2 lg), linezolid (30 lg), trimethoprim (5 lg), chloramphenicol (30 lg), rifampin (5 lg), quinupristin/dalfopristin (15 lg), ciprooxacin (5 lg), gentamicin (10 lg), ampicillin (10 lg), and amoxicillin/clavulanic acid (20/10 lg). Minimum inhibitory concentrations of suspected MRSA isolates were tested using

LIM ET AL. E-test strips (AB BioDisk, Solona, Sweden). The diameter of inhibitory zones surrounding the antimicrobial discs and E-test strips was interpreted according to the CLSI guidelines (2008). S. aureus ATCC 25923 and Escherichia coli ATCC 35218 were used as quality control strains. Molecular characterization of MRSA Staphylococcal cassette chromosome mec (SCCmec) typing of MRSA strains was determined by a multiplex PCR method as described previously (Oliveira and de Lencastre, 2002). Multilocus sequence typing was carried out according to the method described by Enright et al. (2000). Primers specic for seven housekeeping genes, arcC, aroE, glpF, gmk, pta, tpi, and yqiL were used for PCR amplication and sequencing. Puried PCR products were directly sequenced by commercial sequencing company (Macrogen Inc., Seoul, Korea). Allele numbers and sequence types were assigned using the multilocus sequence typing website (http:/ / www.mlst.net). Genomic DNA for pulsed-eld gel electrophoresis (PFGE) was prepared following the procedure reported by McDougal et al. (McDougal et al., 2003). PFGE of Sma I digestedDNA was performed with CHEP-Mapper apparatus (Bio-Rad Laboratories, Hercules, CA). Electrophoresis was performed using 1% SeaKem Gold agarose at a voltage of 6V/cm for 21 h at 14C in 0.5X tris-borate-EDTA (TBE buffer) with pulse time ramped from 5 s (initial switch time) to nal switch time of 40 s. Spa typing was performed as described by Shopsin et al. (1999). Briey, spa gene was amplied and sequenced using primers spa 1095u (5-AGACGATCCTTCGGTGAGC-3) and spa 1517d (CAGCAGTAGTGCCGTTTG). Sequence data were analyzed using Ridom Staph Type software (www .spaserver.ridom.de). The spa repeats and spa types were determined and assigned a numeric code as previously described (Harmsen et al., 2003). Presence of Panton-Valentine Leukocidin toxin genes was determined by PCR as previously described (Lina et al., 1999). Results Prevalence of MRSA A total of 45 MRSA strains were isolated from three (farms G, J, and L) of the 22 (13.6%) dairy farms studied. Thirty-ve of 559 quarter milk samples (6.3%) were MRSA positive, all of which originated from 19 different cattle from a single farm (farm G). Of 86 swab samples of farmers hands and noses, 4 (4.7%) samples originating from two farmers of farms G and J were MRSA positive. Similarly, MRSA was detected from six of 501 (1.2%) environmental samples on two farms (farms G and L), including milk cup (1/102, 1.0%), oor (3/43, 7.0%), fence (1/44, 2.3%), and ventilation instrument (1/42, 2.4%). On farm G, MRSA was isolated from milk and farmers as well as from the environmental samples. In addition, seven isolates of MRCNS were detected in milk samples (5/559, 0.9%) collected from three different farms (farms M, T, and V), and samples of a farmers hand (1/43, 2.3%) and nose (1/43, 2.3%) from a farm (O farm). However, no MRCNS were detected from environmental samples (Table 1). Characterization and persistence of MRSA Twenty-nine of the 45 MRSA isolates: 19 strains originating from different cow milks, four from a farmer, and six from the

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Table 1. Prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) in Samples of Milk, Farmers, and Farm Environment on 22 Dairy Cattle Farms in Korea During 2008 No. of positive samples/no. of tested samples (%) Farma Samples (no.) Milk (n = 559) Quarter milk (n = 559) Human (n = 86) Hand (n = 43) Nasal cavity (n = 43) Farm environment (n = 501) Milk cup (n = 102) Teat swab (n = 210) Floor (n = 43) Fence (n = 44) Ventilation fan (n = 42) Water (n = 30) Feed (n = 30) MRSA 1/22 (4.5)1 2/22 (9.1)1,2 1/22 (4.5)1 1/22 0/22 2/22 1/22 1/22 0/22 0/22 (4.5)1 (0) (9.1)1,3 (4.5)1 (4.5)1 (0) (0) MRCNS 3/22 (13.6)4,6,7 1/22 (4.5)5 1/22 (4.5)5 0/22 0/22 0/22 0/22 0/22 0/22 0/22 (0) (0) (0) (0) (0) (0) (0) MRSA 35/559 (6.3) 3/43 (7.0) 1/43 (2.3) 1/102 0/210 3/43 1/44 1/42 0/30 0/30 (1.0) (0) (7.0) (2.3) (2.4) (0) (0) Sample MRCNS 5/559 (0.9) 1/43 (2.3) 1/43 (2.3) 0/102 0/210 0/43 0/44 0/42 0/30 0/30 (0) (0) (0) (0) (0) (0) (0)

a 1, Farm G; 2, Farm J; 3, Farm L; 4, Farm M; 5, Farm O; 6, Farm T; 7, Farm V. MRCNS, methicillin-resistant coagulase-negative staphylococci.

environment were further characterized in this study. Except for three, all MRSA isolates tested were classied into ST72-spa t324-SCCmec type IV. Two isolates from milk samples (farm G) and one from a oor sample (farm L) were the same ST72-spa t324, but SCCmec type was found to be untypable by our methods. All isolates were Panton-Valentine leukocidin negative. Antimicrobial resistance showed pansusceptibility to nonb-lactam antimicrobials.

Of six MRSA-positive cattle that still remained on a farm (farm G) a year later, two (no. 6 and no. 7) were found to harbor MRSA in their milk. In addition, one (no. 32) of the ve cattle that were newly introduced into the farm was found to be MRSA positive. The genetic type of MRSA strains isolated a year later from the previously MRSA-positive cattle was ST72-spa t324-SCCmec IV, identical with that of MRSA isolated previously in the farms (Table 2). An identical PFGE pattern

Table 2. Genetic Type of Methicillin-Resistant Staphylococcus aureus Isolates from Samples of Milk, Human, and Farm Environment Collected on March 2008 and Those Collected on March 2009 in a Dairy Cattle Farm in Korea March 2008 Source Milk Cow no. 1b Cow no. 4 Cow no. 6 Cow no. 7 Cow no. 8 Cow no. 23 Cow no. 24 Cow no. 28b Cow no. 29b Cow no. 32b Cow no. 33b Farmer Hand Nasal cavity Environment Floor Fence Ventilation fan Milk cup
a b

March 2009 Type Not tested ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 Not tested Not tested Not tested Not tested ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 ST72-IV-t034 Isolation + + + Type Not tested Not tested ST72-IV-t034 ST72-IV-t034 Not tested Not tested Not tested Not tested Not tested ST72-IV-t034 Not tested Not tested Not tested Not Not Not Not tested tested tested tested

Isolationa Not + + + + + + Not Not Not Not + + + + + + tested

tested tested tested tested

+ , detected; , not detected. Newly introduced cow.

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LIM ET AL.

FIG. 1. Pulsed-eld gel electrophoresis patterns of Sma I-digested total DNA of methicillin-resistant Staphylococcus aureus isolates from a farm (farm G). Lanes 1 and 14: Xba I-digested Salmonella Braenderup, and lanes 213: Sma I-digested MRSA isolates. Lane 2: farmers hand; lane 3, farmers nose; lane 4: family 1 hand; lane 5: family 2 hand; lane 6: oor of milking parlor; lane 7: fence of milking parlor; lane 8: ventilation fan of milking parlor; lane 9: milk of cow no. 6 (isolated in 2008); lane 10: milk of cow no. 6 (isolated in 2009); lane 11: milk of cow no. 7 (isolated in 2008); lane 12: milk of cow no. 7 (isolated in 2009); and lane 13: milk of cow no. 32 (newly introduced).

was also observed among all the MRSA strains isolated from samples of milk, farmer, environment of farm G, and the isolates from cattle newly introduced into the farm (Fig. 1). Discussion In this study, we found genetically identical MRSA isolates from samples of milk, farmers, and farm environment in one farm. In addition, the same MRSA strain was found to be persistent in dairy cattle at least for 1 year. Although MRSA is comparatively rare in cattle (Lee, 2006), dairy milk might be one of the potential sources of MRSA because of frequent use of antimicrobials for treatment of rkyilmaz et al., mammary infections (Moon et al., 2007; Tu 2010; Nam et al., 2011). Recently, a high prevalence of MRSA in bovine mastitic milk was reported from some countries sz-Kaszanyitzky et al., 2007), such as Hungary (4.5%) ( Juha Belgium (9.3%) (Vanderhaeghen et al., 2010), and Turkey rkyilmaz et al., 2010). In this study, MRSA was (17.2%) (Tu detected in 6.3% of milk samples tested. Although it is difcult to compare the prevalence in milk samples due to differences in methodologies for sampling or detection, our result was higher than those reported by previous studies in Korea (1.3%, Lee 2006; 0.18%, Kwon et al., 2006; 3.1%, Nam et al., 2011). This is due to the fact that a high rate (79.5%, 35/44) of quarter milk samples originated from a MRSA-positive farm in this study, despite relatively low prevalence of MRSA among farms (6.3%, 35/559). MRSA was reported in the environment associated with animals including dogs (Moodley et al., 2006), horses (Weese et al., 2004), chickens, and pigs (van de Giessen et al., 2009; Friese et al., 2012). Furthermore, identical or closely related MRSA isolates were recovered from animals and their environment (Weese et al., 2004; Friese et al., 2012). In dairy cattle farms, genetically identical S. aureus was isolated from milk and their environments in previous studies (Roberson et al., 1998; Hata et al., 2010). However, data on the

presence or persistence of MRSA in the environment and farm workers in dairy farms are very limited. In this study, MRSA was detected in milking-associated environmental samples such as milk cup, oor, fence, and ventilation instrument of the milking parlor in a MRSA-positive milk farm. Also, all MRSA isolates from the farm environment were genetically identical to those of milk isolates. Although the origin of the MRSA isolates from the environmental samples could not be identied, this result suggests that it may be circulating among cattle and their environment, including the milking parlor. We also found that four MRSA and two MRCNS were isolated from farmers of three different farms (farm G, J, and O). In a farm (farm G), MRSA was detected from the owner of the farm and his family. A similar report from Hungary described the isolation of a shared MRSA strain from dairy cows with sz-Kaszanyitzky subclinical mastitis and a farm worker ( Juha et al., 2007). In the present study, all MRSA isolates except three isolates were classied into ST72-spa t324-SCCmec type IV, the most predominant clonal type among community-associated MRSA in South Korea (Park et al., 2007). Generally, human lineages of S. aureus are rarely found in animals. However, a host shift of a particular S. aureus (sequence type 5) lineage from human to chickens by genetic events was reported (Lowder et al., 2009). Herron-Olson et al. (2007) also report that mobile genetic elements of successful bovine-associated S. aureus were similar to those of S. aureus from infected human hosts. These results suggest the possibility that some lineages of S. aureus have a broader host range. However, further studies are needed to determine whether these animal isolates diverged from a human-associated community-acquired MRSA. S. aureus infection frequently persists for a long time in infected mammary glands in cattle (Atalla et al., 2008). The persistence of MRSA for at least 1 year was demonstrated in this study, although we are not sure whether the MRSA strains persisted in the cattle all the time or were intermittently acquired because we examined them only after a year later. We also found that MRSA identical to the previously

TRANSMISSION AND PERSISTENCE OF MRSA isolated strain in the farm was detected from one of ve cattle newly introduced into the farm. Several studies on the persistence of S. aureus were reported in bovine mastitis cases (Sidhu et al., 2001; Oliveira et al., 2007; Atalla et al., 2008). A possible explanation of persistence may be the occurrence of small colony variants (Atalla et al., 2008) or biolm producing S. aureus (Oliveira et al., 2007) that allow bacterial evasion of the hosts immune system or resistance to antimicrobial therapy. Another explanation may be linkage between qacA/B and the blaZ (b-lactamase gene), which could be selected for penicillinresistant staphylococci by use of b-lactam antimicrobials and vice versa by use of ammonium compounds for prevention of mastitis in dairy herds (Sidhu et al., 2001). In this study, MRSA persisted for at least 1 year in milk samples on one farm but not in the environment or farmer. However, further studies are needed to elucidate the genetic characteristics of the element of the MRSA strain for colonization in cattle. In conclusion, genetically identical MRSA was detected in milk samples as well as in the farmer and the farm environment on a dairy cattle farm. Our results also provide the evidence that MRSA persisted in cattle on the farm and spread to cattle newly introduced into the farm. These observations suggest that measures should be taken to prevent the transmission of MRSA among animals, humans, and the farm environment. Acknowledgments This work was supported by Animal, Plant and Fisheries Quarantine and Inspection Agency, Republic of Korea. Disclosure Statement No competing nancial interests exist. References
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Address correspondence to: Suk-Kyung Lim, DVM, PhD Animal, Plant, and Fisheries Quarantine and Inspection Agency 175 Anyang-ro, Manan-gu, Anyang-si Gyeonggi-do, 430-757, Republic of Korea E-mail: imsk0049@korea.kr