Detection and quantification of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products

Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Detection and quantification of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products J.S. Weese 1, B.P. Avery 2 and R.J. Reid-Smith 2 1 Ontario Veterinary College, University of Guelph, Guelph, ON, Canada 2 Public Health Agency of Canada, Guelph, ON, Canada Keywords antimicrobial(s), food, staphylococci. Correspondence J. Scott Weese, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G2W1, Canada. E-mail: jsweese@uoguelph.ca 2010 0754: received 5 May 2010, revised 15 June 2010 and accepted 5 July 2010 doi:10.1111/j.1472-765x.2010.02901.x Abstract Aims: The objective of the study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) contamination of retail meat and to determine the level of contamination. Methods and Results: Pork (pork chops and ground pork), ground beef and chicken (legs, wings and thighs) were purchased at retail outlets in four Canadian provinces and tested for the presence of methicillin-resistant Staph. aureus using qualitative and quantitative methods. MRSA was isolated from 9Æ6% of pork, 5Æ6% of beef and 1Æ2% of chicken samples (P =0Æ0002). Low levels of MRSA were typically present, with 37% below the detection threshold for quantification and <100 CFU g )1 present in most quantifiable samples. All isolates were classified as Canadian epidemic MRSA-2 (CMRSA-2) by pulsed field gel electrophoresis (PFGE), with two different PFGE subtypes, and were spa type 24 t242. Conclusions: MRSA contamination of retail meat is not uncommon. While CMRSA-2, a human epidemic clone, has been found in pigs in Canada, the lack of isolation of livestock-associated ST398 was surprising. Significance and Impact of the Study: The relevance of MRSA contamination of meat is unclear but investigation is required because of the potential for exposure from food handling. Sources of contamination require investigation because these results suggest that human or animal sources could be involved. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a critically important human pathogen, and attention has been paid recently to the potential role of food animals in human MRSA infection and colonization. This has largely focused on pigs because on widespread reports of MRSA colonization in pigs internationally (Khanna et al. 2007; de Neeling et al. 2007; Dewaele et al. 2008; Smith et al. 2008; Riesen and Perreten 2009) and identification of contact with pigs or veal calves as a significant risk factor for MRSA infection or colonization in some regions (Voss et al. 2005; Graveland et al. 2008; van Rijen et al. 2008; Wulf et al. 2008). Most of this has involved one MRSA clone, sequence type 398 (ST398). This clone has been termed a livestock-associated strain and has predominated in most studies of pigs in Europe and North America (Khanna et al. 2007; van Duijkeren et al. 2008; Smith et al. 2008; Denis et al. 2009; Köck et al. 2009) and appears to be widely disseminated in the pig population internationally. Identification of MRSA in food animals led to logical concerns about foodborne contamination, and MRSA has been identified in retail meat in Europe, Asia and North America (Kitai et al. 2005; Kwon et al. 2006; van Loo et al. 2007; de Boer et al. 2009; Lozano et al. 2009; Pu et al. 2009); however, the role of foodborne contamination in human MRSA infection or colonization is currently unclear and much further study is required to elucidate this potential problem. One aspect that has to be considered is the amount of MRSA in 338 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 338 342

J.S. Weese et al. MRSA in retail meat contaminated meat. Previous studies have used enrichment culture techniques that can have a very low detection threshold; 12 15 CFU per 25 g in one study (de Boer et al. 2009). While the infectious dose of MRSA in meat (if meat is truly a potential source of infection) is not known, it is reasonable to assume that lower-level contamination is less relevant than higher-level contamination, and quantitative studies are required. The objectives of this study were to determine the prevalence of MRSA contamination of retail pork, beef and chicken from Canada, to quantify MRSA contamination and to characterize recovered isolates. Materials and methods Pork (pork chops and ground pork) and ground beef were purchased at retail outlets in four Canadian provinces (British Columbia, Saskatchewan, Ontario, Quebec), and chicken (legs, wings, thighs) was obtained from Ontario between November 2008 and August 2009 inclusive as part of the active retail surveillance component of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). This program performed retail sampling, weighted by population, from chain stores and independent market or butcher shops in a reflection of the shopping behaviour of the subpopulation. Only one sample of each commodity was obtained from each store and, where possible, stores were only sampled once per year. Samples were purchased and shipped via 24-h courier in their original packages, on ice, to the study laboratory and processed within 72 h of receipt. For ground beef and ground pork, 10 g of meat was inoculated into 50-ml enrichment broth (10 g tryptone per l, 75 g sodium chloride per l, 10 g mannitol per l and 2Æ5 g of yeast extract per l). For quantification, serial 10-fold dilutions of the inoculated enrichment broth were performed in phosphate-buffered saline (PBS) and inoculated onto MRSA Chromogenic agar (BBL CHROMagar MRSA; Becton, Dickinson and Company, Sparks, MD, USA). Culture plates were incubated aerobically at 35 C and read at 24 and 48 h. The remaining inoculated broth was incubated at 35 C for 24 h, then inoculated onto MRSA Chromogenic agar. Pork chops and chicken parts were immersed in 50 ml PBS. Serial tenfold dilutions were performed in PBS and inoculated onto MRSA Chromogenic agar. One millilitre of rinse PBS was transferred into 9 ml of MRSA culture broth and incubated as above prior to subculture onto MRSA Chromogenic agar. Culture plates were incubated at 35 C for 48 h. Isolates were identified as Staph. aureus by colony morphology, Gram stain appearance, catalase reaction, coagulase reaction and Staph. aureus latex agglutination test (Pastorex Staph-plus; Bio-Rad, Marnes-la-Coquette, France). Methicillin resistance was confirmed by penicillin-binding protein 2a latex agglutination test (MRSA latex agglutination test; Oxoid Ltd, Hants, UK). Isolates were typed by pulsed field gel electrophoresis (PFGE) as has been described elsewhere (Mulvey et al. 2001). Isolates were also typed by sequencing of the X region of the protein A gene (spa typing) (Shopsin et al. 1999). For spa typing, sequences were analysed using the egenomics software (http://tools.egenomics.com). Ridom database equivalents were identified using the Ridom Spa server website (http://www.spaserver.ridom.de). Real-time PCR was used to detect the lukf and luks components of the Panton-Valentine leukocidin (PVL) (Rankin et al. 2005). Positive and negative controls were included with every PCR run. Categorical comparisons were performed using Fisher s exact or chi-squared tests. A P-value of 0Æ05 was considered significant. Results Culture results are presented in Tables 1 and 2. There was a significant difference in the overall prevalence of contamination of pork (9Æ6%), beef (5Æ6%) and chicken (1Æ2%) (P = 0Æ0002). There was a significant difference in contamination between provinces for pork (P =0Æ024) but not beef (P =0Æ28). Thirteen of 22 (59%) of positive pork samples were positive using both direct (quantitative) and enrichment culture, while seven were positive only with enrichment and two were positive only on direct culture. Of the samples that were quantifiable, 20 CFU g )1 were present in 9 15 (60%). The remaining six samples had 30, 90, 100, Table 1 Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from retail meat Product n MRSA (%) Ground pork 127 8 (6Æ3) Pork chops 103 14 (13Æ6) Total pork 230 22 (9Æ6) Ground beef 198 11 (5Æ6) Chicken 250 3 (1Æ2) Table 2 Prevalence of methicillin-resistant Staphylococcus aureus contamination of retail beef and pork from four Canadian provinces Province Pork Beef British Columbia 2 48 (4Æ2%) 0 38 Saskatchewan 0 27 0 22 Ontario 10 71 (14%) 4 69 (5 8%) Quebec 10 84 (12%) 7 69 (10%) Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 338 342 339

MRSA in retail meat J.S. Weese et al. 110, 340 and 3590 CFU g )1. The two samples that were positive only on direct culture both had only 20 CFU g )1. Similar results were obtained for beef, with 5 11 (45%) positive samples positive with both the direct (quantitative) and enrichment (qualitative) methods. Five (45%) samples were positive only with enrichment culture, while 1 (9%) was positive with direct but not enrichment culture. The six samples that were quantifiable contained 20 (n = 2), 40, 60, 180 and 240 CFU g )1. The sample that was positive on direct but negative on enrichment culture only had 20 CFU g )1. MRSA was only isolated from 3 250 (1Æ2%) chicken samples. Two samples contained 10 CFU g )1, while one contained 30 CFU g )1. All isolates were classified as Canadian epidemic MRSA- 2 (CMRSA-2), a clone that is also known as USA100, by PFGE, with two different PFGE subtypes identified. All isolates were spa type 24 t242 and PVL negative. Discussion The prevalence of MRSA contamination of retail meat in this study is similar to other reports from the Netherlands (various meats, 11Æ9%) (de Boer et al. 2009), the United States (pork 5Æ6%, beef 3Æ3%) (Pu et al. 2009) and Canada (pork, 5Æ8%) (J.S. Weese, Can Vet J, in press). Care should be taken in comparing prevalence data between different studies. The methods used in this study for quantitative and qualitative culture are able to consistently recover small numbers of MRSA in meat (J.S. Weese, unpublished data) but there are no standardized culture methodologies. Therefore, results of this and other studies should mainly be taken to indicate that MRSA can be regularly found in different meat products in many regions, without an attempt to compared prevalences between different studies. The use of different sampling techniques must also be considered, and this study is the first to report the use of a systematic sampling method to reduce the impact of sampling bias and may give a more accurate representation of population prevalence. Similarly, some consideration should be given to the significant difference in prevalence between beef, pork and chicken because of the different types of meat that were tested (i.e. whole meat pieces vs ground meat) and the slightly different methodology that was used for ground vs solid meat. The slight methodological difference, the use of enrichment broth vs PBS for serial dilutions, is unlikely to have resulted in any difference in recovery but must be considered. Comparing results between regions within this study is reasonable, and an explanation for the significant difference in MRSA prevalence in pork between different provinces is not readily apparent. More information about the prevalence of MRSA in pigs across the country, along with issues pertaining to retail meat origins, is required to investigate this further. An important aspect of this study was quantification of contamination. The use of potentially highly sensitive enrichment methods can provide a good estimate of prevalence but does not indicate whether a few bacteria or larger numbers were present. Considering most samples were either only positive on enrichment culture or had <100 CFU g )1, it is apparent that MRSA contamination is typically low level. This must be taken into consideration when evaluating potential risks. However, the infectious dose is not known and while low levels may be less concerning, they should not be dismissed. The presence of a small number of samples that were positive on direct but not enrichment culture was unexpected; however, all those samples had the lowest levels of contamination. Nonhomogenous distribution of low levels of MRSA in meat samples could have accounted for those results. The predominance of USA100 CMRSA-2 was somewhat surprising. This is a common human epidemic clone that is the most common cause of hospital-associated infection in Canada (Christianson et al. 2007) and the most commonly found strain in colonized humans in the United States (Tenover et al. 2008). Further, t242 was the 2nd most common CMRSA-2 spa type in a large study of MRSA from humans in Canada (Golding et al. 2008). USA100 CMRSA-2 has previously been found in pigs in Canada (Khanna et al. 2007) and pork in Canada and the United States (Pu et al. 2009) (JS Weese et al. in press). Further, spa types consistent with USA100 CMRSA-2 were reported, albeit uncommonly, in the large Dutch retail meat study (de Boer et al. 2009). However, while USA100 CMRSA-2 can be found in pigs, ST398 is the most common strain found in pigs in Canada and the United States (Khanna et al. 2007; Smith et al. 2008), yet it was not identified in any sample in this study. If MRSA in meat is a direct reflection of MRSA in food animals, frequent isolation of ST398 strains would be expected, along with other clones that can be found in pigs in a given region, such as USA100 CMRSA-2. This discordance between animal and meat strains is interesting and raises questions about the origin(s) of contamination. Given the predominance of a common human epidemic clone in meat, the potential role of slaughterhouse and food-processing personnel, and the foodprocessing environment require consideration. Laboratory error or contamination is unlikely to account for the strain distribution present here as this clone was not found in other MRSA studies performed concurrently in the same laboratory, and positive samples were identified sporadically throughout the study period. The additional discriminatory power of PFGE also indicates that 340 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 338 342

J.S. Weese et al. MRSA in retail meat contamination should be less of a concern because different PFGE types were identified. Further, while MRSA has commonly been isolated from pigs internationally, there are, to the authors knowledge, no reports of MRSA in beef cattle, and a study of feedlot cattle in Canada did not identify any colonized animals (Weese et al. 2009). Longitudinal farm-to-fork study of the origin of MRSA contamination of food products is indicated to help answer these questions. The relevance of MRSA contamination of retail meat is unknown. While MRSA food poisoning (staphylococcal enterotoxin-associated diarrhoea) has been reported (Jones et al. 2002), there should be no clinical difference in enteric disease caused by MRSA vs methicillin-susceptible Staph. aureus. The likelihood of ingestion of MRSA causing enteric disease is low, given the pathophysiology of staphylococcal food poisoning (ingestion of preformed enterotoxins), although it is plausible that ingestion could result in gastrointestinal colonization and the potential for subsequent extra-intestinal infection or transmission. Touching ones nose after handling contaminated meat could plausibly result in nasal colonization, and contact of contaminated meat with skin lesions could potentially result in infection. The true likelihood of these is unknown but deserves further study. Regardless, proper handling of raw meat, including prevention of crosscontamination, adequate cleaning and disinfection and good personal hygiene practices (especially, hand hygiene) would likely reduce or eliminate any risks. 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