Isolation of Methicillin-Resistant Staphylococcus aureus (MRSA) from Retail Meats in Hong Kong

Introduction

T he emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has led to concerns about its transmission via the food chain. Isolation rates of MRSA from meat have varied greatly between geographical locations and types of meat (van Loo et al., 2007; Normanno et al., 2007; de Boer et al., 2009; Lozano et al., 2009; Weese et al., 2010a; 2010b, Waters et al., 2011; Fessler et al., 2011; O'Brien et al., 2012), with some studies failing to isolate MRSA (Crago et al., 2012). Differences observed may also be attributable to sampling or use of improved enrichment techniques. Although MRSA other than ST398 have been isolated, this clonal type represents the majority of LA-MRSA reported. As Asian LA-MRSA isolates are mainly ST9 (Cui et al., 2009; Guardabassi et al., 2009; Neela et al., 2009; Ho et al., 2012; Vestergaard et al., 2012), this may be reflected in local meat contaminants. In addition to strains of animal origin, meat may be contaminated with human MRSA strains (Kitai et al., 2005; Pu et al., 2009; Lim et al., 2010; Weese et al., 2010a,b; Fessler et al., 2011). Awareness of origins of organisms present may help in devising better contamination-prevention guidelines.

Retail meat may be contaminated with LA-MRSA during slaughter, contributing to the high contamination rates of local pig carcasses in wet markets (Ho et al., 2012), where traditional butchering is performed. Locally slaughtered meat for supermarket sale is prepared in central processing plants. Supermarkets also sell imported frozen and chilled meats and a small amount of meat labeled as organic. There has been limited investigation into differences in contamination rates between fresh and frozen meats and conventional and organic production (de Boer et al., 2009; O'Brien et al., 2012).

This study investigated MRSA contamination of retail meats and compared rates between wet markets and supermarkets, fresh and frozen products, and meats from organically and conventionally reared animals. Isolates were characterized for SCCmec and spa type, presence of genes for enterotoxins and Panton-Valentin Leukocidin (PVL), and antibiotic susceptibility.

Materials and Methods

A total of 1400 fresh and frozen meat samples were purchased at 17 wet markets and from 11 large supermarkets at a range of locations in Hong Kong. The locations were randomly chosen from lists of the wet markets (78) and large supermarkets (49) in the SAR. This represented sampling from approximately 20% of retail outlets in Hong Kong. At the larger wet markets, where there were several butcher's stalls, samples were purchased from all stalls operating. At each location, five portions of each meat type available were purchased. The origin of the meat was noted. Organic products were only available at the supermarkets. Purchases were made over a 3-month period and were transported daily to the laboratory for culture. Meats sampled were as follows: chicken—fresh, organic chilled, chilled, frozen; beef—fresh, chilled, frozen; pork—fresh, chilled, frozen; frozen meatballs—beef, pork. Fresh meats were those that are slaughtered at the central slaughtering facility (pork and beef ) or at the wet market (chicken) and sold on the same day without chilled storage. Chilled meats were those held at 4°C before sale. This includes “jet-fresh” meats (imported pork and beef ) and local chicken killed in a dedicated central facility and then chilled before sale. On purchase, each sample was placed in a separate sterile sealed plastic bag. On arrival at the laboratory, frozen meat in the sealed plastic bag was placed in a cool area until it defrosted. Other meats were sampled immediately on arrival and within 2 h of purchase. A 25-g portion from each piece of meat was placed in a sterile plastic bag with 100 mL of Mueller-Hinton broth and processed for 2 min in a stomacher. The supernatant was aseptically transferred to a clean flask and incubated overnight at 37°C. A 5-mL aliquot was transferred into 20 mL of phenol red broth containing cefuroxime and aztreonam (de Boer et al., 2009) for enrichment of MRSA and incubated overnight. This culture was used to inoculate duplicate MRSAselect® (BioRad) agar plates that were incubated for 24 h and then examined for red colonies. These isolates were confirmed as Staphylococcus aureus by Gram stain, catalase, and coagulase test. Presence of mecA was confirmed by polymerase chain reaction (PCR) (Shittu et al., 2006). SCCmec (Zhang et al., 2005) and spa typing (Harmsen et al., 2003) were performed and susceptibility to a range of antibiotics was determined (CLSI, 2010). The presence of genes encoding for PVL was determined by PCR (Bonnstetter et al., 2007). The presence of both classical and more recently recognized enterotoxins was determined by PCR (Rosec and Gigaud, 2002).

Basic descriptive statistics were used to show the distribution of MRSA with respect to factors such as meat type, meat status, retail outlet type, and origins.

Results

A total of 1400 samples of meat were tested and 127 (9.1%) yielded MRSA. However, the percentage of positive samples differed between the types of meat sampled, with the highest rate observed for fresh pork (47%) (Table 1). MRSA rates in fresh beef (11%) and chicken (6%) were much lower, and MRSA contamination was only found in one sample of pork meatball, while all beef meatballs were negative.

Chicken samples were analyzed as two groups: Parts and whole. “Parts” (n=424) consisted of fresh, frozen, and chilled conventional parts and chilled organic parts. “Whole” (n=31) consisted of frozen whole and chilled organic whole chickens. MRSA was only isolated from chicken parts (7.3%).

There were differences in rates of MRSA contamination of fresh and frozen meat samples with respect to both pork and beef, with contamination of frozen meats averaging 1%. This difference between fresh and frozen meats was not observed for chicken.

Overall MRSA contamination rates of meat samples purchased from wet markets (7.4%) were broadly similar to those from supermarkets (10.5%). At each wet market sampled, there was no difference in MRSA isolation rates between the first (9.1%) and any subsequent butcher (8.9%) sampled. Percentage of MRSA-contaminated samples varied considerably between locations of both the 11 supermarkets (0–20.8%) and 17 wet markets (0–20%) visited. Eight of 28 locations had no MRSA-positive samples. The origin of the food appeared to affect the contamination rate, with 14% of samples originating from China being MRSA-contaminated compared to 3% from Australia and 1% from Brazil and the United States. MRSA isolation was more frequent from organically reared than from conventionally reared chicken.

Spa typing revealed that the majority of isolates belonged to t899, which has been associated with CC9. However, a much wider range of spa types was found in the chicken isolates than from beef or pork (Table 2). Although most chicken isolates were t899, 18% yielded t034, which is associated with CC398, and some samples had spa types associated with clonal clusters found in human isolates. Only three isolates were positive for PVL genes, all being isolated from chicken parts. All of these samples were purchased from wet markets, two from fresh chicken originating in China and belonging to spa type 3590, which is associated with CC338. The third was isolated from a frozen sample imported from the United States, with spa type 034 (CC398). The antibiotic susceptibility patterns of these isolates differed, with the two from China being similar and multidrug resistant, while the United States isolate was resistant to only three agents. Overall, isolates had high rates of antibiotic resistance, with most displaying resistance to more than nine antibiotics, though the antibiograms differed; one pattern, involving resistance to 10 agents, was observed in 73.8% of isolates (Table 3).

None of the isolates were found to harbor genes for the classical enterotoxins, but seg, sei, sej, sem, and sen were present in 97% of isolates, with seo in 82% and sek in 6%.

Discussion

In this large-scale study of retail meat in Hong Kong, rates of MRSA contamination, especially of pork, exceeded rates reported for Western Europe (van Loo et al., 2007; de Boer et al., 2009; Lozano et al., 2009) and North America (Pu et al., 2009; Weese et al., 2010a, 2010b). Elsewhere, studies have reported that contamination in chicken and turkey meat was most common (de Boer et al., 2009; Fessler et al., 2011), but in our study, rates for fresh pork were much higher than for other meats. This may be associated with the butchering methods employed locally in which the pig's head is present while cutting up is performed. MRSA nasal contamination of snouts after slaughter was 39% (Ho et al., 2012), and presence of snouts could act as a reservoir for contamination of the meat, as the same choppers and surfaces are used for the entire process. However, there were differences in MRSA contamination rates between the various wet markets and supermarkets sampled, which may reflect differences in hygienic practices or butchering techniques employed at different sites.

Frozen beef and pork was less frequently contaminated than fresh samples, and it has been suggested that freezing may reduce survival of MRSA on meat (de Boer et al., 2009). However, this may reflect sample origin rather than inhibition by freezing, as contamination of fresh and frozen chickens was similar. Most frozen pork and beef is imported, while frozen and fresh chickens are mainly sourced from mainland China. The high MRSA contamination rates of meat from China may reflect antibiotic use or differences in animal husbandry practices. Only one sample of meatballs yielded MRSA, but these products are boiled prior to freezing, thus eliminating contamination.

Somewhat unexpectedly, MRSA contamination rates of organic chicken were higher than for conventionally reared. In contrast, MRSA was absent from pigs reared without antibiotics in the United States (Smith unpublished findings reported in O'Brien et al., 2012) and in Hong Kong (Boost et al., 2012a). Similarly, MRSA was absent in organic chicken meat, although present in conventionally reared poultry in the Netherlands (de Boer et al., 2009). Although a recent United States study reported MRSA contamination of meat from pigs raised without antibiotic supplements to be higher than conventional pork products (O'Brien et al., 2012), the strains on organic meat were typical human strains. Isolation rates of human MRSA strains from organic meat may vary due to differences in human colonization rates (O'Brien et al., 2012). The organic chicken in our study originated in China, where MRSA incidence in clinical samples is high (Wang et al., 2008), but data on nasal colonization of healthy subjects are limited, with a recent study failing to identify any colonized subjects (Qu et al., 2010). Organic meat may also be contaminated at slaughterhouses or in transit (Broens et al., 2010; Boost et al., 2012a).

Presence of PVL-producing genes remains rare in LA-MRSA (Kadlec et al., 2009; Fessler et al., 2010; Fessler et al., 2011), although they have been reported in human isolates contaminating meat (Pu et al., 2009, O'Brien et al., 2012). Two of the PVL-positive samples from chicken in the current study were t3570, previously reported in human isolates (Ellington et al., 2010; Wu et al., 2010). The remaining positive strain was isolated from frozen chicken originating in the United States and belonged to spa type t899/ST398.

Although most reported MRSA isolates from chicken are ST398 (Nemati et al., 2008; Persoons et al., 2009), spa typing of our isolates revealed that t899 (ST9) predominated in chicken, and all those from pork and beef were associated with CC9, which is the predominant clone colonizing pigs in China (Cui et al., 2009; Guardabassi et al., 2009; Ho et al., 2012; Boost et al., 2012a). ST9 in beef and chicken may be attributable to cross-contamination during butchering and handling, although it has been reported from chicken meat in the Netherlands (de Jonge et al., 2010). In addition to t899 (CC9), one pork isolate belonged to another CC9-asociated type, t4358, previously only reported from Malaysia (Neela et al., 2009). In beef, several isolates were t4312, which has been found in LA-MRSA in Germany. Other types present in chicken included human strains, t002 and t024, from clonal complexes CC5 and CC8, respectively, and t1234 (CC97) reported in isolates from Korea (Peck et al., 2009). It has been suggested that a human CC5 clone has become adapted in poultry (Lowder et al., 2009).

In common with other isolates from this region (Ho et al., 2012), all isolates were multidrug resistant, with most displaying resistance to nine or more agents. These rates are considerably higher than those reported elsewhere (Fessler et al., 2011; O'Brien et al., 2012).

Although the relevance of MRSA contamination of food remains uncertain (Weese et al., 2010b; Kluytmans et al., 2010), its presence on raw meat may increase the risk of wound infection in food handlers and possibly in the home. A Dutch study of food handlers failed to identify MRSA (de Jonge et al., 2010), but this may be reflect handling techniques and hygiene of individual establishments, as local butchers were colonized with LA-MRSA (Boost et al., 2012b). So far it appears that although infections with LA-MRSA occur in personnel exposed to pigs and their families, it has not spread widely to the community. However, as some infections with ST398 were in subjects without livestock contact, it has been suggested that infection could be associated with handling contaminated meat (Golding et al., 2010). Although a few ST9 human infections have been reported, it is not a frequent local isolate (Ip et al., 2005).

Retail meat contamination may also allow MRSA to enter homes and possibly lead to food poisoning as a result of cross-contamination. Enterotoxin genes appear to be rare in LA-MRSA, although strains carrying staphylococcal enterotoxins B, K, and Q have been reported in porcine and bovine ST398 isolates (Kadlec et al., 2009; Huber et al., 2009; Fessler et al., 2010) and the egc enterotoxin group genes (seg, sei, sem, sen, seo, and seu) in those of chicken (Fessler et al., 2011). The majority of our strains yielded the egc enterotoxin group and additionally sej. Other studies have also reported the egc group in ST9 (Monecke et al., 2011; Vestergaard et al., 2012).

Conclusions

Although the role of ST9 in food poisoning and infection remains unclear, the virulence of LA-MRSA may change, which could increase the importance of contamination. There is therefore a need for continued surveillance as well to monitor spread and presence of virulence factors. In addition, attention should be paid to improvements in market and kitchen hygiene to help prevent staphylococcal infections.