High Levels of Staphylococcus aureus Contamination in Chinese-Style Roast Pork

Introduction

The recognition of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and its presence on raw meats (de Boer et al., 2009) led to concern about its spread. Investigation of raw meat has shown that typically human staphylococcal strains may also be present (Boost et al., 2013). S. aureus present in raw meat could facilitate contamination of surfaces and hands of those preparing food. If meat is properly cooked, staphylococci will not survive; however, failure to observe good practices may result in contamination of cooked food, as well as colonization of food handlers (Ho et al., 2014). Keeping cooked foods at room temperature before consumption allows bacterial growth and possible enterotoxin production, leading to subsequent staphylococcal food poisoning (SFP) (Argudín et al., 2010).

Cooked foods favor S. aureus survival as the presence of salt, sugar, and reduced water content may inhibit other organisms (Hennekinne et al., 2012). Approximately one third of SFP outbreaks involve meat and meat products. In Hong Kong, SFP accounts for 18.5% of reported bacterial food poisoning and most commonly involves consumption of sui-mei (Chinese-style roasted pork) (Department of Health, HK, 2010). The source of the staphylococci causing these outbreaks has not been documented, but cross-contamination of cooked meat by raw pork or poor hygienic practices of food handlers are possible causes. Characterization may allow differentiation of S. aureus of animal origin from typically human isolates. This study investigated the presence of S. aureus on sui-mei from food stalls in Hong Kong and characterized isolates with respect to methicillin-resistance, spa type, and carriage of genes for Panton-Valentine leukocidin (PVL) and staphylococcal enterotoxins (SEs).

Materials and Methods

There are 412 licensed sui-mei shops in which the cooked meat is displayed without refrigeration. The area designated for sale of roasted pork must be separated from preparation areas by full-height walls with a separate street entrance to reduce contamination risks from other foods (FEHD, 2012). Roasted pork samples purchased from 50 shops at locations across the territory over 3 days in a 1-month period were stored overnight between 6 and 10°C. Approximately 25 g of each sample was blended with 50 mL saline in a sterile bag for 30 s (Stomacher; Lab Blender 400). After settling, 5 mL was transferred to 5 mL tryptone soy broth (TSB) (Oxoid, Basingstoke, UK) with 6% NaCl and incubated for 24 h. Following enrichment, 0.1 mL was inoculated onto ChromID SA (Biomerieux, Marcy l'Etoile, France) and incubated at 37°C for 24 h. An additional 5 mL of the enrichment broth was transferred to 5 mL TSB containing 5 μg/mL ceftizoxime (Sigma-Aldrich, Gillingham, UK) and 75 μg/mL aztreonam (Sigma-Aldrich) for selection of MRSA (de Boer et al., 2009). Following incubation, 0.1 mL of this supplemented TSB was inoculated onto ChromID MRSA (Biomerieux) and incubated overnight. Typical colonies from ChromID SA and ChromID MRSA were confirmed as S. aureus and tested for susceptibility to cefoxitin. Methicillin resistance was confirmed by mecA presence followed by characterization of the SCCmec cassette and vancomycin MIC determination. If mecA was absent, presence of mecC was investigated (Cuny et al., 2011) and penicillin minimum inhibitory concentration (MIC) determined by Etest. All isolates were characterized for spa type, and presence of PVL and SE genes, sea-see.

Results and Discussion

Of the 25 samples (50%) positive for S. aureus, three yielded two strains, giving a total of 28 isolates. Three displayed phenotypic resistance to methicillin, but one was negative for both mecA and mecC and had a penicillin MIC of 10 mg/L, suggesting it may be a hyper β-lactamase producer. It is important that such strains be differentiated from MRSA to avoid artificial elevation of MRSA prevalence rates. Both mecA-positive isolates had the same SCCmec type (V) and spa-type (t2616), which has been previously reported in methicillin-sensitive strains from nasal carriage (Rijnders et al., 2012). All three methicillin-resistant isolates demonstrated vancomycin susceptibility, but were negative for PVL and enterotoxins, in contrast to other reports that identified SE genes in MRSA strains (Wang et al., 2013).

Only one isolate was PVL positive, demonstrating a low incidence in contaminating strains. In contrast, five isolates (17.9%) carried at least one SE gene. Presence of enterotoxins suggests that prolonged storage of the cooked meat at ambient temperature could lead to proliferation of the organism and toxin production. This may help explain the prominence of sui-mei involvement in SFP locally, which will be exacerbated by the warm and humid conditions in the summer months, when more food poisoning is reported. In this study, only classical enterotoxins were investigated, though there is some evidence that other SEs may play a role in SFP (Hennekinne et al., 2012).

Of the 15 spa types detected, t189 was most common, being present in nine isolates (Table 1). This spa type has been reported from a SFP outbreak in Shenzhen, China (Yan et al., 2012). The Shenzhen study also reported the presence of t701, t091, and t127, which were present in the current study. Spa types t189, t701, and t120 were also reported in Spanish SFP outbreaks (Argudín et al., 2010). All other spa types present have previously been described in human clinical or nasal isolates, with the exception of one methicillin-sensitive strain belonging to t034, which has been associated with ST398, the most common ST type reported in LA-MRSA. The presence of this t034 strain could indicate cross-contamination with raw meat, but it may also have originated from a food handler. Nasal carriage of S. aureus may be elevated in food handlers exposed to raw meat, and strains present in these workers include those of both human and animal origin (Ho et al., 2014). Failure to maintain good hand hygiene by such operatives could lead to contamination of cooked meat. The absence of LA-MRSA suggests that arrangements to prevent raw meat cross-contamination are adequate and that the guidelines for operation of these premises are suitable for this purpose. However, the high rate of S. aureus contamination presumably from food handlers is of concern and indicates a need for improved hygiene measures.

This study has shown high rates of staphylococcal contamination of roasted pork, which has been commonly associated with SFP in Hong Kong (Department of Health, 2010). While the high temperature of roasting makes survival of contaminating organisms unlikely, subsequent contamination by failure to separate cooked and raw meat or use of common chopping boards or knives, followed by prolonged storage at room temperature, as can occur in sui-mei shops, can allow growth and toxin production. As the contamination appeared to be almost entirely with strains of human origin, it was likely to be associated with poor handling of the cooked product rather than cross-contamination with strains from raw meat.

The high rate of staphylococcal contamination of roasted pork with several isolates carrying genes for enterotoxins indicates that improvement of food hygiene practice should be implemented, with regular hand washing and cleaning of surfaces, complemented by further training of staff. Consideration should be given to chilled display areas for the meat before sale and advising consumers to refrigerate their purchases to reduce toxin production. Hands should be washed after contact with roasted pork to reduce contamination and possible nasal colonization. These recommendations should be considered in revised guidelines for preparation and handling of roasted pork.