Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52) of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5%) and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.


Introduction
The term ready-to-eat (RTE) foods refers to prepared food products that are sold as perishable meals for immediate consumption, refrigerated or frozen foods that require minimal cooking, or shelf-stable products [1]. The popularity of the different types of RTE foods differs from country to country, depending on the staple diets of the population. In Malaysia, RTE foods like delicatessen meat and dairy products are unpopular because of their high cost and mainly because rice is the standard diet of Malaysians. Thus, Japanese RTE foods such as sushi (vinegar seasoned rice combined with a variety of toppings) and sashimi (raw meat or fish slivers) are more readily accepted as quick, affordable and tasty meals that require no further cooking.
Although sushi and sashimi are convenient nutritious meals, questions have been raised with regards to their safety and microbiological quality as these RTE foods do not undergo any additional treatment or cooking steps to ensure safety prior to consumption. Food safety issues associated with the consumption of sushi and/or sashimi contaminated with Staphylococcus aureus have been reported in U.S.A. [2], Hong Kong [3], Germany [4], Japan [5] and Italy [6]. Staphylococcal food intoxication results from the ingestion of pre-formed enterotoxins produced by S. aureus [7]. Symptoms show a rapid onset with nausea, vomiting and abdominal cramping, with or without diarrhea [8]. The foodborne illness is usually self-limiting and resolves within 24 to 48 h after onset. However, occasionally the diseases can be severe and hospitalization is needed. The actual incidence of illness may be higher than reported mainly due to misdiagnosis or under-reported cases [9].
S. aureus possesses many virulence factors and the most notable are the five major classical types of staphylococcal enterotoxins (SEs: SEA to SEE), the non-classical SE-like toxins (SEl: SEG to SEU), and other virulence genes such as toxic shock syndrome toxin 1 (TSST-1), exfoliative toxins and cytolytic toxins (leukocidin and hemolysins). Staphylococcal enterotoxins (SEs) are heat stable proteins that are mainly associated with food poisoning outbreaks [7,10], while TSST-1 is a superantigenic exotoxin that causes toxic shock syndrome [11]. The exfoliative toxins are responsible for staphylococcal scalded skin syndrome that typically affects infants and young children [12], lukPV cytotoxin causes leukocytosis with necrotic lesions in the skin or mucosa [13] while hemolysins involve epithelial barrier disruption [14].
Hence, the objectives of the present study were to isolate S. aureus in sushi and sashimi sampled from different food outlets located in the Klang Valley in Malaysia, and subsequently to characterize the presence of virulence gene(s) and antibiotic resistance patterns in these S. aureus isolates.

Sample Collection and Isolation of S. aureus
The Klang Valley, with its population of over 7 million inhabitants, was selected for this study as it comprises the capital city, Kuala Lumpur, and neighboring suburbs with varied businesses, from commercial hypermarkets and shopping malls to smaller chain retail outlets and restaurants. Retail RTE sushi (n = 149) and sashimi (n = 51) were collected from different food outlets in the Klang Valley, Malaysia between August and December 2014. Various types of sushi with different toppings-marine fishes, fish roe, squid, octopus, jellyfish, edible seaweed, scallop, egg, crab stick, cherry shrimp, prawn and clam were selected for this study. Sashimi samples consisted of salmon, tuna, yellow tail, squid and scallop cut into slivers.
The exterior surface of the RTE package was cleaned with 70% (v/v) alcohol before sample isolation. Each sample was aseptically weighed in an analytical balance and twenty-five grams was placed into a sterile plastic bag. The plastic bag then received with 225 mL of buffered peptone water (Oxoid, Hampshire, UK) and homogenized in a Stomacher Bagmixer 400W (Interscience, Saint-Nom, France) for one minute. The mixture was then incubated for 16 h at 37˝C with shaking at 100 rpm. Five milliliter aliquot of the enriched homogenate was transferred into 50 mL trypticase soy broth (Oxoid) containing 7.5% NaCl. After incubation at 35˝C for 18 h, a loopful of the culture was plated onto Baird-Parker agar supplemented with egg yolk tellurite emulsion (Oxoid) and incubated overnight at 37˝C. Three presumptive S. aureus colonies (black shiny colonies surrounded by a clear halo) per food sample were randomly selected for purification using trypticase soy agar (TSA) (Oxoid) containing 0.6% yeast extract.

Virulence Potential
Of the 52 S. aureus isolates, 50 (96.2%) were positive for one or more virulence genes (Table 1) and 20 different virulence gene profiles were confirmed ( Table 2). None of the 52 isolates possessed any of the sed, see, sej, sek, sep, seq, ses, set, seu, etb, etd, lukM and hlg virulence genes. The amplified fragments of the 19 virulence genes corresponded to the expected published amplicon sizes as shown in Supplementary Table S1 and Figure 1.

Susceptibility of S. aureus to Antimicrobials
Overall, 34.6% (18/52) of the S. aureus isolates exhibited resistance phenotypes to at least one antibiotic ( Table 3). Resistance to tetracycline was most commonly observed (28.8%). A small percentage of between 1.9% and 7.7% of the isolates demonstrated resistance to gentamicin, amikacin, cefoperazone, trimethoprim-sulfamethoxazole, chloramphenicol, vancomycin, ciprofloxacin cefoxitin and erythromycin.

Discussion
This study showed that the overall prevalence of S. aureus in the 200 sushi and sashimi samples was high at 26%. S. aureus occurrence from sushi and sashimi samples showed a high rate at 21.5% (32/149) and 39.2% (20/51) respectively; this can be a result from contamination through human contact as S. aureus is not part of the normal microflora of freshly caught marine fishes and fishes bred in farms [28]. Of the 32 contaminated sushi samples, 22 samples were topped with raw seafood products, i.e., salmon (n = 13), fish roe (n = 5), tuna (n = 3) and squid (n = 1). In a report by Vazquez-Sanchez et al. [29], S. aureus was also reported to be responsible for a significant proportion (25.2%) of contamination in fishery products marketed in Northwest Spain. S. aureus is recognized as common resident skin and nasal microbiota in humans, thus these are possible sources of contamination [8]. As the preparation of sushi and sashimi involves a great deal of handling with bare or gloved hands, a possible way to determine definite sources of contamination will be to collect hand and nasal swabs from food handlers.
Investigations carried out to assess the quality and safety of sushi and/or sashimi showed S. aureus contamination in sushi (15.79%) and sashimi (32.79%) samples collected from restaurants in Italy and northern Portugal respectively [6,30]. Recently, Hammad and co-workers [5] demonstrated a high prevalence (87%) of S. aureus contamination in 200 sashimi samples collected in Hiroshima (Japan). The results from this study thus concur with previous published reports.
In Malaysia, there is currently no documented outbreak of foodborne disease due to sushi consumption. The 52 S. aureus isolated in this study were also screened for virulence genes to gain insight into their pathogenic potential. Overall, nearly all of the S. aureus isolates carried some virulence genes, especially the alpha-hemolysin (hla) gene which was detected at 90.4%. Diverse virulence gene profiles were also reported from different food categories worldwide, such as fishery products in Galicia [29], raw and processed food commodities in Shanghai [31], milk products in South Africa [32] and retail chicken in Egypt [33]. Results of the present study and other published reports suggest that various toxins contribute to the pathogenic potential of S. aureus and constitute a risk for consumers' health.
In this study, sixteen S. aureus isolates (30.8%) harboured at least one virulence SE or SEl gene, hence demonstrating the potential toxigenic and pathogenicity of these isolates. SEs classical types (SEA to SEE) were reported in 95% of staphylococcal food poisoning while new identified SEls were responsible for the remaining 5% of food poisoning outbreaks [34]. In addition, the presence of the egc cluster (seg-sei-sem-sen-seo) was detected in 5.8% of S. aureus isolates in this study and this was also observed in food and clinical isolates by other investigators [10,[23][24][25][26][27][28][29][30][31][32][33]. The role of the egc-encoded superantigens has not been fully elucidated, but a remarkable reduced potency in neutralizing capacity compared to classical SEs was reported by Holtfreter et al. [35]. Surprisingly, the study demonstrated that these superantigens were inhibited in less than 10% of human sera, thus suggesting a potential for increased severity of clinical diseases. Recently, food-poisoning outbreaks in Korea and China have been linked to seg-sei positive S. aureus isolates [36]. Thus, the results from this study in combination with reported publications indicate that the pathogenic role of the egc gene cluster should not be underestimated, and further studies should be carried out to elucidate its contribution to pathogenicity of S. aureus infections.
Hemolysin genes-hla (90.4%), hld (59.6%), hlgv (30.8%) and leukocidin lukDE (38.5%) were the most commonly detected genes in this study, and a high incidence of hla-hld combination was confirmed in more than 60% of the strains. These non-enterotoxin hla, hld and lukED genes were reported to be responsible for staphylococcal food-poisoning outbreaks in China [37]. Hemolysin genes were also observed frequently in food isolates in China [38] and Asturias, Spain [39]. It is noteworthy that isolates recovered from milk of cows with subclinical mastitis in Asturias harboured hla (100%), hlgv (100%) and hld (94.2%) genes [39]. These studies indicate the potential of S. aureus in producing cytotoxins (hemolysin and leukotoxin genes) leading to illness in host. S. aureus isolates producing the ETA, TSST-1 and lukPV toxins were also detected in this study, which is in agreement with Cha et al. [40], who reported 12% and 1.2% prevalence of TSST-1 and ETA in S. aureus isolates recovered from stool samples of staphylococcal food-poisoning affected patients in Korea. The presence of these three genes was also reported in RTE sushi, kimbab, California rolls [41] and RTE raw fish [5]. Recently, Song et al. [31] showed the presence of lukPV toxins in fresh meats, raw milk, bean products and frozen foods.
Antibiotic-resistant S. aureus involved in food contamination has been detected worldwide. Resistance to tetracycline is common among S. aureus food isolates but the rates vary greatly [42,43]. In this study, S. aureus resistance to tetracycline was at 28.8% with a higher isolation detected in sushi and sashimi samples. A low percentage of multidrug-resistance (MDR) S. aureus isolates (3.8%, 2/52) was identified based on the MDR criteria (defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories) [44]. One isolate presented a MDR profile in of FOX, TE, VA, E, SXT and C. Another isolate showed resistance to the full panel of antibiotics tested-FOX, CN, AK, TE, VA, E, CIP, SXT, C and CFP. Other investigators have also reported the presence of MDR isolates in ready-to-eat raw fish, infant formula milk and cereal [5,45]. Antibiotic resistant S. aureus that jeopardizes public health has been reported in numerous foods that can be passed to humans through the food chain [46]. With increase in antibiotic resistance, the use of natural occurring compounds as antimicrobials in the food industry has gained attention. In particular, carvacrol, cinnamon oil, and oregano oil were reported to be active against antibiotic resistant S. aureus [47]. However, further evaluation is required before these compounds can be incorporated into food products.
One limitation in this study is that the results only showed the occurrence of S. aureus in sushi and sashimi samples, and did not take into account quantitative assessment of this pathogen. Information from the Food and Drug Administration (2011) states that "S. aureus toxin does not normally reach levels that will cause food poisoning until the number of pathogens reach 10 4 to 10 5 per gram" [48]. Thus, a quantitative contamination level of this pathogen should be carried out in order to provide better microbial risk assessment for public health protection.

Conclusions
The results from this study show S. aureus contamination in 26% (52/200) of the sushi and sashimi samples. A high incidence of virulence genes was identified in 96.2% of the S. aureus isolates with confirmation of 20 different virulence gene profiles. The SEs and SEls genes responsible for staphylococcal food poisoning were detected in 30.8% of the S. aureus, thus confirming the toxigenic and pathogenic capacity of the S. aureus isolates. S. aureus producing hemolysins (hla, hld, hlgv) as well as ETA, TSST-1, lukPV and lukDE toxins were also detected, and this indicates that S. aureus contamination can result in other disorders besides food poisoning. Tetracycline resistance was commonly observed at 28.8%, resistance to at least one antibiotic tested was detected at 34.6% while multiple drug resistance was much lower at 3.8%. There is currently no published data on S. aureus contamination and its virulence gene profile in food items in Malaysia. Raw seafood used in sushi and sashimi preparation in Malaysia is generally harvested from sea locations. There are no directives for seafood collection and different outlets possess individual seafood sources. In addition, guidelines for food preparation vary with different outlets as there are no standard guidelines in Malaysia yet. Thus, in conclusion the results of this study highlight the necessity for more detailed investigations for improved consumer protection and public health safety.