Next Article in Journal
Modified Ideal Cardiovascular Health Status is Associated with Lower Prevalence of Stroke in Rural Northeast China
Previous Article in Journal
Health Impact Assessment for Second-Hand Smoke Exposure in Germany—Quantifying Estimates for Ischaemic Heart Diseases, COPD, and Stroke
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
*
Author to whom correspondence should be addressed.
Int. J. Environ. Res. Public Health 2016, 13(2), 199; https://doi.org/10.3390/ijerph13020199
Submission received: 3 December 2015 / Revised: 28 January 2016 / Accepted: 29 January 2016 / Published: 5 February 2016

Abstract

:
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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. DNA Extraction and Identification of S. aureus

Total genomic DNA was prepared using an adapted in-house boiling method [15] and stored at −20 °C for further investigations. All presumptive colonies were confirmed by DNA amplification using the polymerase chain reaction (PCR) for S. aureus with two sets of primers targeting the 16S rRNA (Staphylococcus genus-specific, 228 bp) [16] and nuc (S. aureus species-specific, 279 bp) genes [17]. The primer sequences used for DNA amplification are—16S rRNA (F): 5′-GTAGGTGGCAAGCGTTATCC-3′, 16S rRNA (R): 5′-CGCACATCAGCGTCAG-3′; nuc (F): 5′-GCGATTGATGGTGATACGGTT-3′ and nuc (R): 5′-AGCCAAGCCTTGACGA-ACTAAAGC- 3′.

2.3. DNA Amplification of S. aureus Virulence Genes

The presence of 32 virulence genes—20 enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, seu), toxic shock syndrome gene (tst), three exofoliative toxin genes (eta, etb, etd), three leukocidin encoding genes (lukM, lukED, lukPV) and five hemolysin encoding genes (hla, hlb, hld, hlg, hlgv)—were investigated by singleplex PCR assay [13,18,19,20,21,22,23,24,25,26]. DNA amplifications were carried out using primers as described in published studies (Supplementary Table S1). Each singleplex PCR was performed using TopTaq Master Mix Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA amplification was carried out in a PCR mixture that contained 25 μL TopTaq Master Mix, 0.2 μM forward primer, 0.2 μM reverse primer, 100 ng of DNA template, RNase-free water PCR products were then electrophoresed in a 1.5% (w/v) agarose gel. One representative of each positive virulence gene isolate was confirmed by direct DNA sequencing.

2.4. Antimicrobial Susceptibility Testing

The susceptibility of the S. aureus isolates to antimicrobials was tested by the disc diffusion method on Mueller-Hinton agar using commercial antibiotic disks (Oxoid) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [27]. S. aureus American Type Culture Collection (ATCC) 29213 and Escherichia coli ATCC 25922 were used as quality control strains in each run. Ten antimicrobials were tested: cefoxitin (30 µg), gentamicin (10 µg), amikacin (30 µg), tetracycline (30 µg), vancomycin (30 µg), erythromycin (15 µg), ciprofloxacin (5 µg), trimethoprim/sulfamethoxazole (1.25/23.75 µg), chloramphenicol (30 µg) and cefoperazone (75 µg). Interpretation of inhibition zones was carried out based on the manufacturers’ and CLSI guidelines [27].

3. Results

3.1. Isolation and Identification of Isolates

Of the 200 food items, the samples consisted of 74.5% (149/200) sushi and 25.5% (51/200) sashimi. S. aureus was detected in 26% (52/200) of the collected retail food items. The contaminated food items consisted of 32 sushi and 20 sashimi samples.

3.2. 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.
Figure 1. Gel electrophoresis of amplified virulence gene products from food isolates. Lane L: 100 bp molecular weight marker; lane 1: sea (102 bp); lane 2: seb (164 bp); lanes 3: sec (451 bp); lane 4: seg (704 bp); lane 5: seh (495 bp); lane 6: sei (630 bp); lane 7: sel (240 bp); lane 8: sem (326 bp); lane 9: sen (680 bp); lane 10: seo (180 bp); lane 11: ser (367 bp); lane 12: tsst (326 bp); lane 13: eta (190 bp); lane 14: lukDE (269 bp); lane 15: lukPV (433 bp); lane 16: hla (209 bp); lane 17: hlb (309 bp); lane 18: hld (111 bp); lane 19: hlgv (390 bp).
Figure 1. Gel electrophoresis of amplified virulence gene products from food isolates. Lane L: 100 bp molecular weight marker; lane 1: sea (102 bp); lane 2: seb (164 bp); lanes 3: sec (451 bp); lane 4: seg (704 bp); lane 5: seh (495 bp); lane 6: sei (630 bp); lane 7: sel (240 bp); lane 8: sem (326 bp); lane 9: sen (680 bp); lane 10: seo (180 bp); lane 11: ser (367 bp); lane 12: tsst (326 bp); lane 13: eta (190 bp); lane 14: lukDE (269 bp); lane 15: lukPV (433 bp); lane 16: hla (209 bp); lane 17: hlb (309 bp); lane 18: hld (111 bp); lane 19: hlgv (390 bp).
Ijerph 13 00199 g001
Table 1. Prevalence of virulence genes in 52 Staphylococcus aureus isolated from sushi and sashimi samples.
Table 1. Prevalence of virulence genes in 52 Staphylococcus aureus isolated from sushi and sashimi samples.
Virulence Gene ProfilesSushi n = 32 n (%)Sashimi n = 20 n (%)Total n = 52 n (%)
One or more 31 (96.9)19 (95.0)50 (96.2)
Classicalsea03 (15.0)3 (5.8)
SE genesseb1 (3.1)0 1 (1.9)
sec3 (9.4)03 (5.8)
Non-classicalseg6 (18.8)0 6 (11.5)
SE genesseh1 (3.1)01 (1.9)
sei4 (12.5)04 (7.7)
sel2 (6.3)02 (3.8)
sem4 (12.5)04 (7.7)
sen3 (9.4)03 (5.8)
seo4 (12.5)04 (7.7)
ser04 (20.0)4 (7.7)
Otherstsst4 (12.5)1 (5.0)5 (9.6)
eta2 (6.3)02 (3.8)
lukDE15 (46.9)5 (25.0)20 (38.5)
lukPV4 (12.5)1 (5.0)5 (9.6)
hla28 (87.5)19 (95.0)47 (90.4)
hlb05 (25.0)5 (9.6)
hld31 (96.9)031 (59.6)
hlgv11 (34.4)5 (25.0)16 (30.8)
No gene 1 (3.1)1 (5.0)2 (3.8)
Table 2. Virulence gene profiles in 52 Staphylococcus aureus isolated from sushi and sashimi samples.
Table 2. Virulence gene profiles in 52 Staphylococcus aureus isolated from sushi and sashimi samples.
Virulence Gene ProfilesSushi n = 32 n (%)Sashimi n = 20 n (%)Total n = 52 n (%)
hla09 (45.0)9 (17.3)
hld1 (3.1)01 (1.9)
pvl, hla01 (5.0)1 (1.9)
ser, hla03 (15.0)3 (5.8)
hla, hld *8 (25.0)08 (15.4)
hld, lukDE2 (6.3)02 (3.8)
eta, hla, hld *1 (3.1)01 (1.9)
hla, hld, lukDE *2 (6.3)02 (3.8)
pvl, hla, hld *1 (3.1)01 (1.9)
seg, hla, hld *2 (6.3)02 (3.8)
tsst, ser, hla01 (5.0)1 (1.9)
hla, hld, hlgv, lukDE *5 (15.6)2 (10.0)7 (13.5)
pvl, hla, hld, hlgv, lukDE *2 (6.3)02 (3.8)
sea, hla, hld, hlgv, lukDE *1 (3.1)2 (10.0)3 (5.8)
seb, seh, pvl, hla, hld *1 (3.1)01 (1.9)
sec, hla, hld, hlgv, lukDE *2 (6.3)02 (3.8)
seg, sei, sen, seo, sem, tsst, hla, hld *1 (3.1)01 (1.9)
seg, sei, sen, seo, sem, tsst, eta, hla, hlgv1 (3.1)01 (1.9)
seg, sei, seo, sem, tsst, sel, hla, hlgv, lukDE1 (3.1)01 (1.9)
sec, seg, sei, sen, seo, sem, tsst, sel, hla, hld, hlgv, lukDE *1 (3.1)01 (1.9)
* hla-hld combination.
Notably, the non-classical SE gene (seg) was most frequently detected at 11.5% followed by sei, sem, seo and ser (7.7% each) (Table 1). The egc cluster (seg-sei-sem-sen-seo) was observed in 3 isolates (5.8%) with other additional genes (Table 2). Isolates carrying non-SE/SEl genes were observed in the following order: hla (90.4%), hld (59.6%), lukDE (38.5%), hlgv (30.8%), tsst (9.6%), lukPV (9.6%), hlb (9.6%) and eta (3.8%). Thirty-one isolates (59.6%) harboured the most common combination of virulence genes—hla and hld either together or in combination with other genes.

3.3. 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.
Table 3. Frequency of antimicrobial resistance in 52 Staphylococcus aureus isolates recovered from sushi and sashimi.
Table 3. Frequency of antimicrobial resistance in 52 Staphylococcus aureus isolates recovered from sushi and sashimi.
Antimicrobial AgentsDisk Concentration (µg)Number of Resistant Isolates (%)
Sushi (n = 32)Sashimi (n = 20)Total (n = 52)
Cefoxitin (FOX)303 (9.4)1 (5.0) 4 (7.7)
Gentamicin (CN)1001 (5.0)1 (1.9)
Amikacin (AK)3001 (5.0)1 (1.9)
Tetracycline (TE)303 (9.4)12 (60.0)15 (28.8)
Vancomycin (VA)302 (6.3)1 (5.0) 3 (5.8)
Erythromycin (E)152 (6.3)2 (10.0)4 (7.7)
Ciprofloxacin (CIP)51 (3.1)2 (10.0)3 (5.8)
Trimethoprim-sulfamethoxazole (SXT)1.25/23.71 (3.1)1 (5.0)2 (3.8)
Chloramphenicol (C)301 (3.1)1 (5.0)2 (3.8)
Cefoperazone (CFP)7501 (5.0) 1 (1.9)
One antibiotic 5 (15.6)13 (65.0)18 (34.6)
Two antibiotics 1 (3.1)01 (1.9)
Six antibiotics 1 (3.1)01 (1.9)
Ten antibiotics 01 (5.0)1 (1.9)

4. 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 104 to 105 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.

5. 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.

Supplementary Materials

The following are available online at www.mdpi.com/1660-4601/13/2/199/s1, Table S1. Primer nucleotide sequences for DNA amplification and amplified amplicon sizes of 32 virulence genes of Staphylococcus aureus.

Acknowledgments

The authors would like to thank Yap Ming Zhi for his assistance. This work was supported by the University of Malaya Research Grant RP039B-15HTM.

Author Contributions

Suat Moi Puah conducted the experiments and wrote the draft of manuscript. Kek Heng Chua and Jin Ai Mary Anne Tan supervised the work and contributed to the writing of the manuscript text.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. FDA Food Code 2009: Chapter 1—Purpose & Definitions. Available online: http://www.fda.gov/Food/GuidanceRegulation/RetailFoodProtection/FoodCode/ucm186464.htm (accessed on 3 December 2015).
  2. Adams, A.M.; Leja, L.L.; Jinneman, K.; Beeh, J.; Yuen, G.A.; Wekell, M.M. Anisakid parasites, Staphylococcus aureus and Bacillus cereus in sushi and sashimi from Seattle area restaurants. J. Food Prot. 1994, 57, 311–317. [Google Scholar]
  3. Food and Environmental Hygiene Department HKSAR. Risk Assessment Studies Report No. 2. Microbiological Hazards Evaluation. Sushi and Sashimi in Hong Kong. Available online: http://www.cfs.gov.hk/english/programme/programme_haccp/files/ss_ras2_eng.pdf (accessed on 3 December 2015).
  4. Atanassova, V.; Reich, F.; Klein, G. Microbiological quality of sushi from sushi bars and retailers. J. Food Prot. 2008, 71, 860–864. [Google Scholar] [PubMed]
  5. Hammad, A.M.; Watanabe, W.; Fujii, T.; Shimamoto, T. Occurrence and characteristics of methicillin-resistant and -susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci from Japanese retail ready-to-eat raw fish. Int. J. Food Microbiol. 2012, 156, 286–289. [Google Scholar] [CrossRef] [PubMed]
  6. Muscolino, D.; Giarratana, F.; Beninati, C.; Tornambene, A.; Panebianco, A.; Ziino, G. Hygienic-sanitary evaluation of sushi and sashimi sold in Messina and Catania, Italy. Ital. J. Food Saf. 2014, 3, 1701. [Google Scholar] [CrossRef]
  7. Hennekinne, J.A.; de Buyser, M.L.; Dragacci, S. Staphylococcus aureus and its food poisoning toxins: Characterization and outbreak investigation. FEMS Microbiol. Rev. 2012, 36, 815–836. [Google Scholar] [CrossRef] [PubMed]
  8. Kadariya, J.; Smith, T.C.; Thapaliya, D. Staphylococcus aureus and staphylococcal food-borne disease: An ongoing challenge in public health. Biomed. Res. Int. 2014, 2014, 827965. [Google Scholar] [CrossRef] [PubMed]
  9. Mead, P.S.; Slutsker, L.; Dietz, V.; Mccaig, L.F.; Bresee, J.S.; Shapiro, C.; Griffin, P.M.; Tauxe, R.V. Food-related illness and death in the United States. Emerg. Infect. Dis. 1999, 5, 607–625. [Google Scholar] [CrossRef] [PubMed]
  10. Argudin, M.A.; Mendoza, M.C.; Gonzalez-Hevia, M.A.; Bances, M.; Guerra, B.; Rodicio, M.R. Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers. Appl. Environ. Microbiol. 2012, 78, 2930–2935. [Google Scholar] [CrossRef] [PubMed]
  11. Fueyo, J.M.; Mendoza, M.C.; Martin, M.C. Enterotoxins and toxic shock syndrome toxin in Staphylococcus aureus recovered from human nasal carriers and manually handled foods: Epidemiological and genetic findings. Microbes Infect. 2005, 7, 187–194. [Google Scholar] [CrossRef] [PubMed]
  12. Ladhani, S. Understanding the mechanism of action of the exfoliative toxins of Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 2003, 39, 181–189. [Google Scholar] [CrossRef]
  13. Lina, G.; Piemont, Y.; Godail-Gamot, F.; Bes, M.; Peter, M.O.; Gauduchon, V.; Vandenesch, F.; Etienne, J. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin. Infect. Dis. 1999, 29, 1128–1132. [Google Scholar] [CrossRef] [PubMed]
  14. Vandenesch, F.; Lina, G.; Henry, T. Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: A redundant arsenal of membrane-damaging virulence factors? Front. Cell Infect. Microbiol. 2012, 2, 12. [Google Scholar] [CrossRef] [PubMed]
  15. Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, USA, 2001. [Google Scholar]
  16. Lovseth, A.; Loncarevic, S.; Berdal, K.G. Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates. J. Clin. Microbiol. 2004, 42, 3869–3872. [Google Scholar] [CrossRef] [PubMed]
  17. Brakstad, O.G.; Aasbakk, K.; Maeland, J.A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 1992, 30, 1654–1660. [Google Scholar] [PubMed]
  18. Mehrotra, M.; Wang, G.; Johnson, W.M. Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J. Clin. Microbiol. 2000, 38, 1032–1035. [Google Scholar] [PubMed]
  19. Maclauchlin, J.; Narayanan, G.L.; Mithani, V.; O’neill, G. The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. J. Food Prot. 2000, 63, 479–488. [Google Scholar]
  20. Monday, S.R.; Bohach, G.A. Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol. 1999, 37, 3411–3414. [Google Scholar] [PubMed]
  21. Srinivasan, V.; Sawant, A.A.; Gillespie, B.E.; Headrick, S.J.; Ceasaris, L.; Oliver, S.P. Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis. Foodborne Pathog. Dis. 2006, 3, 274–283. [Google Scholar] [CrossRef] [PubMed]
  22. Cremonesi, P.; Luzzana, M.; Brasca, M.; Morandi, S.; Lodi, R.; Vimercati, C.; Agnellini, D.; Caramenti, G.; Moroni, P.; Castiglioni, B. Development of a multiplex PCR assay for the identification of Staphylococcus aureus enterotoxigenic strains isolated from milk and dairy products. Mol. Cell Probes 2005, 19, 299–305. [Google Scholar] [CrossRef] [PubMed]
  23. Jarraud, S.; Mougel, C.; Thioulouse, J.; Lina, G.; Meugnier, H.; Forey, F.; Nesme, X.; Etienne, J.; Vandenesch, F. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 2002, 70, 631–641. [Google Scholar] [CrossRef] [PubMed]
  24. Hwang, S.Y.; Kim, S.H.; Jang, E.J.; Kwon, N.H.; Park, Y.K.; Koo, H.C.; Jung, W.K.; Kim, J.M.; Park, Y.H. Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea. Int. J. Food Microbiol. 2007, 117, 99–105. [Google Scholar] [CrossRef] [PubMed]
  25. Ono, H.K.; Omoe, K.; Imanishi, K.; Iwakabe, Y.; Hu, D.L.; Kato, H.; Saito, N.; Nakane, A.; Uchiyama, T.; Shinagawa, K. Identification and characterization of two novel staphylococcal enterotoxins, types S and T. Infect. Immun. 2008, 76, 4999–5005. [Google Scholar] [CrossRef] [PubMed]
  26. Nakaminami, H.; Noguchi, N.; Ikeda, M.; Hasui, M.; Sato, M.; Yamamoto, S.; Yoshida, T.; Asano, T.; Senoue, M.; Sasatsu, M. Molecular epidemiology and antimicrobial susceptibilities of 273 exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan. J. Med. Microbiol. 2008, 57, 1251–1258. [Google Scholar] [CrossRef] [PubMed]
  27. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Second Informational Supplement; CLSI Document M100-S22; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2013. [Google Scholar]
  28. Herrero, M.H.; Saqus, M.; Gerez, H.; Ventura, P. Halotolerant and halophilic bacteria isolated during the ripening of salted products. J. Food Prot. 2003, 61, 318–323. [Google Scholar]
  29. Vazquez-Sanchez, D.; Lopez-Cabo, M.; Saa-Ibusquiza, P.; Rodriguez-Herrera, J.J. Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain). Int. J. Food Microbiol. 2012, 157, 286–296. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Miguéis, S.; Santos, C.; Saraiva, C.; Esteves, A. Evaluation of ready to eat sashimi in northern Portugal restaurants. Food Control 2015, 47, 32–36. [Google Scholar]
  31. Song, M.; Bai, Y.; Xu, J.; Carter, M.Q.; Shi, C.; Shi, X. Genetic diversity and virulence potential of Staphylococcus aureus isolates from raw and processed food commodities in Shanghai. Int. J. Food Microbiol. 2015, 195, 1–8. [Google Scholar] [CrossRef] [PubMed]
  32. Akindolire, M.A.; Babalola, O.O.; Ateba, C.N. Detection of antibiotic resistant Staphylococcus aureus from milk: A public health implication. Int. J. Environ. Res. Public Health 2015, 12, 10254–10275. [Google Scholar] [CrossRef] [PubMed]
  33. Sallam, K.I.; Abd-Elghany, S.M.; Elhadidy, M.; Tamura, T. Molecular characterization and antimicrobial resistance profile of methicillin-resistant Staphylococcus aureus in retail chicken. J. Food Prot. 2015, 78, 1879–1884. [Google Scholar] [CrossRef] [PubMed]
  34. Pinchuk, I.V.; Beswick, E.J.; Reyes, V.E. Staphylococcal enterotoxins. Toxins (Basel) 2010, 2, 2177–2197. [Google Scholar] [CrossRef] [PubMed]
  35. Holtfreter, S.; Bauer, K.; Thomas, D.; Feig, C.; Lorenz, V.; Roschack, K.; Friebe, E.; Selleng, K.; Lovenich, S.; Greve, T.; et al. egc-Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin. Infect. Immun. 2004, 72, 4061–4071. [Google Scholar] [CrossRef] [PubMed]
  36. Yan, X.; Wang, B.; Tao, X.; Hu, Q.; Cui, Z.; Zhang, J.; Lin, Y.; You, Y.; Shi, X.; Grundmann, H. Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China. Appl. Environ. Microbiol. 2012, 78, 6637–6642. [Google Scholar] [CrossRef] [PubMed]
  37. Li, G.; Wu, S.; Luo, W.; Su, Y.; Luan, Y.; Wang, X. Staphylococcus aureus ST6-t701 isolates from food-poisoining outbreaks (2006–2013) in Xi’an, China. Foodborne Pathog. Dis. 2015, 12, 203–206. [Google Scholar] [CrossRef] [PubMed]
  38. Li, G.; Wu, C.; Wang, X.; Meng, J. Prevalence and characterization of methicillin susceptible Staphylococcus aureus ST398 isolates from retail foods. Int. J. Food Microbiol. 2015, 196, 94–97. [Google Scholar] [CrossRef] [PubMed]
  39. Fueyo, J.M.; Mendoza, M.C.; Rodicio, M.R.; Muñiz, J.; Alvarez, M.A.; Martín, M.C. Cytotoxin and pyrogenic toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in daily cows and relationships with macrorestriction genomic profiles. J. Clin. Microbiol. 2005, 43, 1278–1284. [Google Scholar] [CrossRef] [PubMed]
  40. Cha, J.O.; Lee, J.K.; Jung, Y.H.; Yoo, J.I.; Park, Y.K.; Kim, B.S.; Lee, Y.S. Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea. J. Appl. Microbiol. 2006, 101, 864–871. [Google Scholar] [CrossRef] [PubMed]
  41. Kim, N.H.; Yun, A.R.; Rhee, M.S. Prevalence and classification of toxigenic Staphylococcus aureus isolated from refrigerated ready-to-eat foods (sushi, kimbab and California rolls) in Korea. J. Appl. Microbiol. 2011, 111, 1456–1464. [Google Scholar] [CrossRef] [PubMed]
  42. Chao, G.; Zhou, X.; Jiao, X.; Qian, X.; Xu, L. Prevalence and antimicrobial resistance of foodborne pathogens isolated from food products in China. Foodborne Pathog. Dis. 2007, 4, 277–284. [Google Scholar] [CrossRef] [PubMed]
  43. Pu, S.; Wang, F.; Ge, B. Characterization of toxin genes and antimicrobial susceptibility of Staphylococcus aureus isolates from Louisiana retail meats. Foodborne Pathog. Dis. 2011, 8, 299–306. [Google Scholar] [CrossRef] [PubMed]
  44. Magiorakos, A.P.; Srinivasan, A.; Carey, R.B.; Carmeli, Y.; Falagas, M.E.; Giske, C.G.; Harbarth, S.; Hindler, J.F.; Kahlmeter, G.; Olsson-Liljequist, B.; et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 2012, 18, 268–281. [Google Scholar] [CrossRef] [PubMed]
  45. Wang, X.; Meng, J.; Zhang, J.; Zhou, T.; Zhang, Y.; Yang, B.; Xi, M.; Xia, X. Characterization of Staphylococcus aureus isolated from powdered infant formula milk and infant rice cereal in China. Int. J. Food Microbiol. 2012, 153, 142–147. [Google Scholar] [CrossRef] [PubMed]
  46. Marshall, B.M.; Levy, S.B. Food animals and antimicrobials: Impacts on human health. Clin. Microbiol. Rev. 2011, 24, 718–733. [Google Scholar] [CrossRef] [PubMed]
  47. Friedman, M.; Buick, R.; Elliott, C.T. Antibacterial activities of naturally occurring compounds against antibiotic-resistant Bacillus cereus vegetative cells and spores, Escherichia coli, and Staphylococcus aureus. J. Food Prot. 2004, 67, 1774–1778. [Google Scholar] [PubMed]
  48. Food and Drug Administration (FDA). Chapter 12: Pathogen Growth and Toxin Formation (Other Than Clostridium Botulinum) as a Result of Time/Temperature Abuse. Available online: http://www.fda.gov/downloads/Food/GuidanceRegulation/UCM251970.pdf (accessed on 3 December 2015).

Share and Cite

MDPI and ACS Style

Puah, S.M.; Chua, K.H.; Tan, J.A.M.A. 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. Int. J. Environ. Res. Public Health 2016, 13, 199. https://doi.org/10.3390/ijerph13020199

AMA Style

Puah SM, Chua KH, Tan JAMA. 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. International Journal of Environmental Research and Public Health. 2016; 13(2):199. https://doi.org/10.3390/ijerph13020199

Chicago/Turabian Style

Puah, Suat Moi, Kek Heng Chua, and Jin Ai Mary Anne Tan. 2016. "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" International Journal of Environmental Research and Public Health 13, no. 2: 199. https://doi.org/10.3390/ijerph13020199

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop