Characterization of Oxacillin-Susceptible mecA -Positive Staphylococcus aureus from Food Poisoning Outbreaks and Retail Foods in China

Abstract

In this study, we explored the prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) in staphylococcal food poisoning outbreak isolates and foodborne isolates, and then investigated their molecular characteristics, classical staphylococcal enterotoxins (SEs), and drug resistance. Eight (2.9%) of 275 isolates from food poisoning outbreaks and 7 (3.8%) of 184 isolates from retail foods were identified as OS-MRSA isolates. Among the 15 OS-MRSA isolates, the most frequently detected toxin genes were hld (100%), hla (93.3%), pvl (80.0%), and hlb (46.7%) followed by seg and seq (33.3%, each), hlg (26.7%), seb and hlgv (20.0%, each), sec, seh, sel, sep, and tst (13.3%, each), and sei, sem, sen, and seo (6.7%, each). None of isolates carried other tested virulence genes. The most frequently detected classical SEs were SEB and SEC (26.7%, each), followed by SEA and SEE (20.0%, each), and SED (6.7%). Resistance was most frequently observed in ampicillin, penicillin, and cefoxitin (100%, each), followed by trimethoprim/sulfamethoxazole (93.3%), erythromycin (73.3%), amoxicillin/clavulanic acid (46.7%), tetracyclines (26.7%), and ciprofloxacin (6.7%). All isolates were susceptible to other tested antibiotics. A dominant molecular type belonged to ST398-IVa-t034 (26.7%), followed by ST59-IVa-t437 (20.0%), ST88-III-t14340 and ST1-IVa-t114 (13.3%, each), and ST5-II-t002, ST630-t4549, ST5-II, and ST4495-t10738 (6.7%, each). Our findings indicated that OS-MRSA strains had a low prevalence rate among outbreak strains and foodborne strains, which frequently harbored SCCmec IVa, and carried a variety of toxin genes, and also expressed numerous classical SEs. In addition, all OS-MRSA isolates were susceptible to the majority of antibacterial agents except β-lactam. Our study is the first to report that OS-MRSA isolates are associated with food poisoning outbreaks worldwide.

Introduction

S taphylococcus aureus (S. aureus) is an important foodborne pathogen and can produce a wide range of exotoxins in various foods. Among these exotoxins, enterotoxins are known as a leading cause of staphylococcal food poisoning (SFP) outbreaks (Argudin et al., 2010). At present, 23 different enterotoxins have been identified and divided into two groups according to their demonstrated emetic activity: staphylococcal enterotoxins (SEs) and staphylococcal enterotoxin-like proteins (SEls), including the classical SEs (SEA through SEE), the new types of SEs (SEG, SEH, SEI, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, SES, and SET) and SEls (SElJ, SElU, SElV, SElX, and SElY) (Omoe et al., 2013; Alibayov et al., 2014; Ono et al., 2015). Among SEs/SEls, SEA was considered as the main cause of SFP outbreaks, followed by SED, SEC, SEB, and SEE (Argudin et al., 2010).

Methicillin-resistant S. aureus (MRSA) is mediated by the mecA gene encoding penicillin-binding protein 2a (PBP2') with a low affinity for β-lactams antimicrobial agents or phenotypically exhibiting an oxacillin minimum inhibitory concentration (MIC) of ≥4μg/mL (CLSI 2012, 2017). On the contrary, oxacillin-susceptible S. aureus isolates carrying mecA gene have been defined as oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) as previously described (Quijada et al., 2019; Luo et al., 2020). OS-MRSA isolates can easily be misdiagnosed by antibiotic phenotypic laboratory testing, potentially triggering the development of highly new resistant MRSA variants under antibiotic selection, owing to the possession of mecA (Quijada et al., 2019). In recent years, infections caused by OS-MRSA are being increasingly reported in humans and animals in China (He et al., 2013; Pu et al., 2014; Song et al., 2017). However, the transmission of OS-MRSA is still unclear (Luo et al., 2020).

Although OS-MRSA has been widely distributed in humans and animals (He et al., 2013; Pu et al., 2014; Song et al., 2017), few data are reported about OS-MRSA from retail foods and food poisoning outbreaks. Therefore, this study aimed to determine the characteristics of OS-MRSA from food poisoning outbreaks and foods by detecting toxin genes, classical SEs, drug resistance, and molecular typing.

Materials and Methods

Bacterial strains

According to the national standard GB/T 4789-2010 series standard (National Standard of the People's Republic of China, 2010), Salmonella, Shigella, S. aureus, Vibrio parahaemolyticus, Proteus, Diarrheal Escherichia coli and Bacillus cereus were tested and identified in the food poisoning samples from 95 food poisoning outbreaks. The diagnosis of SFP is generally confirmed based on the symptoms of patients and laboratory diagnosis according to the criteria of National Standard WS/T80-1996 (Professional Standard of the People's Republic of China, 1996) and described by Kerouanton et al. (2007) as follows: (1) the symptoms of patients including vomiting, diarrhea, abdominal pain, nausea, or headache between 2 and 4 h after food consumption; (2) laboratory diagnosis of at least one of the following criteria: (i) S. aureus colony count >10⁵ colony-forming unit (CFU)/g from remnant foods, (ii) detection of SEs in remnant foods, or (iii) the strain of S. aureus with the same SEs from both the patients and remnant foods, or (iv) isolate of S. aureus with the same SEs from the different patients. A total of 275 S. aureus isolates were isolated from 95 SFP outbreaks. Of interest, we found that OS-MRSA isolates existed in food poisoning isolates (Supplementary Tables S1 and S2).

We speculated whether the OS-MRSA isolates are also present in foodborne isolates. Therefore, the foodborne and food poisoning isolates were collected in the same region during 2011–2016 and screened for OS-MRSA, to understand the prevalence and distribution of OS-MRSA isolates in food poisoning outbreaks and retail foods. OS-MRSA was identified as previously described, using PCR for detection of nuc gene and mecA genes and phenotypically exhibiting an oxacillin MIC of ≤2 μg/mL (Wang et al., 2014; Quijada et al., 2019; Luo et al., 2020), and OS-MRSA confirmation was repeated three times. Eight OS-MRSA isolates were identified from five SFP outbreaks in Shenzhen and Shanghai in China during 2014–2016 (Table 1). Seven OS-MRSA isolates were identified in 184 S. aureus strains isolated from 4800 food samples of animal origin. Foods of animal origin were collected by Shanghai Municipal Center for Disease Control and Prevention (Shanghai CDC), which were recovered from 20 fresh and frozen food samples taken monthly from June to October over the years 2011–2016 by each district CDC (four district CDCs) in Shanghai (Table 1). All isolates were stored at −80°C in trypticase soy broth (TSB; Beijing Land Bridge Technology Ltd., Beijing, China) plus 20% (v/v) glycerol for further use.

Table 1.

Prevalence of OS-MRSA Isolated from Retail Foods and Staphylococcal Food Poisoning Outbreaks

Source	No. of samples	No. of S. aureus recovered	No. of OS-MRSA	% of positive samples for OS-MRSA
Food poisoning outbreaks	—	275	8	—
Retail foods	4800	184	7	0.15% (7/4800)
Total	—	459	15	—

—, data unknown; OS-MRSA, oxacillin-susceptible mecA-positive S. aureus.

PCR detection of virulence genes

All isolates were analyzed by PCR for virulence genes as previously described (Li et al., 2015). The primers are listed in Supplementary Table S3 and were synthesized by AuGCT DNA-SYN Biotechnology Co. Ltd. (Yangling, China). All isolates were tested by PCR for 5 classical enterotoxin genes (sea, seb, sec, sed, and see), 16 new enterotoxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, seu, and sev), hemolysins genes (hla, hlb, hld, hlg, and hlgv), Panton-Valentine leukocidin gene (pvl), and toxic shock syndrome toxin-1 gene (tst). The PCR products were resolved in 1.0% (w/v) agarose gel electrophoresis in 0.5 × Tris-borate-EDTA buffer.

Antimicrobial susceptibility testing

Antimicrobial susceptibility tests were performed by the agar dilution method for penicillin (PEN) with resistance breakpoint of ≥0.25 μg/mL, ampicillin (AMP, ≥0.5 μg/mL), oxacillin (OXA, ≥4 μg/mL), erythromycin (ERY, ≥8 μg/mL), tetracyclines (TET, ≥16 μg/mL), cefoxitin (FOX, ≥8 μg/mL), cefoperazone (FOP, ≥64 μg/mL), chloramphenicol (CHL, ≥32 μg/mL), gentamicin (GEN, ≥16 μg/mL), ciprofloxacin (CIP, ≥4 μg/mL), rifampin (RIF, ≥4 μg/mL), vancomycin (VAN, ≥16 μg/mL), amikacin (AMK, ≥64 μg/mL), trimethoprim/sulfamethoxazole (T/S, ≥4/76 μg/mL), and amoxicillin/clavulanic acid (A/C, ≥8/4 μg/mL). The antibiotic susceptibility testing results were interpreted according to the guidelines of the Clinical Laboratory Standards Institute (CLSI 2012, 2017). All antibiotics were purchased from Sigma-Aldrich (Shanghai) Trading Co. Ltd. in China. S. aureus ATCC 29213 and E. coli ATCC 25922 were used as quality control isolates in antimicrobial susceptibility experiments. The isolates were identified as multidrug resistant (MDR) with resistance to at least three classes of antibiotics as previously described (Magiorakos et al., 2012).

Staphylococcal protein A (spa) typing

Staphylococcus protein A (spa) consists of Fc-partial region, X-region and C-terminal. Spa typing is a genotyping method based on the presence of gene polymorphism in a variable number of 24 bp repeated sequences in the X-region. The primers were obtained from the website (www.seqnet.org): spa-1113f (5′-TAAAGACGATCCTTCGGTGAGC-3′), spa-1514r (5′-CAGTAGTGCCGTTTG CTT-3′). The sequencing results were analyzed by Ridom Staph software (Ridom GmbH, Wurzburg, Germany).

Multilocus sequence typing

Multilocus sequence typing (MLST) was performed by PCR for seven housekeeping gene (arcC, aroE glpF, gmK, pta, tpi, and yqiL) amplification and sequencing, and the sequencing results were placed at standard length and linked to the MLST website (www.mlst.net) database for comparison. Finally, seven housekeeping gene profiles were obtained. The results were verified on the website and the sequence type (ST) of each strain was obtained. The primers were determined from the website (http://saureus.mist.net) and synthesized by AuGCT DNA-SYN Biotechnology Co. Ltd.

SCCmec typing

According to the method described by Zhang et al. (2005), the Staphylococcal Cassette Chromosome mec (SCCmec) type was determined using a multiplex PCR, which generated a specific amplification pattern for each SCCmec structure type. Then the product sizes were determined by gel electrophoresis.

Enzyme-linked immunosorbent assay for detection of classical SEs

We detected the classical enterotoxin A, B, C, D, and E for all isolates using enzyme-linked immunosorbent assay (ELISA) kit of R-Biopharm following the manufacturer's manual. In brief, these isolates were streaked onto trypticase soy agar (TSA; Beijing Land Bridge Technology Ltd.) and incubated at 37°C for 18–24 h. A single colony from each TSA plate was transferred into 5 mL of TSB and incubated at 37°C with 220 rpm shaking for 24 h. Ten microliters of each overnight culture was transferred into 10 mL of brain–heart infusion (BHI) broth (Beijing Land Bridge Technology Ltd.) and incubated at 37°C with 220 rpm shaking for 24 h. The culture supernatants were collected by centrifugation at 3500 × g at 4°C for 5 min. To determine the production of SEs, 100 μL of each culture supernatant diluted at 1:10 with phosphate-buffered saline (pH = 7.4) was added into an ELISA plate following the manufacturer's instructions. The optical density the each well was determined at 450/630 nm.

Results

Prevalence of OS-MRSA

A total of 15 (3.3%, 15/459) OS-MRSA isolates were detected, including 8 (2.9%) of 275 isolates from food poisoning outbreaks and 7 (3.8%) of 184 isolates from retail foods (Table 1).

Detection of virulence genes

Of the 15 OS-MRSA isolates, 100.0% (15/15) were positive for one or more toxin genes (Table 2). The four most frequently detected toxin genes were hld (100%, 15/15), hla (93.3%, 14/15), pvl (80.0%, 12/15), and hlb (46.7%, 7/15), followed by seg, and seq (33.3%, 5/15 for each), hlg (26.7%, 4/15), seb, and hlgv (20.0%, 3/15 for each), sec, seh, sel, sep, and tst (13.3%, 2/15 for each), sei, sem, sen, and seo (6.7%, 1/15 for each). Ten toxin genes including sea, sed, see, sej, sek, ser, ses, set, seu, and sev) were not detected in these isolates. Overall, the OS-MRSA isolates displayed 10 different toxin gene profiles, toxin genes with 1–9 different combinations for each isolate. The most common toxin gene profile was seg-hla-hld-hlg-pvl (20.0%, 3/15).

Table 2.

Antimicrobial Resistance, Classical Staphylococcal Enterotoxins Production, Toxin Genes, and Molecular Characteristics of OS-MRSA Isolates from Staphylococcal Food Poisoning Outbreaks and Retail Foods

Isolate No.	Source	Sequence type	Spa^a	Types of sample	Classical SEs production^b	Toxin gene profiles	Antimicrobial resistance profiles^c	MecA^d	SCCmec typing^e
3	SPF outbreaks	5	NT	Feces	SEA-SEB-SEC-SED-SEE	Hld	AMP-ERY-FOX-T/S-A/C-PEN	+	II
4		1	t114	Noodles with spicy bean sauce	SEA-SEC-SEE	sec-seh-sel-seq-hla-hld-hlgv-tst-pvl	AMP-ERY-TET-FOX-T/S-A/C-PEN	+	IVa
6		1	t114	Shredded pork with dried bean curd	SEA-SEC-SEE	sec-seh-sel-seq-hla-hld-hlgv-tst-pvl	AMP-ERY-TET-FOX-T/S-A/C-PEN	+	IVa
8		398	t034	Vomit	NT	seg-hla-hld-hlg-pvl	AMP-ERY-FOX-T/S-PEN	+	IVa
9		398	t034	Vomit	NT	seg-hla-hld-hlg-pvl	AMP-ERY-FOX-T/S-PEN	+	IVa
13		398	t034	Pork braised in brown sauce	NT	seg-hla-hld-hlg-pvl	AMP-ERY-FOX-T/S-PEN	+	IVa
15		59	t437	Feces	SEB-SEC	seb-seq-hla-hlb-hld-hlgv	AMP-ERY-TET-FOX-T/S-A/C-PEN	+	IVa
16		630	t4549	Feces	NT	hla-hld-hlg-pvl	AMP-FOX-T/S-PEN	+	NT
18	Retail foods	4495	t10738	Raw egg	NT	hla-hlb-hld-pvl	AMP-FOX-T/S-PEN	+	NT
19		59	t437	Raw pork	SEB	seb-seq-hla-hlb-hld-pvl	AMP-ERY-FOX-PEN	+	IVa
20		88	t14340	Raw duck	NT	sep-hla-hlb-hld-pvl	AMP-FOX-T/S-PEN	+	III
21		88	t14340	Fried diced meat	NT	sep-hla-hlb-hld-pvl	AMP-ERY-FOX-T/S-PEN	+	III
22		398	t034	Raw mutton	NT	seg-hla-hld-pvl	AMP-FOX-T/S-A/C-PEN	+	IVa
23		59	t437	Raw beef	SEB	seb-seq-hla-hlb-hld-pvl	AMP-ERY-FOX-T/S-A/C-PEN	+	Iva
24		5	t002	Raw chicken	NT	seg-sei-sem-sen-seo-hla-hlb-hld	AMP-ERY-TET-FOX-CIP-T/S-A/C-PEN	+	II

a,b,e

NT, no detection; ^bStaphylococcal enterotoxins (SEA, SEB, SEC, SED, and SEE) tested by ELISA; ^cPEN, penicillin; AMP, ampicillin; ERY, erythromycin; TET, tetracyclines; FOX, cefoxitin; CIP, ciprofloxacin; T/S, trimethoprim/sulfamethoxazole; A/C, amoxicillin/clavulanic acid; ^d+, mecA gene positive.

ELISA, enzyme-linked immunosorbent assay.

Antimicrobial susceptibility testing

As given in Table 2, all OS-MRSA isolates (100%) were resistant to ampicillin, penicillin, and cefoxitin, following by 93.3% (14/15) to trimethoprim/sulfamethoxazole, 73.3% (11/15) to erythromycin, 46.7% (7/15) to amoxicillin/clavulanic acid, 26.7% (4/15) to tetracyclines, and 6.7% (1/15) to ciprofloxacin. All OS-MRSA isolates were susceptible to oxacillin, cefoperazone, chloramphenicol, rifampin, amikacin, gentamicin, and vancomycin. Eleven (73.3%, 11/15) OS-MRSA isolates were MDR. In particular, 7 antimicrobial resistance profiles were identified. The most common antimicrobial susceptibility testing profile was AMP-ERY-FOX-T/S-PEN (26.7%, 4/15), followed by AMP-FOX-T/S-PEN and AMP-ERY-TET-FOX-T/S-A/C-PEN (20.0%, 3/15 for each).

Molecular typing

As given in Table 2, 7 different spa types were identified in 15 OS-MRSA isolates, whereas 1 isolate of OS-MRSA spa typing was not detected. Among them, t034 (26.7%, 4/15) and t437 (20.0%, 3/15) were the main spa types, followed by t114 and t14340 (13.3%, 2/15 for each) and t002, t10738, and t4549 (6.7%, 1/15 for each). In the MLST analysis, all OS-MRSA isolates were assigned to seven STs. The most frequently detected ST type was ST398 (26.7%, 4/15), followed by ST59 (20.0%, 3/15), ST5, ST1, and ST88 (13.3%, 2/15 for each) and ST4495 and ST630 (6.7%, 1/15 for each). In this study, three SCCmec types including type IVa (60.0% 9/15), II and III (13.3% 2/15) were detected, and two isolates were nontypeable by SCCmec typing. In addition, two common molecular types of ST59-IVa-t437 and ST398-IVa-t034 were observed between the OS-MRSA from SFP outbreaks and retail food.

Detection of classical SEs

Of the 15 OS-MRSA isolates (Table 2), 6 (40.0%, 6/15) isolates were found to express one or more classical SEs by ELISA test. The two most predominant classical SEs were SEB and SEC (26.7%, 4/15 for each), followed by SEA and SEE (20.0%, 3/15 for each), and SED (6.7%, 1/15). Four enterotoxin profiles were identified, including SEB and SEA-SEC-SEE (13.3%, 2/15 for each), SEA-SEB-SEC-SED-SEE and SEB-SEC (6.7%, 1/15 for each).

Discussion

We have found two common molecular types (ST59-t437 and ST398-t034) were observed between the OS-MRSA from SFP outbreaks and retail food in this study. Reports on OS-MRSA in food poisoning outbreaks and retail foods are scarce. Therefore, we carried out this study to determine the occurrence of OS-MRSA isolates in food poisoning outbreaks and retail foods, and further characterized OS-MRSA isolates by detecting toxin genes, classical SEs, drug resistance, and molecular typing. To our knowledge, this is the first study on the genetic diversity of OS-MRSA isolates from food poisoning outbreaks and retail foods of animal origin.

Among the methods for reliable identification of MRSA of S. aureus strains, detection of mecA gene is often considered the “gold standard” because of its high sensitivity and speed. In addition to molecular detection, phenotype exhibiting an oxacillin MIC of ≥4 μg/mL or a cefoxitin MIC of ≥8 μg/mL is also a common method for screening and detection of MRSA (Corrente et al., 2007). Previously CLSI suggested testing MRSA on oxacillin agar but some MRSA fail to show phenotype on this agar. In 2008, CLSI recommended using cefoxitin for laboratory detection of MRSA when molecular methods are not available. The incongruence between the two antibiotics remains not fully understood, but it is suggested that the better reliability of using cefoxitin over oxacillin may be owing to the fact that cefoxitin is a stronger inducer of mecA gene expression than oxacillin (Chen et al., 2012). To date, cefoxitin-resistant and oxacillin-resistant MRSA (Wang et al., 2014), cefoxitin-resistant and oxacillin-susceptible MRSA (Conceicao et al., 2015; Song et al., 2017), or cefoxitin-susceptible and oxacillin-susceptible MRSA (Cuirolo et al., 2011) have been reported in previous studies. Cefoxitin-resistant and oxacillin-susceptible S. aureus isolates carrying mecA gene were found in animal source foods and SFP outbreaks in this study. Therefore, combining PCR detection of mecA gene with phenotypic resistance test will enable better characterization of MRSA and guide clinical treatment.

OS-MRSA isolates have become one of the most important pathogens in humans and animals worldwide (Guimaraes et al., 2017; Song et al., 2017). However, the detection rate (0.17%. 1/600) of OS-MRSA isolates in foods of animal origin is low (Quijada et al., 2019), which was in agreement with our results in this study (0.15%, 7/4800). These findings indicated that OS-MRSA can be occasionally isolated from animal source foods. Surprisingly, OS-MRSA isolates were also identified among isolates causing SFP outbreaks in this study. Based on the results in this study (Supplementary Tables S1 and S2), some OS-MRSA isolates produced the same SEs as the SEs detected directly from remnant foods (S. aureus colony count >10⁵ CFU/g from remnant foods) in an SFP outbreak, and some OS-MRSA isolates from remnant foods (S. aureus colony count >10⁵ CFU/g from remnant foods) exhibited the same molecular type and SEs genes as the OS-MRSA strains isolated from patients (feces or vomiting) from an SFP outbreak. This indicated that these OS-MRSA isolates could probably be the culprit isolates for food poisoning. Therefore, the possibility to cause food poisoning by OS-MRSA cannot be ignored.

Although those OS-MRSA probably constituted a similar level of risk of causing enteric disease compared with other S. aureus as long as they contained same enterotoxins in this study, the chance for those OS-MRSA to cause disease other than enteric disease could not be neglected, especially for those immunocompromised individuals (Kluytmans et al., 1995; Jones et al., 2002). These extra-intestinal disease may be derived either from direct contact with contaminated foods during food preparation or through ingestion of contaminated food (Kluytmans et al., 1995; Jones et al., 2002). Another concern for those OS-MRSA is their capacity to become highly resistant to methicillin when exposed to β-lactam antibiotics owing to misdiagnosis as methicillin-susceptible S. aureus (MSSA) (Quijada et al., 2019). Therefore, this study indicates another potential transmission route of OS-MRSA through food.

The data in this study revealed that ST1, ST5, ST59, ST88, and ST398 OS-MRSA strains harboring SE-encoding genes and/or expressing classical SEs are associated with food poisoning and foods in this study. It suggests that OS-MRSA may be an important reason to cause food poisoning outbreaks. ST1 OS-MRSA clone presented a high number of enterotoxin genes in this study, which agreed with Cha et al. (2006) who showed that ST1 clone carrying a high number of enterotoxin genes is recognized as a dominant sequence type among SFP. ST5 OS-MRSA from food poisoning was producing all the classical SEs (SEA-SEE) by ELISA in this study. It was different from the findings of Yan et al. (2012) who showed that ST5 clone carried seg and sei. In this study, we also found that only ST5 clone carried seg and sei from foods. Seb-sek-seq was the most common SEs gene profile in the ST59 MRSA strains from Chinese children (Wu et al., 2011). Moreover, seb or seb-seq was detected in the ST59 MSSA clone from SFP (Cha et al., 2006; Li et al., 2015). By contrast, seb-seq was the most common SEs gene profile in the ST59 OS-MRSA strains from SFP and foods in this study. According to previous reports, ST398 OS-MRSA strains carried few SE/SEl genes (Kadlec et al., 2009), which was consistent with our study, in which seg gene only was detected in ST398 OS-MRSA strains from foods and food poisoning. ST398 strains harboring seg gene have also been found in SFP outbreaks in previous reports (Umeda et al., 2017). This suggests that ST398 OS-MRSA strains expressing SEG may be the cause of food poisoning. ST630 strains were not detected any new enterotoxin genes or classical SEs in this study, and so, it is not known whether it could cause SFP. The genetic diversity of OS-MRSA is noteworthy as a potential cause of SFP: it harbors new and/or unknown SE/SEl in addition to classical SEs (Umeda et al., 2017).

Compared with classical enterotoxin A-E by ELISA and classical enterotoxin A-E genes detection by PCR, there was 73.3% (11/15) correlation between enterotoxin types and presence of the respective genes in this study. This was consistent with that in the previous studies (Pereira et al., 2009; Aydin et al., 2011), which demonstrated ∼80% correlation between the enterotoxin types and the presence of respective genes in S. aureus isolates using the ELISA and PCR assays. These findings indicated that ELISA and PCR assays were different in the specificity and sensitivity for the detection of staphylococcal enterotoxins.

Hla is expressed by almost all S. aureus isolates; on the contrary, PVL is secreted only by isolates lysogenized with a bacteriophage carrying lukS-PV and lukF-PV, the structural genes for PVL (Bubeck Wardenburg et al., 2007). Our results showed that the hla gene is often associated with pvl gene in OS-MRSA isolates linked to SFP outbreaks, which is similar to previous reports (Takizawa et al., 2005). Hla and PVL genes are a typical feature of CA-MRSA (Mcclure et al., 2006). Further research is needed to confirm whether these foodborne OS-MRSA isolates are from the community environment.

At present, OS-MRSA isolates have been increasingly reported worldwide, not only among clinical strains but also from foods and animals (Petinaki et al., 2002; Pu et al., 2014; Conceicao et al., 2015; Mistry et al., 2016; Song et al., 2017; Quijada et al., 2019; Luo et al., 2020). These results suggest that foods, humans, and animals may be a reservoir for OS-MRSA. OS-MRSA strains generally carried SCCmec types IV and V in India, Africa, and China, and type III in India and Japan; the sequence typing was ST59/ST338-t437 in China, ST72-t7287, ST168-t7696, and ST239-t037 in India, ST-t4518 or ST88-t786 in Africa in previous studies (Ho et al., 2016; Song et al., 2017). On the contrary, OS-MRSA strains harboring SCCmec types I, II, VI, and VII are less reported (Ho et al., 2016; Song et al., 2017). It was in agreement with the result in our study where the most frequent OS-MRSA strains were SCCmec type IV_a (60.0%, 9/15); the sequence types were ST398-t034 (26.7%, 4/15), ST59-t437 (20.0%, 3/15), and ST1-t114 (13.3%, 2/15). These findings indicate that OS-MRSA strains from SFP overlapped in molecular type (ST59-t437 and ST398-t034) with strains recovered from foods, animals, and humans, and are most likely from them.

Conclusions

We reported the occurrence OS-MRSA from S. aureus isolates associated with foodborne outbreaks and S. aureus isolated from retail foods. These OS-MRSA isolates carried a variety of toxin genes and expressed classical SEs, and some of them exhibited multi-resistance phenotype. Although OS-MRSA existed in food in a low rate, considering it clinical significance, this study still demonstrates that food may not be neglected as a possible transmission route for OS-MRSA. Future research on the relevance of those food-derived OS-MRSA with clinical OS-MRSA are needed.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This research was in supported by the National Natural Science Foundation of China (No. 31871894, U1703119 and 31271858) and Project of science and technology of social development in Shaanxi Province (2018SF-110).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

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