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      Antimicrobial Resistance, Virulence, and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Recovered from Ready-to-Eat (RTE) Food in China: A New Challenge for Food Safety

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            Abstract

            Objective:

            The objective of the present study was to determine the prevalence, antimicrobial resistance, virulence profiles, and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) obtained from ready-to-eat (RTE) foods in China.

            Methods:

            Two hundred seventy-six RTE food-associated S. aureus isolates were collected from 25 provinces across China in 2018, then characterized by antimicrobial susceptibility testing, virulence factors detecting, multilocus sequence typing (MLST), spa typing, SCCmec typing and pulsed-field gel electrophoresis (PFGE).

            Results:

            Two hundred fifty isolates (90.6%) were resistant to at least one antimicrobial agent; 73 (26.4%) isolates were multi-drug resistant (MDR). Thirty MRSA isolates were identified, among which nine toxin genes (sea, seb, sec, sed, seh, selk, sell, selq, and tsst-1) were detected. Sixty percent (18/30) of the MRSA isolates harbored multiple toxin genes. Four virulence gene patterns were identified, with seb-selk-selq (30/30) being the most common pattern. Thirteen sequence types, as well as 13 spa and 4 SCCmec types were found among 30 MRSA isolates. The most prevalent MRSA lineages were CC59-t437-SCCmecIV/V (23.3% [7/30]), CC398-t011-SCCmecV (23.3% [7/30]), and CC1-t114-SCCmecIV (16.7% [5/30]).

            Conclusions:

            Our findings highlight the importance for the identification of prevalent clones, assessment of drug-resistance and virulence, and formulation of food safety measures for public health.

            Main article text

            INTRODUCTION

            Staphylococcus aureus is a common member of the human microbiota within the upper respiratory tract and on the skin, with approximately 30% of the healthy human population being S. aureus carriers [1,2]. S. aureus may also cause a wide range of severe and life-threatening diseases, including skin and soft tissue infections, implanted medical device-related infections [3,4], and bacteremia, which is associated with a high mortality rate [5].

            S. aureus toxigenic strains produce virulence factors, such as toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), and Panton-Valentine leukocidin (PVL) [6]. Heat-stable SEs are regarded as the main cause of staphylococcal food poisoning (SFP) [7,8]. Greater than 20 different SEs and staphylococcal enterotoxin-like (SEls) toxins comprise a superfamily of pyrogenic toxin superantigens (SAgs). The classic enterotoxins (SEA, SEB, SEC1, SECbov, SED, and SEE) have been reported to account for 95% of SFP cases. The clinical characteristics of SFP cases include a short latency (average, 4.4 h), nausea, headaches, violent vomiting, abdominal cramping, and diarrhea [9]. Although SFP is usually a self-limiting illness and seldom causes life-threatening conditions, SFP is associated with significant physical discomfort and a financial burden. Of note, frequent outbreaks of SFP lead to serious public health, food industry, and catering business challenges [10,11]. One hundred twenty-eight foodborne illnesses caused by S. aureus were reported in the US and European Union in 2017, and SEs were responsible for 61 foodborne outbreaks (FBOs), representing 1.5% of all outbreaks reported in 2021 [12,13]. Ninety-four FBOs were attributable to SFP in China between 2003 and 2007, affecting 2223 individual patients and resulting in 1186 hospitalizations [14]; however, the actual incidence of SFP could be much higher because sporadic cases are easily overlooked and not reportable.

            In addition, a growing number of multidrug resistant (MDR) strains [15], especially methicillin-resistant Staphylococcus aureus (MRSA), have been reported from food products in recent years [16]. These MDR or MRSA strains could also lead to human infections via various routes, which limits clinical treatment options and poses a serious clinical threat with persistent high morbidity and mortality rates [17].

            Ready-to-eat (RTE) food products have become popular among consumers for the convenience they offer; however, retail RTE foods are susceptible to microbial contamination due to handling during packaging, sterilization, transportation, storage, and sales. Therefore, food poisoning readily occurs after contamination by foodborne pathogens. It was reported that 4.3% (1150/27,000) of retail foods were contaminated by S. aureus in China in 2015 [18]. The percentage of contaminated foods was much higher in RTE foods, which aroused a major public health concern [1921]; however, the epidemic characteristics of MRSA from RTE foods in China have not been established. The aim of this study was to determine the prevalence, antimicrobial resistance, virulence profiles, and molecular characteristic of MRSA affecting RTE foods in China.

            MATERIALS AND METHODS

            S. aureus isolates

            The 276 RTE food-associated S. aureus isolates used in this study were collected from 25 provinces across China in 2018. The RTE food samples were collected from supermarkets, farm product markets, convenience stores, retailers, fast food restaurants, and take-out restaurants, and included a variety of food types (salads, Chinese dim sum, cereal products, sushi, sashimi, ice cream, and sauced meats). All S. aureus isolates were confirmed using API STAPH test strips (bioMerieux, Marcyl’Etoile, France) and polymerase chain reaction (PCR) amplification of the 16S rRNA and nuc genes, as described previously [22]. The S. aureus isolates were then screened for MRSA by amplifying the mecA gene by PCR, according to a previous method [23]. All confirmed isolates were stored in brain heart infusion broth with 20% glycerol (Land Bridge, Beijing, China) at −80 °C.

            Antimicrobial susceptibility testing (AST)

            AST of all 276 S. aureus isolates were performed using Biofosun® Gram-positive panels (Fosun Diagnostics, Shanghai, China) and the microbroth dilution method [24]. A panel of 13 antimicrobial agents were estimated, including linezolid (LZD [0.25–8 μg/mL]), vancomycin (VAN [0.5–16 μg/mL]), daptomycin (DAP [0.125–8 μg/mL]), gentamicin (GEN [1–32 μg/mL]), trimethoprim-sulfamethoxazole (SXT [0.125/2.4–8/152 μg/mL]), chloramphenicol (CHL [1–64 μg/mL]), ciprofloxacin (CIP [0.125–8 μg/mL]), tetracycline (TET [0.5–16 μg/mL]), clindamycin (CLI [0.125–8 μg/mL]), erythromycin (ERY [0.125–8 μg/mL]), oxacillin (OXA [0.25–16 μg/mL]), cefoxitin (CFX [0.25–16 μg/mL]), and penicillin (PEN [0.06–8 μg/mL]). The S. aureus ATCC™ 29213 strain was used as the quality control for AST.

            DNA extraction

            All MRSA isolates were cultured on brain heart infusion agar (BHA; Land Bridge) at 37 °C overnight. Genomic DNA of the MRSA isolates was extracted using a bacterial DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s procedures. DNA was detected using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The qualified DNA was stored at −20 °C until analysis was performed.

            Detection of virulence factors

            In this study 13 enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sell, selk, and selq), as well as the lukS/F-PV and tsst-1 genes in MRSA isolates were investigated by PCR amplification, as previous described [23,25].

            Multilocus sequence typing (MLST)

            Seven housekeeping genes (arcC, aroE, glp, gmk, pta, tpi, and yqiL) were used in the MLST analysis, as described previously [26]. The alleles and sequence type (ST) were assigned based on the PubMLST database (https://pubmlst.org/). Clonal complexes (CCs) were annotated based on the geoBURST Full MST algorithm using phyloviz software with a primary founder surrounded by single locus variants (SLVs) and known CC type in the MLST database [27].

            spa typing

            The spa typing for all MRSA isolates was analyzed, as described previously [28]. The spa repeats and types were assigned using BioNumerics software (v7.5; Applied Math, Sint-Martens-Latem, Belgium).

            Staphylococcal cassette chromosome mec (SCCmec) typing

            MRSA is endowed with resistance to β-lactam antibiotics by obtaining SCCmec elements. The three basic genetic elements (mec and ccr gene complexes, and J region), SCCmec elements were classified into different types with multiplex PCR methods, as described previously [29].

            Pulsed-field gel electrophoresis (PFGE)

            PFGE was performed as described by Murchan et al. [30] and Salmonella Braenderup H9812 was used as a DNA size standard strain for the PFGE gels [31]. Briefly, the isolates were cultured on BHA (Land Bridge), then the cell suspensions were prepared for PFGE plugs. The genomic DNA of MRSA and H9812 were digested with restriction enzymes (SmaI and XbaI, respectively; New England Biolabs, Ipswich, MA, USA). Electrophoresis was performed through a 1% agarose SeaKem Gold gel in 0.5 × TBE buffer using a CHEF DR III system (Bio-Rad, Hercules, CA, USA) at 14°C for 19 h. The initial and final pulse time were 4.0 s and 40.0 s, respectively. After the gels were stained with GelRed (Biotium, Fremont, CA, USA), images were obtained using a ChemiDoc XRS+ system (Bio-Rad). The PFGE patterns and phylogenetic relationships were analyzed using BioNumerics software (v.7.5).

            RESULTS

            Isolation of S. aureus and MRSA in RTE foods

            A total of 276 S. aureus isolates were cultured from various RTE food items in 25 China provinces (Fig 1A). The food samples included salads, Chinese snacks, cereal products, sushi, sashimi, ice cream, and sauced meats. Of these isolates, 30 strains (10.9% [30/276]) were MRSA.

            FIGURE 1 |

            Map of China showing the location of the 25 provinces where the 276 RTE food-associated isolates were collected in 2018. (A) The provinces in gray were not included in this study, while the other 25 provinces where S. aureus was collected from RTE foods are marked with a rose-red gradient. The numbers enclosed in parentheses are the number of S. aureus isolates (before the comma) and MRSA (after the comma). (B) The AST results of S. aureus against 13 antimicrobial agents. (C) Frequency distribution of S. aureus isolated from different provinces in China completely susceptible or resistant to 1 to > 5 antimicrobial classes. N, total number of S. aureus isolates tested for AST in different provinces; R0->R5, resistance to 0 up to 5 antimicrobial classes.

            Antimicrobial susceptibility of S. aureus and MRSA isolates

            AST was performed for all isolates against 13 antimicrobial agents. Two hundred fifty isolates (90.6% [250/276]) were resistant to at least one antimicrobial, whereas only 26 isolates (9.4% [26/276]) were susceptible or had intermediate susceptibility to all 13 antimicrobials (Fig 1B). Specifically, the greatest resistance was observed for PCN (87.3% [241/276]), followed by ERY (38.4% [106/276]). The resistance rate to CFX, CLI, TET, and OXA ranged from 10.5%-19.2%, while resistance to CIP, CHL, SXT, and GEN was < 10%. Additionally, all isolates were susceptible to drugs of last resort (DAP, LIN, and VAN; Fig 1B).

            Among the 276 S. aureus isolates, 73 (26.1% [73/276]) were MDR (resistant to ≥ 3 classes of antimicrobials; Fig 1C). In addition, 16 (5.8% [16/276]) isolates were resistant to 4 classes of antimicrobials, 13 (4.7% [13/276]) were resistant to 5 classes of antimicrobials, and 3 (1.1% [3/276]) were resistant to ≥ 5 classes of antimicrobials. A total of 34 resistance profiles were identified. The three most prevalent resistant profiles in the cohort were PCN (36.6% [101/276]), PCN-ERY (11.6% [32/276]), and PCN-ERY-CLI (5.1% [14/276]). Moreover, isolates resistant to eight classes of antimicrobials had the following resistant profile: PCN-ERY-CLI-CIP-STX-TET-CHL-GEN.

            All the MRSA isolates were resistant to PCN, and > 73.3% of the isolates (22/30) were MDR (Table 1). Twelve resistance profiles were identified in the MRSA isolates. The most prevalent resistant profiles were PCN-OXA-CFX (23.3% [7/30]), PCN-OXA-ERY-CLI-TET-CFX (20.0% [6/30]), PCN-OXA-ERY-CFX (16.7% [5/30]), and PCN-ERY-CFX (16.7% [5/30]).

            TABLE 1 |

            Antimicrobial susceptibility of MRSA from ready-to-eat foods in China.

            Antimicrobial agentSusceptible
            Intermediate
            Resistant
            n%n%n%
            Daptomycin30100.0%00.0%00.0%
            Linezolid30100.0%00.0%00.0%
            Trimethoprim-sulfamethoxazole30100.0%00.0%00.0%
            Vancomycin30100.0%00.0%00.0%
            Ciprofloxacin2996.7%13.3%00.0%
            Gentamicin2893.3%26.7%00.0%
            Chloramphenicol2790.0%00.0%310.0%
            Tetracycline2066.7%26.7%826.7%
            Clindamycin1963.3%00.0%1136.7%
            Erythromycin826.7%00.0%2273.3%
            Oxacillin723.3%00.0%2376.7%
            Cefoxitin00.0%00.0%30100.0%
            Penicillin00.0%00.0%30100.0%
            Detection of virulence factors among MRSA isolates

            We searched for the presence of 15 virulence genes in the 30 MRSA isolates; 9 virulence genes were detected in 18 isolates (60% [18/30]), including sea (3.3% [1/30]), seb (36.7% [11/30]), sec (23.3% [7/30]), sed (3.3% [1/30]), seh (16.7% [5/30]), selk (50% [15/30]), sell (23.3% [7/30]), selq (50% [15/30]), and tsst-1 (16.7% [5/30]; Fig 2). Most of the MRSA isolates (53.3% [16/30]) harbored multiple virulence genes (≥ 3 virulence genes). With respect to classic SE genes, the seb gene was the most common. For the newly-identified SE and SEl genes, the selk and selq genes had 50%-positive isolates. Four virulence gene patterns were identified among 18 isolates; seb-selk-selq (33.3% [10/30]) was the most prevalent profile, followed by sec-she-selk-sell-selq-tsst-1 (16.7% [5/30]), sec-sell (6.7% [2/30]), and sea-seb-sed (3.3% [1/30]).

            FIGURE 2 |

            Resistant profiles, virulence factors, and genetic relationships of 23 MRSA isolates established by SmaI PFGE analysis. CCs = clone complexes; Sample sites (Sites): I = convenience store, II = farm product market, III = fast food restaurant, IV = retail outlet, V = supermarket, VI = take-out restaurant; food sources (Sources): A = cereal product, B = Chinese salad, C = Chinese dim sum, D = ice cream, E = sauced meats, F = sushi; virulence factors (VFs): purple = present, grey = absent.

            MLST

            The presence of different MRSA lineages was investigated by combining MLST, spa typing, and SCCmec typing. A total of 13 STs were identified based on MLST analysis (Figs 2 and 3). ST59 and ST398 were the most prevalent STs (both 23.3% [7/30]), followed by ST1 (16.7% [5/30]). All 13 STs belonged to 8 CCs, including CC1, CC5, CC8, CC72, CC88, CC59, CC45, and CC398. The most common CC was CC59 (33.3% [10/30]), followed by CC398 (23.3% [7/30]), CC1 (20% [6/30]), and CC88 (10% [3/30]).

            FIGURE 3 |

            Minimum spanning tree of the 30 MRSA isolates by MLST. The size and color of the circle indicate the number of isolates for each ST type. The grey halos surrounding the ST types denote that the ST types belonged to the same complex. The numbers on the straight line indicate the distance of the relationship between neighboring ST types.

            spa typing

            Thirteen spa types were identified among the 30 MRSA isolates (Fig 2); t437 and t011 were the most prevalent 23.3% (7/30), followed by t114 (16.7% [5/30]) and t3622 (6.7% [2/30]). The following spa types were identified in one isolate each: t116; t441; t664; t796; t1107; t3401; t3517; t4549; and t12147. The three most prevalent spa types (t437, t011, and t114) were associated with CC59, CC398, and CC1, respectively.

            SCCmec typing

            Four SCCmec types were identified among the 30 MRSA isolates, including type II, IV, V, and X SCCmec elements (Fig 2); SCCmecIV was the most prevalent 60.0% (18/30), followed by SCCmecV (30.0% [9/30]), SCCmecII (6.7% [2/30]), and SCCmecX (3.3% [1/30]). Combined analysis of CCs, and spa and SCCmec types revealed the presence of 17 different MRSA lineages, of which the most common lineages were CC59-t437-SCCmecIV/V (23.3% [7/30]), CC398-t011-SCCmecV (20.0% [6/30]), and CC1-t114-SCCmecIV (16.7% [5/30]).

            PFGE

            PFGE was performed on the 30 MRSA isolates, among which 7 isolates were not digested by SmaI and were identified as ST398 (not shown in Fig 2). The remaining 23 MRSA isolates were characterized by 18 PFGE patterns, which were further grouped into 6 clusters based on > 76% genetic similarity (Fig 2). The dominant PFGE cluster was cluster III, including 8 PFGE patterns from 10 isolates all associated with CC59; the dominant MRSA lineage was CC59-t437-SCCmecIV/V. Interestingly, the majority of MDR isolates were within this cluster and all isolates had the same virulence gene profile (seb-selk-selq). Cluster II included six isolates belonging to CC1, among which five were CC1-t114-SCCmecIV; all of the isolates collected were from the same province (Guangdong) and all had the same virulence gene profile (sec-she-selk-sell-selq-tsst-1). Three CC88 MRSA were clustered in PFGE cluster IV. No aforementioned enterotoxin genes were identified in the seven ST398 isolates.

            DISCUSSION

            In recent years the consumption of RTE foods in China has markedly increased due to its convenience. The global RTE food market sales were > 194 billion dollars in 2016 and is expected to increase by > 13% between 2018 and 2023 (unpublished data). RTE food sales are growing in China; 500 million people consumed RTE food as meals in 2020. Retail RTE foods are intended for direct human consumption without the need for cooking or other processing; thus, foodborne pathogens are not eliminated or reduced. Once contaminated by foodborne pathogens, the possibility of food poisoning is increased [32,33]. Specifically, S. aureus and MRSA have been frequently reported to cause foodborne outbreaks (FBOs) and are detected in RTE foods [10,11,1618,3436]. Therefore, the presence of S. aureus and MRSA strains in RTE food represents a potential threat to public health. The presence of S. aureus and MRSA strains, the enterotoxin gene repertoires, and molecular characteristics in RTE foods across China were determined in the current study. Our findings will be helpful in assessing the genetic diversity of MRSA and providing the genetic basis for evolutionary and epidemiologic studies of MRSA among various RTE foods.

            We showed that 90.6% of S. aureus isolates were resistant to at least one antimicrobial agent, which was slightly lower than reported for RTE foods in the Sichuan (96.3%) [33] or Shaanxi provinces of China (98.4%) [37]. Because our samples were from 25 provinces across China, our research is more representative of the entire country than the studies that focused on individual provinces. Similarly, we demonstrated a lower MDR prevalence (26.1%) than previous reports from Shaanxi, China (58.6%) [37] and Himachal Pradesh, India (81.3%) [3]. The greatest resistance was to PCN (87.3%), which is in agreement with the findings iin Sichuan province (90.7%) [18] and our previous study (83.7%) [33].

            Our results showed that 10.9% (30/276) of S. aureus isolates were MRSA; the prevalence was similar to previously reported in China (10.1%) and Brazil (8.8%) [32,38], but lower than previously reported in Bangladesh (23.1%) and higher than reported in Japan (2.9%) [39,40]. The relatively low prevalence of MRSA in Japan might be due to that the fact that the samples were mainly marine wild raw fish.

            S. aureus toxigenic strains can produce a wide variety of virulence factors; the SEs are regarded as the main cause of SFP. In the current study 9 virulence genes, including sea, seb, sec, sed, seh, selk, sell, selq, and tsst-1, were found in 60% (18/30) of the MRSA isolates and 53.3% (16/30) of MRSA isolates contained > 3 virulence genes. Notably, the SEB coding gene, seb (36.7% [11/30]), was the most prevalent gene among the classic SE genes, followed by sec, sea, and sed. This result partially contrasts with previous reports in which sea was the the most common cause of SFP worldwide, causing one of the largest SFP outbreaks involving 14,780 individuals in Japan in 2000 [10]. Based on high prevalence of seb among the MRSA isolates in the current study, it is important to understand the role of this toxin in causing more severe poisoning [41]. Consistent with previous reports, we showed that the seb gene was always detected together with selk and selq genes [42]. The coexistence of multiple virulence genes may reflect a potential link of genetic locations.

            ST59, ST398, and ST1 were the three most prevalent ST types among the MRSA isolates in the current study. This finding is agreement with previous studies in which ST59 was the most common ST among community-associated MRSA (CA-MRSA) in China and the predominant clone accounting for 47.7% (51/107) of MRSA isolates in retail foods in China [43,44]. ST59 MRSA were also recovered from humans and pigs in a backyard farm in rural China, indicating a possible bidirectional mode of transmission [45]. ST59 is the most common cause of hospital-acquired CA-MRSA infections, especially in pediatric patients [46,47]. In general, ST59 and ST338 (a single locus variant of ST59) have been shown to be the most common STs associated with community-acquired pneumonia in children in China [48]. Moreover, ST59, ST5711, and ST338 belong to CC59 (33.3% [10/30]), which is the prevalent clone associated with healthy carriers and patients with community-acquired infections in Asia [43]. ST59 was also shown to be the most prevalent clone in food, suggesting the possible high risk of bidirectional transmission between humans and food. In the current study the CC59 isolates were collected from seven provinces, which confirmed the universality of the distribution.

            The second most ST is ST398 (23.3% [7/30]), which was the first detected and most widespread livestock-associated MRSA (LA-MRSA) clone in Europe and North America, and has identified colonizing healthy humans or causing clinical infections [4952]. The ST1 isolates (16.7% [5/30]) are routinely described as CA-MRSA, but have also been collected from animals and has been associated with SFP in Korea and China [53,54]. ST5711 and ST5710 have not been reported, while other STs identified in this study have been reported to cause clinical infections.

            The most frequently observed spa types were t436 and t011. This finding is consistent with previous reports that t437 is the most common type in retail foods in China and the most dominant community-associated clone from Asia across Europe [5557]. Moreover, t011 has been frequently isolated from livestock, especially from pigs [58].

            SCCmecIV and SCCmecV were the top two prevalent types in our cohort, accounting for 90% of the total. Only two isolates typed as SCCmecII belonged to CC88, which is usually reported as hospital-associated MRSA (HA-MRSA). These results are in agreement with previous findings that reported CA-MRSA commonly carries SCCmec types IV and V, while HA-MRSA generally carries SCCmec types I, II, and III [5961].

            The seven MRSA isolates of ST398 all belonged to the same lineage (ST398-t011-V), which is a typical LA-MRSA genotype, frequently found as the predominant type of MRSA isolates from livestock, particularly from pigs and the entire pork production chain [58,62]. Interestingly, we found four ST398-t011-V isolates in non-animal foods (mostly cereal products), suggesting potential cross-contamination of these isolates through food processing. The remaining 23 MRSA isolates were distinguished into 18 PFGE patterns and grouped into 6 clusters (I-VI). The clusters were closely relevant to MLST, spa types, SCCmec types, and virulence gene patterns. All five CC1-t114-SCCmecIV MRSA in PFGE cluster II were identified in Chinese dim sum in Guangdong province. According to a previous report, an MRSA CC1-t114-SCCmecIV clone was isolated from SFP outbreaks in China [63]; however, there are few or no reports about this genotype in other countries up to the present. The dominant MRSA lineage was CC59-t437-SCCmecIV/V in cluster III, which was reported as the major clone among CA-MRSA in hospitals and the predominant genotype in bacteremia in China, and also identified in food poisoning outbreaks [46,47]. In addition, the CC59-t437-SCCmecIV/V clone was found in various food types, suggesting a possible diverse dissemination routes of these isolates. We found that the resistance and virulence genes in most cases exhibited a clonal distribution, and isolate CC59 tended to be resistant to more antimicrobial agents. Moreover, the virulence gene profiles among MRSA isolates had a significant correlation with the genotype lineages, which is different from what was reported in a recent study conducted in Italy [64]. The CC1-t114-SCCmecIV clone was shown to possess the most virulence genes (sec-she-selk-sell-selq-tsst-1), followed by CC59-SCCmecIV/V (seb-selk-selq), whereas no virulence genes were detected in CC398 and CC88 MRSA. Our understanding of the antimicrobial susceptibility and virulence genes among specific MRSA clones is vital for the development of therapeutics to target infections and measures to eliminate these clones in RTE-foods.

            Finally, there were some limitations in our study. Specifically, we could not detect all virulence genes and distinguish strains among the same CCs or STs using PCR. This limitation might benefit from a whole genome sequencing (WGS) approach that could potentially provide additional insights about our data. Specifically, one recent study involving 673 food- and human-associated S. aureus in China based on WGS exhibited a very powerful capacity [65]. In addition to 15 virulence genes detected in the current study, 80 other virulence genes were identified by WGS. Furthermore, the phylogenetic tree could be reconstructed based on 1585 core genes, thus promising much better resolution than the current study.

            CONCLUSIONS

            This study provided a detailed investigation of MDR S. aureus and characterization of MRSA isolates recovered from RTE foods in different geographic regions of China. The prevalence of MDR and MRSA isolates was high, with greater than one-half of MRSA harboring multiple virulence genes. Therefore, the clinic pathogenicity and transmission risk cannot be ignored. The genotype diversity of the MRSA isolates was related to antimicrobial resistance and virulence genes. The MRSA isolates with similar genotype generally had similar antimicrobial resistance patterns and virulence gene profiles. These results suggest that monitoring the genotypes of MRSA in RTE foods would be instrumental in tracing the source of contamination and assessment of antimicrobial resistance and risk of SFP. Therefore, such work will be helpful in assisting the government, food industries, and other stakeholders to improve food safety measures and control the transmission route of this bacterium.

            CONFLICTS OF INTEREST

            The authors declare no conflicts of interest.

            REFERENCES

            1. Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al.. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis. 2005. Vol. 5:751–762

            2. Verhoeven PO, Gagnaire J, Botelho-Nevers E, Grattard F, Carricajo A, Lucht F, et al.. Detection and clinical relevance of Staphylococcus aureus nasal carriage: an update. Expert Rev Anti Infect Ther. 2014. Vol. 12:75–89

            3. Lakhanpal P, Panda AK, Chahota R, Choudhary S, Thakur SD. Incidence and antimicrobial susceptibility of Staphylococcus aureus isolated from ready-to-eat foods of animal origin from tourist destinations of north-western Himalayas, Himachal Pradesh, India. J Food Sci Technol. 2019. Vol. 56(2):1078–1083

            4. Kussmann M, Karer M, Obermueller M, Schmidt K, Barousch W, Moser D, et al.. Emergence of a dalbavancin induced glycopeptide/lipoglycopeptide nonsusceptible Staphylococcus aureus during treatment of a cardiac device-related endocarditis. Emerg Microbes Infect. 2018. Vol. 7(1):202

            5. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev. 2015. Vol. 28(3):603–661

            6. Balakirski G, Hischebeth G, Altengarten J, Exner D, Bieber T, Dohmen J, et al.. Recurrent mucocutaneous infections caused by PVL-positive Staphylococcus aureus strains: a challenge in clinical practice. J Dtsch Dermatol Ges. 2020. Vol. 18(4):315–322

            7. Argudín MÁ, Mendoza MC, Rodicio MR. Food poisoning and Staphylococcus aureus enterotoxins. Toxins (Basel). 2010. Vol. 2:1751–1773

            8. Fisher EL, Michael O, Cheung GYC. Basis of virulence in enterotoxin-mediated staphylococcal food poisoning. Front Microbiol. 2018. Vol. 9:436

            9. Hu DL, Nakane A. Mechanisms of staphylococcal enterotoxin-induced emesis. Eur J Pharmacol. 2014. Vol. 722:95–107

            10. Hennekinne JA, De Buyser ML, Dragacci S. Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation. FEMS Microbiol Rev. 2012. Vol. 36:815–836

            11. Kadariya J, Smith TC, Thapaliya D. Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int. 2014. Vol. 2014:827965

            12. Lee H, Yoon Y. Etiological agents implicated in foodborne illness world wide. Food Sci Anim Resour. 2021. Vol. 41(1):1–7

            13. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union one health 2021 zoonoses report. EFSA J. 2022. Vol. 20(12):e07666

            14. Liang SY, Beekmann SE, Polgreen PM, Warren DK. Current management of cardiac implantable electronic device infections by infectious disease specialists. Clin Infect Dis. 2016. Vol. 63(8):1072–1075

            15. Hu YJ, Zhang CX, Zhang J, Zhang HY, Xiao Y, Dong SJ, et al.. Antimicrobial resistance in non-typhoidal Salmonella from retail foods collected in 2020 in China. Zoonoses. 2023. Vol. 3(1):27

            16. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al.. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol. 2019. Vol. 17(4):203–218

            17. Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. 2009. Vol. 7(9):629–641

            18. Wang W, Baloch Z, Jiang T, Zhang C, Peng Z, Li F, et al.. Enterotoxigenicity and antimicrobial resistance of Staphylococcus aureus isolated from retail food in China. Front Microbiol. 2017. Vol. 8:2256

            19. Kim NH, Yun AR, Rhee MS. 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. Vol. 111(6):1456–1464

            20. Yang S, Pei X, Yang D, Zhang H, Chen Q, Chui H, et al.. Microbial contamination in bulk ready-to-eat meat products of China in 2016. Food Control. 2018. Vol. 91:113–122

            21. Harada T, Taguchi M, Kawahara R, Kanki M, Kawatsu K. Prevalence and antimicrobial susceptibility of bacterial pathogens in ready-to-eat foods retailed in Osaka Prefecture, Japan. J Food Prot. 2018. Vol. 81(9):1450–1458

            22. Louie L, Goodfellow J, Mathieu P, Glatt A, Louie M, Simor AE. Rapid detection of methicillin-resistant Staphylococci from blood culture bottles by using a multiplex PCR assay. J Clin Microbiol. 2002. Vol. 40(8):2786–2790

            23. Wang W, Lin X, Jiang T, Peng Z, Xu J, Yi L, et al.. Prevalence and characterization of Staphylococcus aureus cultured from raw milk taken from dairy cows with mastitis in Beijing, China. Front Microbiol. 2018. Vol. 9:1123

            24. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th edition. Wayne, PA: CLSI. 2020

            25. Alibayov B, Zdenkova K, Sykorova H, Demnerova K. Molecular analysis of Staphylococcus aureus pathogenicity islands (SaPI) and their superantigens combination of food samples. J Microbiol Methods. 2014. Vol. 107:197–204

            26. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . J Clin Microbiol. 2000. Vol. 38:1008–1015

            27. Francisco AP, Vaz C, Monteiro PT, Melo-Cristino J, Ramirez M, Carrico JA. PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods. BMC Bioinformatics. 2012. Vol. 13:87

            28. Harmsen D, Claus H, Witte W, Rothgänger J, Claus H, Turnwald D, et al.. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 2003. Vol. 41(12):5442–5448

            29. Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, et al.. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother. 2007. Vol. 51(1):264–374

            30. Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M, Zinn CE, et al.. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol. 2003. Vol. 41:1574–1585

            31. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, et al.. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis. 2006. Vol. 3(1):59–67

            32. Yang X, Zhang J, Yu S, Wu Q, Guo W, Huang J, et al.. Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in retail ready-to-eat foods in China. Front Microbiol. 2016. Vol. 7:816

            33. Lin Q, Sun H, Yao K, Cai J, Ren Y, Chi Y. The prevalence, antibiotic resistance and biofilm formation of Staphylococcus aureus in bulk ready-to-eat foods. Biomolecules. 2019. Vol. 9(10):524

            34. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, et al.. Food borne illness acquired in the United States–major pathogens. Emerg Infect Dis. 2011. Vol. 17(1):7–15

            35. Macori G, Bellio A, Bianchi DM, Gallina S, Adriano D, Zuccon F, et al.. Molecular typing of Staphylococcus aureus isolate responsible for staphylococcal poisoning incident in homemade food. Ital J Food Saf. 2016. Vol. 5(2):5736

            36. Liao F, Gu W, Yang Z, Mo Z, Fan L, Guo Y, et al.. Molecular characteristics of Staphylococcus aureus isolates from food surveillance in southwest China. BMC Microbiol. 2018. Vol. 18(1):91

            37. Xing X, Li G, Zhang W, Wang X, Xia X, Yang B, et al.. Prevalence, antimicrobial susceptibility, and enterotoxin gene detection of Staphylococcus aureus isolates in ready-to-eat foods in Shaanxi, People’s Republic of China. J Food Prot. 2014. Vol. 77(2):331–334

            38. Rizek CF, Matté MH, Dropa M, Mamizuka EM, de Almeida LM, Lincopan N, et al.. Identification of Staphylococcus aureus carrying the mecA gene in ready-to-eat food products sold in Brazil. Foodborne Pathog Dis. 2011. Vol. 8(4):561–563

            39. Hammad AM, 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. Vol. 156(3):286–289

            40. Islam MA, Parveen S, Rahman M, Huq M, Nabi A, Khan ZUM, et al.. Occurrence and characterization of methicillin resistant Staphylococcus aureus in processed raw foods and ready-to-eat foods in an urban setting of a developing country. Front Microbiol. 2019. Vol. 10:503

            41. Gholamzad M, Khatami MR, Ghassemi S, Vaise Malekshahi Z, Shooshtari MB. Detection of Staphylococcus enterotoxin B (SEB) using an immune chromatographic test strip. Jundishapur J Microbiol. 2015. Vol. 8(9):e26793

            42. Schelin J, Wallin-Carlquist N, Cohn MT, Lindqvist R, Barker GC, Rådström P. The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment. Virulence. 2011. Vol. 2(6):580–592

            43. Chuang YY, Huang YC. Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Asia. Lancet Infect Dis. 2013. Vol. 13(8):698–708

            44. Wu S, Huang J, Zhang F, Wu Q, Zhang J, Pang R, et al.. Prevalence and characterization of food-related methicillin-resistant Staphylococcus aureus (MRSA) in China. Front Microbiol. 2019. Vol. 10:304

            45. Bi Z, Sun C, Borjesson S, Chen B, Ji X, Berglund B, et al.. Identical genotypes of community-associated MRSA (ST59) and livestock-associated MRSA (ST9) in humans and pigs in rural China. Zoonoses Public Health. 2018. Vol. 65(3):367–371

            46. Peng H, Liu D, Ma Y, Gao W. Comparison of community- and healthcare-associated methicillin-resistant Staphylococcus aureus isolates at a Chinese tertiary hospital, 2012-2017. Sci Rep. 2018. Vol. 8(1):17916

            47. Qin Y, Wen F, Zheng Y, Zhao R, Hu Q, Zhang R. Antimicrobial resistance and molecular characteristics of methicillin-resistant Staphylococcus aureus isolates from child patients of high-risk wards in Shenzhen, China. Jpn J Infect Dis. 2017. Vol. 70(5):479–484

            48. Geng W, Yang Y, Wu D, Huang G, Wang C, Deng L, et al.. Molecular characteristics of community-acquired, methicillin-resistant Staphylococcus aureus isolated from Chinese children. FEMS Immunol Med Microbiol. 2010. Vol. 58:356–362

            49. Graveland H, Wagenaar JA, Heesterbeek H, Mevius D, van Duijkeren E, Heederik D. Methicillin resistant Staphylococcus aureus ST398 in veal calf farming: human MRSA carriage related with animal antimicrobial usage and farm hygiene. PLoS One. 2010. Vol. 5(6):e10990

            50. Bouiller K, Gbaguidi-Haore H, Hocquet D, Cholley P, Bertrand X, Chirouze C. Clonal complex 398 methicillin-susceptible Staphylococcus aureus bloodstream infections are associated with high mortality. Clin Microbiol Infect. 2016. Vol. 22(5):451–455

            51. Golding GR, Bryden L, Levett PN, McDonald RR, Wong A, Wylie J, et al.. Livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 in humans, Canada. Emerg Infect Dis. 2010. Vol. 16:587–594

            52. van Cleef BA, Monnet DL, Voss A, Krziwanek K, Allerberger F, Struelens M, et al.. Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe. Emerg Infect Dis. 2011. Vol. 17:502–505

            53. Cha JO, Lee JK, Jung YH, Yoo JI, Park YK, Kim BS, et al.. Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea. J Appl Microbiol. 2006. Vol. 101:864871

            54. Yan X, Wang B, Tao X, Hu Q, Cui Z, Zhang J, et al.. Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China. Appl Environ Microbiol. 2012. Vol. 78:6637–6642

            55. Wang X, Li G, Xia X, Yang B, Xi M, Meng J. Antimicrobial susceptibility and molecular typing of methicillin-resistant Staphylococcus aureus in retail foods in Shaanxi, China. Foodborne Pathog Dis. 2014. Vol. 11:281–286

            56. Wang W, Liu F, Baloch Z, Zhang CS, Ma K, Peng ZX, et al.. Genotypic characterization of methicillin-resistant Staphylococcus aureus isolated from pigs and retail foods in China. Biomed Environ Sci. 2017. Vol. 30(8):570–580

            57. Glasner C, Pluister G, Westh H, Arends JP, Empel J, Giles E, et al.. Staphylococcus aureus spa type t437: identification of the most dominant community-associated clone from Asia across Europe. Clin Microbiol Infect. 2015. Vol. 21(2):163.e1–8

            58. Verhegghe M, Pletinckx LJ, Crombé F, Vandersmissen T, Haesebrouck F, Butaye P, et al.. Methicillin-resistant Staphylococcus aureus (MRSA) ST398 in pig farms and multispecies farms. Zoonoses Public Health. 2013. Vol. 60(5):366–374

            59. Mediavilla JR, Chen L, Mathema B, Kreiswirth BN. Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Curr Opin Microbiol. 2012. Vol. 15:588–595

            60. Liu J, Chen D, Peters BM, Li L, Li B, Xu Z, et al.. Staphylococcal chromosomal cassettes mec (SCCmec): a mobile genetic element in methicillin-resistant Staphylococcus aureus . Microb Pathog. 2016. Vol. 101:56–67

            61. Sunagar R, Hegde NR, Archana GJ, Sinha AY, Nagamani K, Isloor S. Prevalence and genotype distribution of methicillin-resistant Staphylococcus aureus (MRSA) in India. J Glob Antimicrob Resist. 2016. Vol. 7:46–52

            62. Bouchami O, Fraqueza MJ, Faria NA, Alves V, Lawal OU, Lencastre H, et al.. Evidence for the dissemination to humans of methicillin-resistant Staphylococcus aureus ST398 through the pork production chain: a study in a Portuguese slaughterhouse. Microorganisms. 2020. Vol. 8(12):1892

            63. Zhang P, Miao X, Zhou L, Cui B, Zhang J, Xu X, et al.. Characterization of oxacillin-susceptible mecA-positive Staphylococcus aureus from food poisoning outbreaks and retail foods in China. Foodborne Pathog Dis. 2020. Vol. 17(11):728–734

            64. Antonelli A, Giani T, Coppi M, Pilato VD, Arena F, Colavecchio OL, et al.. Staphylococcus aureus from hospital-acquired pneumonia from an Italian nationwide survey: activity of ceftobiprole and other anti-staphylococcal agents, and molecular epidemiology of methicillin-resistant isolates. J Antimicrob Chemother. 2019. Vol. 74(12):3453–3461

            65. Wang W, Baker M, Hu Y, Xu J, Yang D, Maciel-Guerra A, et al.. Whole-genome sequencing and machine learning analysis of Staphylococcus aureus from multiple heterogeneous sources in China reveals common genetic traits of antimicrobial resistance. mSystems. 2021. Vol. 6:e01185–20

            Author and article information

            Journal
            Zoonoses
            Zoonoses
            Zoonoses
            Compuscript (Shannon, Ireland )
            2737-7466
            2737-7474
            02 September 2023
            : 3
            : 1
            : e964
            Affiliations
            [1 ]NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, China
            Author notes
            *Corresponding authors: E-mail: dongyinping@ 123456cfsa.net.cn , Tel: +86-010-67776356 (YD); xiaojing@ 123456cfsa.net.cn , Tel: +86-010-52165413 (JX)
            Article
            10.15212/ZOONOSES-2023-0025
            e1f4f3f2-ecd8-4741-9bd6-029776b9bf8a
            Copyright © 2023 The Authors.

            This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY) 4.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

            History
            : 20 June 2023
            : 25 July 2023
            : 19 August 2023
            Page count
            Figures: 3, Tables: 1, References: 65, Pages: 9
            Funding
            Funded by: National Key Research and Development Program of China
            Award ID: 2018YFC1602703 [the Ministry of Science and Technology of China]
            This work was funded by the National Key Research and Development Program of China (2018YFC1602703 [the Ministry of Science and Technology of China]).
            Categories
            Original Article

            Parasitology,Animal science & Zoology,Molecular biology,Public health,Microbiology & Virology,Infectious disease & Microbiology
            molecular typing,antimicrobial susceptibility,MRSA,virulence factors,ready-to-eat food

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