Molecular characteristics of Staphylococcus aureus isolates from food surveillance in southwest China

Background Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases in the world. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing methods were used to characterize Staphylococcus aureus isolates from food surveillance during 2013–2015 in southwest China, and Staphylococcal cassette chromosome mec (SCCmec) typing was used for methicillin-resistant S. aureus (MRSA). Isolates were also examined for their antibiotic resistance and carriage of virulence genes. Results Isolation rate of S. aureus was 2.60% during the three years’ surveillance and 29.50% of them were MRSA. All the S. aureus had hla genes (100%), 14.34% of the strains had tst, and 16.73% had PVL. 163 PFGE-SmaI patterns, 41 ST types and 36 spa types were obtained for all the S. aureus. Among them, ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the prevalent genotypes. Most of MRSA in this study belonged to SCCmec IV and V, accounted for 74.32% and 20.27% respectively. ST6-SCCmec IV-t701 (36.50%) was the most prevalent clone among isolates from food, followed by ST59-SCCmec V-t437 (20.30%), ST5-SCCmec IV-t002 (12.20%) and ST59-SCCmec IV-t437 (12.20%). Some strains had the identical PFGE patterns, ST and spa types with isolates from patients. Conclusions S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology. Electronic supplementary material The online version of this article (10.1186/s12866-018-1239-z) contains supplementary material, which is available to authorized users.


Background
Food-borne diseases (FBD) represent major public health concern worldwide [1,2]. Staphylococcal food poisoning (SFP) is one of the most common FBD in the world due to the ingestion of staphylococcal enterotoxins (SEs) that are produced by enterotoxigenic strains [3]. In the United States, SFP is the major concern of public health and it is one of the most common causes of reported FBD, causing an estimated 241,000 illnesses per year, imposing a great economic burden [4,5]. In China, 371 outbreaks of bacterial FBDs have been reported from 2008 to 2010, involving 20,062 cases and 41 deaths [6]. Furthermore, 94 outbreaks of SFP were reported from 2003 to 2007 according to the National Monitoring Network with 2223 individual patients and 1186 hospitalizations. During these SFP outbreaks, Staphylococcus aureus was the fifth most isolated pathogenic bacteria followed by Vibrio parahaemolyticus, Bacillus cereus, Bacillus proteus, and Salmonella [7]. Since 2009, Food safety law of People's Republic of China was enacted, and S. aureus was one of the bacteria that must be tested according to this law. Therefore, the systemic surveillance of S. aureus for food products is very important.
S. aureus is a commensal and opportunistic pathogen that can cause wide spectrum of infections, from superficial skin infections to severe and invasive disease [8]. The toxigenic S. aureus strains associated with SFP can express one or more of a family of genes coded for heat-stable enterotoxins. These genes showed high sequence homology in their structure and functions [1]. While, other virulence genes such as toxic shock syndrome toxin 1 (TSST-1), exfoliative toxins and cytolytic toxins (leukocidin and hemolysins) can also cause related diseases [9]. At present, the major molecular typing methods for S. aureus were pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), spa typing and Staphylococcal cassette chromosome mec (SCCmec) typing [10][11][12][13]. PFGE was considered as the golden method for S. aureus typing, showing a high discriminatory power. MLST was suitable for analyzing the evolutionary relation and clone complex of the strains. SCCmec developed by International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, (IWG-SCC: www.sccmec.org) was mainly used for genotyping of MRSA [14].
Yunnan is located in southwest China, including 16 cities. The lifestyle and eating habits are specific, and there is no systemic surveillance program for S. aureus in foods at present. Therefore this study was the first analyzing the characteristics of S. aureus isolates from food surveillance between 2013 and 2015 in this province.

Discussion
SEs produced by toxigenic strains are predisposing causes of SFP, among them, SEA is the most common ones. In this study, sea, seb, sec and sed were the most found toxin genes, 14.34% of the strains had tst, and 16.73% had PVL. Some similar results could be found from other Chinese studies, for example, Chao et al. [15] analyzed 568 isolates from different sources for the prevalence of SEs genes and performed spa typing.
Approximately 54.40% of the isolates from different sources harbored one or more SE genes. Seven genes, sea, seb, seg, seo, sem, seq, and sel were more frequently detected. In Li et al. study, [16] the most detected toxin genes were seu, lukED, hla, hlb and hld, followed by hlg, hlgv, lukPV, sea, see, etd, seb, sec, sed, sej, and sek. Although S. aureus strains in this study were isolated from food surveillance and not from SFP outbreak or patients, the high carriage rates of virulence genes indicated high risk for SFP in case of improper food handling and compromised good hygienic practices during food production and storage. Several studies showed that high levels of MRSA were found on US and European farms and commercially-distributed meats [17][18][19]. The prevalence of MRSA isolated from raw retail meat products was reported, ranging from less than 1% in Asia [20] to 11.9% in the Netherlands [21], with intermediate prevalence in other studies [22,23]. A recent study found that 45% of pork and 63% of beef products were positive for S. aureus in the United States [24].  isolated from raw milk and dairy products in Egypt, and the average isolation rate of MRSA was 53% among all milk and dairy products, with 35, 50, 40, 65 and 75% in yogurt samples, ice cream, Kareish cheese, Damietta cheese, and raw milk, respectively. In our study, 29.50% of isolates were MRSA, being a higher isolation rate to the reported in the above study. ST6-SCCmec IV-t701 was the most prevalent clone among MRSA isolates from food, followed by ST59-SCCmec V-t437, ST5-SCCmec IV-t002 and ST59-SCCmec IV-t437. Most of these strains were from grains and meat products. Grains are the favorite food for Chinese population; it can be processed into a variety of products, such as steamed bread, noodles, bean jelly, rice noodles and rice rolls etc. Among them, rice noodles, rice rolls are the most favorite foods in southwest China, people lived there eat these foods almost every day, and they eat them raw and cold. The comparison between food and patients' isolates, where a high homology was observed for ST6-t701 types isolated from rice noodles and rice rolls, indicated the possible circulation of those types between patients and foods. Therefore, the origin and distribution of MRSA or S. aureus are different between countries probably due to their specific eating habits and the resources of food.
Some studies performed in Europe indicated that ST398 was the most common MRSA type in retail meat, which might be a potential transmission factor of ST398 from farm to the community [28]. Yan et al. [29] analyzed 52 S. aureus isolates from 11 outbreaks in Shenzhen, China. They found that ST6 was the major ST type, and constituted 63.5% of all the strains in seven outbreaks. The second most commonly found ST type was ST943, constituted 23.1% of all the isolates from three outbreaks. The dominant spa types were t701, t091 and t2360, accounting 67.3% of the isolates. Li et al. [16] investigated the molecular epidemiology of S. aureus isolates from seven SFP outbreaks between 2006 and 2013 in Xi'an, northwest China. The results showed that ST6-t701 (71.4%) was the dominant clone, followed by ST5-t002 (14.3%) and ST59-t172 (14.3%). The antibiotic resistance results showed that 71.4% of the strains were resistant to trimethoprim and penicillin, 28.6% resistant to erythromycin, and 14.3% to ampicillin, clindamycin, and tetracycline. All the isolates were sensitive to chloramphenicol, cefoxitin, oxacillin, amikacin, and vancomycin. Furthermore, studies referred to MRSA circulated in the China hospitals revealed that ST59-SCCmec IV-t437 was the most common epidemic clone [30,31]. All these researches mentioned above indicated that ST6-SCCmec IV-t701 and ST59-SCCmec IV-t437 were predominant MRSA genotypes for patients. In this study, ST6-t701, ST7-t091, ST59-t437 and ST5-t002 were the prevalent types of the isolates, accounted for 43.43%. ST6-SCCmec IV-t701, ST59-SCCmec V-t437, ST5-SCCmec IV-t002 and ST59-SCCmec IV-t437 were the most prevalence MRSA clones. The strains showed great heterogenous distributions characteristics; however, ST6-t701 (or ST6-SCCmec IV-t701) was the dominant type in southwest China, because of their homology with strains isolated from patients and the aforementioned outbreaks.
This study was the first systemic S. aureus food surveillance in southwest China, in which the molecular characteristics of strains were determined. However, there were several limitations in this study. Firstly, the study was conducted in urban regions that probably showed a poor representation of the potential S. aureus food surveillance. Secondly, the surveillance time was only for three years, which might not fully reflect the characteristics of local strains. Therefore, further research including urban, rural, and other food sources might be done to comprehensively evaluate the prevalence and features of S. aureus.

Conclusions
Two hundred fifty-one S. aureus for food surveillance were analyzed from 2013 to 2015 in southwest China. Isolation rate of S. aureus was 2.60% during the three years' surveillance and 29.50% of them were MRSA. 163 PFGE-SmaI patterns were obtained for all the strains and the isolates displayed great heterogenous distributions. ST6-t701 (13.15%), ST7-t091 (12.75%), ST59-t437 (9.96%) and ST5-t002 (7.57%) were the most types for all the isolates, scattered distributed in different cities. ST6-SCCmec IV-t701 was the most prevalent clone among MRSA isolates from food, followed by ST59-SCCmec V-t437, ST5-SCCmec IV-t002 and ST59-SCCmec IV-t437. Some strains had the identical PFGE patterns, ST and spa types with isolates from patients. S. aureus isolated from food in southwest China displayed heterogeneity. Isolates had the same genotype profiles with isolates from patients, indicating high homology. Future research including urban, rural, and other food sources might be done to comprehensively evaluate the prevalence and features of S. aureus in southwest China.

Sample collection and isolation of S. aureus
The collection of samples used in this study was complied with national regulation on risk monitoring and management of food safety guidelines, and permitted by Center for Food Safety Risk Assessment of Yunnan Province. A total of 9646 food samples were collected from 16 cities in Yunnan province, from 2013 to 2015. The types of foods included grain products, meat products, milk products, fishery products, drinks, pastry, fruit products and vegetable products etc. The food samples were processed based on S. aureus isolation methods for foods of national standards in China (GB/T 4789. . We used qualitative method to isolate the strains. Each sample was aseptically weighed in an analytical balance, twenty-five grams were placed into a sterile plastic bag and 225 mL of buffered peptone water (Oxoid, UK) was added, then homogenized in a Stomacher Bagmixer 400 W (Interscience, France) for one minute. The subsequent enrichments and isolation process were according to the method described by Puah et al. [32]. All the isolated S. aureus were identified by Vitek Compact 2 (bioMérieux, France). S. aureus isolated from patients from hospital surveillance of Yunnan province were used for comparison to the food isolates. ; S. aureus ATCC 29213 was used as quality control. The mecA gene was detected by PCR, as described by Velasco et al. [33].

Virulence genes detection
The genomic DNA of the bacteria was extracted using Bacterial Genomic Extraction Kit (Tiangen, Beijing) followed the manufacturer's instructions. The virulence genes were detected based on previous study [34]. The enterotoxin, toxic shock syndrome toxin, exfoliative toxin, leucocytic toxin, hemolysin and epidermic cell differential inhibition gene were all amplified (MyCycler, Bio-Rad). Taq premix (TaKaRa, Japan) was used, and the amplification procedures were 94°C 5 min followed by 30 cycles: 94°C 15 s, 55°C 30s, 72°C 30s, and finally 72°C 10 min. Electrophoresis of the amplified products was performed in 1.5% agarose, and detected by Gel Imaging instrument (Bio-Rad, USA).
Pulsed-field gel electrophoresis PFGE was performed according to Coombs et al. [35], and each plug was digested with 50 U SmaI (TaKaRa, Japan) at 37°C for 4 h. CHEF-Mapper (Bio-Rad, USA) was used for electrophoresis, and the pulse time ranged from 4 s to 40 s for 19 h. The gel was stained with Gel Red (Biotium) and visualized using a gel imaging system (Bio-Rad, Gel DocXR). PFGE patterns were analyzed using BioNumerics version 6.6, and a dendrogram was constructed using the Dice coefficient and un-weighted pair group methods with the arithmetic mean algorithm (UPGMA).

Multilocus sequence typing
MLST was performed according to MLST database (http://saureus.mlst.net) and Enright et al. method [12]. The amplification products were sent for bidirectional sequencing by TaKaRa, Japan; and the sequences were analyzed using DNAStar and MEGA 4.0 software. All the sequences were submitted to MLST database for identifying their allele and ST assignments.

Spa typing
The genomic DNA of the bacteria was extracted as described above. PCR amplification and sequencing primers were used according to Harmsen et al. method [36]. The amplification products were sent for bidirectional sequencing by TaKaRa, Japan, and the sequences were submitted to the database for their spa type assignments (http://spatyper.fortinbras.us/).

SCCmec typing
SCCmec typing was performed by discriminating the mec complex and the cassette chromosome recombinase (ccr) genes complex as described previously [37], which was based on a set of multiplex PCR methods. SCCmec types were assigned according to the combination of the mec class and ccr type. MRSA strains which could not be assigned were defined as non-typable (NA).

Statistical analysis
Data analysis was performed by IBM SPSS software (version 19.0 for Windows, Armonk, NY). Non-parameter analysis was performed using a Kruskal-Wallis H test in SPSS statistics package. Two-sided significance was assessed for all variables, applying a cut-off value of P<0.05.