Occurrence, Antibiotic Susceptibility, Biofilm Formation and Molecular Characterization of Staphylococcus aureus Isolated from Raw Shrimp in China

The aim of this study was to determine the prevalence and characterization of Staphylococcus aureus isolated from 145 shrimp samples from 39 cities in China. The results show that 41 samples (28%) from 24 cities were positive, and most of the positive samples (39/41, 95.1%) were less than 110 MPN/g. Antimicrobial susceptibility testing showed that only seven isolates were susceptible to all 24 antibiotics, whereas 65.1% were multidrug-resistant. Antibiotic resistance genes that confer resistance to β-lactams, aminoglycosides, tetracycline, macrolides, lincosamides and streptogramin B (MLSB), trimethoprim, fosfomycin and streptothricin antibiotics were detected. All S. aureus isolates had the ability to produce biofilm and harbored most of the biofilm-related genes. Genes encoding one or more of the important virulence factors staphylococcal enterotoxins (sea, seb and sec), toxic shock syndrome toxin 1 (tsst-1) and Panton–Valentine leukocidin (PVL) were detected in 47.6% (30/63) of the S. aureus isolates. Molecular typing showed that ST15-t085 (27.0%, 17/63), ST1-t127 (14.3%, 9/63) and ST188-t189 (11.1%, 7/63) were the dominant genetic types. The finding of this study provides the first comprehensive surveillance on the incidence of S. aureus in raw shrimp in China. Some retained genotypes found in this food have been linked to human infections around the world.


Introduction
With the development of the economy and improvement in people's living standards in our country, cold chain transportation is continuously improving, and consumers all over our country can eat aquatic products rich in protein. Shrimp is one of the most popular aquatic products, and the estimated level of production from both wild harvest and farm culture is approximately 6624 million metric tons, totaling a value of more than USD 23 billion [1]. According to the China Fishery Statistics Yearbook, the shrimp production in China reached 6,307,300 tons in 2020, and the domestic production of shrimp essentially matches domestic consumption, with significant economic benefits. Shrimp is enjoyed for the uniqueness of its flavor and texture, but one of the major problems faced by the shrimp industry, besides insufficient production and disease outbreaks, is shrimp product safety [1]. In recent years, the prevalence and risk identification of some foodborne pathogens, such as

Isolation and Identification of S. aureus
The shrimp samples were submitted to qualitative and quantitative analysis for S. aureus. Qualitative analysis was performed according to the GB 4789.30-2010 of food microbiological examination of S. aureus (National Food Safety Standards of China) with slight modification, and quantitative analysis was performed with the most probable number (MPN) method. Briefly, approximately 25 g of food sample was added to 225 mL of tryptic soy broth culture with 10% sodium chloride (Huankai, Guangzhou, China). Subsequently, 1 mL, 0.1 mL and 0.01 mL aliquots were transferred to tubes containing 9 mL, 10 mL and 10 mL in triplicate with trypticase soy broth culture (Huankai) supplemented with 10% NaCl. Each tube was incubated at 37 • C for 48 h, respectively.
A portion (10 µL) of enrichment broth culture was streaked onto chromogenic S. aureus agar plates (Huankai), which were incubated at 37 • C for 24 h. Each positive sample selected 1-4 colonies with pink color and was purified on nutrient agar medium at 37 • C for 24 h. The purified colonies were analyzed via coagulase activity test by freeze-dried Rabbit Plasma (Huankai), and the API STAPH test strips (bio Merieux, Marcy-1 Etoile, France). The MPN value was determined based on the number of positive tube(s) in each of the three sets using the MPN table.

Biofilm Formation Assay
The ability of S. aureus isolates to produce biofilm in vitro was assessed by a microtiter plate assay (MPA) as described by Vasudevan et al. (2003) [22] with slight modifications. The isolates were individually cultivated in 37 • C overnight on BHI (Brain Heart Infusion Broth). The overnight culture was diluted 1:100 in fresh BHI, and an aliquot of 200 µL of each prepared suspension were transferred into three wells of 96-well tissue-culturetreated polystyrene microplates (CELLSTAR ® Cell Culture Microplates, Greiner Bio-one, Frickenhausen, Germany). After cultivation at 37 • C for 48 h, the wells were washed three times with 200 mL of sterile phosphate-buffered saline (PBS, pH 7.4) and dried at room temperature. The adherent bacterial cells were fixed with 200 µL of methanol for 15 min, then the plates were emptied and dried overnight. The adherent cells were stained with 1% crystal violet for 10 min and were washed twice with water. The dye bound to the adherent cells was dissolved with 150 mL of 95% ethanol and optical density (OD) was measured at 590 nm using a spectrophotometer (SpectroStar Nano BMG Labtech). The uninoculated wells containing BHI served as negative control. Blank corrected absorbance values of isolates were used for reporting biofilm production. Isolates were considered biofilm producers when their OD values were three times greater than the standard deviation of the mean Dc. Additionally, isolates showing an ability to produce biofilm were classified as weak (Dc < OD ≤ 2* Dc), moderate (2*Dc < OD ≤ 4* Dc) or strong (OD > 4* Dc) biofilm.

Whole-Genome Sequencing and Assembly
Genomic DNA was extracted from shrimp-related S. aureus isolates using a genomic DNA extraction kit (Magen Biotech, Guangzhou, China) according to the manufacturer's instructions. Each DNA sample was fragmented into 400 bp fragments by a Covaris M200 sonicator and prepared for sequencing with the Ion Plus Fragment Library Kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Whole genomes were sequenced on the Life Ion S5 platform with an average coverage of 100×. Clean reads were used for de novo assembly with SPAdes v3.6.2. Only assemblies that harbored ≥ 95% of cg MLST targets were used for further analysis as previously described. If the criteria were not met, the sample was re-sequenced.

Determination of STs, Spa Types, SCCmec Types, Virulence Genes and ARG Genes
The STs of S. aureus was based on 7 housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi and yqil) using multilocus sequence typing [23]. In order to obtain allelic profiles and to determine STs, the full-length sequences of these 7 genes from shrimp-related S. aureus genomes were obtained using BLAST + 2.5.0 [24] and compared at each locus with those of the known alleles in the S. aureus MLST database (https://pubmlst.org/saureus, accessed on 28 March 2023). Spa types (t) were predicted using spaTyper v1.0 webserver [25] from the Centre of Genomic Epidemiology (https://cge.cbs.dtu.dk/services/spatyper, accessed on 25 October 2022). SCCmec types for MRSA isolates were predicted using SCCmecFinder v1.2 from the Center of Genomic Epidemiology (https://cge.cbs.dtu.dk/ services/SCCmecFinder/, accessed on 17 November 2022). The presence of virulence factors and antibiotic resistance factors encoded in the genomes were inferred by comparing all the proteins against the virulence factor database (VFDB) [26], the comprehensive antibiotic resistance database (CARD) [27], and Resfinder [28].

Nucleotide Sequence Accession Numbers
The complete genomic sequences of shrimp-related S. aureus isolates were deposited in the Foodborne Staphylococcus aureus genome database by the Institute of Microbiology, Guangdong Academy of Sciences (http://210.77.86.67/StaphylococcusAureus.html, accessed on 2 September 2022).

Isolation and Identification of S. aureus from Shrimp in China
In total, 41 of 145 shrimp samples were coagulase-positive and 63 isolates were confirmed to be S. aureus using the API test. The qualitative and quantitative results of S. aureus positive samples are shown in Table 1. Overall, the isolation percentage of S. aureus from shrimp samples was 28.28% (41/145), with MPN values showing half of samples (22/41) less than 1 MPN/g, and only two samples were higher than or equal to 110 MPN/g. The prevalence of positive samples was found in 24 of the 39 Chinese cities as follows: 11% in Guangzhou, 9% in Macao, 8% in Hangzhou, Lasa and Nanjing and 6% in Beijing and Huhehaote, and the remaining positive samples were obtained from the other cities.

Prevalence of Virulence-Associated Genes
As shown in Figure 2, only nine SE genes were detected in S. aureus isolates from shrimp, whereas sed, see, seg, sei, sem, seo, seu, sep, sej and ser were not free in the shrimprelated S. aureus isolates. The rates of the SEs were as follows: sel (25.4%), sec (22.2%), seh (20.6%), seb (15.9%), sea (14.3%), sek (9.5%) and seq (7.9%). Among the classical SE genes, 14 isolates carried sec, 10 isolates carried seb, and 9 isolates carried sea, whereas the egc gene group was not detected. Except for SE genes, the WGS analysis found 56 other VFs in S. aureus isolates (Figure 2). All of the conserved VFs were homologous to the VFs identified in S. aureus, including multiple genes related to toxin-related genes

Discussion
As is known, microbial hazards have become one of the most important threats in the field of food safety. Shrimp products are an important food source for many coastal countries in the world. S. aureus and its toxins can cause food poisoning and pose a risk to public health, whether it is due to source pollution in shrimp products, secondary contamination during processing and transportation or cross-contamination in food service links. In our previous study, it was found that 35.0% of retail meat samples were positive for S. aureus in China [29]. In this study, the contamination rate of S. aureus in shrimp products was 28.3%, which showed that the shrimp product was also an important resource for foodborne S. aureus. Fortunately, most of the positive samples had less than 1 MPN/g.
Overall, our result was higher than the results of several years ago in China. In 2013, the prevalence of S. aureus in Shanghai city was 4%; in 2015, the prevalence of S. aureus in Liaoning Province was 5.56%; and in 2016, the prevalence of S. aureus in Huai'an city was 18.2%. Compared with studies from other countries, our result was not lower as well. In Iran, the contamination rate of S. aureus in shrimp was 24.6% [30]. In Nigeria, the percentage of S. aureus-positive samples in ready-to-eat shrimp accounted for 30.97% [31]. The detection rate of S. aureus in prawns which were taken from supermarkets in and around Cochin was high (26.7%) [32]. However, all these reports were subject to district restriction. Therefore, this survey was more full-scale and systematical investigation for the prevalence of S. aureus isolated from shrimp products, especially as the collection site covered most provincial capitals of China and the samples were more country-specific. However, the existing data and our study indicate that S. aureus isolated from shrimp products in China is potentially harmful and deserves our attention. In view of the characteristics of shrimp and other aquatic products, many people like to eat shrimp raw or pickled to taste the freshness of the food itself; once contaminated by S. aureus, the generation of enterotoxin often easily leads to the occurrence of food poisoning events.
Staphylococcal enterotoxins (SEs) are responsible for most staphylococcal food poisoning outbreaks [33]. They can still maintain their biological and immune activity after being treated at 100 • C for 30 min. Currently, there are at least 28 kinds of SEs reported, which are mainly divided into classical enterotoxins (sea-see) and novel enterotoxins [34], of which classical enterotoxins (sea-see) account for more than 95% of confirmed food poisoning cases as the most common enterotoxin. This study examined 23 SE genes in S. aureus isolates that were associated with shrimp products. The results showed that 47.6% of the isolates carried one or more classical SE genes and non-classic SE genes. This suggests a potential risk of S. aureus in shrimp products in China. This rate was like previous research which tested for the prevalence of 18 SE genes of S. aureus isolates from different origins in China, showing that 54.4% of isolates harbored SE genes. In China, sea was the most common gene which was responsible for SFP outbreaks, followed by seb and sed, which were responsible for most outbreaks [35]. Sed, sea and seb were detected in 14.3% and 15.9% of S. aureus isolates in this study, which indicated that these shrimp-related S. aureus isolates have a potential risk of foodborne infections. Fortunately, the highest detection rate of classical SE genes in shrimp-related isolates was in sec (22.22%). However, there were also some outbreaks which have involved sec [36]. The common detection method of SEC was determined using immuno-colloidal gold chromatographic test strips, but the anti-SEC antibody was produced based on SEC1 or SEC2. Thus, this may have caused inaccuracy in SEC detection in previous studies. Additionally, the SFP outbreaks in recent years should also remind people not to ignore the harm caused by new enterotoxins [34]. In this study, seq, seh, sej, sel and sek were detected in S. aureus isolates. Of these enterotoxins, only SEH-producing strains have clearly been involved in SFP outbreaks [37][38][39], but results from different researchers have shown the high incidence of genes encoding new SEs and SEls among food-borne S. aureus [33,40]. It is possible that the corresponding SEs might have been the cause of these outbreaks. Thus, the hazard of these isolates which harbored SE genes should not be ignored.
Except SE genes, VFs include diverse functions (e.g., adhesion, invasion, signaling, conjugation, environmental interaction, immune evasion, etc.) which play a crucial role in cell viability, virulence and evasion of host defenses [41]. In this study, two S. aureus isolates (Sta2404-0 and Sta3706A1) were harbored in the tsst-1 gene. Importantly, previous studies have shown that TSST-1 is a super-antigen that can cause multiple organ dysfunction syndrome and is responsible for an especially high rate of mortality [42,43]. Moreover, the tsst-1 gene, together with sec and sel, have often been found in Staphylococcus aureus Pathogenicity Islands (SaPIs) and play an important role in toxin production [33,42]. Except for TSST-1, one MRSA isolate, Sta705, carried the Panton-Valentine leukocidin (PVL) gene. In recent years, PVL-positive S. aureus has received a significant amount of attention [44]. These strains can cause highly necrotizing pneumonia, necrotizing dermatitis, and other primary diseases in humans [44,45]. In fact, the risk of death associated with PVL-positive S. aureus has been reported to be higher than that associated with non-PVL-producing S. aureus [46]. However, either tsst-1 or PVL were associated with the S. aureus mobile genetic element (SaPIs or bacteriophages, etc.), which may promote the virulence of S. aureus and participate in the pathological process of S. aureus infection in vitro and in vivo [42,47]. The presence of TSST-1-positive S. aureus isolates and PVL-positive S. aureus isolates in shrimp samples is concerning, as they both contain classical SE genes. This poses a potential risk of foodborne infections for consumers and requires further investigation. Therefore, it is important for the public to be aware of these findings and for further attention to be given to S. aureus in shrimp.
As is known, the presence of biofilm on food contact surfaces is considered a health hazard. In fact, S. aureus biofilm on food contact surfaces poses a serious risk of food contamination [48]. It has been frequently found on surfaces of food processing plants responsible for outbreaks related to the consumption of fresh and processed foods worldwide [49][50][51][52].
Overall, the rate of biofilm producer isolates in the present study was higher than previous reports of all S. aureus isolates obtained from shrimp samples which showed the ability to produce biofilm with moderate or strong biofilm production capability [20,[53][54][55][56]. In addition, through whole-genome analysis, it was found that all these isolates carry biofilm-related genes, including ica genes (icaA, icaB, icaC, icaD and icaR) and another set of adhesion genes, such as fnbA, fnbB, cna, map and ebp, as well as clfA and clfB. These genes are both important factors contributing to the initiation of foreign body infection [57][58][59]. Although the frequencies of detection of genes associated with adhesion and biofilm formation reported in S. aureus isolates from food vary according to the geographical area studied, S. aureus isolates from humans found that 100% of them presented these genes and were able to form biofilm [60,61]. Thus, it is necessary to continuously observe the biofilm-forming ability and related genes of S. aureus isolates in food.
In addition, the antimicrobial resistance was tested in shrimp-associated S. aureus in this study. However, many reports have detected antibiotic-resistant strains of S. aureus in various food products [62][63][64], and our data were also not lower. In this study, the rate of multiple drug resistance even reached 65.1%, of which five isolates resisted more than 10 antibiotics. This result was higher than many previous studies: the multidrug resistance rate of S. aureus isolates from aquatic products was 47.9%, from dairy cows was 38.46% and from fresh pork was 50.7%. Interestingly, five isolates resisted more than 10 antibiotics, of which four isolates were confirmed as MRSA. The MRSA isolates showed a broader range of antimicrobial resistance than MSSA. This indicates that resistance to methicillin is just the first step of a multidrug resistance process fostered by the great ability of MRSA to evade antibiotic therapy [65].
Among the different subtyping methods available, both MLST and spa typing have shown a significantly clonal population structure for S. aureus. In this study, most genotypes of S. aureus were related to SPF and clinic infection, such as ST1-t127, ST7-t091, ST6-t701 and ST188-t189 [66][67][68][69], which indicates that these strains of these STs have at least a theoretical pathogenic potential. ST15 was the predominant MLST type of isolate in our study. This lineage is not often listed among the major STs found in humans. In previous studies, it was identified in 2/15 healthy ST15 S. aureus gut carriers in a Spanish population [70], and it was one of the commonest STs among nasal S. aureus strains from children in Ghana [71]. In addition, ST188 and ST6 were identified in 11.1% and 6.3% of isolates in this study, respectively. In China, S. aureus ST6 is the predominant lineage isolated from outbreaks in Shenzhen, Xi'an and Ma'anshan, whereas ST188 has been increasingly linked to hospitalassociated infections and community-associated infections, which may be one of the clones that are associated with SFP [68,72,73]. Fortunately, ST15 (28.6%), ST188 (11.1%) or ST6 (6.3%) were free of any SEs (Table 4). Interestingly, all ST1 isolates harbored the sec gene, whereas most ST7 and ST25 isolates harbored the sea or seb genes (Table 4). This showed that different genetic lineages might have different virulence characteristics. In China, Lv et al. (2021) have found that ST59 was the most prevalent type (32.1%, n = 18) in SFP isolates [69]. Interestingly, this type of isolate was the predominant CA-MRSA lineage in Asia [74][75][76]. In our study, we found that ST59-t437 was the most isolated type of MRSA. Furthermore, all the ST59-t437 MRSA isolates displayed a wider range of antimicrobial resistance and contained classical SE genes sea or seb. This suggests that these isolates have a greater likelihood of causing food poisoning.
In summary, our results provide a comprehensive information on the genetic background of shrimp-derived S. aureus in Chinese aquatic samples. In our study, the contamination level of S. aureus isolated from shrimp was significantly higher than that of other studies, indicating an increased potential contamination risk of S. aureus in shrimp products. These isolates were potentially virulent, and nearly half of them carried virulence factors such as enterotoxin genes. The study of drug resistance could help identify the best treatment after a food poisoning incident. All the isolates had moderate or strong biofilm-producing capacity, and most of the isolates had biofilm-related genes, indicating that these potential virulence genes could persist in food production and circulation chains.
In particular, ST15, ST188 and ST6 were found for the first time to be a retained type in shrimp in this study, which should be drawn to public attention.