Molecular Characterization of Rifampicin-Resistant Staphylococcus aureus Isolates from Retail Foods in China

This study investigated the molecular characteristics of rifampin-resistant (RIF-R) Staphylococcus aureus isolates recovered from 4300 retail food samples covering most provincial capitals in China, from 2011 to 2016. Of the 1463 S. aureus enrolled, 149 isolates (142 MSSA and 7 MRSA) were identified as rifampicin-resistant, including 20 high-level (MICs ≥ 8 μg/mL) and 129 low-level (MICs between 2 and 4 μg/mL) rifampicin-resistant strains. Most of the RIF-R S. aureus isolates were resistant to more than three antibiotics. The mutations in the rifampicin resistance-determining region of the rpoB gene were studied in all RIF-R strains. All of the strains presented the mutational change 481 His/Asn and five isolates presented an additional mutation, including 477 Asp/Tyr, 527 Ile/Met, and 466 Leu/Ser, respectively. Thirteen STs and twenty-one spa types were represented, in which five MRSA showed non-type SCCmec and the remaining MRSA belonged to SCCmec type IV—where, ST1-t127 was the predominant type from all of the isolates, while ST398-t034 was the predominant type for the MRSA isolates. In this study, we found that the food-related RIF-R S. aureus may have a unique genetic background selection. However, the scenario regarding the presence of RIF-R S. aureus, especially MRSA, in retail food in China is not favorable and warrants public attention.


Introduction
Staphylococcus aureus is a versatile pathogens capable of causing nosocomial and community-acquired infections [1]. It is one of the most important foodborne microorganisms, for which its pathogenicity affects human's health through staphylococcal enterotoxins (SEs). The first food poisoning event caused by staphylococci occurred in 1884 [2]. Recently, it has been found in food producing chains, for example the equipment used for the production of food and in contaminated ingredients and so on [3,4]. Between 2000 and 2015, S. aureus was one of the most frequently reported pathogens in ready-to-eat (RTE) food (such as cheeses, cured meats, sausages, smoked fishes, salads, and so on) in Africa and Europe [5][6][7]. In China, the prevalence of S. aureus-contaminated retail food increased over time [8].
Rifampicin (RIF) is the most used broad-spectrum antibiotic against bacterial pathogens. It has a potent bactericidal activity, good tissue penetration and modest activity against nongrowing cells [9]. RIF is currently indicated in combination therapy for implant-associated infections, serious S. aureus infections, and has been shown to broaden the coverage of vancomycin-intermediate S. aureus (VISA) when used in combination with linezolid or daptomycin [10]. However, the long-term use of RIF, as well as, its use as a monotherapy, has been shown to be associated with the development of RIF-resistant (RIF-R) S. aureus. Nowadays, many researchers have suggested that the major reason for RIF resistance is the mutations, mainly single nucleotide polymorphisms (SNPs), in the rpoB gene shown in the Staphylococcus species [11][12][13]. One of the first report suggested that rpoB gene mutations involving eight conserved amino acids were identified in RIF-R Mycobacterium tuberculosis. All mutations were clustered within a region of 23 amino acids [14]. This conclusion was further verified in RIF-resistance Staphylococci [11,15]. The β-subunit of the bacterial RNA polymerase, encoded by the rpoB gene, plays an important role in RIF inhibition [11]. In S. aureus, a number of studies have revealed a worrying link between certain rpoB mutations and decreased susceptibility not only to RIF, but also to other last-line anti-MRSA antibiotics such as vancomycin, daptomycin, β-lactams, and imipenem [16][17][18][19][20].
In our previous study, we collected 4300 retail food samples from supermarkets, fairs, and farmers' markets. We covered most of the provincial capitals of China from July 2011 to June 2016 and collected 1463 S. aureus isolates from 1063 positive samples from all of the sampling sites [21,22]. Furthermore, most of S. aureus isolates from retail food showed severe antibiotic resistance [22][23][24][25]. However, there were a limited number of reported studies on RIF-R S. aureus isolated from retail food. In order to better understand the epidemic background and the molecular characters in RIF-R isolates for S. aureus in food, this study re-identified the RIF-R S. aureus in food-related isolates and further studied RIF-R S. aureus in terms of the following: (1) the mutations in the rpoB gene that contributed to RIF resistance, and (2) the antimicrobial resistance profiles and their genotypic types (MLST and spa typing).

Distribution of Rifampicin-Resistant S. aureus from Retail Foods in China
In total, 149 RIF-R S. aureus isolates, including 7 MRSA strains, were re-identified by the disk diffusion method and were used for the present study. The RIF-R S. aureus isolates represented 10.2% of all S. aureus isolates. The origin of the strains was mainly from raw meat, aquatic products, quick-frozen products, and RTE food, including 35 (8.4%) of the 419 isolates from raw meat, 45 (9.5%) of the 473 isolates from aquatic products, 43 (12.0%) of the 358 isolates from quick-frozen products, 22 (16.8%) of the 131 isolates from ready-to-eat food, 1 (2.4%) of the 41 isolates from edible mushrooms, and 3 (25.0%) of the 12 isolates from pasteurized milk, whereas vegetables were free of RIF-R S. aureus isolates ( Table 1). The RIF-R MRSA isolates were distributed in raw meat (n = 4), aquatic products (n = 1), quick-frozen products (n = 1), and RTE food (n = 1). The prevalence of RIF-R MSSA in foods was significantly higher than RIF-R MRSA (p < 0.001, χ 2 test).

Antibiotic Resistance of Rifampicin-Resistant S. aureus Isolates
The antibiotic susceptibility results of 149 RIF-R S. aureus isolates are shown in Table 2. In this study, most of RIF-R S. aureus isolates were resistant to more than three antibiotics, whereas only 9.4% of isolates were resistant to 0-3 antibiotics. For multidrug resistance isolates, 77.9% of isolates were resistant to 4-10 antibiotics, 10.7% of isolates were resistant to 11-15 antibiotics, and 2.0% of isolates were resistant to 16-24 antibiotics. The isolates were susceptible to linezolid and teicoplanin, and the frequencies of resistance to individual agents were 94.6% for ampicillin and 92.6% for penicillin G, followed by 75.8% for kanamycin, 53.7% for erythromycin, 49% for tetracycline, 30.2% for clindamycin, 28.9% for gentamycin, 26.8% for telithromycin, 26.2% for norfloxacin, 24.8% for fusidic acid, 18.1% for ciprofloxacin, 16.1% for streptomycin, 10.7% for chloramphenicol, 6.7% for amikacin, 6.7% for trimethoprim/sulphamethoxazole 1:19, 5.4% for ceftazidime, 4.7% for cefoxitin, 4.0% for cefepime, 3.4% for quinupristin/dalfopristin, and 2.0% for nitrofurantoin. Only seven RIF-R S. aureus isolates belonged to MRSA, whereas most of RIF-R S. aureus isolates were MSSA (Table 1). Interestingly, the RIF-R MRSA isolates showed multi-drug resistance to more than 10 antibiotics in 71.4% of cases (five out of seven isolates). Rates of resistance to AMC, FEP, CAZ, GEN, CHL, CLI, TEL, SXT, and NIT, and intermediate resistance to RIF were all significantly higher in the MRSA group than in the MSSA group (p < 0.01, χ 2 test) ( Table 2). In total, the MDR rate for MRSA isolates was significantly higher than that for MSSA (p < 0.01, χ 2 test).

Rifampicin Resistance Levels and Associated rpoB Mutations
Among the 149 RIF-R S. aureus isolates, 129 isolates showed a low level of RIF resistance (MICs between 2 and 4 µg/mL) and 20 isolates showed a high level of RIF resistance (MICs ≥ 8 µg/mL). In addition, three out of seven MRSA showed a high level of RIF resistance (MIC > 16 µg/mL). The mutations in the RIF resistance-determining region of the rpoB gene were studied in all 149 RIF-R strains. The results are shown in Table 3. All 149 strains presented the mutational change 481 His/Asn, determined by a mutation in cluster I of the rpoB gene, conferring a low-level of RIF resistance. Three additional mutations were found in five isolates, including 477 Asp/Tyr (n = 3), 527 Ile/Met (n = 1), and 466 Leu/Ser (n = 1). Of these, two isolates with MIC ≥ 16 µg/mL had an additional amino acid substitution: 466 Leu/Ser (n = 1) or 527 Ile/Leu (n = 1), associated with a high level of RIF resistance.

Discussion
In recent years, the frequency of the RIF-R S. aureus isolated from clinics in China has increased rapidly, showing that the percentage of RIF-R MRSA isolates was only 15.5% in 2004, but increased rapidly to 50.2% in 2008 [26,27]. However, studies on RIF-R S. aureus isolated from retail food have rarely been reported in China. In this study, a total of 149 food-related S. aureus isolates (10.2%) were identified as RIF-R isolates, which covered all types of animal-derived food, including raw meat, aquatic products, quick-frozen products, and RTE food. Recently,Şanlıbaba P. (2022) collected 96 S. aureus strains from retail raw beef, sheep and lamb meat in Turkey, and 8.33% of strains were found to be resistant to RIF [28]. In Portugal, only one RIF-R S. aureus was detected in various foods [29]. In north-western Greece, only 1.6% RIF-R S. aureus was found in dairy products [30]. Compared with these studies, the prevalence rate of RIF-R S. aureus isolates in food the food we examined in China was not low, which should be brought to public attention.
RIF acts by interacting specifically with the β-subunit of the bacterial RNA polymerase encoded by the rpoB gene [31,32]. Many previous studies have reported that the mutations of rpoB were a response to RIF resistance in bacteria such as Escherichia coli [33], Mycobacterium leprae [34], Streptococcus pneumoniae [35], and Neisseria meningitides [36]. In this study, we also amplified and sequenced the portions of rpoB of RIF-R S. aureus isolates in food. It was found that all strains presented the mutational change 481 His/Asn. In China, Zhou et al. (2012) collected 88 RIF-R MRSA isolates from clinical specimens in the Microbiology Department of Anhui Provincial Hospital in China, and found that the mutation at 481 His/Asn was the most common in 95.5% of RIF-R isolates [37]. Similarly, this is consistent with Liang's research, showing 481 His/Asn mutations of rpoB in all RIF-R or intermediate-resistant isolates from women and children in Guangzhou, China [38]. Interestingly, as described in previous literature, the position of 481 RpoB presents a hot-spot for amino acid residue replacement and has recently been found to be the strongest genetic marker of increased vancomycin resistance [26,39,40]. Therefore, the scenario regarding the presence of RIF-R S. aureus, especially MRSA in retail food in China, should warrant public attention. Moreover, as previously described, a high-level resistance of RIF may also be attributed to additional mutations within rpoB [41]. Isolates containing multiple mutations also showed high-level RIF resistance [42]. According to Zhou et al. (2012), it was found that 87.5% RIF-R S. aureus had an additional mutation 466 Leu/Ser and the RIF resistance level was higher [37]. In our study, fewer strains had additional mutations, such as 527 Ile/Met and 466 Leu/Ser mutations, which was also associated with a higher-level of resistance of RIF. Although the rate of multiple mutations was not high, it should be paid attention.
In this study, it was found that most RIF-R S. aureus isolated from retail food belonged to ST1-t127. Meanwhile, ST398-t034 was the predominant type of MRSA isolate. This is different from the predominant types of food-related MRSA, which, when isolated simultaneously with RIF-R strains, showed that the predominant type was ST59-t437 [22]. According to previous studies, the predominant types of clinical RIF-R strains were ST239 or ST45 [37,38,43,44]. In 2018, Liang et al. collected 131 S. aureus isolated from patients in four centers for women and children in Guangzhou, and found that CC59 (ST59-IV, 48.8%; ST338-III, 35.7%) was a major clone (84.4%) among the MRSA isolates, but 93.8% of the RIF-R or intermediate resistant isolates belonged to ST45 [38]. Thus, the RIF-R S. aureus in the food may have a unique genetic background selection. However, the reasons for this specific selection need to be further explored.
As we know, RIF is an important antibiotic used in combination therapy for the treatment of deep-seated staphylococcal infections [45]. This review also provided evidence that RIF combined with trimethoprim/sulfamethoxazole and minocycline is associated with a high rate of isolation of S. aureus resistance, whereas RIF combined with other agents is associated with lower rates of resistance [46]. Furthermore, a combination therapy, with an antibiotic such as vancomycin, is often required in order to reach deep-seated infections effectively [47]. Thus, resistance to RIF can make the treatment of S. aureus infection, especially MRSA infection, more difficult. Except for RIF, antibiotics such as ampicillin, penicillin, kanamycin, erythromycin, and tetracycline, are common drugs used for S. aureus infection [48]. However, the resistance rates of these antibiotics for RIF-R S. aureus exceeded 50% in this study. Moreover, most of the RIF-R S. aureus isolates were resistant to more than three antibiotics. Of which, 19 RIF-R S. aureus isolates were resistant to more than 10 antibiotics, namely 5 MRSA and 14 MSSA. Interestingly, ST398 MRSA and MSSA (36.8%, 7/19) were the predominant types of multi-drug resistant isolates in RIF-R S. aureus. As we know, MRSA CC398 is a frequent colonizer of humans with occupational exposure to livestock [49]. Recently, infections caused by MSSA ST398 have been described in humans [50][51][52] and it has been shown to cause infections more frequently than MRSA ST398 [21]. In 2011, Argudín et al. studied the resistance determinants of German S. aureus ST398 isolates from non-human sources, and found that none of the isolates were resistant to RIF [53]. Thus, further investigation is required to elucidate the emergence of multidrugresistant ST398 in RIF-R S. aureus.

Bacterial Strains
A total of 1463 S. aureus were isolated from 4300 retail food products between July 2011 and June 2016. The samples were collected from supermarkets, fairs, and farmers' markets, which covered most of the provincial capitals of China in 39 cities ( Figure S1). One hundred samples were collected from each city, except for Guangzhou, where 500 samples were collected. These strains were isolated from seven types of food products, including 419 isolates from meat and meat products (bacon/sausage, poultry, pork, mutton, and beef), 473 isolates from aquatic products (freshwater fish, shrimp, and seafood), 358 isolates from quick-frozen products (frozen dumplings/steamed stuffed buns and frozen meat), 131 isolates from ready-to-eat (RTE) food (cold vegetable/noodle dishes in sauce, fried rice/sushi, roast meat, sausage, and ham), 41 isolates from edible mushrooms, 29 isolates from vegetables, and 12 isolates from pasteurized milk. These isolates were obtained according to the GB 4789.10-2010 food microbiological examination of S. aureus (National Food Safety Standards of China) and the most probable number (MPN) method [54]. The S. aureus isolates were streaked onto chromogenic S. aureus agar plates (Huankai) and were re-identified by Gram's staining, microscopic examination, coagulase testing, and catalase testing. MRSA was initially screened by the cefoxitin disk diffusion method, and then confirmed by polymerase chain reaction (PCR) detecting mecA.

Detection of Rifampicin Resistance-Associated Mutations
All of the isolates were incubated at 37 • C overnight on BHI (brain heart infusion broth). Genomic DNA was extracted using a Genomic DNA Extraction Kit (Magen Biotech, Guangzhou, China) according to the manufacturer's instructions. The concentration of the genomic DNA was determined at 260 nm using a NanoDrop ND-1000 UV−VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). An internal sequence of gene rpoB of 432 bp (nucleotides 1216 to 1648) was amplified by PCR. This region included the RIF resistance determining cluster I (nucleotides 1384-1464, amino acid number 462-488) and cluster II (nucleotides 1543-1590, amino acid number 515-530). The amplification was carried out in 149 RIF-R strains. Amplification was carried out as previously described [56]. The PCR products were purified and analyzed by DNA sequencing. The nucleotide sequences obtained were compared to the rpoB wild type sequence from S. aureus subsp. aureus (GenBank accession number: X64172) using the clustalw software (http://www.ebi.ac.uk/tools/clustalw/index.html, accessed 5 June 2019).

spa-Typing
Sequence typing of the S. aureus protein A (spa) repeat region was amplified according to a published protocol [57]. All isolates were analyzed using the primers spa-1113f (5 -TAAAGACGATCCTTCGGTGAGC-3 ) and spa-1514r (5 -CAGCAGTAGTGCCGTTTG CTT-3 ). The PCR amplification conditions were as follows: an initial cycle of 80 • C for 5 min; 35 cycles of 94 • C for 45 s, 60 • C for 45 s, 72 • C for 2 min, and a final extension at 72 • C for 10 min. The spa types were randomly assigned using the Spa Server website (http://spaserver2.ridom.de, accessed 10 July 2019).

Multilocus Sequencing Typing
The MLST scheme used to characterize S. aureus isolates is based on the sequence analysis of the following seven housekeeping genes: arcC (Carbamate kinase), aroE (Shikimate dehydrogenase), glpF (Glycerol kinase), gmk (Guanylate kinase), pta (Phosphate acetyltransferase), tpi (Triosephosphate isomerase), and yqil (Acetyle coenzyme A acetyltransferase) [58]. The PCR amplification conditions were as follows: an initial cycle of 94 • C for 5 min, 35 cycles of 94 • C for 30 s, 55 • C for 30 s, 72 • C for 2 min, and a final extension at 72 • C for 10 min. The DNA fragments were purified using a PCR purification kit (Qiagen, Hilden, Germany) and were sequenced in each direction with Big Dye fluorescent terminators on an ABI 3730XL sequencer (Applied BioSystems, Foster, USA). For each MLST locus, an allele number was given to each distinct sequence variant, and a distinct sequence type (ST) number was attributed to each distinct combination of alleles at the seven genes. Sequence types (STs) were determined by using the Staphylococcus aureus MLST database (https://pubmlst.org/saureus/, accessed 20 July 2019). Sequence Type Analysis and Re-combinational Tests software (S.T.A.R.T. ver.2; http://pubmlst.org/software/analysis/ start2, accessed 25 July 2019) was used to analyze the data of MLST.

SCCmec Typing
The SCCmec typing method was performed on the isolates by multiplex PCR, as previously described [59]. The PCR amplification conditions had an initial denaturation step at 94 • C for 5 min, followed by 10 cycles of 94 • C for 45 s, 65 • C for 45 s, and 72 • C for 1.5 min and another 25 cycles of 94 • C for 45 s, 55 • C for 45 s, and 72 • C for 1.5 min, ending with a final extension step at 72 • C for 10 min and followed by a hold at 4 • C. All PCR assay runs incorporated a reagent control (without template DNA). The PCR amplicons were visualized using a UV light box after electrophoresis on a 2% agarose gel containing 0.5 g/mL ethidium bromide.

Statistical Analysis
Statistical analyses were performed using SPSS 17.0 Data Editor (SPSS, Inc., Chicago, IL, USA). Categorical variables were described using frequencies and their proportion, and were compared using the Chi-square (χ 2 ) test. p < 0.05 was considered to indicate a statistically significant difference.

Conclusions
In summary, we investigated food-related RIF-R S. aureus and determined its genetic background in China in this study. RIF-R S. aureus isolates were observed in most animalderived types of food samples and rare in other types of food. Most of the RIF-R S. aureus isolates showed multiple resistance and the reason for RIF resistance in S. aureus is closely associated with the mutations that occur in the rpoB gene. This study indicated that the RIF-R S. aureus in food may have a unique genetic background selection. However, monitoring of the emergence and possible dissemination of the MRSA ST398 strains among food-related infections for RIF treatment is warranted.