Occurrence and Patterns of Enterotoxin Genes, spa Types and Antimicrobial Resistance Patterns in Staphylococcus aureus in Food and Food Contact Surfaces in Singapore

1. Introduction

Staphylococcus aureus has been recognized as a ubiquitous pathogen responsible for Staphylococcal food poisoning (SFP), a gastrointestinal intoxication resulting from the ingestion of food contaminated by enterotoxigenic S. aureus [1]. While S. aureus does not form spores, their opportunistic nature encourages growth in a wide range of temperatures (7 to 48.5 °C) and pHs (4.2 to 9.3) [2]. These characteristics promote the growth and spread of S. aureus in many food products, especially meat and meat products, poultry and egg products, unpasteurized milk, and dairy products [3]. After contamination, improper storage conditions and poor hygiene practices accelerate the growth of S. aureus, allowing it to reach the cell density necessary for enterotoxin production. Hence, it is crucial that proper hygiene standards are adhered to during food processing and storage to minimize the spread and growth of the pathogen and its enterotoxins throughout the food processing chain.

Staphylococcal enterotoxins (SEs) produced by coagulase-positive staphylococci are the main causatives agents of SFP. SEs are resistant to heat, proteolytic enzymes, and other environmental conditions [4]. Due to their stable nature, the detection of SEs is a definite method for the confirmation of outbreaks and the enterotoxigenicity of strains. There are over 20 S. aureus enterotoxins identified. Based on serological classification, they are grouped as classical genes and non-classical genes (new SEs). Classical genes are the top five predominant enterotoxins (sea, seb, sec, sed, and see) that are highly isolated from outbreaks of SFP in more than 90% of cases, while non-classical genes are new enterotoxins that were isolated from 5% of cases [5]. Due to the stable properties of SEs and the low dose required to cause symptoms, consumption of food contaminated with enterotoxigenic S. aureus can easily lead to foodborne outbreaks. There are several reported SFP outbreaks across Asia [6,7,8], though none were reported in Singapore. Nonetheless, contamination of S. aureus with food and food contact surfaces is a public health concern given its virulent and antimicrobial-resistant properties worldwide.

Molecular typing is a useful tool to understand clonal relatedness, genetic diversity, and the spread of pathogens [9]. There are several molecular methods to identify enterotoxigenic S. aureus strains, including pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), whole genome sequencing (WGS), and spa typing. In this study, spa typing was adopted. Spa typing is a single-locus typing method to analyze the highly variable X region of the protein A gene in S. aureus strains. The X region consists of 24-bp tandem repeats flanked by well-conserved regions. Repeats are assigned a unique repeat code, and the spa type of a given strain is derived from the number of tandem repeats and the sequence variation in the X region. Due to its discriminatory power to identify strains based on a single-locus DNA sequence-based marker in the presence of polymorphisms, this method proved to be as effective as other typing methods, including PFGE and MLST [10]. It is also most cost-effective, less time-consuming, and less error-prone compared to other molecular methods [9,11]. While WGS can differentiate closely related strains with greater sensitivity as compared to spa typing, the spa gene is composed of highly variable and similar repeats, which could pose a challenge for WGS since repeated sequences can be misassembled [12]. Since the objective of our study is to understand if there is a possible transfer of S. aureus from food handlers to food, the use of spa typing in our study would be sufficient and more cost-efficient compared to WGS, so spa typing is adopted in this study.

Over the past decades, the increasing use of antibiotics in animal and human medicine has led to an increasing public health concern about antibiotic resistance in pathogens, including S. aureus, [4]. S. aureus also colonizes animals, and transmission between humans and animals has been reported [1]. Once humans acquire it, community transmission is possible. With the increasing use of antibiotics in animals, the emergence of antimicrobial resistance and increasing virulence would be a public health concern [1]. Although SFP is mostly self-limiting in healthy adults, treatment with antimicrobials is necessary for invasive and immunocompromised individuals [13], and the presence of resistance traits can render corresponding antimicrobials ineffective in treating the infection or intoxication, resulting in serious public health issues [13].

To the best of our knowledge, limited research has been done in Singapore on S. aureus in the food chain and its implications for food safety with regards to antimicrobial resistance and enterotoxigenicity. Additionally, SFP is not a notifiable disease in Singapore. Therefore, the data on the incidence of SFP in the population is limited. Hence, the objectives of this study are to evaluate the occurrence and prevalence of S. aureus strains in food and food contact surfaces in Singapore and to examine the antimicrobial susceptibility pattern of these strains. Through an understanding of the molecular epidemiology of these strains in retail food and food handlers, findings from this study will be useful to inform public health and mitigation measures at the retail level, such as good food handling practices among food handlers, and strengthen future surveillance and epidemiological studies.

2. Materials and Methods

2.1. Sample Collection, Isolation and Identification of S. aureus

A total of 1540 retail food and food contact surface samples were obtained from food surveillance and risk assessment studies conducted by the National Environmental Agency between 2009 and 2013.

A ten-gram sample of each food item was placed in a sterile stomacher bag and homogenized with 90 g of Universal Pre-enrichment Broth (UPB) (Acumedia Manufacturers, Lansing, MI, USA) using a stomacher (Seward Stomacher^® 400 Circulator, Seward, West Sussex, UK) at 230 rpm for 30 s. Serial 10-fold dilutions were prepared using 9 mL of Butterfield’s buffer (3M, St. Paul, MN, USA). For the detection of S. aureus, 1 mL of a 10-fold diluted sample was equally distributed between two plates of Baird-Parker agar (Oxoid, Basingstoke, Hants, UK) before incubation at 37 °C for 48 h. Presumptive S. aureus colonies (grey-black colonies with a narrow white margin surrounded by a zone of clearing) were tested for a catalase reaction using 3% hydrogen peroxide (ICM Pharma, Singapore) and confirmed using coagulase rabbit plasma (Remel, Haverhill, MA, USA).

2.2. Detection and Isolation of SE Genes

The detection of SE genes was performed using the following method [14,15,16,17].

DNA was extracted from pure S. aureus colonies grown on Tryptic Soy Agar plates with 5% sheep blood (Acumedia, Baltimore, MD, USA) using the QIAamp^® DNA Mini Kit (Qiagen, Hidden, Germany). Multiplex and singleplex PCR assays were performed to detect virulence genes (sea, seb, sec, sed, see, seg, seh, sei, sej, and sel) and the mecA gene characteristic of methicillin-resistant S. aureus (MRSA).

PCR master mixes were prepared as shown below . Each PCR mix (45 μL) consists of 5× Phusion High-Fidelity Buffer (Thermo Scientific, Vilnius, Lithuania), dNTP mix (1st BASE, Seri Kembangan, Malaysia), 10 μM of each primer (Integrated DNA Technologies, Singapore) , Phusion Hot Start II DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), DNA template, and molecular-grade water. S. aureus 29213, S. aureus 43300, S. aureus ATCC^® 13565, S. aureus ATCC^® 14458, S. aureus ATCC^® 23235, S. aureus ATCC^® 27664, S. aureus ATCC^® 19095, and BAA S. aureus ATCC^® 1761 were used as positive controls, while molecular-grade water was used as a negative control.

Amplification using multiplex PCR was conducted using the following parameters: initial denaturation of the strand at 98 °C for 30 s, followed by 30 cycles of denaturation at 98 °C for 10; annealing at 61 °C for 30 s; extension at 72 °C for 30 s; and final extension for 10 min at 72 °C. For amplification using singleplex PCR, the following parameters were used: initial denaturation of the strand at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 10 s; annealing at 57 °C for 30 s; extension at 72 °C for 30 s; and final extension for 10 min at 72 °C. PCR-positive MSSA samples were confirmed with a latex agglutination test (PBP2) (Oxoid) and a cefoxitin disc (Oxoid) using the disc diffusion method following Clinical and Laboratory Standards Institute (CLSI) guidelines [18,19].

The amplified products were visualized using gel electrophoresis on a 1.5% agarose gel for multiplex PCR 1, singleplex PCR 1 and 2, and a 2% agarose gel for multiplex PCR 2. Detectable PCR bands were confirmed to contain the virulence genes.

2.3. Spa Typing

The spa typing for the S. aureus isolates was performed using the following method [20,21,22,23].

The X region of the protein A gene was amplified using PCR with four primer sets: 1113f and 1514r; 1095f and 1517r; 1084f and 1618r; and 238f and 1717r. If no PCR amplification was detected with one of the primer sets, the other three sets were used for PCR amplification instead.

The PCR mix for spa typing consists of 10 µL of 5× Phusion High-Fidelity Buffer (Thermo Scientific, Vilnius, Lithuania), 1 µL of dNTP mix (10 mM) (1st BASE, Seri Kembangan, Malaysia), 0.5 µL of each forward and reverse primer (Integrated DNA Technologies, Singapore), 0.5 µL of Phusion Hot Start II DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), and 32.5 µL of molecular-grade water. To each PCR mix, 5 µL of DNA template was added.

Amplification was conducted using the following parameters: For primers 1113f and 1514r, initial denaturation of the strand at 98 °C for 30 s is followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 61 °C for 30 s, extension at 72 °C for 30 s, and final extension for 10 min at 72 °C. For primers 1095f and 1517r, initial denaturation of the strand at 98 °C for 30 s is followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 45 °C for 30 s, extension at 72 °C for 30 s, and final extension for 10 min at 72 °C. For primers 1084f and 1618r, 238f and 1717r, initial denaturation of the strand at 98 °C for 30 s is followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and final extension for 10 min at 72 °C. The amplified products were visualized using gel electrophoresis on 1.5% agarose gel. Detectable PCR bands were confirmed to contain the spa gene.

PCR products were purified using the QIAquick^® PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced by capillary electrophoresis using BigDye Terminator chemistry (AIT Biotech, Singapore). Sequences were analyzed using BioNumerics v7.6 to determine spa types.

2.4. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility for S. aureus isolates was determined by the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guideline [18]. All the antibiotics used to determine antimicrobial resistance were grouped into nine classes: Aminoglycosides, Beta-lactams, Cephalosporins, Chloramphenicols, Fluoroquinolones, Glycopeptides, Macrolides, Sulphonamides and Tetracyclines. The antimicrobial agents used were Ciprofloxacin (CIP5), Norfloxacin (NOR10), Amikacin (AK30), Ampicillin (AMP10), Gentamicin (CN10), Tetracycline (TE30), Ceftriaxone (CRO30), Amoxycillin/Clavulanic acid (AMC30), Sulphamethoxazole/Trimethoprim (SXT25), Chloramphenicol (C30), Azithromycin (AZM15), Penicillin G (P10), Vancomycin (VA30), Cefoxitin (FOX30), and Rifampicin (RD5). The zone diameter breakpoints used were obtained from the CLSI standards [18]. Staphylococcus aureus ATCC^® 25923 was used as the quality control strain, while sterile water was used as a negative control.

2.5. Statistical Analysis

All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, LLC, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Non-random associations between categorical variables (spa type and SE gene) were determined using the Fisher’s exact test. Cluster analysis was performed on BioNumerics v7.6 and a maximum distance of 2 was used to determine closely related spa types in the same cluster.

The 95% confidence intervals of proportions were calculated using http://vassarstats.net/prop1.html (accessed on 23 March 2023) Z-scores for two population proportions were calculated using https://www.socscistatistics.com/tests/ztest/default2.aspx (accessed on 1 April 2023).

3. Results

3.1. Occurrences and Distribution of SE Genes

The prevalence of S. aureus in food and food contact surfaces is 15.4% (237/1540 samples). All 237 S. aureus isolates were determined to be methicillin-susceptible S. aureus (MSSA). Of the 237 S. aureus isolates tested, 181 could be typed using spa sequencing. The remaining strains that could not be typed were excluded from the analysis. The frequency of isolates from food and food contact surfaces are shown in .

Of the 181 isolates, 45 (24.9%) [95% CI: 19.1–31.6%] were detected with at least one of the SE genes. A total of 96 SE genes were detected among these 45 isolates.

The occurrence of each SE gene across all isolates is shown in below.

The most common classical SE gene is the sea gene, while the most common non-classical genes are seg and sei. In total, seg and sei genes had the highest occurrence (25/96) (26.0%) across all SE genes. sed and see genes were not detected in any of the isolates tested in this study.

The predominance of classical SE genes was categorized based on their food and food contact surface categories, as shown in Figure 1 below. The sea, seb and sec genes were predominantly found in hand swabs (6/48), meat dishes (3/17) and seafood dishes (2/26) respectively.

Figure 1. Distribution of (a) the sea gene; (b) the seb gene; and (c) the sec gene across categories of food and food contact surface.

3.2. Occurrence and Distribution of Antimicrobial Resistance (AMR) in Food

The percentage of antimicrobial resistance in S. aureus isolates are shown in . All 181 S. aureus isolates were susceptible to ceftriaxone, cefoxitin, chloramphenicol, rifampicin, sulfamethoxazole/trimethoprim, and vancomycin. The occurrence of resistance was 54.7% against ampicillin (99/181), 54.7% against penicillin G (99/181), 14.9% against tetracycline (27/181), and 8.8% against azithromycin (16/181).

shows the classification of antimicrobial agents in different food and food contact surface categories. Noticeably, isolates that were resistant to ampicillin were resistant to penicillin G across all categories. In addition, resistance to tetracycline and azithromycin was observed in most of the categories.

3.3. Distribution of Spa Types

Figure 2 shows the distribution of spa types for the S. aureus isolates. All 181 S. aureus isolates were classified under 39 spa types. The top six predominant spa types were t5078 (73/181, 40.3%), t084 (19/181, 10.5%), t5521 (11/181, 6.1%), t189 (10/181, 5.5%), t6675 (9/181, 5.0%), and t127 (6/181, 3.3%). These spa types were analyzed for the presence of SE genes, as shown in .

Figure 2. Distribution of spa types across all isolates.

The most predominant classical SE gene, sea, was observed to have the highest proportion in t5521, representing 73% (11/15) of all sea-positive isolates. The associations between spa type t5521 and the presence of the sea gene (p < 0.0001) and between spa type t127 and the sea gene (p = 0.0138) were determined to be statistically significant using Fisher’s exact test.

A minimum-spanning tree was constructed to perform spa clustering analysis for all isolates (Figure 3). Clusters were arbitrarily assigned to clustering complexes spa CC01 to spa CC04, according to the four definitive clusters observed. Spa types were partitioned into complexes when the distance between connected nodes was ≤2.

Figure 3. Minimum-spanning tree of spa types from the 181 isolates.

4. Discussion

4.1. Overall Occurrence of SE Genes

In this present study, 24.9% (45/181) of the isolates were detected for the presence of at least one SE gene. The incidence of S. aureus detected with at least one or more SE genes in this present study (24.9%) was relatively lower than that reported in other countries, such as Korea (48.0%), China (54.4%), and Italy (55.5%) [24,25,26], which was expected. One of the most common foods associated with SFP is milk and dairy products [3]. In Singapore, only heat-treated milk is permitted to be sold for direct human consumption [27], hence there is a lower risk of S. aureus contamination as compared to raw milk, which could be a possible reason for the lower incidence compared to other countries. Another possible reason is that food handlers in Singapore are required to undergo a compulsory food safety course to equip them with the basic hygiene knowledge required for handling food, resulting in a lower incidence of S. aureus. The lower incidence of S. aureus with SE genes also correlates with the occurrence of SFP outbreaks, as Singapore has no known reported SFP outbreaks compared to other Asian countries [6,7,8].

Among the ten SE genes tested, the classical SE gene sea (18.8%, 18/96) and the non-classical SE genes sej (26.0%, 25/96) and sei (26.0%, 25/96) were detected at the highest frequencies. For the classical SE gene sea, observation was similar to studies conducted in other countries, such as Taiwan (29.9%) and Iran (25.5%), where the sea gene was most prevalent among the classical genes [5,28]. The sea gene was most commonly isolated in cases of SFP and was frequently isolated in SFP outbreaks in Japan and the United States [3,8]. Enterotoxins sea and seb are known to cause approximately 90% of staphylococcal food poisoning worldwide [29]. The presence of enterotoxin genes in these isolates suggests the isolates’ potential to produce toxins under favorable conditions and cause staphylococcal food poisoning (SFP) if allowed to grow in large numbers in food.

In contrast, several other studies by Hait et al. [30] and Tang et al. [31] have found non-classical genes to be the most predominant genes detected in the isolates investigated. Non-classical genes are new types of genes that have lower expression than classical genes. A non-classical sei gene has been detected in food poisoning-associated S. aureus isolates in Switzerland, the United Kingdom, and Japan [32,33,34]. However, despite the presence of sei genes in these isolates, it remains an open question whether these isolates have produced enterotoxins in sufficient amounts in food to cause SFP. The detection of enterotoxin sei in food should be explored further to make an accurate association between the sei gene and its ability to cause food poisoning.

4.2. Occurrence of SE Genes according to Food and Food Contact Surface Category

Due to the widespread occurrence of classical genes among SFP outbreaks [5], this study will focus on the comparison of the occurrence of classical SE genes across food and food contact surfaces. Of the food and food contact surfaces, hand swabs had the highest incidence of the sea gene (12.5%, 6/48). This is similar to studies conducted in Brazil and Japan, where high occurrences of the sea gene were detected in hand swab samples [35,36]. Without proper hygiene practices, such as wearing gloves during food preparation, S. aureus can be transmitted from human skin to food. This suggests that food handlers without proper hygiene care may increase the risk of contamination in food, as they act as vectors for the spread of enterotoxigenic S. aureus to food [2,35,37], which increases the risk of consumers consuming food contaminated with enterotoxigenic S. aureus. Contamination by food handlers contributes significantly to food poisoning outbreaks. In the United States, 42% of outbreaks between 1975 and 1988 were attributed to contamination by food handlers [2].

Of the food and food contact surfaces, meat dishes had the highest incidence of enterotoxin seb (21.4%, 3/14). Previous studies have reported few or no detections of the seb gene in S. aureus isolates in retail meat samples [38,39], which is interesting to note as the seb gene is directly associated with human contamination [40]. Meat dishes collected in this study could be more susceptible to human contamination, as the dishes, including chicken rice and duck rice, involve post-cooking manipulation, such as cutting and shifting the meat from chopping board to plate. Potential contamination sources include cutting boards, knives, or improper hygiene practices by food handlers [24]. Similar to the sea gene, the seb gene has remarkable stability against heat and proteolytic digestion [29,41,42]. Contamination of food with the seb gene in suitable numbers could result in severe food poisoning as well [19]. The occurrence of the sec gene among S. aureus isolates was highest in seafood dishes (7.7%, 2/26). Similar findings were reported in a study where 12.5% (1/8) of fish products were contaminated with the sec gene [26].

The presence of enterotoxin genes in S. aureus isolates is not necessarily a definitive indication of protein expression in these genes, as these genes may be non-functional or silent due to point mutations [43]. In addition, the level of enterotoxin production is dependent on other factors, including pH, water activity, temperature, and other parameters [34]. Knowledge on the occurrence of enterotoxin genes in this study, therefore, does not reflect the true enterotoxigenic potential of the S. aureus isolates. This limitation calls for greater research into the expression of genes in isolates retrieved from food to inform exposure and quantitative microbiological risk assessment (QMRA). Nonetheless, the presence of S. aureus strains with multiple enterotoxin genes still presents a threat to public health with respect to the consumption of contaminated food and contamination by food handlers.

4.3. Distribution of Spa Types

Molecular characterization by spa typing revealed a wide genetic diversity with the identification of 39 spa types among all the food and food contact surface isolates, with spa type t5078 as the most prominent (40.3%, 73/181), followed by type t084 (10.5%, 19/181), t5521 (6.1%, 11/181), t189 (5.5%, 10/181), t6675 (5.0%, 9/181), and t127 (3.3%, 6/181). While there are several studies associating spa types with food and food contact surfaces [44,45,46], the spa types identified in this current study were more associated with patients and human blood isolates than food or food-related isolates.

Spa type t5078 has been linked to MSSA isolates isolated from patients in different countries. In Singapore, spa type t5078 was discovered in a MSSA isolate that was detected on an infected indwelling graft in a patient suffering from chronic renal failure [47]. In Taiwan, spa type t5078 was isolated from blood samples from patients, which were then discovered to be MSSA isolates [48]. According to Tunsjø et al. [49], S. aureus shares similar virulence genes, pathogenicity islands, and bacteriophages with S. argenteus. This is consistent with a study by Aung et al. [50], where 50% (12/24) of S. argenteus isolates were classified into spa type t5078 and other spa types with similar repeat profiles to t5078. Spa type t084 was found to be the most predominant spa type among MSSA isolates in a children’s hospital in Poland and in the United States, with reports of invasive infections and being present in healthcare-associated and community-onset infections [51,52]. In another study, spa type t084 was also one of the predominant spa types among MSSA isolates among healthcare workers and patients [53]. The third predominant spa type, t5521 (6.1%, 11/181), was not actively studied in many countries. In a study conducted by Uhlemann et al. [54], t5521 was identified as one of eight new spa types isolated from S. aureus isolates from patients in Martinique. However, as t5521 is a relatively new spa type, no further extensive research was conducted.

Spa type t127 was also associated with an MSSA outbreak caused by ice-cream in Germany, with a high concomitance with the sea, in concordance with the results of this study (p = 0.0138) [55]. This links spa type t127 to potential food poisoning events. However, the statistically significant association (p < 0.0001) between spa type t5521 and sea in this present study has not been reported in other studies to the best of our knowledge. Further research is recommended to validate the statistical associations, which can aid in surveillance and epidemiological analysis of S. aureus infections and SFP outbreaks [56].

One limitation of spa typing in this study was the high proportion of non-typable spa types (23.6%), either due to the low quality of tandem repeats or no sequence generated. Future studies could consider using WGS to evaluate the reliability of spa typing by PCR. Spa-typing has been effective in distinguishing S. aureus from various sources, which will be relevant and useful for the epidemiological determination of food sources in outbreak investigations. Although the S. aureus strains in this study were isolated from surveillance and risk assessment studies and not from outbreak investigations, studying the genetic patterns of S. aureus isolates in food and food contact surfaces will be useful to understand the molecular epidemiology of these isolates, which will be useful in cases of improper hygiene practices or food handling during food production and storage.

4.4. General Antimicrobial Resistance Patterns

In this study, resistance to beta-lactams, specifically penicillin G and ampicillin, was observed at the highest frequency (54.7%, 99/181). The results are in agreement with other reports regarding the resistance of S. aureus detected in food to penicillin G in the United States (67.4%), Kuwait (82.0%), China (83.7%), and Western Algeria (60.8%) [57,58,59,60]. Notably, ampicillin and penicillin G resistance occurred at the same frequency (54.7%), similar to the results observed in bovine milk samples in China (91.4%) [61] and MSSA isolates in Trinidad and Tobago (11%) [62]. Penicillin resistance through beta-lactamase is conferred by the blaZ gene, which can be chromosomal or plasmid-encoded [63,64]. Furthermore, the spread of Penicillin G resistance occurs with the spread of resistant strains of S. aureus, where food could act as a vector [63,65]. However, as many clinically relevant S. aureus strains do possess beta-lactamase functions [66], penicillin is unlikely to be used for treatment of SFP, and thus the high resistance to both penicillin G and ampicillin in most sample categories identified in this study could be inherent. However, this study showed limited resistance to amoxycillin/clavulanic acid (0.6%), perhaps owing to the beta-lactamase inhibition activity of clavulanic acid [67]. Therefore, it could be postulated that beta-lactams are resistant. S. aureus isolates in this study were likely due to the presence of beta-lactamase activity.

The findings from this current study also showed that tetracycline resistance was high (27/181, 14.9%). Other studies in the United States (56.4%) and China (24.4%) have reported varied resistance to tetracycline [57,68,69]. The varied resistance to tetracycline in different countries could be explained using the varying usage of tetracycline in animal feeds, and the treatment of bacterial infections in plants, agriculture, and human medicine [70]. While tetracycline resistance in this study is high, compared to other antimicrobial agents tested, the frequency is still considerably low compared to other countries and thus should not be a cause for concern.

To date, there is a limited understanding of the transmission of antimicrobial-resistant S. aureus through food and food-contact surfaces. Food provides a conducive environment for the growth of bacteria. In addition, food chains are important in the spread of antimicrobial resistance between food and the environment [71,72]. These suggest that ready-to-eat food and food contact surfaces can be potential environmental sources for the colonization and circulation of antimicrobial-resistant S. aureus in the community [37,71]. Antimicrobial resistant S. aureus will not be a food safety concern if enterotoxin genes are not expressed and allowed to grow in sufficient numbers in food. However, the consumption of food contaminated with enterotoxigenic S. aureus with antimicrobial resistance could pose a serious food safety and public health risk [59]. In addition, antimicrobial-resistant S. aureus in food could contribute to a larger part of the environmental resistome. Hence, it is crucial to monitor the antimicrobial resistance and enterotoxigenicity of MSSA in retail food to understand epidemiological changes and develop strategies to prevent the contamination of the pathogen in food.

4.5. Antimicrobial Resistance Patterns according to Food and Food Contact Surface Category

The results indicated that tetracycline resistant S. aureus was high in bread products (3/5, 60%). This was reported in China as well (23.3%) [59]. Studies conducted in other countries have shown the possibility of associating antimicrobial resistance with a particular type of food, such as in Iran, where chloramphenicol resistance was identified in food products made from poultry meat, which correlated to the use of chloramphenicol to treat infections in poultry [73]. Due to the limited availability of an equal number of isolates across different sample categories, this study did not have the chance to show that a particular food or food contact surface category was at increased risk of acting as a vehicle for antimicrobial transmission. More data and larger sample sizes are required to calculate risk ratios and draw conclusions about whether an association between antimicrobial resistance and food or contact surfaces is causal in nature.

5. Conclusions

In conclusion, this study analyzed the patterns of SE genes, spa types, and antimicrobial resistance of S. aureus in food and food contact surface samples. This study revealed the occurrence of antimicrobial-resistant or enterotoxigenic S. aureus in food and food contact surface samples, suggesting that food or food contact surfaces can be potential vehicles for spreading S. aureus. Hence, there is a need for constant monitoring of food hygiene. In addition, findings from this study offer epidemiological insights to inform future surveillance and quantitative microbiological risk assessment.

References

1. Bencardino, D.; Amagliani, G.; Brandi, G. Carriage of Staphylococcus aureus among food handlers: An ongoing challenge in public health. Food Control 2021, 130, 108362. [Google Scholar] [CrossRef]
2. Kadariya, J.; Smith, T.C.; Thapaliya, D. Staphylococcus aureus and staphylococcal food-borne disease: An ongoing challenge in public health. BioMed Res. Int. 2014, 2014, 827965. [Google Scholar] [CrossRef] [PubMed]
3. Argudín, Á.M.; María, M.C.; María, R.R. Food poisoning and Staphylococcus aureus enterotoxins. Toxins 2010, 2, 1751–1773. [Google Scholar] [CrossRef] [PubMed]
4. Shanehbandi, D.; Baradaran, B.; Sadigh-Eteghad, S.; Zarredar, H. Occurrence of methicillin resistant and enterotoxigenic Staphylococcus aureus in traditional cheeses in the north west of Iran. Natl. Sch. Res. Not. 2014, 2014, 1–5. [Google Scholar] [CrossRef] [PubMed]
5. Mashouf, R.Y.; Hosseini, S.M.; Mousavi, S.M.; Arabestani, M.R. Prevalence of enterotoxin genes and antibacterial susceptibility pattern of Staphylococcus aureus strains isolated from animal originated foods in West of Iran. Oman Med. J. 2015, 30, 283. [Google Scholar] [CrossRef]
6. Thaikruea, L.; Pataraarechachai, J.; Savanpunyalert, P.; Naluponjiragul, U. An unusual outbreak of food poisoning. Southeast Asian J. Trop. Med. Public Health 1995, 26, 78–85. [Google Scholar]
7. Guo, Y.; Yu, X.; Wang, J.; Hua, D.; You, Y.; Wu, Q.; Ji, Q.; Zhang, J.; Li, L.; Hu, Y. A food poisoning caused by ST7 Staphylococcal aureus harboring sea gene in Hainan province, China. Front. Microbiol. 2023, 14, 1110720. [Google Scholar] [CrossRef]
8. Asao, T.; Kumeda, Y.; Kawai, T.; Shibata, T.; Oda, H.; Haruki, K.; Nakazawa, H.; Kozaki, S. An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: Estimation of enterotoxin A in the incriminated milk and powdered skim milk. Epidemiology Infect. 2003, 130, 33. [Google Scholar] [CrossRef]
9. Oliveira, R.; Pinho, E.; Almeida, G.; Azevedo, N.F.; Almeida, C. Prevalence and diversity of Staphylococcus aureus and staphylococcal enterotoxins in raw milk from Northern Portugal. Front. Microbiol. 2022, 13, 703. [Google Scholar] [CrossRef]
10. Oliveira, D.C.; Tomasz, A.; de Lencastre, H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: Identification of two ancestral genetic backgrounds and the associated mec elements. Microb. Drug Resist. 2001, 7, 349–361. [Google Scholar] [CrossRef]
11. Peacock, S.J.; De Silva, G.D.I.; Justice, A.; Cowland, A.; Moore, C.E.; Winearls, C.G.; Day, N.P.J. Comparison of multilocus sequence typing and pulsed-field gel electrophoresis as tools for typing Staphylococcus aureus isolates in a microepidemiological setting. J. Clin. Microbiol. 2002, 40, 3764–3770. [Google Scholar] [CrossRef] [PubMed]
12. Salzberg, S.L.; Yorke, J.A. Beware of mis-assembled genomes. Bioinformatics 2005, 21, 4320–4321. [Google Scholar] [CrossRef] [PubMed]
13. Sergelidis, D.; Angelidis, A.S. Methicillin-resistant Staphylococcus aureus: A controversial food-borne pathogen. Lett. Appl. Microbiol. 2017, 64, 409–418. [Google Scholar] [CrossRef]
14. Cremonesi, P.; Luzzana, M.; Brasca, M.; Morandi, S.; Lodi, R.; Vimercati, C.; Agnellini, D.; Caramenti, G.; Moroni, P.; Castiglioni, B. Development of a multiplex PCR assay for the identification of Staphylococcus aureus enterotoxigenic strains isolated from milk and dairy products. Mol. Cell. Probes 2005, 19, 299–305. [Google Scholar] [CrossRef] [PubMed]
15. Rosec, J.P.; Gigaud, O. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. Int. J. Food Microbiol. 2002, 77, 61–70. [Google Scholar] [CrossRef]
16. Strommenger, B.; Kettlitz, C.; Werner, G.; Witte, W. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J. Clin. Microbiol. 2003, 41, 4089–4094. [Google Scholar] [CrossRef]
17. Veras, J.F.; do Carmo, L.S.; Tong, L.C.; Shupp, J.W.; Cummings, C.; Dos Santos, D.A.; Cerqueira, M.M.O.P.; Cantini, A.; Nicoli, J.R.; Jett, M. A study of the enterotoxigenicity of coagulase-negative and coagulase-positive staphylococcal isolates from food poisoning outbreaks in Minas Gerais, Brazil. Int. J. Infect. Dis. IJID Off. Publ. Int. Soc. Infect. Dis. 2008, 12, 410–415. [Google Scholar] [CrossRef]
18. CLSI. Zone Diameter and Minimal Inhibitory Concentration Breakpoints for Staphylococcus spp. In Performance Standards for Antimicrobial Susceptibility Testing; CLSI: Wayne, PA, USA, 2017; Volume supplement M100, pp. 56–63. [Google Scholar]
19. Gholamzad, M.; Khatami, M.R.; Ghassemi, S.; Malekshahi, Z.V.; Shooshtari, M.B. Detection of Staphylococcus enterotoxin B (SEB) using an immunochromatographic test strip. Jundishapur J. Microbiol. 2015, 8, e26793. [Google Scholar] [CrossRef]
20. Strommenger, B.; Kettlitz, C.; Weniger, T.; Harmsen, D.; Friedrich, A.W.; Witte, W. Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J. Clin. Microbiol. 2006, 44, 2533–2540. [Google Scholar] [CrossRef]
21. Shopsin, B.; Gomez, M.; Montgomery, S.O.; Smith, D.H.; Waddington, M.; Dodge, D.E.; Bost, D.A.; Riehman, M.; Naidich, S.; Kreiswirth, B.N. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 1999, 37, 3556–3563. [Google Scholar] [CrossRef]
22. Mellmann, A.; Weniger, T.; Berssenbrügge, C.; Rothgänger, J.; Sammeth, M.; Stoye, J.; Harmsen, D. Based Upon Repeat Pattern (BURP): An algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms. BMC Microbiol. 2007, 7, 98. [Google Scholar] [CrossRef] [PubMed]
23. Hallin, M.; Friedrich, A.W.; Struelens, M.J. spa typing for epidemiological surveillance of Staphylococcus aureus. Methods Mol. Biol. 2009, 551, 189–202. [Google Scholar] [CrossRef] [PubMed]
24. Su Kyung, O.; Nari, L.; Young Sun, C.; Dong-Bin, S.; Soo Young, C.; Minseon, K. Occurrence of toxigenic Staphylococcus aureus in ready-to-eat food in Korea. J. Food Prot. 2007, 70, 1153–1158. [Google Scholar]
25. Guoxiang, C.; Guangyu, B.; Yongzhong, C.; Wenguang, Y.; Yan, W.; Xiaorong, Z.; Liping, Z.; Yantao, W. Prevalence and diversity of enterotoxin genes with genetic background of Staphylococcus aureus isolates from different origins in China. Int. J. Food Microbiol. 2015, 211, 142–147. [Google Scholar]
26. Normanno, G.; Firinu, A.; Virgilio, S.; Mula, G.; Dambrosio, A.; Poggiu, L.; Decastelli, L.; Mioni, R.; Scuota, S.; Bolzoni, G.; et al. Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy. Int. J. Food Microbiol. 2005, 98, 73–79. [Google Scholar] [CrossRef]
27. Singapore Statutes Online (Ed.) Food Regulations, 2005th ed.; Singapore Statutes Online: Singapore, 2005. [Google Scholar]
28. Yu Cheng, C.; Wan Wen, L.; Chin Ming, F.; Wan Yu, P.; Chien-Shun, C.; Hau-Yang, T. PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan. Int. J. Food Microbiol. 2008, 121, 66–73. [Google Scholar] [CrossRef] [PubMed]
29. Pinchuk, I.V.; Ellen, B.J.; Victor, R.E. Staphylococcal Enterotoxins. Toxins 2010, 2, 2177–2197. [Google Scholar] [CrossRef]
30. Hait, J.; Tallent, S.; Melka, D.; Keys, C.; Bennett, R. Prevalence of enterotoxins and toxin gene profiles of S taphylococcus aureus isolates recovered from a bakery involved in a second staphylococcal food poisoning occurrence. J. Appl. Microbiol. 2014, 117, 866–875. [Google Scholar] [CrossRef]
31. Tang, J.; Tang, C.; Chen, J.; Du, Y.; Yang, X.-N.; Wang, C.; Zhang, H.; Yue, H. Phenotypic characterization and prevalence of enterotoxin genes in Staphylococcus aureus isolates from outbreaks of illness in Chengdu City. Foodborne Pathog. Dis. 2011, 8, 1317–1320. [Google Scholar] [CrossRef]
32. Johler, S.; Petra, G.; Marco, J.; Jörg, H.; Andreas, B.; Roger, S. Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins. Toxins 2015, 7, 997–1004. [Google Scholar] [CrossRef]
33. McLauchlin, J.; Narayanan, G.L.; Mithani, V.; O'neill, G. The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction. J. Food Prot. 2000, 63, 479–488. [Google Scholar] [CrossRef] [PubMed]
34. Omoe, K.; Ishikawa, M.; Shimoda, Y.; Dong-Liang, H.; Ueda, S.; Shinagawa, K. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. J. Clin. Microbiol. 2002, 40, 857–862. [Google Scholar] [CrossRef]
35. Rall, V.L.M.; Sforcin, J.M.; Augustini, V.C.M.; Watanabe, M.T.; Fernandes Jr, A.; Rall, R.; Silva, M.G.; Araújo Jr, J.P. Detection of enterotoxin genes of Staphylococcus sp isolated from nasal cavities and hands of food handlers. Braz. J. Microbiol. 2010, 41, 59–65. [Google Scholar] [CrossRef]
36. Shimamura, Y.; Kidokoro, S.; Murata, M. Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts. Biosci. Biotechnol. Biochem. 2006, 70, 1571–1577. [Google Scholar] [CrossRef] [PubMed]
37. Aung, K.T.; Hsu, L.Y.; Koh, T.H.; Hapuarachchi, H.C.; Chau, M.L.; Gutiérrez, R.A.; Ng, L.C. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in retail food in Singapore. Antimicrob. Resist. Infect. Control 2017, 6, 1–4. [Google Scholar] [CrossRef] [PubMed]
38. Abdalrahman, L.S.; Wells, H.; Fakhr, M.K. Staphylococcus aureus is more prevalent in retail beef livers than in pork and other beef cuts. Pathogens 2015, 4, 182–198. [Google Scholar] [CrossRef]
39. Velasco, V.; Vergara, J.L.; Bonilla, A.M.; Munoz, J.; Mallea, A.; Vallejos, D.; Quezada-Aguiluz, M.; Campos, J.; Rojas-Garcia, P. Prevalence and characterization of Staphylococcus aureus strains in the pork chain supply in Chile. Foodborne Pathog. Dis. 2018, 15, 262–268. [Google Scholar] [CrossRef]
40. Şanlıbaba, P. Prevalence, antibiotic resistance, and enterotoxin production of Staphylococcus aureus isolated from retail raw beef, sheep, and lamb meat in Turkey. Int. J. Food Microbiol. 2022, 361, 109461. [Google Scholar] [CrossRef]
41. Berger, T.; Eisenkraft, A.; Bar-Haim, E.; Kassirer, M.; Aran, A.A.; Fogel, I. Toxins as biological weapons for terror—Characteristics, challenges and medical countermeasures: A mini-review. Disaster Mil. Med. 2016, 2, 1–7. [Google Scholar] [CrossRef]
42. Gill, D.M. Bacterial toxins: A table of lethal amounts. Microbiol. Rev. 1982, 46, 86–94. [Google Scholar] [CrossRef]
43. Blaiotta, G.; Ercolini, D.; Pennacchia, C.; Fusco, V.; Casaburi, A.; Pepe, O.; Villani, F. PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802. J. Appl. Microbiol. 2004, 97, 719–730. [Google Scholar] [CrossRef] [PubMed]
44. Felix, B.; Vingadassalon, N.; Grout, J.; Hennekine, J.-A.; Guillier, L.; Auvray, F. Staphylococcus aureus strains associated with food poisoning outbreaks in France: Comparison of different molecular typing methods, including MLVA. Front. Microbiol. 2015, 6, 882. [Google Scholar]
45. Alni, R.H.; Mohammadzadeh, A.; Mahmoodi, P. Molecular typing of Staphylococcus aureus of different origins based on the polymorphism of the spa gene: Characterization of a novel spa type. 3 Biotech 2018, 8, 1–7. [Google Scholar]
46. Johler, S.; Macori, G.; Bellio, A.; Acutis, P.L.; Gallina, S.; Decastelli, L. Characterization of Staphylococcus aureus isolated along the raw milk cheese production process in artisan dairies in Italy. J. Dairy Sci. 2018, 101, 2915–2920. [Google Scholar] [CrossRef] [PubMed]
47. Teo, J.W.; Kum, S.; Jureen, R.; Lin, R.T. Molecular characterization of a catalase-negative Staphylococcus aureus blood culture isolate. J. Clin. Microbiol. 2015, 53, 3699–3701. [Google Scholar] [CrossRef]
48. Ho, J.; O’Donoghue, M.M.; Boost, M.V. Occupational exposure to raw meat: A newly-recognized risk factor for Staphylococcus aureus nasal colonization amongst food handlers. Int. J. Hyg. Environ. Health 2014, 217, 347–353. [Google Scholar] [CrossRef]
49. Tunsjø, H.S.; Kalyanasundaram, S.; Charnock, C.; Leegaard, T.M.; Moen, A.E. Challenges in the identification of methicillin-resistant Staphylococcus argenteus by routine diagnostics. Apmis 2018, 126, 533–537. [Google Scholar] [CrossRef]
50. Aung, M.S.; Urushibara, N.; Kawaguchiya, M.; Sumi, A.; Takahashi, S.; Ike, M.; Ito, M.; Habadera, S.; Kobayashi, N. Molecular epidemiological characterization of Staphylococcus argenteus clinical isolates in Japan: Identification of three clones (ST1223, ST2198, and ST2550) and a novel staphylocoagulase genotype XV. Microorganisms 2019, 7, 389. [Google Scholar] [CrossRef]
51. Ilczyszyn, W.M.; Sabat, A.J.; Akkerboom, V.; Szkarlat, A.; Klepacka, J.; Sowa-Sierant, I.; Wasik, B.; Kosecka-Strojek, M.; Buda, A.; Miedzobrodzki, J.; et al. Clonal structure and characterization of Staphylococcus aureus strains from invasive infections in paediatric patients from South Poland: Association between age, spa types, clonal complexes, and genetic markers. PLoS ONE 2016, 11, e0151937. [Google Scholar] [CrossRef]
52. Miko, B.A.; Hafer, C.A.; Lee, C.J.; Sullivan, S.B.; Hackel, M.A.; Johnson, B.M.; Whittier, S.; Della-Latta, P.; Uhlemann, A.-C.; Lowy, F.D. Molecular characterization of methicillin-susceptible Staphylococcus aureus clinical isolates in the United States, 2004 to 2010. J. Clin. Microbiol. 2013, 51, 874–879. [Google Scholar] [CrossRef]
53. Saffari, F.; Radfar, A.; Sobhanipoor, M.H.; Ahmadrajabi, R. Spa gene-based molecular typing of nasal methicillin-susceptible Staphylococcus aureus from patients and health-care workers in a dialysis center in southeast Iran. Pathog. Glob. Health 2020, 114, 160–163. [Google Scholar] [CrossRef] [PubMed]
54. Uhlemann, A.C.; Dumortier, C.; Hafer, C.; Taylor, B.S.; Sánchez, E.; Rodriguez-Taveras, C.; Leon, P.; Rojas, R.; Olive, C.; Lowy, F.D. Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones. Eur. J. Clin. Microbiol. Infect. Dis. 2011, 31, 505–511. [Google Scholar] [CrossRef] [PubMed]
55. Fetsch, A.; Contzen, M.; Hartelt, K.; Kleiser, A.; Maassen, S.; Rau, J.; Kraushaar, B.; Layer, F.; Strommenger, B. Staphylococcus aureus food-poisoning outbreak associated with the consumption of ice-cream. Int. J. Food Microbiol. 2014, 187, 1–6. [Google Scholar] [CrossRef]
56. Strommenger, B.; Braulke, C.; Heuck, D.; Schmidt, C.; Pasemann, B.; Nübel, U.; Witte, W. spa Typing of Staphylococcus aureus as a frontline tool in epidemiological typing. J. Clin. Microbiol. 2008, 46, 574–581. [Google Scholar] [CrossRef]
57. Ge, B.; Mukherjee, S.; Hsu, C.-H.; Davis, J.A.; Tran, T.T.T.; Yang, Q.; Abbott, J.W.; Ayers, S.L.; Young, S.R.; Crarey, E.T.; et al. MRSA and multidrug-resistant Staphylococcus aureus in US retail meats. Food Microbiol. 2017, 62, 289–297. [Google Scholar] [CrossRef] [PubMed]
58. Udo, E.E.; Al-Mufti, S.; Albert, M.J. The prevalence of antimicrobial resistance and carriage of virulence genes in Staphylococcus aureus isolated from food handlers in Kuwait City restaurants. BMC Res. Notes 2009, 2, 1–6. [Google Scholar] [CrossRef]
59. Wang, W.; Baloch, Z.; Jiang, T.; Zhang, C.; Peng, Z.; Li, F.; Fanning, S.; Ma, A.; Xu, J. Enterotoxigenicity and antimicrobial resistance of Staphylococcus aureus isolated from retail food in China. Front. Microbiol. 2017, 8, 2256. [Google Scholar] [CrossRef]
60. Chaalal, W.; Chaalal, N.; Bourafa, N.; Kihal, M.; Diene, S.M.; Rolain, J.-M. Characterization of Staphylococcus aureus isolated from food products in Western Algeria. Foodborne Pathog. Dis. 2018, 15, 353–360. [Google Scholar] [CrossRef]
61. Zhang, L.; Li, Y.; Bao, H.; Wei, R.; Zhou, Y.; Zhang, H.; Wang, R. Population structure and antimicrobial profile of Staphylococcus aureus strains associated with bovine mastitis in China. Microb. Pathog. 2016, 97, 103–109. [Google Scholar] [CrossRef]
62. Akpaka, P.E.; Roberts, R.; Monecke, S. Molecular characterization of antimicrobial resistance genes against Staphylococcus aureus isolates from Trinidad and Tobago. J. Infect. Public Health 2017, 10, 316–323. [Google Scholar] [CrossRef]
63. Lowy, F.D. Antimicrobial resistance: The example of Staphylococcus aureus. J. Clin. Investig. 2003, 111, 1265–1273. [Google Scholar] [CrossRef] [PubMed]
64. Olsen, J.E.; Christensen, H.; Aarestrup, F.M. Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci. J. Antimicrob. Chemother. 2006, 57, 450–460. [Google Scholar] [CrossRef] [PubMed]
65. Aydin, A.; Muratoglu, K.; Sudagidan, M.; Bostan, K.; Okuklu, B.; Harsa, S. Prevalence and antibiotic resistance of foodborne Staphylococcus aureus isolates in Turkey. Foodborne Pathog. Dis. 2011, 8, 63–69. [Google Scholar] [CrossRef] [PubMed]
66. Da Costa, T.M.; De Oliveira, C.R.; Chambers, H.F.; Chatterjee, S.S. BP4: A new perspective on Staphylococcus aureus β-lactam resistance. Microorganisms 2018, 6, 57. [Google Scholar] [CrossRef] [PubMed]
67. Reading, C.; Cole, M. Clavulanic acid: A beta-lactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 1977, 11, 852–857. [Google Scholar] [CrossRef]
68. Achek, R.; Hotzel, H.; Cantekin, Z.; Nabi, I.; Hamdi, T.M.; Neubauer, H.; El-Adawy, H. Emerging of antimicrobial resistance in staphylococci isolated from clinical and food samples in Algeria. BMC Res. Notes 2018, 11, 1–7. [Google Scholar] [CrossRef]
69. Xu, J.; Shi, C.; Song, M.; Xu, X.; Yang, P.; Paoli, G.; Shi, X. Phenotypic and genotypic antimicrobial resistance traits of foodborne Staphylococcus aureus isolates from Shanghai. J. Food Sci. 2014, 79, M635–M642. [Google Scholar] [CrossRef]
70. Ardic, N.; Ozyurt, M.; Sareyyupoglu, B.; Haznedaroglu, T. Investigation of erythromycin and tetracycline resistance genes in methicillin-resistant staphylococci. Int. J. Antimicrob. Agents 2005, 26, 213–218. [Google Scholar] [CrossRef]
71. Chajęcka-Wierzchowska, W.; Zadernowska, A.; Nalepa, B.; SIERPI´ NSKA, M.A.G.D.A.; Łaniewska-Trokenheim, L. Retail ready-to-eat food as a potential vehicle for Staphylococcus spp. harboring antibiotic resistance genes. J. Food Prot. 2014, 77, 993–998. [Google Scholar] [CrossRef]
72. Tatini, S.R. Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins. J. Milk Food Technol. 1973, 36, 559–563. [Google Scholar] [CrossRef]
73. Mesbah, A.; Mashak, Z.; Abdolmaleki, Z. A survey of prevalence and phenotypic and genotypic assessment of antibiotic resistance in Staphylococcus aureus bacteria isolated from ready-to-eat food samples collected from Tehran Province, Iran. Trop. Med. Health 2021, 49, 1–12. [Google Scholar] [CrossRef] [PubMed]