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

Background Resistant Staphylococcus aureus (S. aureus) bacteria are considered among the major causes of foodborne diseases. This survey aims to assess genotypic and phenotypic profiles of antibiotic resistance in S. aureus bacteria isolated from ready-to-eat food samples. Methods According to the previously reported prevalence of S. aureus in ready-to-eat food samples, a total of 415 ready-to-eat food samples were collected from Tehran province, Iran. S. aureus bacteria were identified using culture and biochemical tests. Besides, the phenotypic antibiotic resistance profile was determined by disk diffusion. In addition, the genotypic pattern of antibiotic resistance was determined using the PCR. Results A total of 64 out of 415 (15.42%) ready-to-eat food samples were contaminated with S. aureus. Grilled mushrooms and salad olivieh harbored the highest contamination rate of (30%), while salami samples harbored the lowest contamination rate of 3.33%. In addition, S. aureus bacteria harbored the highest prevalence of resistance to penicillin (85.93%), tetracycline (85.93%), gentamicin (73.43%), erythromycin (53.12%), trimethoprim-sulfamethoxazole (51.56%), and ciprofloxacin (50%). However, all isolates were resistant to at least four antibiotic agents. Accordingly, the prevalence of tetK (70.31%), blaZ (64.06%), aacA-D (57.81%), gyrA (50%), and ermA (39.06%) was higher than that of other detected antibiotic resistance genes. Besides, AacA-D + blaZ (48.43%), tetK + blaZ (46.87%), aacA-D + tetK (39.06%), aacA-D + gyrA (20.31%), and ermA + blaZ (20.31%) were the most frequently identified combined genotypic patterns of antibiotic resistance. Conclusion Ready-to-eat food samples may be sources of resistant S. aureus, which pose a hygienic threat in case of their consumption. However, further investigations are required to identify additional epidemiological features of S. aureus in ready-to-eat foods.


Discussion
Contaminated ready-to-eat food samples, especially those of an animal origin, are considered probable causes of S. aureus transmission to the human population [12].
The present survey was performed to evaluate the prevalence as well as phenotypic and genotypic profiles of antibiotic resistance in S. aureus bacteria isolated from the samples of hamburgers, salami, grilled mushrooms, falafel, salad olivieh, chicken nuggets, and Mexican corn. The prevalence of S. aureus was 15.42% in the examined samples. Besides, a higher prevalence was observed in grilled mushrooms (30%) and salad olivieh (30%), while a lower prevalence was found in salami samples (3.33%). This finding could have been due to the different levels of water activity (a w ) and pH values in diverse food samples. Furthermore, the use of low-quality and contaminated raw ingredients might be the reason for the high prevalence of bacteria in ready-to-eat food samples. However, the transmission of S. aureus from infected staff to food samples should be recognized as well. Foodstuff contamination with S. aureus may be directly caused by infected food animals, or their products, such as meat, or by poor hygiene throughout their processing. The low prevalence of S. aureus in salami samples could have been due to the high temperature used in their processing. A similar survey was conducted by Safarpoor Dehkordi et al. [13], in which they showed the prevalence of S. aureus in raw meat, raw chicken, grilled meat, grilled chicken, soup, salad, and rice samples collected from hospital kitchens was 26.31%, 27.02%, 16.12%, 8.53%, 6.38%, 7.14%, and 4.20%, respectively. Besides, they showed that food sample manipulation by the infected staff of hospital kitchens was the main causative factor for development of S. aureus. Hasanpour Dehkordi et al. [7] reported that the prevalence of S. aureus bacteria in diverse kinds of food samples was within the range of 10.00 and 24.00%. A high prevalence of S. aureus bacteria was also reported in diverse kinds of foodstuffs from the continents of Asia [12,14], Europe [15][16][17][18][19][20], Africa [21], America [22,23], and Australia [24]. In the same vein, Wu et al. [25] reported that the prevalence of S. aureus in raw meat, pork, beef, poultry and mutton, sausages, frozen meat,  Table 3 Genotypic profile of antibiotic resistance in S. aureus isolates recovered from diverse kinds of ready-to-eat food samples   Table 4 Combined genotypic profile of antibiotic resistance in S. aureus isolates recovered from diverse kinds of ready-to-eat food samples      11.80% and 0%, respectively. In addition, they reported that the prevalence of S. aureus in ready-to-eat food samples was relatively lower than that in raw products, which could have been due to processing operations, such as heating as well as the adding of additives to ready-to-eat food samples. Achi and Madubuike (2007) reported that the prevalence of S. aureus in ready-to-eat fish sausage, meat sausage, fried fish, fried meat, suya, moin moin, wash water, and rinse water samples was 23.60%, 29.70%, 8.30%, 6.60%, 17.20%, 13.40%, 27.60%, and 18.10%, respectively [26]. Similarly, they introduced water and ready-to-eat food samples as the sources of S. aureus. Findings of the present survey showed a high prevalence of resistance to diverse classes of antibiotic agents, particularly penicillins, tetracyclines, aminoglycosides, macrolides, fluoroquinolones, phenicols, and ansamycins. Additionally, some strains harbored antibiotic resistance genes. Chloramphenicol is a forbidden antibiotic agent, which is sometimes used to treat infections in poultry. Emergence of resistance to this antibiotic may imply its unauthorized prescription.
Besides, development of antibiotic resistance to chloramphenicol could indirectly imply the poultry-based origin of the isolated S. aureus bacteria. Most of the examined samples, particularly salad olivieh, chicken nuggets, and hamburgers were made from poultry meat and its products. Additionally, S. aureus bacteria isolated from salad olivieh, chicken nugget, and hamburger samples had a moderate prevalence of chloramphenicol resistance (16.66%, 80%, and 14.28%, respectively). Moreover, the prevalence of the cat1 chloramphenicol encoding gene among the S. aureus bacteria isolated from salad olivieh, chicken nugget, and hamburger samples was high, having been 27.77%, 40%, and 28.57%, respectively. Thus, the findings could indirectly verify the origin of the S. aureus isolates.

Conclusion
In conclusion, the high prevalence of S. aureus in the examined samples, particularly in grilled mushrooms and olivieh salad and a high prevalence of resistance to diverse classes of antibiotic agents and different antibiotic resistance genes were reported in this study. A high prevalence of resistance to penicillin, tetracycline, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, and ciprofloxacin antibiotic agents as well as the presence of tetK, blaZ, aacA-D, gyrA, and ermA antibiotic resistance genes were reported in the present survey. Furthermore, the high prevalence of multi-drug resistant bacteria and the presence of aacA-D + blaZ, tetK + blaZ, aacA-D + tetK, aacA-D + gyrA, and ermA + blaZ genes together may indicate the leading role of ready-to-eat food samples in the transmission of antibiotic-resistant S. aureus to human populations. Accordingly, the use of high-quality raw materials, proper hygienic foodprocessing conditions, the adequate cooking of food samples, cross-contamination prevention, and antibiotic prescription rendering the outcomes of disk diffusion could diminish the risk of antibiotic-resistant S. aureus in the examined samples. Further surveys are recommended to be conducted to illuminate other epidemiological aspects of antibiotic-resistant S. aureus in ready-to-eat food samples.

Samples
From April to November 2018, a total of 415 different kinds of ready-to-eat food samples, such as hamburgers (n = 75), chicken nuggets (n = 70), salad olivieh (n = 60), salami (n = 60), falafel (n = 50), grilled mushrooms (n = 50), and Mexican corn (n = 50) were randomly collected from the fast-food restaurants of the Tehran province, Iran. Sampling was performed in highly consumed ready-to-eat food samples. According to the low diversity of ready-to-eat food-producing restaurants in Iran, sampling was done in all of them. To this end, simple stratified sampling was performed according to the production volume of each fast food unit. Besides, the samples (100 g) were directly delivered to the Food Hygiene Research Center. In addition, the food samples were transported in cool boxes with ice packs.

S. aureus isolation and identification
As many as 20 g of each collected ready-to-eat food sample was blended with 225 mL of buffered peptone water (Merck, Germany). Next, the solutions were homogenized using a stomacher (Interscience, Saint-Nom, France  [13].

Statistical analysis
Statistical analysis was performed by SPSS Statistics 21.0 (SPSS Inc., Chicago, IL, USA). Chi-square and Fisher's exact two-tailed tests were performed to assess any significant relationship between the prevalence of S. aureus