Methicillin-resistant Staphylococcus aureus isolates from Iranian restaurant food samples : Panton-Valentine Leukocidin , SCCmec phenotypes and antimicrobial resistance

Purpose: To assess the distribution of Panton-Valentine Leukocidin, SCCmec types and antimicrobial resistance pattern of methicillin resistant Staphylococcus aureus isolated from restaurant food. Methods: Five-hundred and eighty food samples were collected and directly transported to the laboratory. Samples were cultured and S. aureus strains were confirmed using biochemical tests. MRSA strains were determined using polymerase chain reaction (PCR)-based amplification of mecA and femA genes. MRSA strains were then subjected to disk diffusion methods. Results: One-hundred and nineteen out of 580 samples (20.51 %) were positive for S. aureus. Eightythree out of 119 S. aureus (69.74 %) were methicillin-resistant. Thirty-nine out of 83 MRSA samples (46.98 %) harbored PVL gene. Cooked chicken (37 %) had the highest prevalence of S. aureus. Marked seasonality was observed for the prevalence of bacteria. MRSA strains exhibited high resistance against penicillin G (100 %), tetracycline (92.77 %), oxacillin (83.13 %) and azithromycin (71.08 %). All MRSA bacteria were resistant to at least 2 antibiotics (100 %). TetK (80.72 %), linA (67.46 %), aadA1 (62.65 %), and msrA (55.42 %) were the most frequently identified resistance genes. SCCmec V (57.83 %), SCCmec Iva (55.42 %) and SCCmec IVb (30.12 %) were the most frequent. Conclusion: Based on the high prevalence of resistant MRSA strains and also high consumption rate of restaurant foods in Iran, it is essential to exercise control over the hygienic conditions of restaurant foods to minimize MRSA strains.


INTRODUCTION
Food containing pathogenic agents, causes more than two-hundred diseases al-around the world [1,2].Foodborne diseases cause around 75 million diseases, 330,000 hospitalizations, and 5,000 deaths in the United States annually [3].Most of these food-borne diseases outbreaks occurred in public places like restaurants.Probable presence of children, elders, sick persons and even immune-suppressive persons in restaurants caused staffs of restaurant to seriously considered principles of food hygiene [1][2][3].
Staphylococcus aureus (S. aureus) is a coccal bacterium that is a member of the Firmicutes, and is commonly originate from the respiratory system and the skin [2][3][4].This bacterium is a causative agent of nosocomial infections and foodborne diseases globally [2][3][4].Meat is one of the most important food stuffs related to the Staphylococcal foodborne diseases [4,5].

Staphylococcal
food-borne and acquired infections are primarily related to the rise of antibiotic resistance [6,7].S. aureus has the highest points of resistance against methicillin and other beta-lactam antimicrobial agents [6,7].Prevalence of methicillin-resistant S. aureus (MRSA) strains is 50 % in various types of hospital infections [6,7].Resistance of bacterial strains against antibiotics mainly occurred by presence of antibiotic resistance genes [8].
MecA is a specific gene responsible for occurrence of resistance against methicillin.This geneis carried on a 21-to 67-kb element, the staphylococcal chromosomal cassette mec (SCCmec), which participates at a preserved location in the S. aureus genome [9].SCCmec elements are typically divided into I, II, III, IV and V types [9].Type IV is classified to IVa, IVb, IVc and IVd alleles [9].Data revealed that Panton-Valentine Leukocidin (PVL) is the most important virulence factor detected in the MRSA strains isolated from the cases of foodborne and clinical infections [10].Inappropriately, major principles of food hygiene have not been addressed in most Iranian restaurants.Therefore, from the microbiological and epidemiological perspectives, it is important to know the exact hygienic conditions of restaurant foods especially for MRSA which has a global emergence.The current research was done to study the prevalence of MRSA in various types of restaurant foods as well as study the distribution of PVL gene, SCCmec types and antibiotic resistance properties of bacterial isolates.

EXPERIMENTAL Ethical considerations
The study was permitted by the Moral Board of Exploration of the Baqiyatallah University of Medical Sciences, Tehran, Iran (Consent ref no.110523741).Confirmation of this project and the authorizations related to sampling procedure were obtained from Professors Reza Ranjbar and Ebrahim Rahimi (approval ref no.Med 3932017).

Sample collection and S. aureus identification
From February 2015 to February 2016, a total of 580 samples were randomly collected from various types of raw and cooked restaurant foods including raw meat (n = 70), raw chicken (n= 70) and raw fish (n = 70) and cooked meat (n= 100), cooked chicken (n = 100), cooked fish (n= 100), and soup (n = 70).Cooked samples were produced by quick-cooking of meat on charcoal (barbecue).Samples were collected under sterile hygienic conditions and were immediately transported to the laboratory at 4 °C.Samples were collected from the various regions of Isfahan province, Iran.
A 25 g crushed food sample was homogenized using a grinder under aseptic conditions and it was added to 225 mL of sterile Buffered Peptone Water (Merck, Germany) and incubated at 37 ˚C for 24 h.Thereafter, 0.1 mL of sample was plated onto Baird-Parker agar complemented with egg yolk telluride emulsion and incubated at 37 ˚C for 24 to 48 h.Colonies showing characteristic phenotype of S. aureus (circular, black, convex colonies surrounded by 2-to 5-mm clear zones) were sub-cultured on 5 % sheep blood agar to isolate single colonies.S. aureus was identified through a characteristic hemolysis pattern on sheep blood agar, Gram staining results, catalase reaction (using 0.3 % hydrogen peroxide), mannitol fermentation and coagulase tests.

Identification of methicillin-resistant Staphylococcus aureus
Identification of MRSA strains was done using the PCR-based method.Bacterial strains were sub-cultured in Tryptic Soy Broth (TSB, Merck, Germany) and further incubated for 48 h at 37 o C. DNA was extracted from bacterial colonies using the DNA extraction and purification kit (Fermentas, Germany) according to manufacturer's instruction.The DNA concentration has been determined by measuring absorbance of the sample at 260 nm using spectrophotometer [11].Those DNA samples which were concurrently positive for femA and mecA genes were considered as MRSA.For this purpose, PCR method which was introduced by Jonas et al was used [12].

Detection of antibiotic-resistance genes, SCCmec types and Panton-Valentine Leukocidin
Table 1 indicates the list of primers and PCR circumstances used for amplification of antibiotic resistance genes, SCCmec types and PVL gene in the MRSA [14,15].The DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) were used in all PCR reactions.Fifteen microliters of the PCR products were subjected to electrophoresis in a 1.5% agarose gel in 1X TBE buffer at 80 V for 30 min, stained with SYBR Green (Fermentas, Germany).PCR grade water was used as a negative control and S. aureus ATCC 6538 were used as positive control.

Statistical analysis
Statistical analysis was performed using the SPSS 21.0 software.The chi-square and Fisher's exact tests were performed on obtained data to identify any significant differences for the prevalence of S. aureus, PVL genes, SCCmec types and antibiotic resistance properties.Statistical significance was regarded at p < 0.05.

Prevalence of S. aureus, MRSA and PVL gene
Table 2 shows the total prevalence of S. aureus, MRSA and also the PVL gene in various types of restaurant food samples.One-hundred and nineteen out of 580 restaurant food samples (20.51 %) were positive for S. aureus.Eightythree out of 119 S. aureus strains isolated from restaurant food samples (69.74 %) were methicillin-resistant.Thirty-nine of 83 MRSA strains (46.98%) harbored the PVL gene.Cooked chicken (37 %) had the highest prevalence of S. aureus, followed by raw chicken (35.71 %) and cooked meat (31 %).There was no positive results for the raw fish.Statistically significant difference was seen between the incidence of S. aureus and type of food samples (p < 0.05) and also between the prevalence of MRSA and type of food samples (p < 0.05).

Seasonal distribution of MRSA
Figure 1 shows the seasonal distribution of MRSA isolated from various types of restaurant food samples.We found that the prevalence of MRSA strains in all types of restaurant food samples were entirely higher in winter seasons than other seasons of the year.Statistically significant difference was seen for the prevalence of MRSA between cold and warm seasons (P <0.05).

Antibiotic resistance of MRSA
Figure 2 exhibits the incidence of multi-drug resistance in the MRSA strains of restaurant food samples.All of the MRSA strains were resistant to at least 2 antibiotics (100 %), while prevalence of resistance against ten, eleven, twelve, thirteen, fourteen, fifteen and more than fifteen antibiotics were 26.50, 19.27, 14.45, 9.63, 7.22, 3.61 and 1.20 %, respectively.Table 3 shows the antibiotic resistance pattern of MRSA strains isolated from various types of restaurant food samples.MRSA strains of our study exhibited the highest levels of resistance against penicillin G (100 %), tetracycline (92.77 %), oxacillin (83.13 %), azithromycin (71.08 %), trimethoprimsulfamethoxazole (67.46 %), vanco-mycin (61.44 %) and cotrimoxazole (61.44 %).Statistically substantial variance was seen for the prevalence of antibiotic resistance between raw and cooked food samples (p < 0.05).MRSA strains of cooked chicken samples had the highest and also the most diverse antibiotic resistance pattern.
Table 4 indicates the total frequency of antibiotic resistance genes in the MRSA strains isolated from different types of restaurant food samples.We obtained that tetK (80.72 %), linA (67.46 %), aadA1 (62.65 %), and msrA (55.42 %) were the most routinely identified antibiotic resistance genes in the MRSA strains of restaurant food samples.Statistically significant differences were seen between the prevalence of tetk and tetM (p = 0.019), ermA and ermC (p = 0.027), vatA and vatC (p = 0.036) and msrA and msrB (p = 0.022).MRSA strains of cooked chicken samples had the highest and also the most diverse antibiotic resistance genes.

Profile of SCCmec types
Table 5 represents the total distribution of SCCmec types in the MRSA strains isolated from various types of restaurant food samples.SCCmec V (57.83 %), SCCmec Iva (55.42 %), SCCmec IVb (30.12 %) and SCCmec IVc (20.48 %) were the most commonly detected types of

DISCUSSION
The results of the present investigation showed that resistant and virulent strains of MRSA had a high prevalence, antibiotic resistance and pathogenicity in restaurant food samples.As far as we know, this is the first prevalence report of the MRSA and their antibiotic resistance properties in the restaurant food samples in Iran.We found that 20.51 % of food samples were contaminated with S. aureus and among the bacteria isolates 69.74 % were MRSA.High prevalence of MRSA and also the PVL gene (46.98 %) showed an important public health problem regarding the Iranian restaurants.Using from low quality ingredients, high prescription of antimicrobial agents in veterinary and medicine fields, nonconformity of individual and public hygiene in kitchen and also staffs of kitchens in restaurants and lack of adequate time and temperature for cooking of foods are some key factors which may cause high prevalence of MRSA in restaurant food samples of our investigation.
Low prevalence of MRSA in fish meat samples of our investigation may be due, in part, to the presence of specific primary bacterial flora in fish and low ability of S. aureus to compete with them for survival.Higher prevalence of MRSA in chicken meat samples may be due, in part, to the high prescription of human-based antibiotics and especially methicillin in the poultry farms in Iran.In addition, higher temperature of poultries body caused the destruction of weak bacteria and survival of more resistant bacteria like S. aureus.Another important reason could be the fact that veterinarian used various types of antibiotics in poultry farms as a therapeutic agent and also growth factor.Therefore, other pathogenic bacteria cannot survive well and in this situation S. aureus which has the highest levels of resistance can survive without any competitor.
Cooked food had the higher prevalence of S. aureus, MRSA and the PVL gene than raw foods.The main reason for this finding in addition to cross-contamination from kitchen staff and environment during the production process into the food samples.Unfortunately, chiefs do not use high enough and adequate temperature for cooking of barbecues.Therefore, the central parts of barbecue samples are not cooked well.In addition, new strains of S. aureus can transmit from the infected staff of the kitchens and especially their skin infections and respiratory secretions (which are considered as a main sources of S. aureus) to cooked foods which are free from any competitor bacteria.Therefore, MRSA strains can survive without any rival.Indiscriminate, unauthorized and irregular prescription of antibiotics in veterinary and medical fields caused occurrence of high antibiotic resistance in the MRSA strains of our study.We found that more than 70 % of MRSA strains of our study were resistant to several antibiotics, together.We found that resistance of MRSA strains of cooked samples against human-based antibiotics was completely higher than animal-based antibiotics which can indirectly established the transmission of MRSA strains from the chef of the kitchen and also kitchen staff to food stuffs.In addition, MRSA strains of restaurant food samples and especially cooked samples harbored the high levels of resistance against human-based antibiotics such as cephalothin, imipenem, ciprofloxacin, norfloxacin, cotrimoxazole, clindamycin, trimethoprim-sulfamethoxazole, oxacillin and azithromycin which can indirectly confirm their anthropogenic origin.The prevalence of antibiotic resistance genes and especially those that encode resistance against human-based antibiotics were also higher among MRSA strains isolated from cooked restaurant food samples which also can indirectly confirm the broadcast of anthropogenic MRSA strains possibly from staffs of restaurant kitchens to foods after cooking process.
Several studies have conducted in this field.For instance Puah et al [16] reported that 26 % of restaurant food samples in Malaysia were infected with S. aureus which was higher than our results.They showed that 28.80 % of bacterial strains were resistant against tetracycline and the prevalence of multi-drug resistance was 3.80 % which was lower than our findings; (92.77 % resistance against tetracycline and more than 70 % of strains were resistant against multiple antibiotics).Udo et al [17] showed that S. aureus had a high prevalence in the restaurant food samples of Kuwait.They reported that S. aureus strains were resistant to penicillin G (82.0 %), tetracycline (19.0 %), erythromycin (2.5 %), clindamycin (2.0 %), trimethoprim (7.5 %), kanamycin (2.5 %), streptomycin (1.5 %), ciprofloxacin (1.5 %), fusidic acid (1.0 %) and cadmium acetate (68.0%) which was similar to our findings.

Occurrence
of resistance against chloramphenicol in the MRSA strains of our examination were 18.07 % which was completely higher in chicken-based samples.Chloramphenicol is a restricted antibiotic.The high prevalence of resistance against chloramphenicol displayed its unequal and illegal use in veterinary treatment and especially in the field of poultry in Iran.Therefore, antibiotic resistance will occur against this antibiotic especially in poultry and chicken samples.There were no additional chloramphenicol-resistant MRSA strains in cooked samples compared to the raw foods which showed that all of these strains had a primary and/or animal-based origin.High prevalence of resistance against chloramphenicol was seen in studies conducted by Momtaz et al [18], Dehkordi et al [19], and Osman et al [20].
Gundogan et al [21] reported that the S. aureus strains of food samples had high levels of resistance against penicillin G (53.8%), while the levels of resistance against erythromycin was low (7.5 %) which was in harmony with our findings.They showed that all of the S. aureus strains were susceptible to vancomycin, sulbactam-ampicillin, ciprofloxacin and cefaperazonesulbactam which was in contrast with our results.
We found that 46.98 % of MRSA strains of our study harbored the PVL gene.This gene encodes one of the major exotoxins of the S. aureus.High prevalence of this gene showed an important public health problem facing Iranian restaurant foods.Previously published data reported that majority of PVL positive strains of S. aureus were accompanying with skin and soft tissue infections [21].Therefore, PVL positive strains of the S. aureus isolates of our investigation maybe transmitted from the handmanipulation of staff into the foods.Absence of the PVL gene in the cases of food poisoning has been reported previously [22].
Additional portion of our inquiry was attentive on the distribution types in MRSA strains of various types of food samples.We found that the distribution of SCCmec I, SCCmec II, SCCmec III, SCCmec Iva, SCCmec IVb, SCCmec IVc, SCCmec IVd and SCCmec V in various types of food samples were 8.43, 4.81, 10.84, 55.42, 30.12, 20.48, 8.43 and 57.83 %, respectively.Jackson et al [23] reported similar results for the high prevalence of SCCmec IV in retail meat samples.They reported that all of the MRSA strains of beef and pork samples were positive for SCCmec IV which was higher than our findings.In a study which was conducted by Vossenkuhl et al [24] most of the MRSA strains of turkey meat samples carried SCCmec V (58.1 -71.9 %) followed by type IVa (19 -27.0 %) which was similar to our findings.In addition to the results of our study and that of Vossenkuhl et al [24], Zhang et al [15] reported high prevalence of SCCmec III in their food samples.High prevalence of SCCmec IVa and V were also detected in the retail meat samples of Bhargava et al [25].

CONCLUSION
As far as we know, this is the first prevalence report of the molecular characterization and antimicrobial resistance properties of MRSA strains isolated from restaurant food samples in Iran.An overall incidence of 20.51 % and a relatively high prevalence of PVL gene, SCCmec types, antibiotic resistance genes as well as the multi-drug resistance pattern of S. aureus show a possible public health threat from Iranian restaurant food.Each MRSA isolate, regardless of their origin, harbor at least one of the main antibiotic resistance genes, indicating their pathogenicity, which must be considered a serious health hazard.Standard hygiene protocols and control measures need to be instituted to mitigate serial health hazards.

Figure 1 :Figure 2 :
Figure 1: Seasonal distribution of MRSA in various types of restaurant food

Table 1 :
Oligonucleotide primers and PCR conditions used for detection of antibiotic resistance genes in MRSA strains isolated from various types of restaurant food

Table 2 :
Total distribution of S. aureus, MRSA and PVL gene in various types of restaurant food samples

Table 3 :
Antibiotic resistance pattern of MRSA strains isolated from various types of restaurant food

Table 4 :
Total prevalence of antibiotic resistance genes in MRSA strains isolated from various types of restaurant food