It is indispensable to distinguish the exact routes of transmission of antibiotic-resistant bacteria to the human population from the epidemiological perspective. Foods have a critical role in transmitting some types of antibiotic resistant-bacteria to humans [31–33]. Vegetables and salad are two of the most important and highly consumed foodstuffs worldwide [3, 34]. They are considered a ubiquitous source of antibiotic-resistant-S. aureus [35, 36].
An existing survey was aimed to assess the molecular typing and phenotypic and genotypic assessment of antibiotic resistance and virulence factors of the MRSA bacteria isolated from vegetable and salad samples. The total prevalence of MRSA bacteria amongst the vegetable and salad samples was 4.44% (8/180) and 10.58% (18/170), respectively. The present research is the first report of isolation of MRSA bacteria from vegetable and salad samples. Cesar et al. (2015) [37] described that the total prevalence of coagulase-negative and -positive staphylococci amongst the lettuce salad samples collected from Brazil were 77.80% and 5.60%, respectively. Osiriphun, Poonlarp, and Boonyakiat [38] stated that S. aureus is one of the most prevalent causes of contamination of mixed salad vegetables in Thailand. Total prevalence of S. aureus amongst the vegetable and salad samples collected from Argentina [39], Brazil [40], Zambia [41], Korea [42], Pakistan [11], Saudi Arabia [43], and India [44] were 7.81%, 43.80%, 60.%, 11.30%, 54%, 51.50%, and 86%, respectively. Close contact of the vegetable samples with the contaminated animal- and even human-based fertilizers and water used for their growth and irrigation and contaminated soil is a probable reason for MRSA presence. Survival of bacteria from farm to fork and the transmission of MRSA bacteria from contaminated restaurants to salad samples are probable reasons for the higher prevalence of bacteria in salads. S. aureus is present in soil, feces, sewage, and water, and characteristically originates from contact with vegetables by means of the water used for their growing. The bacterium can contaminate salads on account of poor hygienic circumstances of farmers, retailers, and food processer and vendors. Furthermore, the bacterium can distribute by straight contact throughout harvesting, processing, and packing. Another probable reason for the higher prevalence of S. aureus in salad samples is that S. aureus cannot compete well with indigenous microflora of vegetables. Both vegetable and salad samples have optimum growth circumstances for S. aureus (pH (4.8–9.3) and temperatures (7–43 oC)). Thus, it is not surprising that 12.85% of samples were contaminated with S. aureus.
Our survey revealed that MRSA bacteria isolated from vegetable and salad samples harbored high resistance toward cefoxitin, ceftaroline, penicillin, tetracycline, gentamicin, trimethoprim-sulfamethoxazole, and erythromycin antibiotic agents, which was assisted with a high prevalence of blaCTX-M, blaZ, aacA-D, tetK, dfrA1, and vanA antibiotic resistance genes. Otherwise, phenotypic assessment of antibiotic resistance of MRSA bacteria was confirmed by the genotypic evaluation of the resistance gene. Furthermore, the presence of multidrug resistant-MRSA was found in some isolates. Irregular and unauthorizing antibiotic agents' prescription is the probable reason for the high prevalence of resistance and high distribution of antibiotic resistance genes. Findings disclosed that some MRSA bacteria isolated from salad samples exhibited a higher prevalence of resistance toward antibiotic agents used to treat human clinical infections, which can indirectly signify that they may transmit from infected staff and workers of restaurants. The high prevalence of resistance of MRSA bacteria isolated from vegetable samples toward antibiotic agents used for the treatment of animal clinical infections may show their transmission from animal-based fertilizers and even contaminated water used for the growth of vegetable samples. According to the literature, the current research is the first report of the phenotypic and genotypic assessments of antibiotic resistance among MRSA bacteria isolated from vegetable and salad samples. Wu et al. (2018) [45] described that the antibiotic resistance of S. aureus bacteria isolated from vegetable in China against amoxicillin/clavulanic acid, ampicillin, cefepime, cefoxitin, penicillin, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, linezolid, rifampicin, trimethoprim/sulphamethoxazole, quinupristin/dalfopristin, teicoplanin, nitrofurantoin and fusidic acid were 26.70%, 93.30%, 10%, 16.70%, 93.30%, 16.70%, 3.30%, 16.70%, 33.30%, 23.30%, 23.30%, 23.30%, 40%, 20%, 23.30%, 16.70%, 43.30%, 0%, 0%, 0%, 3.30%, 0%, 0% and 10%, respectively. Similarly, SergeliDiS et al. (2012) [46] stated that the prevalence of resistance of S. aureus bacteria isolated from ready-to-eat salads in Greece against ampicillin, chloramphenicol, clindamycin, erythromycin, fosfomycin, gentamicin, penicillin, quinupristin/dalfopristin, rifampin, and trimethoprim/sulfamethoxazole antibiotic agents were 94%, 3%, 1.50%, 89.40%, 82.60%, 4.50%, 59.10%, 1.50%, 1.50%, and 1.50%, respectively. Similar to our findings, a high prevalence of resistance of S. aureus bacteria isolated from vegetables and salads and other foodstuffs and clinical infections against cefoxitin, ceftaroline, penicillin, tetracycline, gentamicin, trimethoprim-sulfamethoxazole, and erythromycin antibiotic agents were reported from Iran [9, 47–50], Nigeria [51], China [52], Korea [42], and the United States [53]. The presence of antibiotic resistance genes was not examined in the MRSA bacteria isolated from vegetable and salad samples previously. Dehkordi, Gandomi, Basti, Misaghi and Rahimi [54] revealed that the prevalence of aacA-D, tetK, tetM, msrA, ermA, ermC, vatA, vatB, vatC, and linA amongst the MRSA bacteria isolated from hospital food samples were 62.16%, 72.97%, 27.02%, 64.86%, 72.97%, 27.02%, 45.94%, 18.91%, 5.40%, and 43.24%, respectively. In the same way, Rahi et al. (2020) [55] disclosed that the prevalence of blaZ, aacA-D, ermA, ermB, msrA, msrB, mefA, tetK, tetM, gyrA, grlA, linA, dfrA1, cfr, and rpoB amongst the MRSA bacteria isolated from raw milk samples were 100%, 67.85%, 50%, 25%, 35.71%, 10.71%, 35.71%, 85.71%, 35.71%, 42.85%, 28.57%, 28.75%, 71.42%, 25%, and 10.71%, respectively. Our findings were also released a higher prevalence of phenotypic profile of resistance than the genotypic pattern. For instance, all of the glycopeptides-resistant MRSA bacteria didn't harbor vanA antibiotic resistance gene. This matter has also existed for other antibiotic agents and resistance genes. This finding is maybe because antibiotic resistance genes are one of the known procedures for the occurrence of antibiotic resistance in MRSA strains. Otherwise, different mechanisms have been recognized to induce antibiotic resistance in bacteria, including efflux antibiotic's active pumps to out of the bacterial cell, reduced permeability of bacteria to antibiotics, inactivation of antibiotics through hydrolysis or changes in their structure, change in antibiotic target site, access of bacteria to the secondary metabolic pathways that compensate the antibiotic-inhibited reactions and occurrence of genetic mutations. Discoveries also showed the high prevalence of multidrug resistant-MRSA strains amongst examined samples, particularly salads. In the same way, high prevalence of multidrug-resistant bacteria has been reported in herbal product samples in Cameron [56], Kuwait [57], and Tanzania [58]. Altogether, high prevalence of antibiotic resistant-MRSA which was accompanying with the high distribution of antibiotic resistance genes and presence of multidrug resistance disclosed an imperative public health issue regarding the consumption of vegetable and salad samples. Given that these antibiotics have been progressively utilized in human and animal treatments and the exchange of antibiotic-resistant genes by the mobile genetic elements, it is not astonishing that resistant bacteria become more mutual nowadays. Nevertheless, the high antimicrobial resistance of MRSA observed in this research should receive much attention. Furthermore, controlled use of antimicrobials would limit the emergence of drug-resistant bacteria.
Findings described the considerable prevalence of virulence factors amongst the MRSA bacteria isolated from vegetable and salad samples. Alpha-hemolysin (hla) toxin is the most emphasized virulence factor of the S. aureus, which is considered a candidate of vaccine to inhibit the dissemination of infections [59]. Most S. aureus bacteria recovered from clinical infections and, more recently, food samples, harbored the coa factor [60]. It mainly acts as a blood coagulase factor in the pathogenesis of staphylococcal infections [60]. PVL is cytotoxin responsible for severe tissue necrosis and leukocyte destruction [61]. PVL, coa, and hla virulence factors also had high prevalence amongst the S. aureus bacteria isolated from food and clinical samples collected from Egypt [62], Myanmar [63], China [59], Iran [64], and Brazil [65]. The high prevalence of identified virulence factors in the MRSA bacteria of this research may show high virulence and pathogenicity of MRSAS bacteria, which poses an imperative public health hazard rendering the consumption of contaminated vegetable and salad samples. Assess the PVL gene's distribution is one of the essential practical methods to find the presence of healthcare-associated (HA-) or community-associated (CA) MRSA bacteria. Findings of epidemiological investigations revealed that the CA-MRSA bacteria mainly carry the PVL gene [63]. In the present research, 37.50% of MRSA bacteria isolated from vegetable samples and 66.66% of those of salad samples harbored the PVL gene. Thus, majority of MRSA bacteria isolated from vegetable samples may categorized as HA-MRSA, while majority of those of salad samples may categorized as CA-MRSA. However, assessment of the presence of Staphylococcal Cassette Chromosome mec (SCCmec) may clear the exact type of MRSA isolates.
Molecular typing of MRSA bacteria showed that they had a similarity lower than 80% (except isolate No 10), which may show that they have the same genetic cluster. This matter may show the expected contamination of vegetable and salad samples with MRSA bacteria with the same molecular cluster.