Molecular Typing, Phenotypic and Genotypic Assessment of Antibiotic Resistance and Virulence Factors amongst the Staphylococcus aureus Bacteria Isolated from Raw Chicken Meat

The present research was performed to evaluate the phenotypic and genotypic properties of antibiotic resistance, virulence factor profiles and molecular typing of Staphylococcus aureus strains isolated from chicken meat. A total of 36 S. aureus strains were isolated from 300 raw chicken meat samples. Phenotypic pattern of antibiotic resistance was assessed by disk diffusion. Distribution of antibiotic resistance and virulence genes was evaluated by PCR. Molecular typing of isolates was performed by the ERIC-PCR. Considering the over than 80% similarity, 36 S. aureus isolates were classified in 9 different profiles with 43 to 100% similarities. S. aureus strains showed the highest incidence of resistance against penicillin (100%), tetracycline (91.66%), cephalothin (100%), ciprofloxacin (75%), erythromycin (75%), mupirocin (63.88%), clindamycin (61.11%) and trimethoprim/sulfamethoxazole (61.11%). The most commonly detected antibiotic resistance genes amid the S. aureus isolates were mecA (100%), tetK (80.55%), tetM (66.66%), aacA (6'')/aphD (2'') (61.11%), msrA (55.55%) and ermA (55.55%). Total distribution of etB, etA, tsst-1, clfA and coa virulence factors amongst the S. aureus strains was 61.11, 58.33, 13.88, 75 and 100%, respectively. Genetic cluster of bacteria affected the antibiotic resistance and virulence characters of S. aureus strains. S. aureus strains with the same ERIC-genetic cluster had similar antibiotic resistance and virulence characters which may show their similar origins. Presence of one or more virulence factors and antibiotic resistance genes amongst the resistant S. aureus strains signifies an important public health threat rendering the consumption of raw or undercooked chicken meat.


INTRODUCTION
Poultry meat is a rich source of various nutrients, especially protein, carbohydrates, fats, minerals and vitamins that are essential for the human body. Thus, their regular daily consumption has been widely recommended. On the other hand, human involvement in the production, inspection and processing of chicken meat increased the risk of microbial contamination and occurrence of foodborne diseases [23,37,39,50].
Staphylococcus aureus (S. aureus) is a bacterium characteristically originate from nose and respiratory tract and on the skin. This microorganism can cause diseases of the skin (abscesses, acne and wound and burn infections), respiratory system (pharyngitis, bronchitis and lung inflammation), urinary tract diseases (urethritis and bladder inflammation), bone inflammation and sepsis. S. aureus reservoirs are humans and animals in which it temporarily or permanently colonizes the mucous membranes, mainly in the nasopharynx. S. aureus also contaminates food products, being one of the causes of food poisoning in people in Iran and in the world [1,19,39,58].
A major problem related to nutritional bacterial food poisoning is the antibiotic resistance of microorganisms'. S. aureus strains resistant to antibiotics and the possibility of their elimination from the environment of animals and food are a frequent topic of conferences in research centers [19,46,58].
Epidemiological surveys have recognized that the S. aureus bacteria isolated from diverse kinds of foodstuffs, particularly those with animal origins, harbored considerable incidence of resistance toward frequently used antibiotic agents, especially aminoglycosides, penicillins, tetracyclines, fluoroquinolones, cephalosporins, and macrolides [15,19,58,70].
Some potential virulence factors are accompanied in the pathogenesis of clinical infections and in some cases foodborne diseases caused by S. aureus [8,36]. Toxic shock syndrome toxin-1 (tsst-1), coagulase encoding gene (coa), exfoliative toxins A and B (etA and etB) and clumping factor (clfA) are frequently detected in S. aureus strains isolated from human clinical infections and foodstuffs [9,39,47].
Genotypic-based molecular analysis of bacterial strains has an imperative role in their classifications especially in outbreaks of nosocomial infections and foodborne diseases. Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) is a technique with high ability to generate DNA fingerprints that differentiate between bacterial strains [10]. Application of diverse kinds of ERIC-PCR methods has been used for molecular typing of S. aureus isolates [11].
Notwithstanding the great number of reports and researches on the dynamics of S. aureus, supplementary investigations is desirable, particularly in some area like Iran where there is a comparative scarcity of data on the epidemiology of S. aureus's foodborne diseases. Thus, an existing survey was performed to assess the phenotypic and genotypic properties of antibiotic resistance, distribution of virulence factors and molecular typing of S. aureus bacteria isolated from raw chicken meat samples in Iran.

Samples and Bacteria Isolation and Identification
From March 2019 to November 2019, a total of 36 S. aureus isolates were recovered from 300 raw chicken meat samples. Meat samples were randomly collected (simple random sampling procedure) from 30 different shopping center of different parts of the Isfahan province, Iran. Samples were immediately (2-4 h after sampling) were transferred to Microbiology Research Center of the Islamic Azad University, Shahrekord Branch using ice-packs (4°C). Isolation of S. aureus was performed according to method described by Momtaz et al. [39]. Briefly, 25 g of each collected samples were blended with 225 mL of buffered peptone water (Merck, Germany). At that moment, solutions were homogenized using Stomacher (Interscience, Saint-Nom, France). At that time, five milliliters of the achieved solution was transferred into 50 mL Trypticase Soy Broth (TSB, Merck, Germany) supplemented with 10% NaCl and 1% sodium pyruvate and incubated for 18 h at 35°C. At that moment, a loopful of the culture was transferred into Baird-Parker agar supplemented with egg yolk tellurite emulsion (Merck, Germany) and incubated at 37°C for about 24 h. Black shiny colonies enclosed with significant zones were identified using biochemical tests including Gram staining, oxidase test, catalase activity, resistance to bacitracin (0.04 U), coagulated test (rabbit plasma), urease activity, glucose O/F test, voges-proskaver (Merck, Germany) test, nitrate reduction, phosphatase, deoxyribonuclease (DNase, Merck, Germany) test, mannitol fermentation, hemolysis activity on blood agar (Merck, Germany) and carbohydrate (xylose, sucrose, trehalose and maltose, fructose, lactose, mannose) fermentation tests [39].

Phenotypic Evaluation of Antibiotic Resistance
Patterns of antibiotic resistance of the S. aureus bacteria was assessed using the simple disk diffusion according to the Kirby-Bauer technique. The Mueller-Hinton agar (Merck, Germany) was used for this goal. Susceptibility of S. aureus isolates were examined toward 11 diverse antibiotic disks including penicillin (10 μg/disk), trimethoprim/sulfamethoxazole (5 μg/disk), tetracycline (30 μg/disk), ciprofloxacin (5 μg/disk), nitrofurantoin (300 μg/disk), azithromycin (15 μg/disk), rifampin (5 μg/disk) and erythromycin (15 μg/disk). clindamycin (10 μg/disk), mupirocin (5 μg/disk) (Oxoid, UK), and cephalothin (30 μg/disk) (Padtanteb, Iran). Instructions of the Clinical and Laboratory Standards Institute [10] was used to assess the susceptibility of bacteria toward antibiotic agents. The plates containing the discs were allowed to stand for at least 30 min before incubated at 37°C for 24 h. The diameter of the zone of inhibition produced by each antibiotic disc was measured and interpreted using the CLSI zone diameter interpretative standards [10]. Staphylococcus aureus ATCC 25923 was used as quality control organism in antimicrobial susceptibility determination.

Genotypic Assessment of Antibiotic Resistance and Virulence Factors
S. aureus isolates were sub-cultured on TSB media (Merck, Germany) and further incubated for 48 h at 37°C. Genomic DNA was extracted from bacterial colonies using the DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany) according to manufacturer's instruction. Purity (A260/A280) and concentration of extracted DNA were then checked (NanoDrop, Thermo Scientific, Waltham, MA, USA). The truth of the DNA was assessed on a 2% agarose gel stained with ethidium bromide (0.5 μg/mL) (Thermo Fisher Scientific, St. Leon-Rot, Germany). Table 1 represents the list of primers and PCR conditions used for amplification of antibiotic resistance genes and virulence factors in the S. aureus bacteria isolated from raw chicken meat [1,30,33,39]. A programmable DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions. Amplified samples were analyzed by electrophoresis (120 V/208 mA) in 2.5% agarose gel. The gel was stained with 0.1% ethidium bromide (0.4 μg/mL). The UVI doc gel documentation systems (Grade GB004, Jencons PLC, London, UK) was applied for analysis of images. All runs included negative PCR-grade water (Thermo Fisher Scientific, St. Leon-Rot, Germany)) and 1ЈA positive (DNA samples which were positive for each gene achieved from our previous studies) controls.

Molecular
Typing DNA samples were amplified by PCR for the repetitive element sequence using the primer ERIC-PCR: ERIC1R: 5'-ATGTAAGCTCCTGGGGAT-TCAC-3', ERIC2: 5'-AAGTAAGTGACTGGGGT-GAGC-3' [34,64]. Electrophoretic patterns were analyzed either visually or by using the Bionumerics software (Applied Maths, Sint-Martems-Latem, Belgium). The BioNumerics analysis was performed using the Dice coefficient and the unweighted pair group method of averages (UPGMA) with a 1% tolerance limit and 1% optimization. Isolates that clustered with ≥80% similarity were considered to belong to the same ERIC type, respectively.

Statistical Analysis
Statistical analysis was done using the SPSS 21.0 statistical software (SPSS Inc., Chicago, IL, USA). Chi-square test and Fisher's exact two-tailed test were used to assess any significant relationship between the phenotypic and genotypic properties of antibiotic resistance, virulence factors and molecular typing of S. aureus bacteria. P value <0.05 was considered as statistical significant level.

Profile of Virulence Factors
The analysis of the presence of the etB, etA, tsst-1, clfA and coa genes, which determine the production of virulence factors by S. aureus strains, showed their presence in 61.11, 58.33, 13.88, 75 and 100% of the examined strains respectively (Table 4). Statistically significant difference was found between ERIC-type of S. aureus isolates and incidence of virulence factors (P < 0.05).

Molecular Typing of S. aureus Strains
The present survey was conducted to assess the phenotypic and genotypic properties, virulence factor profiles and molecular typing of a total of 36 S. aureus strains isolated from chicken meat. Figure 1 reveals the ERIC-based molecular typing of S. aureus strains isolated from chicken meat samples. Considering the over than 80% similarity, 36 S. aureus isolates were classified in 9 different profiles with 43 to 100% similarities. Isolate nos. 2, 11, 20, 24, 35, 5, 12, 13 and 36 were classified in diverse profiles. The lowest similarity (43%) was found for isolate nos. 20, 11, 2 and 24. An achieved major clonal cluster was ERIC-cluster no. 3 with 7 S. aureus isolates. Diverse ERIC-types were further analyzed for phenotypic and genotypic properties of antibiotic resistance and distribution of virulence factors.

DISCUSSION
From the epidemiological prospective, it is essential to know the exact ways of transmission of antibiotic resistant-bacteria to human population. Foods with animal origin have a critical role in transmission of antibiotic resistant-bacteria to human [52][53][54]56]. Chicken meat is considered as a ubiquitous source of antibiotic resistant-S. aureus [23,67].
An existing survey was carried out to assess the phenotypic and genotypic properties of antibiotic resistance, characterization of virulence factors and molecular typing of a total of 36 S. aureus bacteria isolated from raw chicken meat samples. Our findings described that the S. aureus bacteria exhibited the highest incidence of resistance toward penicillin, tetracycline, cephalothin, ciprofloxacin, erythromycin,  mupirocin, clindamycin and trimethoprim/sulfamethoxazole which was assisted with attendance of mecA, tetK, tetM, aacA (6'')/aphD (2''), msrA and ermA antibiotic resistance genes. Widespread and unauthorized administration of antimicrobials and disinfectant solutions in both medicine and veterinary have been considered to be a major factor in the emergence of antibiotic resistance amongst S. aureus bacteria. Sim-      ilarly, considerable incidence of resistance of S. aureus bacteria recovered from chicken meat samples toward penicillin, tetracycline, cephalothin, ciprofloxacin, erythromycin, mupirocin, clindamycin and trimethoprim/sulfamethoxazole antibiotic agents was conveyed from China [67], Bangladesh [6], Nepal [8], Pakistan [4], United States [20] and Iran [50].
Toxins are usually regarded as one of the major factors in the virulence of S. aureus globally, and hence, it is significant to evaluate their distribution amongst isolates from food with respect to measuring public health risks. Results of an existing survey revealed that the distribution of coa, clfA, etB, etA and tsst-1 virulence factors amongst the S. aureus isolates were 100, 75, 61.11, 58.33 and 13.88%, respectively. Presence of virulence S. aureus amongst the chicken meat samples exhibited an imperative public health threat regarding the consumption of raw or undercooked chicken meat samples. In the same way, these genes were predominant in the S. aureus strains isolated from foods with animal origins [12,21,40]. The genes encoding exfoliative toxin isoforms (etA and etB) were the most important factors associated with the pathogenesis of the S. aureus infections. Exfoliative toxins play a role in host colonization and the invasion of injured mucosa and skin. Thus, the high distributions of etA and etB genes in examined chicken meat samples could be due to the transmission of S. aureus strains from the skin of workers and staffs of slaughterhouses [29]. Li et al. [31] reported that the S. aureus strains isolated from poultry meat samples didn't harbor etA and etB virulence factors, while the distribution of the tsst-1 gene was 3.40%. The tsst-1 gene is a super-antigen that can cause a diversity of clinical complications. Yang et al. [69] reported that the distribution of tsst-1, etA and etB virulence factors amongst the S. aureus strains isolated from foods with animal origins were 7.24, 10.14 and 10.14%, respectively. It has been reported that S. aureus clones containing eta, etb, and tsst are increasingly responsible for severe infections. Waryah et al. [65] reported that the incidence of clfA gene amongst the S. aureus strains isolated from human origins on Australia was 83.87%. Additionally, coa and clfA virulence genes were detected in 63.41 and 76.82% of S. aureus strains isolated from chicken meat in previous Iranian survey [39]. Clumping factor is a significant adhesion protein of S. aureus that is governed by clfA gene. This virulence factor is critical for colonization and establishment of infections caused by the S. aureus. It contributes in the pathogenesis of diseases caused by S. aureus by facilitating bacterial binding via soluble or immobilized fibrinogen as fibrinogen plays an important role in platelet thrombus formation [28]. Coagulase protein has the capabil-ity to convert fibrinogen to fibrin and has enough potentials to be a virulence factor in infections caused by the S. aureus. It is codified by the coa gene that holds a preserved and a recurrent polymorphic region that can be used to evaluate relatedness amid S. aureus isolates [60]. High distribution of clfA and coa virulence factors amongst the S. aureus strains isolated from foods with animal origins was reported from Brazil [12], Thailand [63], Switzerland [62] and India [68].
Our survey also showed that the S. aureus strains of the same molecular cluster (ERIC-type) had the same profiles of antibiotic resistance and virulence factors. This matter maybe show the common source of contamination of chicken meat samples with S. aureus with the same molecular cluster.

CONCLUSIONS
In conclusion, presence of S. aureus in examined samples which was accompanied with the high incidence of resistance toward diverse classes of antibiotic agents and also dissimilar antibiotic resistance genes and virulence factors was reported in the current survey. High incidence of resistance of S. aureus bacteria toward penicillin, tetracycline, cephalothin, ciprofloxacin, erythromycin, mupirocin, clindamycin and trimethoprim/sulfamethoxazole which was assisted with attendance of mecA, tetK, tetM, aacA (6'')/aphD (2''), msrA and ermA antibiotic resistance genes may pose an imperative menace regarding the role of raw or undercooked chicken meat consumption on transmission of antibiotic-resistant S. aureus. Simultaneous presence of one or more antibiotic resistance genes and virulence factors in the antibiotic-resistant S. aureus bacteria specify an imperative public health threat about the consumption of raw or undercooked chicken meat samples. Thirty-six S. aureus strains were classified into the 9 diverse genetic clusters according to the ERIC-PCR. S. aureus strains with similar genetic cluster had similar phenotypic and genotypic properties of antibiotic resistance and distribution of virulence markers. Thus, they may have the same origin. Our research highlights the importance of control the antibiotic susceptibility of S. aureus in the foodstuffs such as food producing animals, retail foods, and even human beings, and these information could be used proactively to assist Iranian industries to progress better-quality food safety measures. Otherwise, on the basis of these observations, we recommend that attention should be paid by governments and individuals to prevent the further spread of antibiotic-resistant S. aureus. However, supplementary surveys are essential to determine more epidemiological features of the S. aureus bacteria in raw chicken meat.