Molecular Characterization and Genotypic and Genotypic Evaluation of Antibiotic Resistance of Methicillin Resistant-staphylococcus Aureus Isolated From Raw Meat


 Methicillin-resistant Staphylococcus aureus (MRSA) is considered to be one of the most important causes of food-borne diseases. The present investigation was done to assess the phenotypic and genotypic characterization and distribution of Staphylococcal cassette chromosome mec types and Panton–Valentine leukocidin gene in the MRSA strains isolated from raw meat samples. Six-hundred and eighty meat samples were collected and cultured. MRSA strains were subjected to disk diffusion and Polymerase Chain Reaction. One-hundred and thirty-five out of 680 (18.38%) raw meat samples were positive for S. aureus. Seventy-nine out of 125 (63.20%) S. aureus strains were determined as MRSA. Raw sheep meat samples (75%) had the highest prevalence of MRSA, while raw camel had the lowest (50%). Fifty-eight out of 79 (73.41%) MRSA strains harbored the PVL gene. SCCmec IVa (39.65%), V (22.41%) and III (10.34%) were the most commonly detected types in the MRSA strains. MRSA strains harbored the highest prevalence of resistance against penicillin (100%), tetracycline (100%), gentamicin (65.51%) and erythromycin (56.89%). AadA1 (58.62%), tetK (56.89%), msrA (41.37%) and vatA (36.20%) were the most commonly detected antibiotic resistance genes. Simultaneous presence of PVL and antibiotic resistance genes in multi-drug resistant MRSA strains specifies significant public health problem.


Introduction
Staphylococcus aureus(S. aureus) is Gram-positive, catalase positive, and cocci-shaped bacterium typically originate from nose and respiratory tract and on the skin. It is mainly associated with nosocomial and community-acquired infections and foodborne diseases (Kadariya et al. 2014). S. aureus is a causative agents for about 241,000 food-borne illnesses per year in the United States (Scallan et al. 2011). Meat is one of the most important food stuffs related to the Staphylococcal food-borne diseases all around the world (Beneke et al. 2011;Hanson et al. 2011;Pu et al. 2009;Weese et al. 2010). High prevalence of S. aureus strains has also been reported in different types of meat samples (Hasanpour Dehkordi et al. 2017;Madahi et al. 2014;Momtaz et al. 2013;Safarpoor Dehkordi et al. 2018;Safarpoor Dehkordi et al. 2017a).
Staphylococcal foodborne diseases are primarily related to the emergence of antibiotic resistance (De Boer et al. 2009). It has been documented that about 50% of strains of this bacterium were considered to be methicillin-resistant S. aureus (MRSA) (De Boer et al. 2009;Johnson 2011;Morell and Balkin 2010;Shen et al. 2013). A survey which was conducted on the United States revealed an annual estimate of 94,000 MRSA infections with nearly 20% mortality rate (Klevens et al. 2007). Presence of certain antibiotic resistance genes such as mecA (methicillin), linA (lincosamides), msrA and msrB (macrolides), vatA, vatB and vatC (acetyl transferase genes and streptogramin A), ermA, ermB and ermC (macrolide-lincosamide-streptogramin B), tetK and tetM (tetracycline) and aacA-D (aminoglycosides) is one of the most important mechanisms for occurrence of antibiotic resistance (Argudín et al. 2010;Momtaz and Hafezi 2014). The gene for methicillin resistance, mecA, is carried on a 21-to 67-kb element, the staphylococcal chromosomal cassette mec (SCCmec), which integrates at a conserved location in the S. aureus genome (Shukla et al. 2012). SCCmec genetic element characterized by the presence of two essential genetic markers (the mec and the ccr gene complexes). SCCmec elements are classically classi ed into types I, II, III, IV and V according to the nature of the mec and ccr genes (Ferreira et al. 2013;Turlej et al. 2011). Type IV of the SCCmec genetic element is divided to IVa, IVb, IVc and IVd alleles (Ferreira et al. 2013;Turlej et al. 2011). The frequent recovery of staphylococcal isolates that produce leukocidal toxins from the cases of foodborne diseases and clinical infections, suggests that the Panton-Valentine Leukocidin (PVL) is a virulence marker with signi cant role in the pathogenicity of staphylococcal diseases (Ogata et al. 2012;Shrestha et al. 2014;Velasco et al. 2014).
Rendering the high importance of MRSA strains as a foodborne pathogen, the current research was done to study the prevalence rate, distribution of SCCmec types and PVL gene and antimicrobial resistance properties of the MRSA strains isolated from raw ruminant, poultry and sh meat samples in Iran.
Isolation and identi cation of S. aureus and MRSA strains Each sample was aseptically weighed in an analytical balance and twenty-ve grams were transferred into a sterile plastic bag. Then, 225 mL of buffered peptone water (Merck, Germany) was added and homogenized in a Stomacher Bagmixer 400W (Interscience, Saint-Nom, France) for two min. Five milliliter aliquot of the enriched homogenate was transferred into 50 mL Trypticase Soy Broth (TSB, Merck, Germany) supplemented with 10% NaCl and 1% sodium pyruvate. After incubation at 35 °C for 18 h, a loopful of the culture was plated onto Baird-Parker agar supplemented with egg yolk tellurite emulsion (Merck, Germany) and incubated overnight at 37 °C. Black shiny colonies surrounded by 2 to 5-mm clear zones were further identi ed on the basis of some biochemical tests (Fijałkowski et al. 2016).
Cefoxitin (30 µg) and oxacillin (1 µg) susceptibility tests were used to distinguish the MRSA strains from S. aureus isolates of meat samples. All tests were performed using the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (CLSI 2007). MRSA isolates were identi ed another time using the Polymerase Chain Reaction (PCR)-based ampli cation of mecA gene (Safarpoor Dehkordi et al. 2017a). MRSA strains were sub-cultured on TSB (Merck, Germany) and further incubated for 48 h at 37 o C. Genomic Deoxyribonucleic acid (DNA) was extracted from bacterial colonies using the DNA extraction kit (Thermo Fisher Scienti c, Germany) according to manufacturer's instruction. After extraction, the DNA samples were quanti ed (NanoDrop, Thermo Scienti c, Waltham, MA, United States (USA)), their purity checked (A260/A280), and their concentrations adjusted to 50 nanogram per microliter (ng/μL).
Detection of antibiotic resistance genes and SCCmec types Table 1 represents the oligonucleotide primers and PCR conditions used for ampli cation of antibiotic resistance genes and SCCmec types amongst MRSA strains isolated from various types of raw meat (Lina et al. 1999;Strommenger et al. 2003;Zhang et al. 2005). Additional PCR method was used to detect the PVL gene amongst the MRSA strains isolated from different types of meat samples (Yamasaki et al. 2005). A programmable DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions. All runs included a negative DNA control consisting of sterile PCR grade water (Thermo Fisher Scienti c, Germany) and positive DNA control consisting of positive DNA of each target gene. Fifteen microliters of ampli ed PCR products were subjected to electrophoresis in a 2% agarose gel in 1× Tris-borate-Ethylenediamine tetraacetic acid (EDTA) (TBE) buffer at 90 V for 30-40 min, stained with SYBR Green (Thermo Fisher Scienti c, Germany).

Statistical analysis
Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) 25.0 statistical software (SPSS Inc., Chicago, IL, USA). Chi-square test and Fisher's exact two-tailed test were used to assess any signi cant relationship for prevalence of MRSA strains and their antibiotic resistance and molecular characters between different types of samples. P value <0.05 was considered as statistical signi cant level.

Results
Incidence of S. aureus, MRSA and PVL gene Table 2 represents the distribution of S. aureus, MRSA strains and PVL gene in different types of raw meat samples. Onehundred and thirty-ve out of 680 (18.38%) raw meat samples were positive for S. aureus strains. Raw sheep samples (28%) had the highest prevalence of S. aureus strains, while raw sh samples had the lowest (1.66%). Statistically signi cant difference was seen between type of samples and prevalence of S. aureus strains (P <0.05). MecA gene was identi ed in all MRSA strains. Seventy-nine out of 125 (63.20%) S. aureus strains were determined as MRSA strains. There were no MRSA strains in sh samples. Raw sheep samples (75%) had the highest prevalence of MRSA strains, while raw camel meat samples had the lowest (50%). Statistically signi cant difference was also seen between type of samples and prevalence of MRSA strains (P <0.05).
Fifty-eight out of 79 (73.41%) MRSA strains harbored the PVL gene. MRSA strains isolated from raw sheep meat samples had the highest prevalence of the PVL gene (95.23%), while those of quail had the lowest (33.33%). Statistically signi cant difference was also seen between type of samples and distribution of the PVL gene (P <0.05). Table 3 represents the distribution of SCCmec types in MRSA strains isolated from various types of raw meat samples. SCCmecIVa (39.65%), V (22.41%) and III (10.34%) were the most commonly detected types in the MRSA strains isolated from various types of raw meat samples. Raw sheep meat samples had the highest and most diverse distribution of SCCmec types. Statistically signi cant difference was also seen between type of samples and distribution of the SCCmec types (P <0.05).

Distribution of SCCmec types
Antibiotic resistance pattern of MRSA strains Table 4 represents the antibiotic resistance pattern of MRSA strains isolated from various types of raw meat samples. MRSA strains harbored the highest prevalence of resistance against penicillin (100%), tetracycline (100%), gentamicin (65.51%), erythromycin (56.89%) and trimethoprim-sulfamethoxazole (55.17%). MRSA strains exhibited the lowest prevalence of resistance against chloramphenicol (25.86%) and clindamycin (29.31%). Statistically signi cant difference was seen in the prevalence of antibiotic resistance between different types of raw meat samples (P <0.05).
Distribution of antibiotic resistance genes Table 5 represents the distribution of antibiotic resistance genes amongst the MRSA strains isolated from various types of raw meat samples. AadA1 (58.62%), tetK (56.89%), msrA (41.37%) and vatA (36.20%) were the most commonly detected antibiotic resistance genes amongst the MRSA strains isolated from various types of raw meat samples. Distribution of vatC (3.44%), msrB (10.34%) and vatB (12.06%)were lower than other detected antibiotic resistance genes. Statistically signi cant difference was seen in the distribution of antibiotic resistance genes between different types of raw meat samples (P <0.05).
The present research was done to assess the phenotypic and genotypic characterization of antibiotic resistance and study the distribution of SCCmec types and PVL gene in the MRSA strains isolated from ruminants, poultries and sh meat samples. Zero prevalence rate of MRSA in sh meat samples is may be due, in part, to the presence of speci c primary bacterial ora in sh and low ability of S. aureus to compete. Low prevalence of MRSA strains in camel meat samples is may be due the fact that camel's slaughter is done in speci c slaughterhouses with high hygienic conditions. Higher pH level of sheep and goat meat than beef (Lazzaroni et al. 2007) is probably caused higher ability of S. aureus strains to growth and survival. Similar prevalence rate of MRSA strains was reported by Pu et al. (2009) (United States) (Pu et al. 2009), Huber et al. (2009 (Bhargava et al. 2011) reported that 65 out of 289 raw meat samples (22.5%) collected from Michigan, USA were positive for S. aureus: 32 beef (20.5%), 19 chicken (25.0%), and 14 turkey (24.6%). They showed that 6 samples, consisting of 2 beef (1.30%), 3 chickens (3.90%) and 1 turkey (1.70%), were positive for MRSA which was entirely lower than our results. Febler et al. (2011) (Feßler et al. 2011) showed that the prevalence of MRSA strains in turkey, chicken, pork and beef samples were 35.30%, 16%, 15.20% and 10.60%, respectively. Additionally, prevalence of MRSA strains in raw meat samples collected from Turkey (Gundogan et al. 2005), Egypt (Karmi 2013), Germany (Richter et al. 2012) and Denmark (Tang et al. 2017) were 30%, 40.80%, 71.50% and 52.00%, respectively. Role of food handlers in transmission of MRSA strains was reported previously by Agwu Ulu et al. (2014) (Agwu Ulu et al. 2014).
High prevalence of PVL gene in the MRSA strains poses an important public health threat. As far as we know, the present study is the rst report of detection of the PVL gene in the MRSA strains of beef, sheep and chicken meat samples. Previously published data reported that majority of PVL-positive S. aureus strains were associated with skin and soft tissue infections (Holmes et al. 2005). Thus, PVL-positive MRSA strains may originate from infected staffs and meat inspectors in slaughterhouses. Presence of the PVL gene in the S. aureus strains isolated from food samples has been reported previously (Abdalrahman et al. 2015;Holmes et al. 2005;Lozano et al. 2009). SCCmec IV and V had the highest distribution in the MRSA isolates. MRSA is often sub-categorized as Health-care Associated MRSA (HA-MRSA) and Community Associated MRSA (CA-MRSA). CA-MRSA strains carry SCCmec type IV or V, whereas the majority of HA-MRSA strains carry SCCmec type I, II or III (Asghar 2014). Thus, majority of MRSA strains isolated from raw meat samples of the present investigation were CA-MRSA. However, some of them were originate from the hospital environment and are recognized as HA-MRSA. Higher prevalence of SCCmec types IV in beef and pork samples was also reported by Jackson et al. (2013) (Jackson et al. 2013). In a study which was conducted by Vossenkuhl et al. (2014) (Vossenkuhl et al. 2014) most of the MRSA strains of turkey meat samples carried SCCmec V (58.10-71.90%), followed by type IVa (19-27%) which was similar to our results. High prevalence of SCCmec IV and V in foods with animal origin have also been reported previously (Argudín et al. 2010;Bhargava et al. 2011;Kreausukon et al. 2012). Therefore, raw meat samples may be the sources of CA-MRSA with high prevalence of SCCmec IV and V.

Conclusion
To put it in a nutshell, we identi ed high prevalence of antibiotic resistance, antibiotic resistance genes, SCCmec types and PVL gene in the MRSA strains isolated from raw ruminants, poultries and sh meat samples. MRSA strains had the higher prevalence in raw sheep and goat meat samples. Additionally, MRSA strains isolated from sheep meat samples harbored higher prevalence of PVL gene, antibiotic resistance gens and SCCmec types. MRSA strains harbored the highest prevalence of resistance against penicillin, tetracycline, gentamicin, erythromycin and trimethoprim-sulfamethoxazole antibiotic agents. Higher prevalence of aadA1, tetK, msrA and vatA antibiotic resistance genes is another important nding. Some MRSA strains exhibited simultaneous resistance against more than one antibiotic agents. Additionally, some of the harbored more than one antibiotic resistance genes. Furthermore, our research demonstrated that most MRSA were found to be resistant to commonly used antimicrobial agents which raised concerns regarding transmission risk following the consumption of raw or undercooked meat. Moreover, raw meat samples were the main sources of CA-MRSA strains with higher prevalence of SCCmec IV and V types. MRSA strains isolated from poultry meat samples harbored the highest prevalence of resistance against chloramphenicol antibiotic agents. Prevalence of resistance against human-based antibiotic was also high. Simultaneous presence of PVL gene, SCCmec types and antibiotic resistance genes in the MRSA strains pose an important public health threat regarding the consumption of raw or undercooked meat samples. As far as we know, the present study is the rst report in its eld on MRSA strains isolated from camel, turkey and quail meat samples. Our research highlights the importance of monitoring the antimicrobial susceptibility of MRSA in the food chains including raw meat and especially sheep, goat and poultry meat samples and these data could be used proactively to assist government and industries in Iran to develop improved food safety measures, designed to reduce the contamination and transmission of this bacterium. Moreover, a future large-scale and multipopulation-based study must be conducted to obtain more comprehensive data on the prevalence and distribution of MRSA strains in different Iranian ethnic populations. All data generated or analyzed throughout this research are included in this published article.

Competing interests
Authors declared that they have no con ict of interest.

Funding
Funding is not applicable for this research.

Authors' contributions
Sample collection, microbial analysis, PCR alignment, writing and drafting of the manuscript were performed by the RC. Table 1. Target enterotoxin genes, oligonucleotide primers, PCR programs and volumes used for detection of antibiotic resistance genes and SCCmec types amongst the MRSA strains isolated from various types of raw meat samples.