Molecular Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) Isolates from Ruminants

Methicillin resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that causes severe morbidity and mortality worldwide. Staphylococcus aureus, a unique bacterium, has adaptive power for antibiotics that lead to the emergence of Methicillin Resistant Staphylococcus aureus (MRSA) in the 1960s. Recent studies have documented the increased costs associated with MRSA infection. Pneumonia and bacteremia account for the majority of MRSA serious clinical infections, but intra-abdominal infections, osteomyelitis, toxic shock syndrome, food poisoning and deep tissue infections are also important clinical. It is a frequent cause of both subclinical and clinical mastitis in dairy cattle, which causes significant financial losses for dairy farmers all over the world (Seeger et al., 2003). It results in both subclinical and clinical mammary gland infections. Moreover, enterotoxigenic S. aureus is the cause of almost 10% of food-borne illnesses linked to dairy products (Zigo et al., 2021).
 
Staphylococcus species showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans and other species of Staphylococcus; however, the differences were not sufficient for reliable differentiation of Staphylococcus species by the SDS-PAGE method Berber et al., (2003). Abid et al., (2019) compared two novel S. aureus surface protein extraction methods with the biotinylation method and evaluated the immune-reactivity of extracted proteins. The presence of different band patterns among MRSA isolates has been shown by SDS-PAGE and its importance in epidemiology has also been implied (Gaston et al., 1998; Costas et al., 1990). The conventional methods are time consuming and difficult so molecular diagnosis of MRSA is increasingly important for rapid detection. During PCR the number and size of the fragments generated are the basis for typing an isolate (Hussain et al., 2018; Xia and Wolz, 2014). The polymerase chain reaction is a molecular technique that can rapidly detect the mec A gene (Jonas et al., 2002; Oliveira et al., 2002; Rocchetti et al., 2018; van Belkum and Rochas, 2018; Abed et al., 2020). Developing it for MRSA has been hampered because mec A is highly conserved in all staphylococcal species.

Collection of samples
 
A total of 725 samples of nasal swab and milk samples were collected for the study. The samples from nasal passage (375) were collected from cattle (100), buffalo (100), sheep (100) goat (75) and The composite milk samples (350) were collected from cattle (100), buffalos (100), sheep (75) and goats (75) maintained at the Livestock Farm Complex (LFC), from the animals presented at Veterinary Clinical Complex (VCC) of College of Veterinary Science and Animal Husbandry and animals maintained by local farmers around the university campus.
 
Isolation and identification of S. aureus was done as per the recommendations of the National Mastitis Council (NMC, 1990) and the National Committee for Clinical Laboratory Standards (NCCLS, 1997). Mannitol salt agar mixed with oxacillin resistance selective supplement was used for isolation of MRSA from nasal swab and milk samples. MRSA standard culture obtained from Veterinary Type Culture Collection (VTCC) ICAR-NRC on Equines, Hisar was used as standard. The organism on slants was subjected to Gram staining and biochemical tests viz. catalase test, coagulase test, methyl red test (MR Test), Voges-Proskaur test (VP-Test), sugar fermentation test, haemolysis on blood agar, DNase test and latex agglutination test. 
 
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
 
Genomic DNA was extracted by the procedure described by Ausubel et al., (1989). Denatured whole cell proteins were analyzed by SDS-PAGE according to the method described by Laemmli (1970). 
 
Preparation of samples
 
Isolates of MRSA were plated out on nutrient-agar plates and incubated at 37°C for 24 h. A sweep of colonies from these plates sufficient to give approximately 2 µg bacterial dry weight/ml was inoculated into 150 ml volumes of nutrient broth and incubated at 37°C overnight in an orbital incubator for 3 min at 12100 rpm. The collected cells were washed three times with sterile distilled water and stirred after adding 25 µl SDS sample buffer (0.06 M Tris, 2.5% glycerol, 0.5% SDS, 1.25% β-mercaptoethanol and 0.001% bromophenol blue). The proteins were denatured in boiling water for 5 min. The supernatant was then centrifuged again for 3 min at 12100 rpm, collected in an eppendorf tube and kept at -50°C until the electrophoresis was carried out.

Denatured proteins were analyzed by SDS-PAGE according to Laemmli (1970). This method used a 2 cm layer of 4% acrylamide stacking gel and a 10 cm layer of 10% acrylamide separating gel. Sigma wide range marker was used as molecular weight standard in SDS-PAGE. Electrophoresis was performed with a discontinuous buffer system in a BRL gel apparatus model V16-2BRL Gaithersburg MD, USA. The gel was run at a constant current of 35 mA until the bromophenol blue had reached the bottom. Gels were then stained with Coomassie Brilliant Blue R 250 (Sigma).
 
Polymerase chain reaction
 
Subsequent to biochemical characterization, the S. aureus isolates that appeared methicillin resistant in diffusion test were subjected to amplification of mec A gene and fem A gene using protocol with specific primers sets. The amplified products were imaged by running them in 1.5% agarose containing 0.5% µg/ml ethidium bromide.
 
Preparation of DNA template by heating
 
All the clinical samples that appeared methicillin resistant in disc diffusion test were cultured in the Brain heart infusion (BHI) broth culture by incubating overnight at 37°C and subsequently, subjected for confirmation of Staph. aureus by targeting the mec A and fem A gene based specific assay. Briefly, 1 ml of overnight broth was centrifuged at 4000 rpm for 15 min. the supernatant was discarded and the pellet was resuspended in 200 µl of nuclease free water (NFW). The Suspension was heated to 94°C for 10 min and immediately chilled on ice to release the DNA after the  breakage of the cell. After centrifugation at 8000 rpm, the supernatant was used as a DNA template.
 
Primers
 
The following oligonucleotide primers specific for mec A and fem A gene sequences of methicillin resistant S. aureus from published primer sets were synthesized by Merck India and were used in the present study (Table 1).


 
Reaction mixture for PCR
 
The Amplification reaction was performed in a total volume of 25 µl for one PCR reaction as given below (Table 2). The reaction mixture was performed by two different ways as follows:


 
Gene amplification conditions
 
The amplification of target sequences was carried out under the following conditions using a thermal cycler (ProFlex PCR machine) (Table 3).

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Confirmation of PCR products
 
The amplified PCR products was confirmed for its excepted size in 1.5% agarose gel prepared in 0.5X TBE buffer as per the method of Sambrook et al., (1989) using horizontal submarine electrophoresis apparatus (Torson). A 1.5% agarose gel prepared in 0.5X TBE buffer was boiled for 2 min and allowed to cool down to 50°C. Ethidium bromide was then added in the gel to a final concentration of 0.5 µg/ml and mixed thoroughly. The gel casting platform was then placed on a leveled surface and the open sides were sealed with adhesive tape was removed. 
 
Confirmation of PCR products
 
The amplified PCR products were confirmed for their excepted size in 1.5% agarose gel prepared in 0.5X TBE buffer as per the method of Sambrook et al., (1989) using horizontal submarine electrophoresis apparatus (Bio-Rad). A 1.5% agarose gel prepared in 0.5X TBE buffer was boiled for 2 min and allowed to cool down to 50°C. Ethidium bromide was then added in the gel to a final concentration of 0.5 µg/ml and mixed thoroughly. The gel casting platform was then placed on a leveled surface and the open sides were sealed with adhesive tape was removed. The set gel with gel casting platform was then submerged in a sufficient quantity (about 1 mm above the gel level) of electrophoresis buffer (TBE) in the electrophoresis tank keeping the wells towards cathode end.
 
10 ul of PCR product was loaded into respective wells. The molecular weight marker (100 bp DNA ladder, Thermo scientific) was also mixed with bromophenol blue dye in similar quantities and loaded into the first well of gel. Electrophoresis was carried out at 5 volt/cm current and the progress of motility was monitored by migration of the dye. At the end of electrophoresis, the gel was visualized and analyzed using a Gel documentation system (Bio-Rad) for an amplified product of the desired length.

In the present study, samples  which included nasal swabs (375) and milk samples (350) were collected randomly from cattle, buffalo, sheep and goats. Out of 725 samples, 96 nasal and 75 milk samples (total 171 samples) were found positive for Oxacillin resistant S. aureus. These 171 MRSA isolates were confirmed by Gram’s Staining and biochemical tests. All the positive isolates were subjected to Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for the identification of major polypeptides in whole cell protein. All the isolates irrespective of animal species and site of collected sample revealed almost similar molecular weight polypeptides in size and number. The gel showed polypeptides ranging between 20 and 200 kDA. Depending upon the intensity of bands 17 major polypeptides of molecular weight  13 kDA, 17 kDA, 20 kDA, 24 kDA, 26 kDA, 28 kDA, 33 kDA, 36 kDA, 39 kDA, 43 kDA, 47 kDA, 59 kDA, 64 kDA, 72 kDA, 86 kDA, 97 kDA and 121 kDA were observed in whole cell protein of all the S. aureus isolates (Fig 1and 2).




 
The DNA was extracted from all positive S. aureus isolates that exhibited methicillin resistance in disc diffusion method and extracted DNA was subjected to Polymerase Chain Reaction PCR for the amplification of mec A and fem A genes which are considered to be responsible for methicillin resistance. Out of 171 methicillin resistance S. aureus isolates recovered from cattle, buffalo, sheep and goat during this study, mec A and fem A genes were amplified in 137 and 133 isolates with an amplicon 533 kb (Fig 3) and 510 kb (Fig 4) respectively. The MRSA isolates were positive for mec A gene, 19 out of 23 (82.61%), 27 out of 39 (69.23%), 19 out of 23 (82.61%), 18 out of 20 (90.00%), 25 out of 29 (89.66%), 10 out of 11 (90.91%), 9 out of 14 (64.28%), 10 out of 12 (83.33%) in cattle nasal swab sample, cattle milk, buffalo nasal swab, buffalo milk, sheep nasal swab, sheep milk, goat nasal swab and goat milk respectively (Table 4).






 
The MRSA isolates were positive for fem A gene, 19 out of 23 (82.61%), 25 out of 39 (64.10), 18 out of 23 (78.26%), 17 out of 20 (85.00%), 25 out of 29 (89.66%), 10 out of 11 (90.91%), 9 out of 14 (64.28%), 10 out of 12 (83.33%) in cattle nasal swab sample, cattle milk, buffalo nasal swab, buffalo milk, sheep nasal swab, sheep milk, goat nasal swab and goat milk respectively (Table 4).
 
The presence of different band patterns among MRSA isolates has been shown by SDS-PAGE and its importance in epidemiology has also been implied (Gaston et al., 1998). In this study depending upon the intensity of bands 17 major polypeptides were of molecular weight 13 kDA, 17 kDA, 20 kDA, 24 kDA, 26 kDA, 28 kDA, 33 kDA, 36 kDA, 39 kDA, 43 kDA, 47 kDA, 59 kDA, 64 kDA, 72 kDA, 86 kDA, 97 kDA and 121 kDA  observed in whole cell protein of all the S. aureus isolates from cattle, buffalo, sheep and goat. It is supported by the finding of Jayshree, (2018) that analysed human and animal isolates were subjected to SDS-PAGE for the identification of major polypeptides in whole cell protein of isolates with the ranged from 20-200 kDA. Depending upon the intensity of bands out of 23 polypeptides 12 major polypeptides were of molecular weight 20 kDA, 28 kDA, 33 kDA, 39 kDA, 43 kDA, 59 kDA, 64 kDA, 72 kDA, 86 kDA, 97 kDA, 121 kDA and 200 KDA. Additionly 11 minor polypeptides were also observed in whole cell protein of all the S. aureus isolates.  It has been used for taxonomic and typing tools analysis (Manikandan et al., 2009). 
 
The DNA was extracted from all S. aureus isolates that exhibited methicillin resistance in disc diffusion method and extracted DNA was subjected to PCR for the amplification of mec A and fem A genes which are considered to be responsible for methicillin resistance. Out of 171 methicillin resistance S. aureus isolates recovered during this study, mec A and fem A genes could be amplified in 137 and 133 isolates with an amplicon of 533 kb and 510 kb respectively. The data indicated a high prevalence of MRSA in cattle. (80.11% for mec A and 77.78% for mec A).
 
Singh et al., (2015) reported that 18 (24%) isolates from 75 samples of mastitic milk and 10 (28.57%) isolates from 35 samples of nasal swab that appeared to be resistant to methicillin in disc diffusion method showed amplification of mec A gene. Similarly, Shanehbandi et al., (2014) also reported that all 110 MRSA isolates that screened positive for coagulase, also positive for mec A gene. In the present study, fem A gene was exhibited by 15 out of 18 MRSA isolates from mastitic milk and 8 out of 10 MRSA isolates from nasal swabs. Maniknandan et al.  (2011) reported all MRSA isolates from clinical pus samples to be positive for fem A gene.
 
Singh et al., (2016) used the disc diffusion method to determine the methicillin resistance of 57 isolates from 240 milk samples. The PCR amplification process revealed that 53 (92.98%) of the isolates were positive for mec A and 42 (73.68%) for fem A genes, with amplicon sizes of 510 bp and 533 bp, respectively.

All authors declared that there is no conflict of interest.