Methicillin-resistant Staphylococcus aureus (MRSA) is a critically important human nosocomial pathogen worldwide that is also an emerging concern in veterinary medicine, being an important cause of a wide variety of hard-to-treat pyogenic infections (Otto, 2012). Methicillin resistance is chromosome mediated and is related to the mobile genetic element Staphylococcal Cassette Chromosome mec (SCCmec) that includes mecA gene specifying the production of an abnormal penicillin binding protein 2a (PBP 2a) (Hartman and Tomasz, 1981). Another mechanism of resistance to penicillin in S. aureus is production of β-lactamase enzymes (encoded by blaZ gene) (Hartman and Tomasz, 1981). Recent studies on MRSA suggest that dogs may act as a significant reservoir for MRSA (Walther et al., 2012). Pet associated persons, pet breeders and veterinarians encompass the primary risk groups that may become colonized from MRSA of canine origin (Loeffler and Lloyd, 2010). Genetic characterization of MRSA isolated from dogs also suggests transmission between humans and dogs, since they carry the same strains that are prevalent in humans (Baptiste et al., 2005 and Weese and van Duijkeren, 2010).

Molecular typing of MRSA is important particularly for the purposes of tracing of outbreaks and subsequent infection control. There are a number of techniques available to type MRSA like Pulsed field Gel Electrophoresis (PFGE), Multi Locus Sequence Typing (MLST), Staphylococcus surface protein A (Spa) typing, Polymerase Chain Reaction (PCR)-Amplified Ribosomal DNA spacer Polymorphism (RS-PCR-ribotyping) etc. (Moodley et al., 2006 and Walther et al., 2012). Data regarding epidemiology of MRSA of canine origin and its molecular characterization is lacking in India. Hence the present study was carried out with an objective of molecular typing and assessment of genetic diversity of MRSA isolated from nasal swabs of apparently healthy dogs, corresponding dog owners and veterinary students attending canine wards in Andhra Pradesh by RS-PCR ribotyping.

Materials and Methods                          

Ethical Approval

Ethical approval is not necessary to pursue this study. However, samples were collected without harming dogs. Informed consent was obtained from corresponding dog owners.

Bacterial Reference Strain

The reference strain MRSA (ATCC 25923) was obtained from Hi-Media Laboratories (Mumbai) and maintained at the bacteriology laboratory of Department of Veterinary Public Health and Epidemiology, NTR College of Veterinary Science, Gannavaram, Andhra Pradesh.

Source of Bacterial Isolates

A total of 13 MRSA isolates recovered from nasal swab samples of dogs (4/40), corresponding dog owners (6/40) and veterinary students (3/40) attending canine wards of Teaching Veterinary Clinical Complex (TVCC), NTR College of Veterinary Science (Gannavaram) and College of Veterinary Science (Tirupati), Andhra Pradesh, were used in the present study. The identification of each MRSA isolate was carried out by cultural and biochemical tests (Fig. 1) viz. gram positive cocci with yellow colonies on mannitol salt agar, catalase (positive), oxidase (negative), Voges-Proskauer (positive), haemolysis (positive) and coagualse activity (positive), blue colour colonies on MeReSa CHROM agar, cefoxitin and oxacillin resistance (Sneath and Holt, 2001 and Velasco et al., 2005). Microbiological culture media, buffers and all other chemical reagents were procured from M/s. HiMedia Laboratories (Mumbai). All the 13 isolates were confirmed as MRSA by PCR targeting mecA and blaZ gene (Vannuffel et al., 1995 and Martineau et al., 2000). Oligonucleotide primers used in the present study were custom synthesized from M/s. Bioserve Biotechnologies Pvt. Ltd. (Hyderabad).

Ribosomal DNA Spacer PCR (RS-PCR Ribotyping)

Oligonucleotide primers used for amplification of 16S-23S ribosomal DNA spacer region of MRSA from different sources were as described by Jensen et al. (1993) and custom synthesized from M/s. Bioserve Biotechnologies Pvt. Ltd. (Hyderabad). A 3.0 µl aliquot of DNA was combined with 2.5µl of PCR reaction buffer with 15mM MgCl₂, 1.0 µl of a dNTP mixture (10mM), 1.25µl of each of two 15 base oligonucleotide primers (G1-GAA GTC GTA ACA AGG and L1- CAA GGC ATC CAC CGT) [10 pmol/μl] and 40.0 µl of nuclease free water. This mixture was heated to 94^oC for 5 min and 1.0 U of thermostable DNA polymerase was added. PCR assay was performed in Eppendorf (Germany) thermal cycler with heated lid under the following standardized cyclic conditions: 1 min of denaturation at 94^oC; 2 min annealing at 55^oC; and extension at 72^oC for 1 min for 34 cycles and a final cycle of elongation at 72^oC for 7 min. The amplified PCR products were subjected to 2% agarose gel electrophoresis under 110V for 2 h (Sambrook and Russell, 2001) and bands were visualized under UV trans-illumination using Bio-Rad Gel documentation system.

Scoring of RS-PCR Ribotypes

The PCR amplicons were photographed and analyzed using BIO-RAD Gel Documentation image analysis system (USA) and the supplied image lab software. The position of bands was compared using 100 bp and 1 kb DNA ladder (Genei^TM, Bengaluru) as an external reference. Binary matrix was generated using binary coding based on presence (1) or absence (0) of a particular band in the given isolate. The binary data was analyzed using dollop programme of PHYLIP (Phylogeny Inference Package) software (version 3.6) with default options and dendrograms were constructed to establish genetic relationship among the MRSA strains from different sources. Clusters were considered at a 70% similarity cut-off and the similarity of band patterns was calculated using the Pearson’s correlation coefficient.

Results and Discussion

Over the past few decades, MRSA has emerged as an important pathogen in veterinary medicine. Significant epidemiological and genetic differences exist between MRSA from humans and different animal species (Loeffler and Lloyd, 2010). In order to evaluate the strain diversity and genetic relatedness of MRSA isolates from different sources viz. dogs, dog owners and veterinary students, a total of 13 CoPS isolates were typed by RS-PCR-ribotyping. The resultant DNA amplicons ranged in size from 150 bp to 1000 bp, with 1 to 6 resolved fragments per isolate (Fig. 2).

Fig. 2: RS-PCR-DNA finger printing patterns of MRSA isolates from dogs (D4, D9, D20, D28), their owners (H4, H6, H9, H13, H21, H31), veterinary students (S15, S22, S29) and reference strain MRSA, ATCC 25923 (S8).

RS-PCR ribotypes and genetic diversity of MRSA from different sources detected in the present study was given in Table 1. Polymorphism was observed with 13 ribotypes identified among the 13 MRSA strains tested. Molecular typing is essential for differentiation and characterization of MRSA strains from diverse sources (Mulligan and Arbeit, 1991).

Table 1: Genetic diversity of MRSA strains of canine and human origin

Accurate epidemiological typing is of primary importance for detecting routes of transmission of MRSA. Further, the 16S-23S intergenic sequence has been shown to be highly conserved and direct indicator of evolutionary divergence of MRSA (Gurtler and Barrie, 1995). In a study on the comparison of ribosomal spacer DNA amplicon polymorphisms and PFGE for differentiation of MRSA strains, PCR-ribotyping was shown to have almost as discriminatory power as PFGE and suggested for investigation of MRSA outbreaks, being a rapid inexpensive technique that is highly reproducible (Kumari et al., 1997). The RS-PCR Ribotyping for characterization of staphylococcal isolates was successfully used earlier by many workers from India and abroad (Oliveira and Ramos, 2002; Dubey et al., 2009 and Reshma et al., 2017). In the present study, phylogenetic analysis discriminated MRSA isolates from different sources of the study into three clusters (Cluster I, Cluster II and Cluster III) for 70% similarity cut-off (Fig. 3).

Fig. 3: Cluster analysis of RS-PCR-ribotyping patterns of MRSA isolates from dogs (D4, D9, D20, D28), their owners (H4, H6, H9, H13, H21, H31), veterinary students (S15, S22, S29) and reference strain MRSA, ATCC 25923 (S8) generated using dollop programme of PHYLIP version 3.6.

All the four MRSA isolates of dog origin (D4, D9, D20, D28) along with MRSA isolate from a dog owner (H4) and a veterinary student (S22) were grouped under Cluster I; while cluster II comprised exclusively MRSA isolates from dog handlers (H9, H13, H21). Cluster III is intermixed with MRSA isolates from both veterinary students (S15, S29), dog handlers (H6, H31) and reference strain of MRSA (ATCC 25923). Interestingly, MRSA isolates of one of the dog (D4) and its corresponding owner (H4) were present within the same cluster (Cluster I) indicating possibility of transmission of MRSA between dogs and humans (although the direction of transmission could not be proven). These results were in accordance with other study findings that had shown that people and pets can harbor identical strains of MRSA when they share an environment (Morris et al., 2010; Loeffler et al., 2011 and Tarazi et al., 2015).

Also, several studies revealed a strong association between human and animal strains of MRSA (Morris et al., 2010 and Tarazi et al., 2015). A limited number of studies have characterized the MRSA isolated from healthy canine samples and have found that the strains found in dogs are characteristics of the strains isolated in human hospitals (O’Mahony et al., 2005; Moodley et al., 2006 and Van Duijkeren et al., 2011). In a study by Zhang et al. (2011), MRSA isolates originating from dogs and veterinary staff in Beijing shared similar PFGE patterns, suggesting possibility of cross-transmission of MRSA between pet animals and veterinary staff. This makes the scenario of transfer of MRSA strains from humans to their pets or other animal contacts and subsequent colonisation or infection of the dog. Therefore, while dogs may not be a primary reservoir of MRSA for humans, they do present an important secondary reservoir for re-infection or re-colonisation of humans. Further, intensive daily contact between dogs and their owners was shown to increase the likelihood of interspecies-transmission of CoPS for both sides (Manian, 2003 and Van Duijkeren et al., 2011).

The increasing indiscriminate antibiotic usage has also made canine population a reservoir of MRSA. At the same time, the evolution of new MRSA clones has emphasized the need for infection control practices in animals and humans in close contact. Medical and veterinary staff should appreciate that animals can carry MRSA and cooperate in eliminating infections by implementing guidelines for dealing with MRSA.

Conclusion

The current study demonstrated a wide genetic diversity and little host specificity of MRSA strains among dogs, dog owners and veterinary students. MRSA isolates from one of the owner and his associated pet dog were present within the same cluster indicating the possibility of zoonotic transmission. No genetic relatedness was observed between MRSA isolates from other dogs and humans. In this context, our report is the first from India exploring diversity of MRSA from dogs, owners and professionals. The study emphasized the utility of RS-PCR-ribotyping in the epidemiological investigations for the detection of polymorphism and to elucidate the genetic relatedness of MRSA strains. Other molecular techniques like Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), Staphylococcal protein A (Spa) typing and whole genome sequencing are required to establish zoonotic transmission of these strains from animals to animal handlers and vice versa.

Acknowledgements

The Authors thank Sri Venkateswara Veterinary University (SVVU), Andhra Pradesh and Department of Science and Technology (DST), Government of India for the financial support extended in conducting research.

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