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Identification of Staphylococcus pseudintermedius and MRSP in Dogs with Pyoderma by PCR

Dermatological disorders in dogs are of major concern due to its multiple etiological factors, higher cost of treatment and the long duration of therapy and management required. Therefore appropriate diagnosis and identification of exact etiology is the key for successful therapeutic management (Scott and Paradis, 1990). Pyoderma is one of the most common dermatological disorders encountered in small animal practice. Almost ninety per cent of the cases of pyoderma in dogs are caused by coagulase positive International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 8 (2020) Journal homepage: http://www.ijcmas.com


Introduction
Dermatological disorders in dogs are of major concern due to its multiple etiological factors, higher cost of treatment and the long duration of therapy and management required. Therefore appropriate diagnosis and identification of exact etiology is the key for successful therapeutic management (Scott and Paradis, 1990). Pyoderma is one of the most common dermatological disorders encountered in small animal practice. Almost ninety per cent of the cases of pyoderma in dogs are caused by coagulase positive

ISSN: 2319-7706 Volume 9 Number 8 (2020)
Journal homepage: http://www.ijcmas.com Pyoderma is one of the most common dermatological disorders encountered in small animal practice. Almost ninety per cent of the cases of pyoderma in dogs is caused by bacteria belonging to the genus Staphylococcus and now Staphylococcus pseudintermedius is recognized as the main etiological agent responsible for pyoderma Further, Methicillin Resistant S pseudintermedius is being reported worldwide and represents a serious threat to the health of dogs. However not much work on S pseudintermedius has been done in India. Hence the study, with the primary objective being isolation, identification and genotypic characterization of staphylococcal organisms responsible for pyoderma in dogs along with detection of genotypic resistance to methicillin in these organisms. Dogs presented to the Veterinary College Hospital, Bangalore with clinical signs suggestive of pyoderma were chosen as subjects for the study. Materials from lesions from these dogs were collected, processed and cultured in Mannitol Salt Agar. DNA extracted from these isolates were subjected to multiplex PCR using species specific primers and were also tested for methicillin resistance genotypically by PCR targeting the mecA gene. All the twenty isolates were staphylococci and eleven of the isolates were found to be S pseudintermedius and nine were S aureus. Sixteen of the twenty isolates were resistant to methicillin genotypically on PCR. The study conclusively proved the occurrence of S pseudintermedius in dogs with pyoderma which probably has not been reported from India. Further, it also proved the occurrence of methicillin resistant S pseudintermedius and S aureus in dogs with pyoderma which could be important from the perspective of therapeutic management of pyoderma Staphylococci. In the the early 70s and 80s, Staphylococcus aureus was believed to be the main organism responsible for pyoderma. More recently, biotyping methodology for coagulase positive staphylococcal species indicated that the pathogenic coagulase positive staphylococcus in the dog was Staphylococcus intermedius (Berg et al., 1984). Of the staphylococcal species, Staphylococcus intermedius has been implicated in approximately 90% of cases of pyoderma in dogs. However, in actuality, isolates based on phenotypical characteristics originally identified as S. intermedius have been found to be from three different species, Staphylococcus intermedius, Staphylococcus pseudintermedius and Staphylococcus delphini. For definitive identification of these species, molecular diagnostic methods, such as polymerase chain reaction (PCR) techniques are required (Bannoehr et al., 2009) and the term Staphylococcus intermedius Group (SIG) is now used to refer to the three previously mentioned isolates (S. intermedius, S. pseudintermedius and S. delphini) as a group (Bannoehr et al., 2007 andSasaki et al., 2007).
Consequently, since the reclassification of the species, it has been proposed that all canine isolates belonging to the SIG should be considered as S. pseudintermedius unless proven otherwise by genetic typing methods (Devriese et al., 2008). Staphylococcus pseudintermedius is a canine commensal and opportunistic pathogen, which is analogous to S. aureus in human beings. Antibiotic resistance in staphylococci is of great concern due to increasing incidence of methicillin resistance among staphylococci. Of late, several reports are emerging of methicillin resistance in SIG. Also, a high rate of multidrug resistance is being reported among Methicillin Resistant S. pseudintermedius (MRSP) strains in dogs (Schwarz et al., 2008).
Methicillin resistance in S pseudintermedius similar to S aureus is mediated by the mecA gene which encodes the penicillin binding protein 2a (PBP 2a) which has low affinity for beta lactam antimicrobials and therefore confers resistance to staphylococcus (Moon, 2012). In 2013, Videla stated that methicillin resistance and resistance to other antimicrobials regularly used by veterinarians is common among S. intermedius which can complicate treatment. The first report of mecA gene responsible for methicillin resistance in S. intermedius was in 1999 and MRSP was first reported in 2005. Since then, resistance to methicillin and to other antimicrobials has become increasingly common, making this bacterium a possible reservoir for antimicrobial resistance genes.
Thus, taking into consideration the fact that Staphylococcus pseudintermedius is recognized as the main etiological agent responsible for pyoderma and that not much work has been done in India to detect the presence of this bacteria, this study was taken up. Further, Methicillin Resistant S pseudintermedius (MRSP) is being reported worldwide and represents a serious threat to the health of dogs and yet not much work on MRSP has been done in India.
The primary objectives of the study were to detect the occurrence of different species of staphylococci with emphasis on S pseudintermedius and to detect methicillin resistance in different species of staphylococci causing pyoderma in dogs.

Culture, Coagulase, Catalase testing and Gram's staining
Twenty animals presented to Veterinary College, Bangalore with clinical signs suggestive of pyoderma such as papules, pustules, erythema, alopecia, pruritus and epidermal collarettes were selected as subjects for bacterial culture, coagulase and catalase testing, Grams staining and molecular studies. Samples were collected from the lesions using sterile cotton swabs and subjected to bacterial culture, primarily using nutrient broth or brain heart infusion broth and subcultured using Mannitol Salt Agar. All plates were incubated aerobically at 37˚C for 18-24 hrs for observation of characteristic growth and tentative identification was done based on the morphology of colonies. Individual colonies were selected and catalase and coagulase testing (tube coagulase) was done as per standard procedure (Elmer et al, 1988) and Gram's staining was done and biochemical characterization was done using a commercially available kit (HiStaph kit, Himedia, Mumbai, India)

DNA extraction and PCR
The isolates from culture were subjected to DNA extraction as per the standard protocol prescribed by the manufacturer using a kit procured commercially (AMpurE Bacterial gDNA Mini Spin, Amnion Biosciences Pvt Ltd, Bangalore, India) and Multiplex PCR (m-PCR) was carried out for identification of staphylococcus to the species level targeting the nuc gene loci for differentiation of species using the primer sequences given in Table 1 (Sasaki et al, 2010).
Multiplex PCR was carried out using a programmable master cycler with a reaction mixture volume of 25 ul. The DNA samples were subjected to thermal cycling conditions as given below 1. Initial denaturation at 95° C for 10 min followed by 2. Denaturation at 95° C for 30 sec 3. Annealing at 56° C for 1 min 4. Extension at 72° C for 1 min 5. Final extension at 72°C for 10 min 6. Hold at 4° C After completion of PCR, 3 ul of the amplified products along with 100 bp DNA ladders mixed with 6X gel loading dye were subjected to electrophoresis on 2 % agarose gel (prepared using TAE buffer, agarose and Ethydium Bromide). One positive control and one negative control were used with the test samples. The PCR product size was determined by comparing with a standard molecular marker (DNA ladder) and the images were captured using Gel Documentation system.

Detection of methicillin resistance by PCR
The extracted DNA from all the twenty isolates were utilized for molecular studies on methicillin resistance targeting the mecA gene. PCR was carried out using a programmable master cycler (Eppendorf, Hamburg, Germany).The reaction mixture volume was 25 ul. (using 10X Taq buffer, Taq polymerase, dNTPs, Primers, Template and Nuclease Free Water) PCR was carried out with the following thermal cycler conditions 1. Initial denaturation at 94° C for 1 min followed by 2. Denaturation at 94° C for 30 sec 3. Annealing at 50° C for 1 min 4. Extension at 72° C for 2 min 5. Final extension at 72°C for 10 min 6. Hold at 4° C The steps 2, 3, 4 were repeated (programmed) for 30 cycles. After completion of PCR, 3 ul of the amplified products along with 100 bp DNA ladders mixed with 6X gel loading dye were subjected to electrophoresis on 2 % agarose gel (prepared using TAE buffer, agarose and Ethydium Bromide).One positive control and one negative control were used with the test samples. The primers used for the study i.e Staph mecA gene (F-TGGCTATCGTGTCACAATCG and R-CTGGAACTTGTTGAGCAGAG) were designed at the Department of Microbiology, Veterinary College, Bangalore for an earlier study (Sundareshan, 2012). The PCR product size was determined by comparing with a standard molecular marker (DNA ladder) and the images were captured using Gel Documentation system.

Culture, Coagulase, Catalase testing and Gram's staining
All the twenty isolates showed growth on Mannitol Salt Agar, were catalase and coagulase positive and showed the presence of Gram positive cocci on Gram's staining.

Identification of species by Multiplex PCR
Eleven (55%) of the twenty isolates were found to be S.pseudintermedius which corresponded to 926 bp and nine (45%) were S.aureus that corresponded to 359 bp in the DNA ladder. The results are depicted in Table  2, Fig I

Detection of Methicillin Resistance by PCR
All the twenty Staphylococci isolates were subjected to PCR. Sixteen out of 20 isolates yielded 304 bp corresponding to mecA gene specific for methicillin resistance. Of the sixteen mecA positive isolates, 8 (50%) were S. aureus and 8 (50%) were S. pseudintermedius as depicted in Table 3   our knowledge this is probably the first report on the occurrence of S pseudintermedius and MRSP in India.

, Fig II
In the present study, of the 20 isolates examined, 55% (11) proved to be S. pseudintermedius and 45% (9) were S. aureus on PCR. It is now well established that S pseudintermedius is the major staphylococci causing pyoderma in dogs. Devriese et al (2005) following necropsy examination of dogs and cats demonstrated the occurrence of a new species of staphylococci with a distinct taxonomy closely related to S intermedius and S delphini. Fitzgerald (2009) in a review on Staphylococcus intermedius Group (SIG) of organisms discussed about how S intermedius has long been regarded as the cause of pyoderma in dogs and how genetic diversity of S intermedius has resulted in the reclassification of S intermedius and identification of S pseudintermedius as the common canine pathogen. Bannoehr et al (2009) reported that a diagnostic technique involving PCR-RFLP which allows for differentiation of S pseudintermedius from closely related members of SIG which may not be possible by biochemical methods. Kadlec et al (2010) stated that S pseudintermedius was the most frequent causative agent of canine pyoderma and that it may also be associated with wound infections, urinary tract infections and otitis externa in dogs. Bannoehr & Guardabassi (2012) in their review on S pseudintermedius stated that dog is the natural host for S pseudintermedius and that definitive identification of this organism relies on molecular methods. This is primarily because S. pseudintermedius cannot be distinguished from S. intermedius by phenotypic methods. Further, due to the lack of standardized and specific phenotypic tests, the routine presumptive identification of S.pseudintermedius is based on the fact that it is the only member of the SIG that has been isolated from dogs. The results of the present study was based on multiplex PCR with species specific primers for various coagulase positive staphylococcal species i.e S aureus, S intermedius, S pseudintermedius and S schlefieri all of which have been reported in dogs with pyoderma by other workers. Sasaki et al., 2007 andVan Hoovels et al., 2006 have stated that conventional microbiological diagnostic tests often fail to distinguish between S pseudintermedius and S intermedius, leading to S. pseudintermedius being frequently misidentified as S. intermedius or S. aureus. This has also been corroborated by other workers (Sasaki et al., 2010 andVidela 2013).
Antibacterials represent one of our most effective therapeutic defenses against infectious diseases. However, the continuous use of antibacterials is under enormous threat due to bacterial resistance. The development of antibacterial resistance is a major issue that can compromise the treatment of infectious diseases as well as other advanced therapeutic procedures (Videla, 2013). Methicillin resistant staphylococci strains have emerged as serious pathogens over the last decade. These strains are usually multidrug resistant thus making successful therapy difficult. Besides they are now a major cause of hospital and community acquired infections associated with high morbidity and mortality. Further, because of the close association between man and animals especially dogs, the threat of transmission of diseases from man to animals and animals to man cannot be over emphasized.
One of the primary causes of resistance to beta-lactamase resistant penicillins (methicillin being the prototype) in staphylococcal isolates is presence of the mecA gene, which encodes a supernumerary penicillin binding protein (PBP2a) with reduced affinity for beta-lactams. Resident PBPs play important roles in the formation of the bacterial cell wall peptidoglycans. These PBPs can be inactivated by the presence of beta-lactam antimicrobials, leading to abnormal cell wall synthesis and bacterial death. However, the poor affinity for betalactams associated with the carriage of the mecA gene, serves as a mechanism of protection for the bacteria, evading disruption of the peptidoglycan layer and preventing bacterial death.
In present investigation, 16 out of 20 isolates yielded 304 bp corresponding to mecA gene specific for methicillin resistance. In the 16 mecA positive isolates, 8 (50%) isolates were S. aureus and 8 (50%) isolates were S. pseudintermedius. This is in agreement with Feng, et al., (2012)