Detection of Staphylococcus pseudintermedius in Dogs and Cats in Selangor, Malaysia

Corresponding Author: Saleha Abd Aziz Department Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Email: saleha@vet.upm.edu.my Abstract: Staphylococcus pseudintermedius is an emerging coagulase gram-positive staphylococci in dogs and cats. It has now become a threat to animal health due to its multidrug resistance leading to very limited therapeutic options particularly in the treatment of small animals and therefore, requires urgent action to control its spread. The organism is currently recognised as a significant pathogen in veterinary medicine causing skin, ear and wound infections in dogs and cats. So far, there is limited coverage in relation to research studies concerning on S. pseudintermedius in dogs and cats in Malaysia. Therefore, this study was conducted to determine the presence of S. pseudintermedius in dogs and cats as well as to evaluate the best site for isolation of the organism. A total of two hundred dogs (n = 100) and cats (n = 100) were randomly sampled. Swab samples were taken from the nasal cavity, buccal cavity, rectum and perineal skin aseptically from both pet and stray dogs and cats. The samples were cultured and isolates were identified by biochemical tests and confirmed using a species-specific PCR assay. Our results revealed that the proportion of detection of S. pseudintermedius was higher in stray dogs and cats than pet animals. Staphylococcus pseudintermedius was detected in 6.5% (13/200) stray dogs, 6% (12/200) stray cats, 1.5% (3/200) pet dogs and 0% in pet cats. Our findings also demonstrated that nasal cavity was the ideal site for isolation of the organism (5.5%), followed by buccal cavity (3.5%), skin (2.5%) and rectum (2.5%), but statistically, the difference was not significant (p = 0.174). In conclusion, this study provided an empirical evidence in relation to the detection of S. pseudintermedius in stray and pet dogs and cats in Selangor, Malaysia. Future research is urgently needed to better understand the epidemiology of S. pseudintermedius in field settings.


Introduction
Staphylococcus pseudintermedius (S. pseudintermedius) is an emerging opportunistic coagulase gram-positive pathogen in dogs and cats. The organisms are commensals on the skin and mucous membranes of humans and a wide range of mammalian species. In most cases, the infection is manifested as topical infections and otitis externa in pet animals (Weese and van Duijkeren, 2010).
The clinical significance of Staphylococcus species is quite variable, with some being important causes of diseases and other minimally pathogenic organisms. Among the important species of Staphylococcus in animals are Staphylococcus aureus, Staphylococcus intermedius and Staphylococcus pseudintermedius.
Until recently, Staphylococcus pseudintermedius was described as a new coagulase-positive Staphylococcus species based on 16S rRNA gene sequence analysis of isolates from a wide range of animals that include cats, dogs, horses and parrots (Devriese et al., 2005;Sasaki et al., 2007a). The organism was grouped and classified as a member of a genetically heterogeneous group of bacteria called Staphylococcus Intermedius Group (SIG) which consists of three related species: S. intermedius, S. pseudintermedius and S. delphini (Sasaki et al., 2007b). Since then, S. pseudintermedius is recognized as the species which colonizes and causes infections in pet dogs and cats more than the other staphylococcal species (Perreten et al., 2010). A number of animal body sites can be colonized by the organism, especially the nasal passages, oral cavity, skin and perineal mucosa (Hartmann et al., 2005;Fazakerley et al., 2010). In Malaysia, there is limited coverage in relation to research studies concerning on S. pseudintermedius in dogs and cats. Therefore, this study was undertaken with the objectives of determining the prevalence of S. pseudintermedius along with evaluating the ideal sites for the isolation of the organism in pet and stray dogs and cats.

Samples Collection
A total of 200 animals consisting of 100 pet and stray dogs and 100 pet and stray cats were sampled. The stray dogs and cats were from a municipality council animal pound and an animal shelter. The pet dogs and cats were from a University Veterinary Hospital (UVH) and private clinics. For pet animals, owners were individually approached with a consent form to take samples from their pets. A total of 50 stray dogs were sampled from animal pound and 50 pet dogs were from private clinics. A total of 50 stray cats were sampled from an animal shelter and 50 pet cats were from the hospital. In each animal, samples were taken from the skin, nasal cavity, buccal cavity and rectum using different sterile cotton swabs moistened with normal saline for each of the site on/in the animal. A cotton swab was rubbed gently on the skin at the perineal part of the body. Each nasal swab was taken by rubbing the swab against the nasal cavity for at least 5 sec. Rectal swab was taken by inserting a swab into the rectum by touching and rubbing the mucous membrane. The sample from oral cavity was done by inserting a swab into the buccal cavity and rubbing the mucous membranes. Each swab was placed in a Bijou bottle containing Tryptone Soya broth (Oxoid) as enrichment media and kept cool during transportation to the laboratory. All the enrichment broth containing the swabs were incubated at 37°C overnight.

Bacterial Isolation and Identification
Of 10 µL of enriched culture was streaked onto Mannitol salt agar plate (Oxoid) and incubated overnight at 37°C for 24 h. The suspected colonies of Staphylococcus on the agar plates were examined by observing the colonial morphology and colour (yellow colonies). Presumptive Staphylococcus colonies were sub-cultured onto Columbia blood agar (Oxoid) with 5% horse blood added and were incubated at 37°C for 24 h. Suspected S. pseudintermedius isolates were identified on the basis of colony characteristics, pigment production, Gram-stained appearance and hemolysis. All suspected S. pseudintermedius isolates were further tested for catalase, coagulase, DNase, O-Nitrophenyl-beta-D-Galactopyranoside (ONPG), Arginine Dihydrolase (ADH) and acetoin production. All the suspected isolates were stored in skim milk powder broth (Oxoid) at -80°C until used.

DNA Extraction
Stored frozen isolates were thawed and each was streaked onto Columbia Blood agar plate with 5% horse blood added and incubated for 24 h at 37°C. DNA was extracted by using boiling method. In brief, 1 mL of sterile distilled water was transferred to a sterile 1.5 mL Eppendorf tube and a loopfull of the isolate was picked from a plate and transferred into the Eppendorf tube. The mixture was vortexed for 1 min and then heated in a dry bath at 96°C for 10 min. The mixture was cooled down for 5-10 min to room temperature and centrifuged at 13000×g for 3 min. The pellet was used as DNA template for multiplex PCR (mPCR) assay.

Multiplex PCR Assay Procedure
The mPCR assay was performed as described by Sasaki et al. (2010). The reaction mixture for PCR consisted of 5 µL DNA extract, 25 µL MyTaq Red Reaction Buffer (Bioline), 18 µL deionized water and 2 µL of the primers (forward and reverse) in a total volume of 50 µL. The following PCR conditions were used: heat denaturation at 95°C for 2 min, 30 cycles with denaturation at 95°C for 30 s, annealing at 56°C for 35 s, extension at 72°C for 1 min and a final extension at 72°C for 2 min. the PCR amplification was done as described earlier by Sasaki et al. (2010), with the following sequences: NucA1, 5'-TRGGCAGTAGGATTCGTTAA-3' and NucA2, 5'-CTTTTGTGCTYCMTTTTGG-3'. The reference strain S. pseudintermedius (CCUG 49543) was used as a positive control. In addition, 5 µL of sterile deionized distilled water was used as a negative control. DNA fragments were analysed by electrophoresis in 1_ Trisacetate-EDTA on a 1% agarose gel stained with ethidium bromide.

Data Analysis
Data was analysed using statistical package SPSS software 20 ((IBM SPSS Version 20) and Openepi software (http://www.openepi.com/). The differences in the proportion of positive samples between stray and pet animals and also between the different sites of isolation were analysed using Chi-square test. The results were considered statistically significant at p≤0.05 on 95%CI.

Discussion
To our knowledge, this is the first study carried out on S. pseudintermedius in dogs and cats in Malaysia. A total of 28 (3.5%) S. pseudintermedius isolates were identified from 200 dogs and cats sampled. The result showed a high number of stray animals were colonized with S. pseudintermedius compared to pet animals, 6.5% in stray dogs, 6% in stray cats, 1.5% in pet dogs and 0% in pet cats. This is probably because the stray animals are more exposed to open environment and thus frequently in contact with other animals from many sources including animal shelter that suffer from high density, suboptimal cleaning and disinfection of its facilities as well as the stress of the environment (Miller, 2004).
The colonization of S. pseudintermedius in companion animals, especially dogs varies greatly on the parts of the body (Hanselman et al., 2009). According to Fazakerley et al. (2010) and Hartmann et al. (2005), various body sites can be colonized, particularly the nasal passages, oral cavity, skin and perineal mucosa. Up to date, only few studies, if any, determined the S. pseudintermedius colonization pattern on four parts of the animal body. Sampling from different sites on the same animal is time consuming and resource demanding particularly if large numbers of animals are involved. This study showed that the nasal cavity (5.5%) and buccal cavity (3.5%) were the most frequently colonized body sites compared to perineal skin (2.5%) and rectum (2.5%). The occurrence of S. pseudintermedius in nasal cavity in this study was less than that reported in Spain (23%) (Gómez-Sanz et al., 2013), Germany (13.9%) (Walther et al., 2012), but more than that reported in healthy dogs in Canada (0.7%) (Rubin and Chirino-Trejo, 2011). The prevalence of S. pseudintermedius in the buccal cavity was less than that in the study of Paul et al. (2012) which was 65%. The prevalence on the perineal skin was considered very low compared to that reported by Fazakerley et al. (2010) in both dogs having atopic dermatitis (87%) and the healthy ones (37%). However, it should be considered that S. pseudintermedius causes skin infections such as pyoderma mostly in dogs and cats. The prevalence of S. pseudintermedius in rectum is very low compared to the work of Rubin and Chirino-Trejo (2011) who reported at 11.1%. The difference in the reported prevalence in this and other studies were due to different geographical location, sampling and isolation procedures and animal breed. The knowledge of common sites of colonization is essential for conducting surveillance; this is because failing to sample relevant sites may result in falsenegatives and thus underestimation of its prevalence. Conversely, the increased expense and time required for excessive sampling are undesirable.
Although the prevalence was low compared to other studies, the presence of S. pseudintermedius in dogs and cats could be a potential source of zoonotic infection in humans as evidenced by recent outbreaks in which veterinary personnel and pet owners involved (Paul et al., 2011). Therefore, we strongly recommend strict sanitary measures with the highest level of international excellency to be maintained when contacting infected animals.