Indian Journal of Animal Research

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Characterization of Staphylococcus pseudintermedius Isolates from Skin Infections of Dogs

Hridya Susan Varughese1, Murugesan Ananda Chitra1,*
1Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Vepery, Chennai-600 007, Tamil Nadu, India.
Background: Staphylococcus pseudintermedius is a part of the canine skin microflora and an opportunistic pathogen. It plays a central role in canine pyoderma, otitis and surgical wound infections. These conditions correlate with virulence genes distributed in the bacterial genome. These genes determine strain variability on typing, in turn aiding epidemiological surveillance. The aim of this study was to isolate, identify and characterize Staphylococcus pseudintermedius (SP) and Methicillin resistant Staphylococcus pseudintermedius (MRSP) from dogs with skin infections in Chennai, India.

Methods: SP and MRSP positive isolates were identified by multiplex PCR for nuc and mecA genes respectively. Characterization of the isolates for virulence genes responsible for biofilm formation (icaA, icaD), cell wall adherence (SpsO, SpsK, SpsP, SpsQ, SpsF), toxins (ExpA, ExpB, SIET, Sel, Se-int, LukS, LukF) and gene regulation (Agr, SarA) was performed.

Result: Out of 275 samples, 120 SP and 8 MRSP positive isolates were identified. Only one isolate could be typed as SCCmec Type V whereas other MRSP isolates were non typeable. Agr typing of MRSP isolates revealed type II in 7 isolates and type III in one isolate. Our study revealed that there was no significant difference in the detection of virulence genes between MSSP and MRSP.
Staphylococcus pseudintermedius (SP) is a commensal bacterium of dogs and cats skin and mucous membranes. It is usually present in pharynx, rectum, nares, conjunctiva, perineum, perioral and axilla region. SP is responsible for pyoderma, otitis externa and post-operative infections in dogs. S. pseudintermedius can also colonize the nasal cavity of pet owners, veterinarians and animal handlers (Bannoehr and Guardabassi, 2012) and it is generally harmless in healthy individuals. However, individuals with compromised immune systems (e.g. HIV/AIDS, transplant and cancer patients) are more susceptible to SP infection and there have been reports of dermatitis and septicemia in human patients (Somayaji et al., 2016). Consequently S. pseudintermedius and especially methicillin resistant (MRSP) strains, are of public concern as potentially emerging zoonotic bacteria (Paul et al., 2011). Contamination with S. pseudintermedius in the environment in animal hospitals and households has been reported (Laarhoven et al., 2011; van Duijkeren et al., 2011) enabling re-infection and transfer of antimicrobial resistance genes (Frank et al., 2009).

The pathogenic potential of the organism can be explored through study of virulence factors such as cell adhering factors, toxins and enzymes. Particular genomic regions like the OriC in SP, controls virulence genes for adherence like SpsK, SpsP, SpsQ, SpsL, SpsM, SpsG and SpsJ genes. After adherence, epithelial colonization can be attributed to biofilm formation coded by the ica operon (Gerke et al., 1998). Following colonization, bacterial cell survival is dependent on toxin production for defense. Hence, this study was aimed to isolate, identify and determine the distribution and frequencies of various virulence factors in SP and MRSP isolates from skin infections of dogs in Chennai, India.
Isolation and Identification
Skin swabs from pyoderma, otitis, allergy, demodicosis, dermatitis and other cases (hypothyroidism etc.) were collected during September 2017 to February 2018 from dogs presented to Dermatology ward of Teaching Veterinary Clinical Complex of Madras Veterinary College, Chennai, India. SP and MRSP isolation and identification were carried out as described previously (Ananda Chitra et al., 2016).

Table 1: Details of the primers used for identification and characterization of SP isolates.

Virulence genes detection
Molecular characterization for the presence of different virulence genes was performed by conventional PCR and primer details along with reference for these genes are given in Table 1. PCR was performed in a reaction volume of 10 µl containing approximately 100-150 ng of genomic DNA, 5 pmol of each primer in 2X master mix (Ampliqon, Denmark). Cycling conditions were 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at appropriate temperature for 30 s, extension at 72°C for 30/45 s and a final extension cycle of 5 min at 72°C. PCR products were loaded on a 2% agarose gel for electrophoresis, visualized with ethidium bromide and documented.
Typing of MRSP isolates
The SCCmec cassette of MRSP isolates was typed using previously described multiplex PCR methods developed by Zhang et al., (2012) and Perreten et al., (2010) for type II-III and VII. Agr typing was carried out using published primers (Ananda Chitra et al., 2015) and sequencing by Sanger’s sequencing technique.
Isolation and Identification
A total of 275 swab samples were collected from various skin infections of dogs. Predominant samples (n=164) were collected from superficial bacterial folliculitis (pyoderma), followed by otitis (n=38), equal number of demodicosis and dermatitis cases (n=23 each). Out of 275 samples, 128 (46.5%) samples were found to be positive for SP by amplifying 780 bp amplicon of SP specific thermonuclease gene in PCR as shown in Fig 1. The prevalence of SP infection in various skin infections is given in Fig 2. SP has also been identified at varying rates with 26.6% incidence in Lithuanian dogs (Ruzauskas et al., 2016), 59% Chennai, India (Ananda Chitra et al., 2016) and 59.6% in Brazil (Scherer et al., 2018).

Fig 1: Agarose gel electrophoresis showing the results of PCR amplified products of nuc and mecA genes of SP isolates.

Fig 2: Case wise prevalence of MSSP and MRSP isolates

Among the 128 SP, eight isolates (6.25%) were identified as MRSP by detecting mecA gene (Fig 1). Six out of eight isolates were from pyoderma cases and two were from otitis cases. Higher prevalence of MRSP infection with 48% in China (Wang et al., 2012), 40.5% in Canada (Beck et al., 2012) and 28% in Chennai, India (Ananda Chitra et al., 2016). However, low MRSP prevalence rates have also been recorded such as 2% in Sweden (SWEDRES-SVARM, 2016) and 13.4% in Canada (Saab et al., 2018).
Detection of genes encoding cell wall associated proteins
The ability of bacteria to colonize and to cause infection is initiated by attachment to the host cells using surface proteins or cell wall anchored proteins. SpsK gene was present in all the SP isolates in this study and the same was observed in various studies (Bannoehr et al., 2011; Phumthanakorn et al., 2017). Prevalence of various virulence genes in MSSP and MRSP isolates are given in Fig 3 and 4 respectively.

Fig 3: Prevalence of virulence genes in MSSP isolates.

Fig 4: Prevalence of virulence genes in MRSP isolates.

SpsF gene was detected in 38/128 (29.68%) isolates which varied from 17.7% (Phumthanakorn et al., 2017) to 71.4% (Latronica et al., 2014) worldwide. SpsO was the one which was present in the least number of isolates (11/128=8.6%) in this study. It has been reported that the SpsO gene was detected in 28.6% of isolates by Latronica et al., (2014) and 40% by Phumthanakorn et al., (2017).

SP organism has two orthologues (SpsP and SpsQ) of staphylococcal protein A. SpsQ was more frequently present in 47/128 (36.71%) isolates than SpsP gene (36/128=28.12%). Both the orthologues (SpsP and SpsQ) were identified in 23 strains of SP and 69 isolates were negative for both the genes. In general, incidence of detection of cell wall protein genes was in a greater number of MRSP isolates than MSSP isolates. Phumthanakorn et al., (2017) and Latronica et al., (2014) reported the concomitant presence of SpsP and SpsQ genes in SP isolates whereas, Bannoehr et al., (2011) reported the differential presence of genes with more prevalence of SpsQ genes (60%) than SpsP (40%) as seen in the present study.
Detection of Biofilm forming genes
Staphylococci species have ica operon which contains icaADBC genes for biofilm formation and regulatory function. The icaA gene was present in 69/128 isolate (53.9%) and icaD gene was present in 86/128 (67%) SP isolates. Higher prevalence of icaD than icaA gene observed in the present study is contrary to the presence of 75.7% icaD and 77.9% icaA genes in SP isolates of Canada and USA reported by Singh et al., (2013). However, transcriptome analysis of SP isolates showed that MSSP isolates had an increased ability to form biofilm under acidic circumstances through up-regulation of the entire arc operon (Couto et al., 2016).
Detection of virulence regulatory genes
All staphylococcal species examined to date have been shown to encode AIP (Auto-inducible protein) peptides that are unique to each species based on AgrD gene. Accessory gene regulator genes were detected in all SP isolates in this study. Agr typing of 5 MSSP isolates were previously reported in India and type I and III AIP were produced by two strains of each and one isolate produced type II AIP (Ananda Chitra et al., 2015). Type IV agr AIP was the majority type found in MRSP isolates of USA (Black et al., 2009) whilst type III was predominantly seen in MRSP than MSSP isolates in Portugal (Couto et al., 2016). The present study identified Agr II in seven MRSP isolates and Agr III type in one MRSP isolate.

SarA is a DNA-binding protein, which binds to the agr promoter region affecting control of several virulence genes in S. aureus. SarA like gene is also present in SP and it was detected in 5/8 (62.5%) and 43/120 (35.8%) of MRSP and MSSP strains respectively. To the best of our knowledge, there is no available literature either to support or contradict this report. However, Couto et al., (2016) reported that higher expression of transcription of regulatory genes in MRSP isolate than MSSP isolate and, in MSSP isolates agrD regulatory genes had higher transcriptional expression.
Detection of exfoliative toxin genes
Exfoliative toxins (ET) - serine proteases, produced by staphylococci are involved in cutaneous infections of mammals. All the isolates of this study were detected to have SIET gene which concurs with previous studies (Ananda Chitra et al., 2016; Couto et al., 2016; Melter et al., 2017). In the present study, ExpA isoform of ET was detected in more number (14.84%) of isolates than ExpB isoforms (6.25%) as opposed to 4.7% ExpA and 9.52% ExpB in another study (Walther et al., 2012). The occurrence of ExpB was found to be 23.2% of isolates from dogs with superficial pyoderma and 6.1% of SP isolates from healthy dogs while ExpA gene was detected in 23.3% of SP isolates from canine pyoderma (Iyori et al., 2010).
Detection of Panton-Valentine Leucocidin (PVL) Like Toxin (Luk-I) in SP isolates
Panton-valentine Leucocidin (PVL) found in certain strains of S. aureus is a bi component-LukS-PV and LukF-PV, pore forming leukotoxin that causes leukocyte damage and tissue necrosis (Gillet et al., 2002). A similar bi component leukotoxin Luk-I, encoded by two genes, LukS/F, was detected in SP. All the SP isolates characterized in the present study as well as in the previous study (Ananda Chitra et al., 2016) possessed Luk-I genes. In other studies, 96.2% and 29.4% prevalence were reported (Melter et al., 2017; Ruzauskas et al., 2016). Luk-I gene was highly expressed in the MRSP isolate than MSSP isolate under transcriptome analysis (Couto et al., 2016).
Detection of enterotoxin se-int and superantigen like toxin (sel) genes in SP isolates
Staphylococcal enterotoxins (SE) are pyrogenic proteins associated with food poisoning and toxic shock syndrome. They are considered as superantigens as they bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells releasing a cytokine bolus leading to an acute toxic shock. S. pseudintermedius produces two unique SEs - SECcanine which is an SEC variant (Cardona et al., 2006) and the other one is SE-int (Futagawa-Saito et al., 2004). In the present study, se-int gene was detected in 37.5% SP isolates where as 100% se-int gene prevalence was reported by Couto et al., (2016), Melter et al., (2017) and Futagawa-Saito et al., (2004). In another study, 37.5% and 75.9% of SP isolates from pyoderma and healthy dogs were found to possess se-int gene respectively (Tanabe et al., 2013).

In the present study, superantigen like protein (sel) gene was detected in all the SP isolates. 73.4% of the SP isolates from skin infection of Czech Republic was identified with sel gene (Melter et al., 2017).
SCCmec typing of MRSP isolates
In the present study, out of eight MRSP strains, only one isolate was identified as having SCCmec Type V by using multiplex PCR. Other seven strains were not typeable by the multiplex PCR methods. A 31% of the MRSP isolates from Italy were reported to be non-typeable (Gronthal et al., 2017).

SCCmec V has gained prominence in the UK (Maluping et al., 2014), Europe and North America (Perreten et al., 2010). In Asia, type V is dominant in Thailand, South China and Korea (Chanchaithong et al., 2014; Feng et al., 2012) and type II–III in Japan and North China (Ishihara et al., 2016; Wang et al., 2012). Therefore, SCCmec dissemination in Thailand and South China display a closer genetic relationship with Korea than Japan and North China.
This study showed the prevalence of various virulence genes in SP and MRSP in Chennai, India. Our study also revealed that there was no significant difference in the detection of virulence genes between MSSP and MRSP. However, it is possible that all the studied virulence factors may work in concert with other virulence factors to worsen skin infections of dog. Further studies are required to identify prevalent clonal types in this region to aid in molecular epidemiology.
The authors are thankful to DST-SERB, Government of India for funding this study (EMR/2016/006141) and Tamil Nadu Veterinary and Animal Sciences University Chennai, India for providing necessary infrastructure to carry out the research work.
Ethical approval was not required for this study as there were no invasive procedures. However, oral consent from the owners and complete confidentiality of the results were maintained.
The authors declare that there are no conflicts of interest.

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