Journal of Applied Microbiology, 2022, 134, 1–9 https://doi.org/10.1093/jambio/lxac041 Advance access publication date: 14 December 2022 Research Article Staphylococcus coagulans possesses many virulence factors of Staph. aureus and Staph. pseudintermedius Ananda Chitra Murugesan 1,* , Maya Ramachandran 1 , Hridya Susan Varughese 1 , Karthik Kumaragurubaran 2 1 Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600007, Tamil Nadu, India 2 Central University Laboratory, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai-600051, Tamil Nadu, India ∗ Corresponding author. Central University Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai-600051, Tamil Nadu, India. Tel: +91-44-25551581; E-mail: anandachitra.m@tanuvas.ac.in Abstract Aims: To understand the Staphylococcus coagulans prevalence in causing skin infections in dogs and detection of various virulence genes in Staph. coagulans isolates. Methods and Results: Staph. coagulans was isolated from pus swabs collected from dogs with skin infection and identifed by detecting ther- monuclease, coagulase, and urease genes. The presence of methicillin-resistant gene (mecA) was performed by PCR. Antimicrobial susceptibility test was carried out by disc diffusion method. In total, 38 Staph. coagulans clinical isolates and 42 Staph. coagulans genomes available in NCBI database were screened for 19 virulence genes by PCR and in silico prediction, respectively. A prevalence of 13.8% (38/275) of Staph. coagulans dog skin infection was observed and 15.8% (6/38) of Staph. coagulans isolates carried mecA gene. Many Staph. coagulans isolates were suscep- tible to all tested antimicrobials. Twenty nine per cent isolates were resistant to ciprofoxacin. Genes encoding leukotoxins, DNase, exfoliative toxin, superantigen-like exotoxin, immunoglobulin-binding proteins, fbrinogen-binding proteins, autolysin, and rod shape-determining protein were detected in almost all the Staph. coagulans clinical isolates and genomes from NCBI database, whereas anti-adhesin plasma-sensitive protein genes were present in relatively lesser number of Staph. coagulans clinical isolates and genomes from NCBI database. Conclusions: Staph. coagulans possesses many virulence factors that are present in other coagulase-positive staphylococci, such as Staph. aureus and Staph. pseudintermedius. The presence of two bi-component leukotoxin genes in tandem with other virulence factor genes in a single pathogenic island in the Staph. coagulans genomes explained their eminence in the virulence of Staph. coagulans causing infections. Staph. coagulans was classifed as a separate species in the year 2020 and primarily causes skin infections in dogs. Identifcation of this species is not included in any of the automated bacterial identifcation systems. Hence, many veterinary laboratories do not have a strategy to identify this bacterium. This study will help in the identifcation of Staph. coagulans in veterinary laboratories by PCR apart from detecting various virulence factors present in this pathogen. The existence of many virulence factors and prevalence in different animals in varied geographical locations suggest that Staph. coagulans is an important coagulase-positive staphylococcal pathogen in animals. Keywords: Staphylococcus coagulans, animal staphylococci, dog skin infection, prevalence, virulence genes 1. Materials and methods 1.1. Ethical and institutional biosafety committee approval We acquired informed consent from all the pet owners involved in this study, and diagnostic results were main- tained confdentially. There was no invasive procedure in- volved in this study, and skin swabs were collected for the clinical diagnostic purposes only. This study has been approved by Institutional Biosafety Committee vide VIII- Approval Lr.No.2286/DFBS/4/IBSC/2017 dated 10 October 2017. 1.2. Sampling and identifcation of Staphylococcus coagulans isolates Pus samples from dogs suffering with skin infections brought to the Dermatology Unit of Madras Veterinary College Teach- ing Hospital during September 2017 to June 2018 were col- lected using a sterile cotton swab (HiMedia, India). Samples were collected from dogs with primary or secondary skin in- fections and also included recurrent pyoderma cases. A total of 275 samples were collected during the study period from various skin infections of dogs of different breeds, ages, and sex. Details of the source of Staph. coagulans isolates are given in Supplementary Table S1 Samples were inoculated within 3–5 h of collection, on the mannitol salt agar, and colonies were initially identifed by Gram’s staining, catalase tests, and other basic biochemical tests. DNA extraction was carried out as described previously (Ananda Chitra et al. 2015). Coagulase-positive staphylococci were identifed by multiplex PCR of Sasaki et al. (2010), which targets thermonuclease gene (nucA). Coagulase and urease- positive Staph. schleiferi isolates were identifed as Staph. schleiferi subspecies coagulans (Igimi et al. 1990, Costa et al. 2021). In the present study, Staph. coagulans was identifed by detecting coagulase and urease alpha-subunit genes by PCR (SC-Coa-F/R and SC-UreA-F/R primers details are given in Table 1) and methicillin resistance of the isolates was deter- mined by detecting mecA gene by PCR. Briefy, PCR reaction consisted of 10 μl of 2X PCR master mix (Ampliqon, Den- mark), 5 pmol of each primer, 150–200 ng template DNA ob- tained by the boiling method, and the total reaction volume Received: June 6, 2022. Revised: October 6, 2022. Accepted: November 1, 2022 C  The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com Downloaded from https://academic.oup.com/jambio/article/134/1/lxac041/6902068 by guest on 19 March 2023