Characterisation of Staphylococcus felis isolated from cats using whole genome sequencing

Highlights

• Staphylococcus felis isolates were examined using whole genome sequencing and phenotypic tests.

• Gene sequences of putative virulence factors were found in all isolates.

• Ninety two percent of isolates were susceptible to all antimicrobials tested.

• One isolate caused coagulation of feline plasma.

Abstract

This study used phenotypic tests and whole genome sequencing to characterise a collection of 37 clinical Staphylococcus felis isolates from cats. Samples were isolated from a range of diseases including feline lower urinary tract disease (n = 15), otitis externa (n = 13), and ocular disease (n = 2). Isolates were identified using MALDI-TOF MS and by BLASTn analysis of S. felis-specific 16S rRNA, rpoB and nuc genes in whole genome sequence-based contigs. Phenotypic antimicrobial resistance was determined using disk diffusion and broth microdilution. Coagulase activity was assessed using feline and rabbit plasma. Genomes were screened for putative virulence and antimicrobial resistance genes using the sequences of known genes from other staphylococci as homologous references. Phylogenetic relationships were inferred using single nucleotide polymorphisms. One isolate was coagulase-positive when tested with feline plasma but all isolates were rabbit plasma coagulase-negative. No genetic determinant of coagulase activity was identified in this isolate. A range of putative virulence genes were found amongst isolates including genes associated with adhesion, toxin production and immune evasion. Ninety two percent of isolates were fully susceptible to all antimicrobials tested, which was reflected by a general absence of resistance genes. Clustering within the phylogenetic tree suggested a multiclonal population structure; this clustering did not correlate with disease syndrome or geographic origin of the isolate. Future studies of veterinary staphylococci will benefit from the publicly available S. felis draft genomes that were generated in this study.

Introduction

The resident staphylococcal species for cats, Staphylococcus felis, was first identified as a new coagulase-negative Staphylococcus species in 1989 (Igimi et al., 1989). The species, found on the skin of around 25% of healthy cats (Lilenbaum et al., 1998), is the most frequently isolated Staphylococcus species carried on the skin (Lilenbaum et al., 1998) and in the saliva of cats (Lilenbaum et al., 1999). S. felis is coagulase-negative when tested using rabbit plasma and produces incomplete haemolysis on sheep blood agar. Although phenotypically similar to S. simulans and S. sciuri (Devriese et al., 1984; Igimi et al., 1989), it can be definitively differentiated from other staphylococci by DNA-DNA hybridization. Due to its phenotypic similarity to other coagulase-negative staphylococci (CNS), the prevalence of S. felis may have been underestimated in early studies that did not use advanced molecular methods. While it is now generally agreed that S. felis is a common cat commensal, its role as a feline pathogen is more contentious (Higgins and Gottschalk, 1991; Patel et al., 2002; Litster et al., 2007, Litster et al., 2011; Kwaszewska et al., 2015). S. felis-positive urine samples have significantly higher pH and are more likely to contain urine crystals (Litster et al., 2007). Like many staphylococcal species, S. felis produces urease (Igimi et al., 1989), which is the likely cause of the high pH seen in S. felis-positive urine. As a skin commensal, S. felis could be dismissed as a contaminant of urine samples. However, Litster et al. (2007) concluded the production of urease and the subsequent increase in urine pH suggest S. felis is a potential feline urinary pathogen. A subsequent study which examined a single S. felis isolate from a cat’s subcutaneous wound identified virulence factors such as biofilm and proteolytic enzyme production, demonstrating the organism’s potential as an opportunistic skin pathogen (Kwaszewska et al., 2015).

Overall, there is a scarcity of literature regarding the epidemiology and potential virulence factors of S. felis, and the species population structure in cats is unknown. Furthermore, there have been divergent reports on its antimicrobial susceptibility profile (Igimi et al., 1989; Litster et al., 2007; Kwaszewska et al., 2015). While methicillin resistance and multidrug resistance have been identified amongst S. felis isolates (Lilenbaum et al., 1998; Muniz et al., 2013), other studies have reported low levels of antimicrobial resistance (Litster et al., 2007, Litster et al., 2011). Typing methods such as multilocus sequence typing (MLST), as well as whole genome sequencing, have already shed light on the population structure of other veterinary Staphylococcus species (spp.) such as S. aureus (Enright et al., 2000) and S. pseudintermedius (Solyman et al., 2013), but similar genomic epidemiology approaches have not been applied to S. felis. Given the current scarcity of research characterising S. felis, the three aims of this study were to: 1) investigate potential virulence factors that may aid S. felis in its colonization and formation of opportunistic infection; 2) investigate the phenotypic and genotypic antimicrobial profiles of a collection of clinical S. felis isolates; and 3) use whole genome sequencing to infer the population structure of S. felis amongst Australian cats.