Staphylococcus caledonicus sp. nov. and Staphylococcus canis sp. nov. isolated from healthy domestic dogs

Two strains, H8/1T and H16/1AT, of Gram-stain-positive, coagulase-negative staphylococci were isolated from separate healthy domestic dogs in Scotland. Both strains were genome sequenced and their inferred DNA–DNA hybridisation indicates that H8/1T and H16/1AT represent two novel species of the genus Staphylococcus. On the basis of the results of genome sequence analysis (genome blast distance phylogeny and single nucleotide polymorphism analysis) H8/1T is most closely related to Staphylococcus devriesei and H16/1AT most closely related to Staphylococcus felis. Also, average nucleotide identity distinguished H8/1T and H16/1AT from S. devriesei and S. felis as did minor phenotypic differences. On the basis of these results, it is proposed that H8/1T and H16/1AT represent novel species with the respective names Staphylococcus caledonicus and Staphylococcus canis. The type strain of S. caledonicus is H8/1T (=NCTC 14452T=CCUG 74789T). The type strain of S. canis is H16/1AT (=NCTC 14451T=CCUG 74790T)

At the time of writing the genus Staphylococcus [1,2] of Gram-stain-positive, coccus-shaped bacteria consists of 55 species (https:// lpsn. dsmz. de/ genus/ staphylococcus, accessed 20th March 2020) [3]. Typically, staphylococci are found as commensal inhabitants of the skin and mucous membranes in a wide range of animal hosts, particularly in humans, other mammals and birds. The genus includes several important human and veterinary opportunistic pathogens, such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis and Staphylococcus pseudintermedius. Two canine staphyloccocal strains H8/1 T and H16/1A T have been characterised, for which the respective names of Staphylococcus caledonicus sp. nov. and Staphylococcus canis sp. nov. are proposed.
S. caledonicus H8/1 T and S. canis H16/1A T were isolated in 2018 from multisite swabs (a single swab sampling the nares, axilla, groin and perineum) from separate healthy adult dogs at the Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, UK. These samples were collected as part of an epidemiological study to isolate and genome sequence canine commensal bacteria. The dogs had no clinical signs or history of skin disease and had not received antimicrobial treatment for at least 12 months prior to sampling. Swabs were cultured initially by salt broth enrichment, statically at 37 °C for 24 h [tryptone soya broth (Oxoid) plus 6.5 % w/v sodium chloride] before plating onto mannitol salt agar (Oxoid) and then incubation at 37 °C for 24 h. Both isolates are Gram-stain-positive, catalase-positive cocci and were whole-genome sequenced using HiSeq technology (Illumina) with 2×250 bp paired-end reads, read trimming and assembly (performed by Microbes NG, Birmingham, UK). Reads were trimmed using Trimmomatic version 0.30 [4], using a sliding window quality cut-off of 15. Genome assembly was done de novo using SPAdes version 3.7 [5], with default parameters for 250 bp Illumina reads. Assemblies were annotated using the NCBI Prokaryotic Genome Annotation Pipeline [6].
To identify these two strains to the species level their genome sequences were uploaded onto the Type (Strain) Genome OPEN ACCESS Server (TYGS) (https:// tygs. dsmz. de/) [7]. The results of this genome-based analysis support the proposal of the two strains as each representing a novel species of the genus Staphylococcus. The TYGS phylogenetic tree generated by the Genome blast Distance Phylogeny approach (GBDP) indicated that H8/1 T is most closely related to, but distinct from Staphylococcus devriesei CCUG 58238 T with H16/1A T most closely related to, but distinct from Staphylococcus felis DSM 7377 T Fig. 1. Balanced minimum-evolution tree containing Staphylococcus caledonicus H8/1 T and Staphylococcus canis H16/1A T (highlighted in bold type) and all other type strains of members of the genus Staphylococcus. Tree generated using the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de) [7] and inferred with FastME 2.1.6.1 [22] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d 5 with pseudo-bootstrap support values shown of >50 % from 100 replications. Mcrococcus canis KM45013 T was included as the outgroup to root the tree. Accession numbers for all genomes in the analysis are provided in Table S1 (available with the online version of this article). Tree generated using the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de) [7] and inferred with FastME 2.1.6.1 [22] from GBDP distances calculated from 16S rRNA gene sequences. Staphylococcus caledonicus H8/1 T and Staphylococcus canis H16/1A T are highlighted in bold type. The branch lengths are scaled in terms of GBDP distance formula d 5 . The numbers below branches are GBDP pseudo-bootstrap support values >50 % from 100 replications. Macrococcus canis KM45013 T was included as the outgroup to root the tree. Accession numbers for all isolates in the analysis are provided in Table S1.
( Fig. 1). A 16S rRNA gene phylogeny, produced by TYGS, also indicated that H8/1 T is most closely related to S. devriesei and H16/1A T most closely related S. felis (Fig. 2). Although, 16S rRNA gene sequences often lack sufficient variation to differentiate between species of the genus Staphylococcus in pairwise comparisons [8][9][10][11]. Indeed, neither H8/1 T nor H16/1A T could be differentiated from S. devriesei or S. felis type strains respectively on the basis of 16 rRNA gene sequence, Table 1. Nonetheless, designation of both H8/1 T and H16/1A T as representing two novel species is supported by digital DNA-DNA hybridization (dDDH) values calculated with the Type (Strain) Genome Server using the recommended settings of the Genome-to-Genome Distance Calculator (GGDC) 2.1 [12] and by ANI values calculated by blast (ANIb) [13], MUMmer (ANIm) [13] and the tetranucleotide signature correlation index [13] (Table 1). Analysis of the latter three parameters was performed using JSpeciesWS [14]. With one exception, the values in each case were less than the threshold which is used to circumscribe strains as belonging to the same species [15] (Table 1). The one exception which did not fulfill the criteria for differentiating bacterial species was the tetranucleotide signature correlation index value of 99.7 % calculated in the comparison of S. caledonicus H8/1 T with S. devriesei CCUG 58238 T . Divergence between ANI and tetranucleotide signature correlation index has been described previously with a possible explanation being that evolutionary or environmental forces may impede modifications in this genome signature despite genetic drift occurring [13]. Furthermore, the utility of the tetranucleotide signature correlation index and its correlation with dDDH and ANI in circumscribing or differentiating among staphylococci has received little attention to date. A further genome-based phylogeny of type strains of species of the genus Staphylococcus was generated using CSI Phylogeny 1.4 [16] applying the default setting as follows: minimum depth at SNP positions: 10×; minimum relative depth at SNP positions: 10 %, minimum distance between SNPs (prune): 10 bp; minimum SNP quality: 30; minimum read mapping quality: 25 and minimum Z-score: 1.96. Using Staphylococcus aureus DSM 20231 T as the reference genome, this analysis produced a single-nucleotide polymorphism tree comprising 19637 nucleotide positions (Fig. 3). In agreement with the GGDC-based Staphylococcus phylogeny, the results of this analysis indicate that H8/1 T is most closely related to, but distinct from, Staphylococcus devriesei; with H16/1A T most closely related to, but distinct from, Staphylococcus felis. Before the advent of accessible whole-genome sequencing, the partial sequences of various housekeeping genes such as dnaJ [17], tuf [18], sodA [19] and rpoB [20] had been proposed to discriminant staphylococcal species. Whereas each of these single-gene approaches could distinguish H16/1A T from S. felis DSM 7377 T , none of them could be used to separate H8/1 T from S. devriesei CCUG 58238 T , Table 1. The draft genome of H8/1 T is 2 503 367 bases in length with a DNA G+C content of 33.6 mol%, while the draft genome of H16/1A T is 2 229 149 bases in length with a DNA G+C content of 34.8 mol%. These values are similar to those of their nearest relatives and consistent with the average and range values of the rest of the members of the genus Staphylococcus (Table 2).
Phenotypic characterisation of H8/1 T and H16/1A T was performed using the API Staph system (bioMérieux) according to the manufacturer's instructions alongside the type strains of S. devriesei DSM 25293 T and S. felis DSM 7377 T (Table 3). H8/1 T is distinguished from the related S. devriesei by the inability of the former to ferment lactose while the lack of arginine dihydrolase activity differentiates H16/1A T from the related S. felis, (Table 3). Additionally, H8/1 T and H16/1A T were tested for clumping factor and coagulase activity using rabbit plasma (with EDTA) and for DNAse activity using DNAse agar (Oxoid). In each case, both H8/1 T and H16/1A T tested negative for these activities.
Antimicrobial sensitivity testing was performed using the Vitek2 system (bioMérieux) according to the manufacturer's instructions. Using the AST-GP80 card and applying the CLSI 2017 interpretations for coagulase-negative staphylococci,  Table S1.
Gram-stain-positive, non-spore forming, facultative anaerobe, forms non-pigmented, smooth, circular colonies about 1-2 mm in diameter with entire margins on Columbia horse blood agar after 18 h incubation at 37 °C. Able to produce acid from d-glucose, d-fructose, maltose, trehalose, d-mannitol and sucrose but not from d-mannose, lactose, xylitol, melibiose, raffinose, d-xylose, methyl α-d-glucopyranoside or N-acetylglucosamine. Has arginine dihydrolase activity and is able to reduce nitrates to nitrites. Catalase-positive and negative for clumping factor, coagulase and DNAse.

SP. NOV.
Staphylococcus canis (ca′nis L. gen. masc./fem. n. canis, of a dog, in reference to the host from which the type strain was isolated).