Comparison of phenotypic classification based on coagulase testing, and genotypic detection of <i>coa</i>, <i>clfb</i>, <i>spa</i>, <i>and fnB</i> genes.

Comparison of phenotypic classification based on coagulase testing, and genotypic detection of coa, clfb, spa, and fnB genes.</p

Ligand-binding assays.

The amount of fibrinogen-binding protein (upper panel), fibronectin-binding protein (middle panel), and cell wall-associated protein A (lower panel) on the surface of S. schleiferi, and S. coagulans isolates of human (H) and canine (C) origin was measured by flow cytometry using FITC-conjugated fibrinogen HiLyte Fluor TM 488-conjugated fibronectin, and chicken anti-protein A antibody, respectively. Dashed columns are S. coagulans, light gray columns are S. schleiferi and white columns are control species. Numbers in parentheses indicate the number of fibronectin tandem repeats (middle panel), and the number of IgG binding domains (lower panel). The values represent the medians from three independent experiments (*p0.05).</p

Protein A binding domains.

A) Binding site of spa primers used in this study showed variation in the length of IgG binding domains of protein A among the S. schleiferi published sequences. B) CLUSTALW alignment of the amino acid sequences of the IgG binding domains of protein A among 7 S. schleiferi and S. coagulans isolates of human (H) and canine (C) origin in comparison with S. aureus Newman protein A. The annotation label indicates the number of IgG binding domains (I-V). Gray shading indicates 80–100% similarity between sequences. (PDF)</p

List of primers used in this study.

Staphylococcus schleiferi and Staphylococcus coagulans are opportunistic pathogens of animals and humans. They were previously classified as Staphylococcus schleiferi subs. schleiferi and Staphylococcus schleiferi subs. coagulans, respectively, and recently reclassified as separate species. S. coagulans, is frequently associated with dogs, whereas S. schleiferi is more commonly isolated from humans. Coagulase activity status is a defining characteristic of the otherwise closely related species. However, the use of coagulase tests originally developed to distinguish S. aureus from non-coagulase-producing staphylococci, for this purpose is questionable and the basis for their host preference has not been elucidated. In the current study, a putative coa gene was identified and correlated with coagulase activity measured using a chromogenic assay with human and bovine prothrombin (closely related to canine prothrombin). The results of the tests performed with human prothrombin showed greater reactivity of S. coagulans isolates from humans than isolates obtained from dogs with the same substrate. Our data suggest that unlike S. coagulans isolates from humans, isolates from dogs have more coagulase activity with bovine prothrombin (similar to canine prothrombin) than human prothrombin. Differences in nuc and 16s rRNA genes suggest a divergence in S. coagulans and S. schleiferi. Phenotypic and genotypic variation based on the number of IgG binding domains, and the numbers of tandem repeats in C-terminal fibronectin binding motifs was also found in protein A, and fibronectin-binding protein B respectively. This study identified a coa gene and associated phenotypic activity that differentiates S. coagulans and S. schleiferi and identified key phylogenetic and phenotypic differences between the species.</div

Coagulase activity.

Coagulase activation of human (Panel A) and bovine (Panel B) prothrombin measured using a chromogenic assay. The amounts of coagulase activity in the bacterial supernatant of S. coagulans isolates from human (H) and canine(C) are indicated. Dashed columns are S. coagulans, light gray columns are S. schleiferi and white columns are control species. The values represent the medians from three independent experiments (*p0.05).</p

16S rRNA multiple sequence alignment.

Clustal W alignment of 16S rRNA gene sequence of S. schleferi (sequence profile I) and S. coagulans (sequence profile II) of human (H) and canine (C) origin. The single nucleotide difference between the two sequence profiles is highlighted by gray shading. (TIF)</p

<i>nuc</i> gene multiple sequence alignment.

Clustal W alignment of nuc gene sequence of S. schleiferi (sequence profile I) and S. coagulans (sequence profile II) of human (H) and canine (C) origin. The 16 nucleotide difference between the two sequence profiles is highlighted by gray shading. (TIF)</p

Coagulase gene phylogenetic comparison.

Phylogenetic tree based on complete coagulase (coa) protein sequences showing the relationships among 19 species of the genus Staphylococcus including 13 S. coagulans of human (H) and canine (C) origin. The scale bar indicates the sequence divergence. Bootstrap probabilities are expressed as percentages and are shown at diverging points of branches. The following staphylocoagulase protein sequences were obtained from the GenBank database (accession numbers): S. aureus subsp. aureus strain Newman (WP_000744074), S. intermedius ATCC 29663(WP_019169028.1), S. schleiferi 1360-13(WP_050345467.1), S. schleiferi 2317-03(WP_050330609.1), S. delphini 8086(WP_019166910.1), and S. pseudintermedius 081661 (WP_037544060.1).</p

Fibronectin binding protein.

A) Binding site of FnbpB primers used in this study showed variation in the length of C-terminal fibronectin binding motifs among the S. schleiferi published sequences B) CLUSTALW alignment of the amino acid sequences of the fibronectin tandem repeats of FnB protein among six S. schleiferi and S. coagulans isolates of human (H) and canine (C) origin in comparison with S. aureus NCTC 8325 FnB. The annotation label indicates the fibronectin repeat region (1–5). Numbers between braces indicate the number of fibronectin tandem repeats. Gray shading indicates 80–100% similarity between sequences. (PDF)</p

Thermonuclease gene phylogenetic comparison.

Panel A) The binding site of sch-nuc primers used in this study compared with previously published primer pairs. Panel B) Sequence logos of the S. schleiferi and S. coagulans thermonuclease (nuc) genes. Sequence logos created by Geneious 2019.2.1 software show a 16 nucleotide difference between the two species. Panel C) Phylogenetic tree based on complete nuc sequences in staphylococci showing the relationships among S. schleiferi and S. coagulans of human (H) and canine (C) origin. The scale bar indicates the amount of sequence divergence over the length of the bar as a decimal percentage (0.2 equals 20%).Bootstrap probability is expressed as percentages indicated at diverging points of branches. Braces indicate the two nuc sequence profiles; sequence profile I is similar to the S. schleiferi and sequence profile II is similar to the S. coagulans nuc sequence. The following nuclease gene sequences were obtained from the GenBank database (accession numbers): S. aureus subsp. aureus strain Newman (CP023391.1), S. epidermidis strain ATCC 12228 (CP022247.1,), S. intermedius ATCC 29663(AB327165.1), S. schleiferi subsp. coagulans JCM 7470T(AB465334), S. schleiferi TSCC54 (AP014944.1), S. schleiferi 2317-03(CP010309.1), S. delphini LMG 22190 (AB327167.2), S. pseudintermedius 081661 (CP016073.1), and S. pseudintermedius LMG 22219T (AB327164).</p