General features of S. caprae genomes
We determined complete whole-genome sequences of three methicillin-resistant S. caprae strains isolated from humans. Two draft genome sequences of S. caprae 9557 and M23864:W1 strains were recently determined, and genome comparison analysis of S. epidermidis, S. capitis, and S. caprae was carried out using two complete genomes of S. epidermidis and S. capitis and two draft genomes of S. capitis and S. caprae [17, 18]. However, it is difficult to elucidate the complete features of Staphylococcus chromosome structures and genes containing repeats with draft genome sequence analysis. For example, the draft genome sequence of S. caprae M23864:W1 strain could not clarify the complete sequences of biofilm-associated genes encoding Embp/EbphA and ClfB-like genes since they might contain repeat sequences. In the case of the draft sequence, assembly is incomplete; there may be errors in the sequences; and annotation is incomplete. Determined complete whole-genome sequences of three methicillin-resistant S. caprae strains are shown in Fig. 1. The strains of S. caprae JMUB145, JMUB590, and JMUB898 contained one single circular chromosomes, and one, five, and seven plasmids, respectively. The respective chromosome sizes of S. caprae JMUB145, JMUB590, and JMUB898 were 2,618,380, 2,629,173, and 2,598,513 bp, with almost identical G + C content (33.6%). A total of 2,489, 2,510, and 2,476 protein-CDSs were annotated on the chromosomes of S. caprae JMUB145, JMUB590, and JMUB898, respectively. S. caprae JMUB145, JMUB590, and JMUB898 strains were also found to contain six, five, and five rRNA-encoding gene (rDNA) clusters, as well as 59, 59, and 58 tRNA genes, respectively.
Figure 1 shows the blastn identities of CDSs among individual genomes of S. caprae JMUB898, JMUB145, and JMUB590 chromosomes, with JMUB898 as the central reference chromosomal sequence [19]. Most of the differences between S. caprae genomes were found on genomic islands including SCCmec, prophages, and transposons (Fig. 1). JMUB145 carried a type V SCCmec element. The entire structure of SCCmec in the JMUB145 genome was almost identical with that of S. aureus JCSC5952, which was isolated from children with impetigo in Japan in 2002 (Fig. 2) [20]. The SCCmec of JMUB145 also showed high similarity with that of S. pseudointermedius 06–3228, which was isolated from a dog [21]. The SCCmec of JMUB145 possessed two ccrC genes: ccrC1 allele 2 (ccrC2) and ccrC1 allele 8 (ccrC8). Both JMUB590 and JMUB898 carried type IVa SCCmec elements, which are closely related to type IVa SCCmec of the S. aureus USA300 TCH1516 strain (Fig. 2) [22].
Phylogenetic relationship among S. epidermidis, S. capitis and S. caprae
To date, the genus Staphylococcus has been classified into more than 47 species and 23 subspecies, comprising 15 cluster groups, based on a phylogenetic analysis performed using DNA sequence data from multiple loci, such as the 16S rRNA gene and dnaJ, rpoB, and tuf gene fragments [7, 15]. S. caprae, together with S. epidermidis, S. capitis subsp. capitis, S. capitis subsp. urealyticus, and S. saccharolyticus, belongs to the epidermidis cluster group [7, 15]. Species of the epidermidis cluster group showed oxidase-negative, novobiocin-susceptible, and coagulase-negative phenotypes. To confirm the phylogeny of the genus Staphylococcus, we constructed a genome-wide phylogenetic SNP tree using three S. caprae chromosome sequences determined in this study and 80 complete genome sequences of 24 Staphylococcus species available on GenBank (Additional file 1 Table S1). We also included two S. caprae draft genome sequences, which are also available on GenBank, into the analysis. The phylogenetic analysis confirmed that S. epidermidis, S. capitis, and S. caprae strains of the epidermidis cluster group fell into a single clade (Fig. 3). Based on the SNP tree, the epidermidis cluster group is most closely related to S. haemolyticus strains (Fig. 3).
To elucidate the genome structure of the epidermidis cluster group, we compared the entire chromosome sequences among S. epidermidis, S. capitis, and S. caprae strains. The chromosome sequence of S. caprae JMUB898 was compared with those of S. caprae JMUB145 and JMUB590 by dot plot analysis (Fig. 4). Moreover, the S. caprae JMUB898 chromosome sequence was also compared with the complete genome sequences or complete chromosome sequences of seven S. epidermidis and three S. capitis strains. Dot plot analysis revealed that genome structures were relatively conserved among S. caprae and S. capitis strains (Fig. 4). However, as compared with the S. caprae JMUB898 chromosome, three of seven S. epidermidis chromosomes (ATCC12228, PM221 and BPH0622) were inverted around oriC. The S. aureus chromosome was also inverted around oriC when chromosome sequences were compared between S. caprae JMUB898 and S. aureus N315 (Fig. 4).
Identification of species-specific genes in S. epidermidis, S. capitis and S. caprae and conserved genes among the epidermidis cluster group
In order to uncover the genetic diversity of the epidermidis cluster group, we first determined the core/pan-genome of each species to estimate the number of shared genes between every S. epidermidis, S. capitis, and S. caprae strain. Although more than 400 genome sequences of the epidermidis cluster group are available on GenBank, including draft genome sequences, we focused on the complete genome-sequenced strains. Complete genome sequences are considered to be more reliable and allow identification of precise strain-specific genes on each chromosome. In this core/pan-genome analysis, genes harbored on genomic islands, prophages, SCCmec, and transposons were separated from the category of conserved genes in each species. There were a total of 1,945 conserved genes identified among all S. epidermidis strains (Fig. 5 and Additional file 2 TableS2). Similar to S. epidermidis, 2,064 and 2,313 conserved genes were shared between every S. capitis and S. caprae strain, respectively. The conserved genes showed high sequence similarity among the three species. More than 95% of conserved genes displayed > 95% sequence identities by interspecies comparison (Additional file 2 TableS2).
Next, we sought to identify both conserved and species-specific gene sets of the epidermidis cluster group using the conserved gene sets of each species. As shown in Fig. 5, 1,719 CDSs were conserved among the three species, while 141, 59, and 263 species-specific genes were identified in S. epidermidis, S. capitis, and S. caprae, respectively. We also found that 21, 265, and 64 genes were shared between species when comparing S. epidermidis vs S. capitis, S. capitis vs S. caprae, and S. caprae vs S. epidermidis, respectively (Fig. 5d).
To analyze the genome robustness or plasticity of the epidermidis cluster group, the gene sets specific to each species, shared between two species, and conserved among all three species were mapped on S. epidermidis RP62a, S. capitis TW2795, and S. caprae JMUB898 chromosomes (Fig. 6). The gene synteny was found to be well retained in the chromosome of the epidermidis cluster group. However, the chromosomal regions where some species-specific genes were clustered in each species seem to be the remnants of genomic islands.
The downstream regions of SCCmec elements also were not conserved among the three species. Our analysis revealed that the species-specific genes of S. capitis and S. caprae were typically mapped to the downstream of SCC elements, similar to that of S. aureus, S. epidermidis, and S. haemolyticus genomes [23] (Fig. 6). Some remnants of prophages or genomic islands were also found (Fig. 6). These species-specific genomic regions are considered likely to contribute to the evolution and differentiation of Staphylococcus species differentiation [23].
Although the downstream regions of the SCCmec elements were divergent among species, we found that the type V SCCmec identified in the S. caprae JMUB145 genome was closely related to those of S. aureus JCSC5952 and S. pseudointermidius 06–3228 (Fig. 2) [20, 21], while type IVa SCCmec of S. caprae JMUB590 and JMUB898 was closely related to that of the S. aureus USA300 TCH1516 strain (Fig. 2) [22]. The SCCmec of methicillin-resistant S. caprae has rarely been analyzed. Previous studies showed that S. caprae stains carried a variety of mec gene complexes, for example, one S. caprae strain isolated from pig was reported to carry class A mec and type I ccr [24, 25], and in S. epidermidis and S. capitis strains, type I–V SCCmec elements (originally identified in MRSA) were identified by PCR-based methods [7]. It might be interpreted as the results of co-evolution and interspecies exchange of the SCCmec elements over long-term evolution.
Genes conserved across three species
Our analysis identified 1,719 conserved CDSs among S. epidermidis, S. capitis, and S. caprae. Most of the conserved genes are involved in fundamental biological processes. Compared with the highly virulent S. aureus, the three species possessed fewer known and putative virulence factors. Well-known virulence factors of S. aureus such as coagulase, protein A, leukocidins, α-toxin, and staphylococcal enterotoxins were missing from these genomes. Nevertheless, the three species shared virulence factors involved in biofilm formation and protection against the innate immune system. Furthermore, teichoic acid biosynthesis genes (tagAHGBXD) encoding wall teichoic acid (WTA) were conserved among the three species. This gene cluster confers positive charge to bacterial cells, thereby mediating primary adherence to polystyrene surfaces as well as host organisms to initiate biofilm formation [7]. The dltABCD genes were also conserved among the three species. dltABCD genes function as the modification system for WTA by incorporating d-alanine into WTA [26]. Our genome analysis also confirmed that all analyzed genomes from the three species (S. epidermidis, S. capitis, and S. caprae) shared poly-γ-dl-glutamic acid (PGA) genes. The PGA capsule is another Staphylococcus extracellular virulence factor found in S. epidermidis, which facilitates bacterial growth and survival in the human host [27]. Kocinaova et al. described that all tested S. epidermidis strains and the reference strains of the epidermidis cluster group, including S. capitis subsp. capitis, S. capitis subsp. ureolyticus, and S. caprae, produced PGA, and carried capB and capD genes [27]. The other extracellular proteins such as secreted thermonuclease [28] and Clp protease [29] involved in biofilm formation were also found to be conserved among the three species.
The three S. caprae strains carried two phenol soluble moduline (PSM) gene clusters, PSMβ and PSMα/PSMδ. The former was located upstream of Arginine-tRNA and was conserved among the Staphylococcus species, including S. epidermidis, S. capitis, and S. caprae. The number of genes of a PSMβ cluster has been shown to vary from two to six [17]. Consistent with this previous report [17], the PSMβ cluster of the three S. caprae strains contained five genes, while that of S. epidermidis RP62a and S. capitis TW2795 consisted of four genes. The PSMα/PSMδ cluster of S. caprae was located upstream of the NADH dehydrogenase gene and contained single copies of PSMα and PSMδ. PSMα and PSMδ were conserved in S. epidermidis RP62a and S. capitis TW2795 strains.
Divergent interspecies evolution of cell surface extracellular proteins
Cell surface extracellular proteins mediate bacterial adherence to abiotic surfaces and host tissues as well as intercellular adhesion during biofilm formation [7]. By analyzing the conserved genes among the three species, S. epidermidis, S. capitis, and S. caprae, we found that most cell surface proteins showed substantially lower sequence identities between species compared with the other conserved proteins. The average of amino acid identities for total conserved genes were 80.4, 90.9, and 84.8% among S. epidermidis RP62a vs S. capitis TW2795, S. capitis TW2795 vs S. caprae JMUB898, and S. caprae JMUB898 vs S. epidermidis RP62a, respectively. In contrast, the identities for cell surface proteins were less than 50%. Since we set a cutoff value of 50% for amino acid identity in this analysis, proteins with amino acid identities of less than 50% must be categorized as species-specific proteins. However, even though most cell surface proteins had less than 50% identities among the three species, they could be considered as homologues of each other, because they were judged to share the same ancestors or origin by analyzing gene structure, location on the genome, and functional motifs. For example, S. capitis and S. caprae carried SesC-like proteins. The amino acid sequence identities of SesC/SesC-like proteins were 45.0% between S. epidermidis RP62a and S. capitis TW2795 and 46.1% between S. epidermidis RP62a and S. caprae JMUB898. The SesC-like proteins are postulated to have a similar function as SesC in S. epidermidis because SesC-like proteins in S. capitis and S. caprae contain the LPXTG motif, as does SesC in S. epidermidis. Moreover, the genome loci of the genes were considered to be the same since the upstream region of every sesC in these three species contained a gene encoding an organic hydroperoxide resistance-like protein. Therefore, SesC-like proteins in S. capitis and S. caprae might function as host cell factors and/or abiotic surface-binding proteins owing to the fact that SesC in S. epidermidis is one of the fibrinogen-binding proteins containing a wall-anchoring LPXTG motif that mediates biofilm formation [30] although the amino acid identities of the three proteins were shown to be less than 50%. These lower identities seemed to reflect their divergent interspecies evolution.
In this study, we also identified the cell surface proteins involved in adhesion and biofilm formation and created a heat map of these proteins based on their amino acid identities (Fig. 7). We found that 17 of the 26 cell surface proteins were well conserved in the three species. Eleven cell-wall-anchored proteins, SesA-I and SdrFG, which contained an N-terminal secretion signal sequence and wall-anchoring LPXTG motif, were predicted from the S. epidermidis RP62a genome sequence [31]. These proteins are involved in bacterial attachment to host tissue or cells and biofilm formation. Although 5 (sesABCEH) of the 11 genes encoding the cell wall-anchored proteins were identified at the similar genome locus tags of the three species, there were few interspecies similarities. The amino acid identities of SesA, SesB, SesC, SesE, and SesG between S. epidermidis RP62A and S. capitis TW2795 were 53.2, 75.0, 45.0, 34.6, and 58.0%, respectively, while those between S. epidermidis RP62A and S. caprae JMUB898 were 58.3, 75.5, 46.1, 35.9, and 37.2%, respectively. In addition to SesA-I and SdrFG, S. epidermidis strains carry an extracellular matrix-binding protein Embp/EbhA that is similar to Ebh identified in S. aureus [32, 33]. S. capitis and S. caprae also carry Embp/EbhA-like proteins with the amino acid identities of ~ 60.3% compared with those of S. epidermidis.
S. epidermidis carries three multifunctional autolysin/adhesins, AtlE, Atl, and Aae, which mediate both cell lysis and attachment to materials including host tissue or cells, abiotic surfaces, and extracellular DNA [27, 34]. Similar to other cell surface proteins containing an LPXTG motif, these proteins are conserved among the three species, but the amino acid identities were less than the average amino acid identities of conserved proteins. The most analyzed staphylococcus autolysin is AtlE in S. epidermidis, and the atlE gene disseminates among staphylococcal species. AtlE homologues, such as Atl in S. aureus, Atlwm in S. warneri, AtlC in S. caprae, Aas in S. saprophyticus, and AtlL in S. lugdunensis, have been reported as fibronectin-binding proteins involving in biofilm formation [35,36,37].
Most organisms colonizing human skin possess lipolytic activity to hydrolyze lipids found on the surface of human skin [38]. Other than cell wall-anchored proteins, extracellular lipases, GehCD, Lip, and LipA, which mediate bacterial colonization on human and animal skins, were conserved in S. epidermidis, S. capitis, and S. caprae with high interspecies diversity [39, 40]. Every strain in the epidermidis cluster group also possesses an extracellular metalloprotease with elastase activity, such as SepA (SE2219) that plays a role in conferring resistance to the antimicrobial peptide dermicidin [41].
The elastin-binding protein (ebpS) or ebpS-like genes were also found to be conserved among S. epidermidis, S. capitis, and S. caprae strains. Similar to extracellular lipases, EbpS also showed high diversity among the three species. Our analysis categorized S. epidermidis EbpS as a different protein from the EbpS of S. capitis and S. caprae since the amino acid identity of EbpS proteins between S. epidermidis and S. caprae was less than 39.9%, and the length of the homologous region between EbpS of S. epidermidis and S. capitis was less than 50% of the entire region. As reported in S. aureus, EbpS was first identified as an adhesin for extracellular matrix elastin of host cell tissue [42]. However, EbpS in S. aureus shows relatively weak binding potential to elastin [43]. The adherence of some S. aureus strains to immobilized elastin is mediated by fibronectin-binding proteins FnBPA and FnBPB, but not by EbpS, and the inactivation of ebpS in S. aureus strains has only a minimal effect on the binding of S. aureus to elastin peptide [43]. Regardless, it was recently reported that EbpS regulates bacterial growth rate in liquid culture and promotes biofilm maturation in a zinc concentration-dependent manner [44]. Therefore, EbpS and EbpS-like proteins of the epidermidis cluster group might play a role in biofilm maturation.
Genes shared by S. capitis and S. caprae but not by S. epidermidis
Based on the phylogenetic analysis, S. capitis is more closely related to S. caprae than S. epidermidis. Therefore, a larger set of shared genes could be identified between S. capitis and S. caprae than when comparing S. epidermidis with S. capitis or S. epidermidis with S. caprae (Fig. 5 and Additional file 2 TableS2). Four major biofilm formation-related factors were shared by S. capitis and S. caprae. These include polysaccharide intracellular adhesin (PIA), elastin-binding protein (EbpS), SesC-like proteins, and SdrH-like proteins. Since EbpS, SesC, and SdrH of S. capitis and S. caprae (EbpS-like, SesC-like, and SdrH-like proteins) showed lower similarities (add number %) to those of S. epidermidis, they were categorized as shared by S. capitis and S. caprae.
PIA, also known as poly-N-acetylglucosamine, mediates biofilm formation and plays an important role in immune evasion [45,46,47]. PIA production is regulated by the ica operon (icaADBC) [45, 48], and the ica locus has been identified in many staphylococcal species including S. aureus, S. capitis, and S. caprae [49, 50]. We found that all S. capitis and S. caprae strains analyzed in this study possessed the ica operon. However, only three S. epidermidis strains (RP62a, 1457, and BH0622) carried the ica operon. This is in concordance with a previous study where a number of ica negative clinical isolates of S. epidermidis were reported [51].
SesG is one of the cell surface–anchored proteins identified in the S. epidermidis RP62a genome. Although four of the seven S. epidermidis strains lack the sesG gene, every S. capitis and S. caprae strain analyzed in this study carried the sesG gene, which had amino acid identities of 53.2–54.6% (S. epidermidis RP62a vs S. capitis) and 47.2–53.2% (S. epidermidis RP62a vs S. caprae).
S. capitis and S. caprae also shared the same mannitol metabolic pathways. Mannitol utilization has been adapted for species classification among CoNS [52]. Our genome analysis confirmed that mannitol acquisition and utilization pathways are conserved in mannitol-positive species such as S. capitis and S. caprae, while mannitol-negative S. epidermidis species lacks this system. Arginase, which catalyzes the fifth and final steps in the urea cycle, resulting in the conversion of l-arginine into l-ornithine and urea, was conserved in S. capitis and S. caprae but not in S. epidermidis. It is interesting that urease genes were conserved in all three species, yet urease activity, being one of the key phenotypes for classification of CoNS, was shown to be negative in S. capitis subsp. capitis [52]. S. capitis and S. caprae also shared staphyloxanthin biosynthesis genes crtOPQMN. These genes regulate the production of orange carotenoid, conferring the characteristic golden-yellow color of colonies, which aids in distinguishing S. aureus from S. epidermidis [53]. A clumping factor B-like protein gene, located at the identical locus downstream of the arcR gene was identified in S. capitis and S. caprae genomes but not in the S. epidermidis genome.
Genes shared by S. epidermidis and S. capitis but not by S. caprae
S. epidermidis and S. capitis shared 21 genes (Fig. 5), among which are the accessory Sec systems. The canonical Sec system, which translocates the majority of proteins across the cytoplasmic membrane, is present in all bacteria including staphylococcal species. In addition to the canonical Sec system, accessory Sec systems were found in S. epidermidis and S. capitis but not in S. caprae. The accessory Sec systems are conserved in staphylococci and streptococci, facilitating the transportation of serine-rich repeat glycoproteins [54, 55]. In S. aureus, the accessory Sec system secretes the serine-rich glycoprotein SraP, which mediates staphylococcal binding on human platelets [54]. S. capitis possesses a SraP homologue (JMUB0001_172). Although the SraP homologue was not conserved in all S. epidermidis strains, we identified it in S. epidermidis RP62a, PM221, and 14.1.R1 strains. This finding indicated that the additional Sec system might contribute to glycoprotein translocation.
Our genome comparison analysis showed that 5 of the 11 cell wall-anchored proteins identified in the S. epidermidis RP62a genome were conserved among S. epidermidis, S. capitis, and S. caprae (Fig. 7 and Additional file 2 TableS2). Another two genes, sesF and sdrG, were identified only in some S. epidermidis and S. capitis strains but not in S. caprae. The sesF gene, encoding the accumulation-associated protein Aap, had been identified as an essential protein for biofilm accumulation on glass or polystyrene surfaces in certain S. epidermidis strains [56, 57]. The giant extracellular protein mediates biofilm formation and accumulation via fibronectin-binding and intercellular adhesion abilities [58, 59]. Four of the seven genome-sequenced S. epidermidis strains and one of the three S. capitis strains carried the aap gene on their chromosomes. In contrast, five of the seven genome-sequenced S. epidermidis strains carried the sdrG/fbe gene, while 1457 and 14.1.R1 lacked this gene. S. capitis CR01 also had the SdrG/Fbe-like protein with an amino acid identity of 38.4% between RP62a and CR01. SdrG, one of the three SD repeats-containing serine asparagine-rich (Sdr) proteins identified in S. epidermidis RP62a (also known as fibrinogen-binding protein [Fbe]), was suggested to promote device-related infection due to the observation of that an sdrG/fbe deletion mutant was attenuated in an intravascular catheter-associated rat infection model [60,61,62].
Genes shared by S. epidermidis and S. caprae but not by S. capitis
We found that S. epidermidis shared 64 genes with S. caprae strains (Fig. 5). Every sequenced S. epidermidis and S. caprae strain carried serine V8 protease GluSE, also known as Esp/SspA (SE1543) [63,64,65]. When S. epidermidis and S. aureus co-exist in an organism, Esp produced by commensal S. epidermidis strains inhibits S. aureus biofilm formation and subsequently its colonization in the anterior nares [66]. The esp gene was not found in the three sequenced S. capitis strains.
All sequenced S. epidermidis and S. caprae strains also carried the arginine synthesis pathway genes argJBCD. l-Arginine is required for the growth of most Staphylococcus species, including S. aureus, S. epidermidis, and S. capitis [67]. The presence of argJBCD genes in S. epidermidis and S. caprae is consistent with a previous study where the mutants not requiring l-arginine were generated by in vitro selection from S. epidermidis strains but not from S. capitis strains [67]. This implies that S. epidermidis strains might have the arginine biosynthesis enzymes, but the enzymes were not expressed in the wild-type strains [67].
Genes carried by S. epidermidis but not by S. capitis and S. caprae
Each of the seven S. epidermidis strains tested carried 380–789 strain-specific genes. The conserved gene analysis identified 141 genes that were conserved in all S. epidermidis strains but not in S. capitis or S. caprae. Most of these S. epidermidis-specific genes encode virulence factors that have been known to mediate biofilm formation or be involved in the central metabolic pathways.
The cell wall-anchored protein SdrF was identified only in four of seven S. epidermidis strains. The full-length sdrF gene, which encodes a type I collagen-binding protein, was identified in SEI, 1457, and BH0622 genomes. Nevertheless, truncated sdrF genes were found in S. epidermidis RP62a, PM221, and 14.1R1. S. capitis AYP1020 also carries the truncated SdrF homologue with 73.3% amino acid identity, but the S. capitis sdrF gene was fragmented by a frameshift mutation.
The genome comparison revealed that S. epidermidis has a specific central metabolism pathway that does not exist in the genomes of S. capitis and S. caprae. S. epidermidis possesses the glycerol dehydrogenase gene gldA, which mediates glycerol metabolism. A previous study showed that S. epidermidis could ferment glycerol into succinate, and the resulting succinate could in turn inhibit the growth of another skin colonizer P. acnes in vitro and in vivo [68]. Moreover, S. epidermidis strains, but not S. capitis and S. caprae strains, carried fumarate reductase-mediating succinate fermentation and accumulation [69]. S. epidermidis also carried biosynthesis genes for biotin, which is a cofactor of acetyl-CoA carboxylase involved in fatty acid metabolism.
Genes carried by S. capitis but not by S. epidermidis and S. caprae
Each of the three S. capitis strains tested carried 199–357 strain-specific genes, in which 59 were identified by our analysis to be S. capitis specific. Many species of Gram-positive bacteria, including staphylococci, produce lantibiotics and small cationic antimicrobial peptides to provide antimicrobial activities in facilitating niche compensation [70]. Some CoNS strains produce unique lantibiotics such as epidermin (from S. epidermidis) and gallidermin (from S. Gallinarum) [71,72,73]. Kumar et al. reported an identification of an S. capitis strain TE8 that carried both epidermin and gallidermin gene loci [17]. In our analysis, all three S. capitis strains tested carried lantibiotics genes on their chromosomes, but they were similar to that of S. aureus RF122 with an amino acid identity of 47% [74].
All three S. capitis strains and S. epidermidis RP62A, PM221, and 14.1.R1 carried serine-rich adhesin for platelet (SraP) homologues. SraP homologues have been widely identified among staphylococci including S. aureus and S. gordonii [75]. SraP mediates bacterial binding to platelets, whereby the binding is considered the key step during infective endocarditis [75, 76].
Genes carried by S. caprae but not by S. epidermidis and S. capitis
Our genome analysis identified a total of 265 genes that were carried by S. caprae but not by any of S. epidermidis and S. capitis strains, of which 163–197 were carried by five individual strains of S. caprae tested. Among those, biofilm-associated protein (Bap), capsular polysaccharide, and type VII secretion factor recognized as virulence factors were included. Bap is known to mediate bacterial attachment to polystyrene and biofilm formation in S. aureus and some CoNS [77, 78].
Capsular polysaccharide production is an important virulence determinant in many invasive bacterial pathogens. Capsular polysaccharides are produced by ~ 90% of S. aureus strains, and encapsulated S. aureus strains are more resistant to phagocytosis than the nonencapsulated strains [79]. Among the CoNS, S. haemolyticus, S. hyicus, and S. lentus produce a capsular polysaccharide-like surface antigen that cross-react serologically with S. aureus type 5 capsular polysaccharide [80].
In our genome analysis, three S. caprae strains carried a capsular gene operon composed of 16 genes. Even though the first four genes in this operon showed high amino acid identities to S. aureus cap 5ABCD, S. caprae’s capsular polysaccharide might form a different structure compared with S. aureus’s capsular polysaccharide. The S. caprae cap operon contained capHIOM homologues, and the cap operon encoded putative polysaccharide modification enzymes such as glycosyltransferases, O-acetyltransferase, and aminotransferase.
The type VII secretion system is encoded by the genomes of diverse bacterial species across the Firmicutes and Actinobacteria phyla, including S. aureus and Mycobacterium tuberculosis. The S. aureus type VII secretion system exports several effector proteins including EsxABCD and nuclease toxin EsaD, enabling long-term survival in abscesses [81,82,83,84,85]. The gene component of the type VII secretion system cluster varies among S. aureus strains, but the gene cluster of S. caprae showed high similarity to those of S. aureus, e.g. there were 60% identity and 94% similarity of EsaA between S. caprae JMUB898 and S. aureus MRSA252. The S. caprae type VII secretion system contained eight genes encoding four membrane-associated proteins (EsaA, EssAB, and EssC), two soluble cytosolic proteins EsaBC, and two secreted virulence factors EsxAB.
The utilization of trehalose as a carbon source is a key characteristic of S. caprae because most S. epidermidis and S. capitis strains cannot use trehalose as a carbon source [9, 86]. Our genome analysis confirmed that the trehalose-specific PTS system was found only in S. caprae genomes but not in S. epidermidis and S. capitis.
Biofilm formation capacity of S. caprae
In order to assess whether S. caprae strains can produce biofilms, we carried out biofilm assays in comparison with other staphylococcal species that are well known as biofilm producers. Four S. epidermidis, four S. capitis, five S. caprae, and two S. aureus strains were compared (Fig. 8). All strains tested formed biofilm on a plastic surface and the biofilm formation was induced by adding 1% glucose. However, the levels of biofilm formation varied among strains. Two S. aureus strains (N315 and MW2), two S. epidermidis strains (RP62a and JMUB051), and one S. capitis strain (JMUB603) produced higher amounts of biofilm mass on a plastic surface than the other strains. The S. caprae strains produced detectable but weaker biofilms.