Reclassification of Phenotypically Identified Staphylococcus intermedius Strains

Bacterial strains.We selected 117 S. intermedius strains, which were derived from animals for which the isolation of S. intermedius had previously been reported (2, 5, 6, 10-12, 14, 36) and tentatively identified by Rapid ID32 Staph (bioMérieux, Marcy-l′Etoile, France) from our coagulase-positive staphylococcus collections from various animal sources, which we designated the S. intermedius group (SIG) (Table 1). These strains were stored in 10% skim milk at −85°C until use and maintained on Trypticase soy agar II with 5% sheep blood (BD Japan Co., Ltd., Tokyo, Japan).

Phenotypic characterization.Coagulase was determined by tube coagulase test using rabbit serum (Denka Seiken Co., Ltd., Tokyo, Japan) for all strains tested. Acid production from carbohydrates was detected under aerobic conditions, except d-mannitol, using a broth microplate method (16). Each carbohydrate of d-galactose, d-mannitol (in anaerobic culture for 20 h), arbutin, α-lactose, d-mannose, β-gentiobiose, and d-turanose was added in purple broth base (24 g/liter; Difco, MD) and thioglycolate medium without glucose or indicator (12 g/liter; Difco) at a final concentration of 0.5% (wt/vol). Each plate was incubated at 37°C and was evaluated at 20 and 48 h. MIC testing for polymyxin B using Etest (AB Biodisk, Solna, Sweden) was performed. An acetoin production (Vogues-Proskauer) test was carried out according to a method reported previously (9).

Thermonuclease production was detected by the metachromatic agar diffusion method using DNA and o-toluidine blue by using a modified method described previously (23), which was conducted as follows: culture supernatants in brain heart infusion broth (Difco) were filtrated and heated (at 100°C for 15 min), and disks with the filtrate were applied to DNase test agar (Becton Dickinson Co., Ltd., France) with o-toluidine blue. Agars were incubated at 37°C for 12 h.

DNA extraction.A single colony was suspended to a McFarland standard of 1.0 in 100 μl of TE buffer (20 mM Tris, 2 mM EDTA [pH 7.5]) with 10 U of achromopeptidase (Wako Chemical Co., Ltd., Osaka, Japan), and the suspension was incubated at 55°C for 10 min. After centrifugation at 18,500 × g for 5 min, the supernatants were used as crude DNA extracts for PCR (17). Purified DNA extracts for DNA-DNA hybridization were made according to methods reported previously (7).

Genetic identification of the SIG.Direct sequencing of sodA and hsp60 genes was performed for the differentiation of SIG strains as described previously (21, 22, 25, 27). Multiple alignment was carried out by using the CLUSTAL X program (32). The construction of the unrooted phylogenetic tree was performed by the neighbor-joining method (27).

Genetic characterization of the thermonuclease (nuc) gene.Detection of the nuc gene, encoding thermonuclease, was carried out for SIG strains by PCR (PCR1) as previously described (2). PCR products were sequenced directly. Another PCR (PCR2) was conducted for PCR1-negative strains. The primer set of Nuc-dF (5′-GAA GGC ATA TTG TAG AAC AA-3′) and Nuc-eR (5′-NCK YTC CCA DAT RTT NAR YTK-3′) was designed to amplify the conserved region of the nuc gene by multiple alignment of amino acid sequences of the staphylococcal nuc gene from those of PCR1-positive strains and the staphylococcal species whose genomes had previously been wholly sequenced through the GenBank database. The size of PCR products was 724 bp including the coding sequence of the nuc gene. The reaction mixture for the PCR consisted of 2 μl of DNA extract in a total volume of 50 μl composed of 2 U of Ex Taq (Takara Shuzo Co., Ltd., Kyoto, Japan), 10 pmol of each primer, 0.2 mM deoxynucleoside triphosphate mixture, 1.5 mM MgCl₂, and 1× reaction buffer (Takara Shuzo Co., Ltd.). Reaction mixtures were thermally cycled once at 95°C for 5 min; 35 times at 95°C for 1 min, 44°C for 1 min, and 72°C for 1 min; and then once at 72°C for 7 min. These PCR products were also sequenced directly.

DNA-DNA hybridization.DNA-DNA hybridization was performed by microplate methods using a Benchmark Plus microplate reader (Bio-Rad, Tokyo, Japan) (7). Hybridization was performed at 45°C for 2 h in a hybridization mixture containing 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5× Denhardt solution, 3% dextran sulfate, 50% formamide, 200 ng/ml denatured herring DNA, and 1,500 ng/ml biotinylated probe DNA.

Molecular phylogenetic analysis of sodA and hsp60 gene sequences among SIG strains.As shown in Fig. 1 and 2, all 78 strains from dogs were very close to S. pseudintermedius LMG 22219^T, with high sequence homology of both the sodA (98.8 to 100%) and hsp60 (98.7 to 99.5%) genes. All the strains from cats and humans also belonged to the same cluster. Thus, these 83 strains were identified as being S. pseudintermedius strains. All the 11 wild pigeon-derived strains except strain P-50 showed high sequence similarities to both the sodA (99.7 to 100%) and hsp60 (100%) genes of S. intermedius ATCC 29663^T. Consequently, these strains were identified as being S. intermedius strains. On the other hand, strains from 13 domestic pigeons, 1 wild pigeon (P-50), 6 horses, and a mink showed close sequence similarities to sodA (97.7 to 99.3%) of S. delphini LMG22190^T. However, unlike the S. intermedius and S. pseudintermedius clusters, hsp60 sequences among these strains were more divergent, and similarities to S. delphini LMG22190^T were 95.7 to 98.9%.

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FIG. 1.

Phylogenetic tree (unrooted) based on partial sodA gene sequences of SIG strains used in the present study. The tree was constructed by the neighbor-joining method using CLUSTAL X. It is unknown whether S. intermedius ATCC 29663^T is derived from a wild or domestic pigeon.

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FIG. 2.

Phylogenetic tree (unrooted) based on partial hsp60 gene sequences of SIG strains used in the present study. The tree was constructed by the neighbor-joining method using CLUSTAL X. It is unknown whether S. intermedius ATCC 29663^T is derived from a wild or domestic pigeon.

Detection and phylogenetic characterization of nuc genes among SIG strains.For a better differentiation of S. pseudintermedius, S. intermedius, and S. delphini, we carried out two nuc PCRs and direct sequencing of the nuc genes (Table 2).

PCR1 could detect predictive fragments among all the S. pseudintermedius and S. intermedius strains and 5 of 22 S. delphini strains. The remaining S. delphini strains were not amplified by PCR1 in spite of the ability to produce thermonuclease. PCR2 successfully amplified predictive fragments with 17 PCR1-negative strains.

In the phylogenetic tree of the nuc gene, SIG strains were classified into four clusters (Fig. 3). S. intermedius and S. pseudintermedius strains constituted a single cluster that confirmed their sodA and hsp60 phylogeny. Interestingly, S. delphini strains were divided into two clusters: one (S. delphini group A) was similar to S. delphini LMG22190 ^T, and the other (S. delphini group B) was closer to the S. pseudintermedius cluster than S. delphini LMG22190^T.

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FIG. 3.

Phylogenetic tree (unrooted) based on partial nuc gene sequences of SIG strains used in the present study. The tree was constructed by the neighbor-joining method using CLUSTAL X. It is unknown whether S. intermedius ATCC 29663^T is derived from a wild or domestic pigeon.

DNA-DNA hybridization among SIG strains.To elucidate the taxonomic position of S. delphini groups A and B, We performed DNA-DNA hybridizations among SIG strains (Table 3). Given a threshold level of 70% DNA-DNA relatedness for the definition of a bacterial species (37), our reclassification based on phylogenetic analysis of the nuc gene corresponded to the species distinction. Among S. delphini group strains and those of the other clusters, S. delphini group A had the highest level (77%) of DNA similarity to S. delphini LMG22190^T. On the other hand, S. delphini group B strains showed less than 70% similarities with those of any clusters. DNA similarities of greater than 70% were observed among S. delphini groups A and B, respectively (data not shown). These results suggest that S. delphini group B constitutes a novel species.

Phenotypic characteristics among SIG strains.To search for phenotypic characteristics that distinguish the genetically distinct SIG strains, we compared their biochemical characteristics. As shown in Table 4, S. intermedius could be differentiated from the other SIG strains by 100% negative arginine dihydrolase and 100% positive acid production from β-gentiobiose aerobically and d-mannitol anaerobically. There was no obvious difference in biochemical reactions among S. pseudintermedius, S. delphini group A, and S. delphini group B. There were some variations in the abilities of acid production from d-turanose: S. pseudintermedius and S. delphini group B showed weak or delayed reaction, S. delphini group A strains were negative, and S. pseudintermedius and S. delphini group A strains did not use β-gentiobiose, whereas about 60% of the S. delphini group B strains fermented β-gentiobiose. In standard acetoin production tests (9), 66.3% (55/83) of S. pseudintermedius strains were positive. The abilities were not as strong as with S. aureus, and most of them were not detected by the Rapid ID32 Staph test. S. intermedius and S. delphini group A (except for S. delphini LMG22190^T) and group B strains were negative for this characteristic.

All the S. pseudintermedius strains produced thermonuclease; the level of production was stronger than that for the other species. Although 66% (8/12) of S. intermedius strains showed a positive reaction, the activities were weaker than those of S. pseudintermedius. All S. delphini group A strains except S. delphini LMG22190^T and strain CCUG 51769 showed a positive reaction.