BacteriologyThe sigH gene sequence can subspeciate staphylococci
Introduction
The Staphylococcus genus is currently classified into 39 species (Euzeby, 1997). The precise species identification of staphylococci is important in etiologic and epidemiologic studies. Phenotypic tests are broadly used in today's clinical laboratories, but precise discrimination of the species often requires additional information (Grant et al., 1994, Perl et al., 1994, Renneberg et al., 1995). Several gene sequences such as 16S rRNA (Becker et al., 2004, Fujita et al., 2005, Skow et al., 2005, Takahashi et al., 1999), tuf (Martineau et al., 2001), rpoB (Drancourt & Raoult, 2002, Mellmann et al., 2006), hsp60 (Goh et al., 1997, Kwok & Chow, 2003), gap (Yugueros et al., 2000), femA (Vannuffel et al., 1999), and sodA (Giammarinaro et al., 2005, Poyart et al., 2001, Sivadon et al., 2004) have been used in the identification of staphylococcal species. However, highly conserved genes are not suitable for the discrimination of closely related species.
The sigH gene encodes a sigma factor that constitutes a large alternative sigma factor group in Firmicutes, and it is located within an evolutionarily conserved gene organization (Morikawa et al., 2003). In sporulating bacteria such as Bacillus subtilis and Clostridium perfringens, SigH regulates the initiation of sporulation and is highly conserved even among the distinct genera. In contrast, SigH in nonsporulating bacteria such as Staphylococcus and Streptococcus shares low homology among species.
In this study, we analyzed the sigH sequences of 39 staphylococcal species, aiming to show that the sigH gene is a promising molecular target for discriminating between closely related species. In addition, bioinformatic analysis revealed that there are similar gene clusters with high local diversity, suggesting that such syntenic regions may be promising molecular targets for species identification.
Section snippets
Bacterial strains
Staphylococcus strains used in this study are shown in Table 1. Clinical isolates of Staphylococcus saprophyticus candidates were obtained as described previously (Higashide et al., 2006). Chromosomal DNA was extracted using conventional methods.
Polymerase chain reaction amplification of the sigH locus and sequencing analysis
Polymerase chain reaction (PCR) primers (Table 1) were designed for the conserved region outside the sigH coding sequence (Fig. 1). The sigH locus was amplified by PCR using Ex-taq polymerase (Takara, Otsu, Japan). The PCR products were purified by
The sigH gene is located between conserved genes
The sigH gene has a low homology among species but is embedded in a conserved gene cluster (Fig. 1) (Morikawa et al., 2003). The genes adjacent to sigH in Staphylococcus epidermidis, Staphylococcus haemolyticus, and S. saprophyticus share more than 70% identity with those in Staphylococcus aureus, whereas the sigH gene shares less than 40% identity. We supposed that such a local diversity would be useful for identifying species; the high divergence of the target gene is necessary to
Discussion
We established the sigH sequence data set of all 39 staphylococcal species. Its high divergence is useful for discriminating between closely related species that cannot be assigned by conventional phenotypic testing or by using more highly conserved genes. Among the genes tested so far, the 1 most successfully used for discriminating between closely related species is sodA (Poyart et al., 2001, Sivadon et al., 2004, Sivadon et al., 2005). It was used to design hybridization probes and enabled
Acknowledgment
The authors thank Mr. Masato Higashide (Kotobiken Medical Laboratories, Japan) for clinical isolates and Mr. Yu Ohki for DNA sequencing. We are also grateful to Dr. Flaminia Miyamasu for correction of the manuscript. This work was supported by a Grant-in-Aid for Young Scientists of the Japan Society for the Promotion of Science (to K.M.).
References (31)
- et al.
Evaluation of two commercial systems for identification of coagulase-negative staphylococci to species level
Diagn. Microbiol. Infect. Dis.
(1994) - et al.
Evaluation of a cefoxitin disk diffusion test for the detection of mecA-positive methicillin-resistant Staphylococcus saprophyticus
Int. J. Antimicrob. Agents
(2006) - et al.
Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species
Diagn. Microbiol. Infect. Dis.
(1994) - et al.
Use of sodA sequencing for the identification of clinical isolates of coagulase-negative staphylococci
Clin. Microbiol. Infect.
(2004) - et al.
Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species
Res. Microbiol.
(1999) Eubacterial sigma-factors
FEMS Microbiol. Rev.
(1998)- et al.
Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species
J. Clin. Microbiol.
(2004) - et al.
Staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals
Int. J. Syst. Evol. Microbiol.
(2005) - et al.
rpoB gene sequence-based identification of Staphylococcus species
J. Clin. Microbiol.
(2002) List of bacterial names with standing in nomenclature: a folder available on the Internet
Int. J. Syst. Bacteriol.
(1997)
PHYLIP—Phylogeny Inference Package version 3.2
Cladistics
Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis
J. Clin. Microbiol.
Development of a new oligonucleotide array to identify staphylococcal strains at species level
J. Clin. Microbiol.
Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain
J. Bacteriol.
Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization
J. Clin. Microbiol.
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