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Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Japan
Correspondence
Takayuki Ezaki
tezaki{at}gifu-u.ac.jp
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ABSTRACT |
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. An analysis of dnaJ gene sequence similarity among the staphylococcal type strains used in this study is presented in a supplementary table in IJSEM Online.
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MAIN TEXT |
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Sequence-based identification of micro-organisms is becoming a useful and reliable alternative to phenotypic methods. Analysis of 16S rRNA gene sequences is the most commonly used method for identification and classification of bacteria, including staphylococci; however, the usefulness of 16S rRNA gene sequences is limited because of the high degree of sequence similarity between closely related species (Gribaldo et al., 1997; Becker et al., 2004; Skow et al., 2005). Recently, partial sequences of the highly conserved hsp60 (Kwok et al., 1999; 2003), sodA (Poyart et al., 2001) and rpoB (Drancourt & Raoult, 2002; Mellmann et al., 2006) genes have been found to be useful in the identification of Staphylococcus at the species level. However, a universal DNA target with well-conserved DNA sequences within a given species, but with sufficient sequence variation to discriminate between species and subspecies, is needed.
DnaJ, also known as Hsp40, is a member of the heat-shock protein (Hsp) family, distributed ubiquitously in Eukarya, Bacteria and Archaea (Ang et al., 1991; Macario et al., 1993). The dnaJ gene has been shown to be more discriminative than the 16S rRNA gene for identifying species of the genus Streptococcus (Itoh et al., 2006), Legionella pneumophila serogroups (Liu et al., 2003) and Mycobacterium species (Takewaki et al., 1993; Victor et al., 1996). In the present study, we developed a Staphylococcus-specific dnaJ gene sequence-based assay for the identification and establishment of phylogenetic relationships among (sub)species of the genus Staphylococcus.
A total of 45 staphylococcal type strains (Table 1), 20 isolates of two clinically important species, Staphylococcus aureus subsp. aureus (n=10) and Staphylococcus epidermidis (n=10) and eight non-staphylococcal strains as negative controls (Macrococcus caseolyticus, Bacillus subtilis, Micrococcus luteus, Enterococcus faecalis, Streptococcus pyogenes, Lactococcus lactis, Escherichia coli and Pseudomonas aeruginosa) were used in this study. Some recently described species (Euzéby, 1997; http://www.bacterio.cict.fr/, updated February 2006) were not available for our study. All strains were grown on brain–heart infusion agar at 37 °C for 24 h under aerobic conditions, with the exception of S. aureus subsp. anaerobius and Staphylococcus saccharolyticus, which were grown on Columbia blood (5 % defibrinated sheep blood) agar under anaerobic conditions.
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). PCR amplification and DNA sequencing were performed with the following pair of dnaJ degenerate primers: SA-(F) 5'-GCCAAAAGAGACTATTATGA-3' and SA-(R) 5'-ATTGYTTACCYGTTTGTGTACC-3'. The PCR mixture was prepared with the Takara Ex Taq Hot Start version kit (Takara) according to the manufacturer's instructions. Reaction mixtures were first incubated at 94 °C for 3 min, followed by five cycles of 94 °C for 30 s, 45 °C for 30 s and 72 °C for 60 s and were then subjected to 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 60 s and completed with a final extension at 72 °C for 3 min. PCR products were purified with a Wizard SV Gel and PCR clean-up system (Promega) and sequenced with a BigDye Terminator v3.1 cycle sequencing kit on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). To exclude the possibility of sequencing errors attributable to misincorporation by Taq DNA polymerase, all PCR amplicons were sequenced twice with PCR products obtained from two independent rounds of PCR. For purposes of comparison, approximately 1450 bp of the 16S rRNA gene of ten Staphylococcus species were amplified and sequenced with 16S rRNA gene universal primers. Additional 16S rRNA and available conserved gene sequences (hsp60, sodA and rpoB) of corresponding Staphylococcus species were downloaded from the GenBank/EMBL/DDBJ database (Table 1).
Multiple sequence alignments were performed with CLUSTAL W software (Thompson et al., 1994). Evolutionary distances were calculated by Kimura's two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods, with MEGA3 software (Kumar et al., 2004) and with bootstrap values based on 1000 replications (Felsenstein, 1985). Tree figures were drawn by TREEVIEW software (Page, 1996).
In this study, an approximately 920 bp internal fragment of the dnaJ gene from 45 staphylococcal type strains was amplified with the use of dnaJ universal primers. Amplification products were not obtained from non-staphylococcal species. After we omitted the primer sequences, approximately 883 bp sequences were used for analysis and the resulting sequences were deposited in the DNA Data Bank of Japan (DDBJ) and assigned accession numbers as listed in Table 1. Phylogenetic analysis by the neighbour-joining (Fig. 1) and maximum-parsimony methods (data not shown) produced similar trees with the exception of minor differences in the tree topology of the basal branches.
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; Mellmann et al., 2006), hsp60 (mean similarity 82 %) (Kwok et al., 1999; 2003) and sodA (mean similarity 81.5 %) (Poyart et al., 2001). In addition, the evolutionary substitution rate of the dnaJ sequence was much faster than that of the 16S rRNA gene sequence.
However, at the interspecies level, the dnaJ gene sequence showed remarkable discrimination, (71.1–88.8 %), except for the most similar pairs such as S. condimenti and S. carnosus (90.9 %), S. intermedius and S. delphini (92.3 %) and S. pulvereri and S. vitulinus (99.3 %). Recently, S. pulvereri was reclassified as a later heterotypic synonym of S. vitulinus (
vec et al., 2004) and this was also inferred in our dnaJ gene sequence analysis (see Supplementary Table S1). All staphylococcal species showed more than 90 % similarity in their 16S rRNA gene sequences. S. epidermidis, S. muscae, S. saccharolyticus, S. sciuri and S. vitulinus species showed 90.1–98.6 % similarity in their hsp60 gene sequences (Kwok et al., 1999; 2003). The species S. capitis, S. caprae, S. carnosus, S. cohnii, S. condimenti, S. delphini, S. equorum, S. gallinarum, S. intermedius, S. kloosii, S. pasteuri, S. piscifermentans, S. saprophyticus, S. sciuri, S. simulans, S. vitulinus, S. warneri and S. xylosus showed 90–96.8 % similarity in their sodA gene sequences (Poyart et al., 2001). When the rpoB gene sequence was examined, S. auricularis, S. capitis, S. caprae, S. carnosus, S. cohnii, S. condimenti, S. chromogenes, S. equorum, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis, S. hyicus, S. kloosii, S. nepalensis, S. pasteuri, S. piscifermentans, S. saprophyticus, S. sciuri, S. succinus, S. vitulinus, S. warneri and S. xylosus showed 90–96.5 % gene sequence similarity (Drancourt & Raoult, 2002; Mellmann et al., 2006). These results indicate that the dnaJ sequence may constitute a more useful target sequence than that of other conserved genes, including the 16S rRNA gene, in the discrimination of closely related species.
At the subspecies level, dnaJ gene sequences can discriminate only S. cohnii subsp. cohnii and S. cohnii subsp. urealyticus (7 % sequence divergence) and S. aureus subsp. aureus and S. aureus subsp. anaerobius (3.7 % sequence divergence). The remaining subspecies, S. capitis subsp. capitis, S. capitis subsp. ureolyticus, S. carnosus subsp. carnosus, S. carnosus subsp. utilis, S. hominis subsp. hominis, S. hominis subsp. novobiosepticus, S. saprophyticus subsp. bovis, S. saprophyticus subsp. saprophyticus, S. schleiferi subsp. coagulans, S. schleiferi subsp. schleiferi, S. sciuri subsp. rodentium, S. sciuri subsp. sciuri and S. sciuri subsp. carnaticus, showed more than 97.3 % gene sequence similarity and did not allow for complete discrimination at the subspecies level. This finding was consistent with that for other conserved genes, including the hsp60 gene which showed 9 % sequence divergence between S. capitis subsp. capitis and S. capitis subsp. ureolyticus and 7 % sequence divergence between S. cohnii subsp. cohnii and S. cohnii subsp. urealyticus; the sodA gene which showed 4.4 % sequence divergence between S. cohnii subsp. cohnii and S. cohnii subsp. urealyticus and the rpoB gene which showed 12.7 % sequence divergence between S. aureus subsp. aureus and S. aureus subsp. anaerobius (Drancourt & Raoult, 2002), although it was recently reported that obligately anaerobic S. aureus could not be identified on the basis of rpoB sequences (Peake et al., 2006). However, the remaining subspecies of Staphylococcus showed more than 98 % gene sequence similarity for the hsp60, sodA and rpoB genes (Poyart et al., 2001; Drancourt & Raoult, 2002; Kwok & Chow, 2003; Mellmann et al., 2006).
In addition, 20 isolates of Staphylococcus representing two clinically important species, S. aureus subsp. aureus and S. epidermidis, were used for the analysis of intraspecies sequence variation. Sequence divergence was 0.7 % among S. aureus isolates and 0.4 % among S. epidermidis isolates (data not shown) indicating that a dnaJ gene sequence database would be useful in the identification of clinical isolates.
Use of the dnaJ and other conserved genes to compare phylogenetic relationships among Staphylococcus species
Taxonomic studies based on DNA–DNA reassociation and 16S rRNA and hsp60 gene sequence analysis have indicated that genealogically distinct species groups exist in the genus Staphylococcus (Kloos & Schleifer, 1986; Takahashi et al., 1999). However, in many cases, low resolution and lack of congruity was observed to delineate these groupings. For example, DNA–DNA reassociation studies indicated the existence of nine staphylococcal species groups, whereas 16S rRNA and hsp60 gene sequence-based analyses indicated twelve and six genogroups, respectively (Takahashi et al., 1999; Kwok & Chow, 2003). Moreover, both DNA–DNA reassociation and 16S rRNA gene sequence-based studies included Staphylococcus caseolyticus in their groups. Staphylococcus caseolyticus has since been reclassified as Macrococcus caseolyticus (Kloos et al., 1998), thus limiting the number of species included in DNA–DNA reassociation studies and 16S rRNA gene sequence-derived genogroups.
Species groupings have also been observed on the basis of phenotypic characteristics. For example, novobiocin resistance and oxidase activity can differentiate the S. saprophyticus and S. sciuri groups from other groups. However, other well-described phenotypic characteristics are not shared by all species due to their diverse phenotypic relationships (Kloos et al., 1991).
In the present study, dnaJ gene sequence analysis was compared with 16S rRNA gene sequence and DNA–DNA reassociation studies in an effort to determine more reliable species relationships in the genus Staphylococcus. Phylogenetic analysis based on dnaJ gene sequences yielded eight distinct species groups with relatively high bootstrap values (80–100 %); the S. sciuri group, S. simulans group, S. saprophyticus group, S. hyicus–intermedius group, S. haemolyticus group, S. epidermidis group, S. aureus group and S. lugdunensis group (Fig. 1). Interestingly, these groups showed nearly identical relationships with those from 16S rRNA gene sequence and DNA–DNA reassociation studies, with the exception of some minor differences in the basal branches. In the dnaJ gene sequence tree, the S. simulans species group (S. carnosus subsp. carnosus, S. carnosus subsp. utilis, S. condimenti, S. piscifermentans and S. simulans) produced a monophyletic clade with a high bootstrap value of 99 % at each node, except for the branching of S. auricularis. This finding is inconsistent with the data from the 16S rRNA gene sequence and DNA–DNA reassociation studies and requires further analysis. In addition, S. warneri and S. pasteuri produced a deep subline within the S. epidermidis group consistent with sodA gene sequence analysis and DNA–DNA reassociation studies, whereas S. simulans and S. carnosus and S. warneri and S. epidermidis produced separate species groups in the 16S rRNA gene sequence tree, with low resolution. However, in a previous study (Takahashi et al., 1999) 16S rRNA gene sequence-based analysis yielded twelve genogroups, possibly due to the limited number of species used or the low discriminatory power to produce reliable branches. Moreover, similar to the 16S rRNA gene, the dnaJ gene sequence analysis data also showed minor differences with those of DNA–DNA reassociation studies, inferring close relationships of the S. hyicus–intermedius and S. epidermidis species groups.
In contrast to the topologies of the dnaJ and 16S rRNA gene sequence trees, hsp60 gene sequences yielded two cluster groups. Cluster 1 contained the S. aureus group (group a), the S. epidermidis group (group b), the S. haemolyticus group (group c), the S. saprophyticus group (group d) and the S. intermedius group (group e). Cluster 2 contained the S. sciuri group (Kwok & Chow, 2003). However, in the hsp60 gene sequence tree, species relationships in the S. aureus and S. haemolyticus groups were not apparent compared with those of other species groups and showed discordance with the dnaJ and 16S rRNA gene sequence trees. In the sodA gene phylogenetic tree, S. sciuri, S. simulans, S. epidermidis, S. saprophyticus, S. hyicus–intermedius and S. lugdunensis species groups showed good concordance with the dnaJ gene sequence tree topology, with the exception of S. haemolyticus, S. hominis and S. schleiferi (Poyart et al., 2001). In the rpoB tree, the positions of S. cohnii, S. haemolyticus, S. hominis and S. warneri inferred an inconsistent relationship with those of the dnaJ and 16S rRNA gene sequence trees (Drancourt & Raoult, 2002). Nevertheless, the dnaJ gene sequence-based phylogenetic analysis showed remarkable concordance with the DNA–DNA reassociation, 16S rRNA, hsp60 and sodA gene sequence-based analysis and showed greater power to discriminate species relationships in the genus Staphylococcus.
In conclusion, our dnaJ gene sequence-based assay offers a reliable alternative to currently used methods, including the 16S rRNA gene sequence-based assay, for the identification and phylogenetic analysis of almost all staphylococci at the species level and, with some exceptions, at the subspecies level. We are currently studying the utility of this dnaJ gene sequence-based assay in the identification and phylogenetic analysis of other bacterial genera.
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