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Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S–23S intergenic spacer region

¹ Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan, ROC

² School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan, ROC

³ Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium

⁴ Division of Clinical Microbiology, Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan, ROC

Correspondence
Tsung Chain Chang
tsungcha{at}mail.ncku.edu.tw

Received 25 October 2006
Accepted 15 December 2006

Abbreviations: ITS, intergenic spacer region; NVS, nutritionally variant streptococci.

The phylogenetic trees and table of ITS sequences submitted to GenBank are available as supplementary material in JMM Online.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Enterococci, nutritionally variant streptococci (NVS) – of which most have been allocated to the genera Abiotrophia and Granulicatella – and streptococci are Gram-positive, catalase-negative bacteria causing a wide variety of opportunistic and invasive infections (Facklam, 2002; Teixeira & Facklam, 2003). No single system of classification suffices for the differentiation of this heterogeneous group of organisms. Instead, classification depends on a combination of features including patterns of haemolysis on blood agar plates, antigenic composition, growth characteristics and biochemical reactions (Facklam, 2002). The routine procedures based on phenotypic tests do not allow unequivocal identification of many species of these bacteria (Hoshino et al., 2005; Ruoff, 2003). In addition, some recently described species such as Streptococcus cristatus, Streptococcus infantarius and Streptococcus gallolyticus (Arbique et al., 2004; Poyart et al., 2002) have complicated the problems with identification of these micro-organisms.

A variety of molecular methods have been developed for the identification of enterococci, NVS and streptococci to species level. The targets used for molecular diagnoses include genes encoding rRNA (Clarridge et al., 2001), RNA polymerase (Drancourt et al., 2004), the D-alanine-D-alanine ligase (Garnier et al., 1997), the ß-subunit of the elongation factor (Picard et al., 2004), the manganese-dependent superoxide dismutase (sodA_int) (Poyart et al., 1998, 2000), the heat-shock proteins (groESL) (Teng et al., 2002) and the tRNA gene intergenic spacer region (ITS) (Baele et al., 2000, 2001). Recently, optimal identification of non-haemolytic streptococci was performed by phylogenetic sequence analysis of four housekeeping genes (ddl, gdh, rpoB and soda) (Hoshino et al., 2005).

The ribosomal 16S–23S ITS sequence has been suggested as a good candidate for bacterial identification and strain typing (Gürtler & Stanisich, 1996; Hassan et al., 2003; Roth et al., 1998). Sequences of the ITS region have been found to have low intraspecies variation and high interspecies divergence (Hassan et al., 2003; Whiley et al., 1995). In our previous study, the feasibility of using the ITS sequence to identify 11 species of viridans streptococci was established (Chen et al., 2004). This study aimed to expand the results of this technique to cover a total of 57 species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains. As GenBank currently lacks ITS sequence entries for many species of enterococci, NVS and streptococci, the ITS sequences of 38 type strains were determined and submitted to GenBank (see Supplementary Table S1 available in JMM Online) to facilitate ITS sequence comparison. Following this, a total of 217 strains comprising 84 reference and 133 clinical isolates (Table 1) was analysed by ITS sequencing. Reference strains were obtained from the American Type Culture Collection (ATCC; USA), the Bioresources Collection and Research Center (BCRC; Taiwan) and the Culture Collection of the University of Göteborg (CCUG; Sweden). Clinical isolates were obtained from the National Taiwan University Hospital (Taiwan), the National Cheng Kung University Medical Center (Taiwan) and the Ghent University Hospital (Belgium). Most of the clinical isolates were phenotypically identified using the Rapid ID 32 STREP system (bioMérieux) or by tRNA gene PCR (Baele et al., 2000, 2001). A limited number of clinical isolates of NVS were tested in this study, as the feasibility of ITS sequence analysis for identification of these bacteria has been reported previously (Chen et al., 2004). All strains were cultured on sheep blood agar, except for strains of Abiotrophia and Granulicatella, which were cultured on chocolate agar.

View larger version (68K): [in this window] [in a new window]   Fig. 1. Amplification of the ITS regions and separation of PCR products by 2 % agarose gel electrophoresis. Lane M, 100 bp ladder. (a) Lanes: 1, S. agalactiae; 2, S. alactolyticus; 3, Streptococcus australis; 4, S. bovis; 5, Streptococcus canis; 6, S. cristatus; 7, S. downei; 8, S. dysgalactiae subsp. equisimilis; 9, S. equi subsp. equi; 10, S. equi subsp. zooepidemicus; 11, S. equinus. (b) Lanes: 1, Streptococcus gallolyticus subsp. gallolyticus; 2, S. gallolyticus subsp. macedonicus; 3, S. gallolyticus subsp. pasteurianus; 4, S. lutetiensis; 5, S. infantarius subsp. infantarius; 6, S. infantis; 7, S. iniae; 8, S. pneumoniae; 9, Streptococcus porcinus; 10, S. pyogenes; 11, Streptococcus ratti. (c) Lanes: 1, Streptococcus sobrinus; 2, Streptococcus suis; 3, S. thermophilus; 4, S. vestibularis; 5, Streptococcus urinalis. (d) Lanes: 1, E. avium; 2, E. casseliflavus; 3, Enterococcus cecorum; 4, E. columbae; 5, Enterococcus dispar; 6, E. durans; 7, E. faecalis; 8, E. faecium; 9, E. flavescens; 10, Enterococcus gallinarum; 11, Enterococcus gilvus. (e) Lanes: 1, E. hirae; 2, Enterococcus mundtii; 3, Enterococcus pallens; 4, Enterococcus pseudoavium; 5, E. raffinosus; 6, Enterococcus saccharolyticus; 7, Enterococcus villorum. (f) Lanes: 1, A. defectiva; 2, G. adiacens; 3, Granulicatella balaenopterae; 4, G. elegans.

Species identification by ITS sequencing. A total of 217 strains including 84 reference strains and 133 clinical isolates was analysed (Table 1). The ITS region of each strain was amplified by PCR. The amplicons were sequenced with forward primer 13 BF and, in case the forward sequence was not readable, reverse primer 6R was also used for generating the sequences. PCR products of all species of streptococci (Fig. 1a–c) were sequenced directly, except for Streptococcus downei, which apparently contained two ITS fragments (Fig. 1a, lane 7). For S. downei, enterococci and NVS that possessed multiple ITS fragments (Fig. 1d–f), only the smallest amplicon was eluted from the agarose gel and sequenced for species identification. Species identification was done by searching databases using the BLAST (http://www.ncbi.nlm.nih.gov/blast/) sequence analysis tool. Species identification was determined from the best-scoring reference sequence of the BLAST output and whether the best-scoring reference sequence in the databases had a sequence identity of 98 % with the query sequence. Strains producing discrepant species names by ITS sequence analysis were analysed further by sequencing of the near-complete 16S rRNA gene (Relman, 1993).

Phylogenetic analysis. Unrooted phylogenetic trees were constructed by the neighbour-joining method listed in the MEGA (version 3.0) analytical package (Kumar et al., 2004). For neighbour-joining analysis, the distance between two ITS sequences was calculated using Kimura’s two-parameter model (Li & Tsoi, 2002). The robustness of the neighbour-joining method was evaluated statistically by bootstrap analysis with 1000 bootstrap samples.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Amplification and sequencing of ITS fragments

The ITS fragments of Abiotrophia, Enterococcus, Granulicatella and Streptococcus strains were amplified by PCR. A single amplicon was observed for each species of Streptococcus except for S. downei, which possessed two ITSs (Fig. 1a, lane 7). Multiple PCR products were observed for strains of Abiotrophia, Enterococcus and Granulicatella (Fig. 1d–f). For a strain that possessed multiple ITSs with different lengths and sequences, only the shortest fragment (usually the dominant band on the agarose gel) was eluted and sequenced. However, the amplicons eluted from E. raffinosus (Fig. 1e, lane 5) still contained two fragments with lengths of 224 and 246 bp, respectively, as revealed by cloning of the PCR products (see Supplementary Table S1 available in JMM Online). In addition, the PCR product eluted from G. adiacens (Fig. 1f, lane 2) contained two amplicons of identical size (222 bp) but which differed in sequence, as revealed by cloning and sequencing of the eluted amplicons. As specific primers were used for ITS amplification, multiple bands on agarose gels implied the presence of different rRNA operons in species of the three genera. The presence of multiple ITS operons has also been found in many species of Acinetobacter (Chang et al., 2005). To simplify sequence comparison among species, only the shortest ITS fragments of the three genera were used for sequence determination.

The size of the ITS ranged from 189 bp (Enterococcus columbae) to 601 bp (Streptococcus equi subsp. equi) (see Supplementary Table S1 available in JMM Online). At the start of this study, GenBank contained ITS sequences of the following 22 species: Abiotrophia defectiva, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, G. adiacens, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus bovis, Streptococcus constellatus subsp. constellatus, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus parauberis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis and Streptococcus uberis. After deposition of the 38 ITS sequences determined in this study (see Supplementary Table S1 available in JMM Online), most clinically relevant species of the four genera Abiotrophia, Enterococcus, Granulicatella and Streptococcus could be identified by ITS sequencing and BLAST search analysis.

Interspecies sequence similarity of the ITS region

Pairwise comparisons of the ITS sequences between the type strains of any two given enterococcal species showed that sequence similarity ranged from 0.42 (Enterococcus avium vs E. faecium) to 0.996 (Enterococcus casseliflavus vs Enterococcus flavescens) (data not shown). In general, interspecies ITS sequence similarities were less than 0.90. The interspecies ITS sequence similarities of streptococci ranged from 0.36 (S. anginosus vs Streptococcus iniae) to as high as 0.95–1.0 (S. pneumoniae vs S. mitis) (Chen et al., 2004) and 1.0 (S. bovis vs Streptococcus equinus). However, based on the 16S rRNA gene sequence and total DNA–DNA hybridization, S. equinus and S. bovis were proposed to form a single DNA group, i.e. the same species (Facklam, 2002; Schlegel et al., 2003). In addition, a low level of interspecies ITS sequence homology was observed among the four species of NVS, with sequence similarities ranging from 0.37 (A. defectiva vs Granulicatella elegans) to 0.57 (G. adiacens vs G. elegans) (data not shown).

Signature sequences of some enterococcal and streptococcal species

Only one nucleotide difference was observed in the ITS regions (229 bp) in strains of E. casseliflavus and E. flavescens. Multiple ITS sequence alignment revealed that nt 220 was T in strains of E. casseliflavus, but A in strains of E. flavescens (Fig. 2a). E. casseliflavus and E. flavescens are intrinsically vancomycin resistant (possessing the vanC genotype), motile and capable of producing pigments. However, Baele et al. (2000) pointed out that E. casseliflavus and E. flavescens are most probably synonymous, as revealed by tRNA ITS PCR and other evidence (Descheemaeker et al., 1997; Teixeira et al., 1997).

View larger version (52K): [in this window] [in a new window]   Fig. 2. Sequence alignment of the partial ITS regions or the 3' end regions of the 16S rRNA gene to reveal species- or subspecies-specific signature sequences: (a) E. casseliflavus (CCUG 18657T, ATCC 12755, ATCC 12817) and E. flavescens (CCUG 30567T, CCUG 30568, CCUG 30569); (b) S. bovis (ATCC 33317T, CCUG 4214), S. equinus (ATCC 9812T, CCUG 4213) and S. lutetiensis (CCUG 43822T, CCUG 43823, CCUG 38926); (c) S. gallolyticus subsp. gallolyticus (ATCC 43143T, CCUG 35224, CCUG 46101, CCUG 46667) and S. gallolyticus subsp. macedonicus (CCUG 39970T, CCUG 39969, CCUG 43003); (d) S. salivarius (ATCC 7073T, 9624-2, ATCC 13419, ATCC 25975), S. thermophilus (BCRC 13869, ATCC 19987, CCUG 35458, LMG 18311, CNRZ 1066) and S. vestibularis (CCUG 24893T); (e) The 3' end sequences of 16S rRNA genes of S. pneumoniae (ATCC 33400T, ATCC 49619, b5, r6, TIGR, NCTC 7978) and S. mitis (ATCC 49456T, sk34, sm91, 3788-2, 7538); (f) S. oralis (ATCC 35037T, ATCC 9811, ATCC 55229, ATCC 700233, ATCC 700234) and S. mitis (ATCC 49456T, sk24, 3788-2, 7538); (g) the two short ITS fragments of strains of G. adiacens (ATCC 49175T, CCUG 27811, CCUG 44407 and CCUG 44408). Type strains are indicated with a superscript T.

S. salivarius, Streptococcus thermophilus and Streptococcus vestibularis are members of the S. salivarius group and are closely related micro-organisms. Multiple ITS sequence alignment revealed that nt 193 and 264 were T and A, respectively, in strains of S. salivarius, whilst both nucleotides were G in strains of S. thermophilus and S. vestibularis (Fig. 2d). In addition, there was a single nucleotide (A) insertion at position 271 in strains of S. thermophilus and this insertion could be used to differentiate this species from S. vestibularis.

S. mitis and S. pneumoniae are members of the S. mitis group and have a high level of ITS sequence homology (Chen et al., 2004). However, sequence analysis of the co-amplified 3' end of the 16S rRNA gene revealed that the nucleotides at positions 1444 and 1446 were G and A, respectively, in strains of S. pneumoniae, whilst both nucleotides were T in strains of S. mitis (Fig. 2e). Moreover, there was a single nucleotide deletion at position 1447 in strains of S. pneumoniae. The ITS sequences of S. mitis and S. oralis also had high similarity (Chen et al., 2004). In comparison with S. oralis, two single nucleotide insertions at positions 202 (A) and 217 (T) were found in strains of S. mitis (Fig. 2f). In addition, S. pneumoniae, S. oralis and S. mitis are difficult to differentiate by sequencing of the 16S rRNA gene (Bosshard et al., 2004; Kawamura et al., 1995). Phylogenetic trees constructed using the genes encoding 16S rRNA (Bentley et al., 1991; Kawamura et al., 1995), sodA (Poyart et al., 2000, 2002), groESL (Teng et al., 2002) and the ITS region (Chen et al., 2004) grouped S. mitis, S. oralis and S. pneumoniae together. This is the first report indicating the presence of signature sequences in some species of enterococci and streptococci. These signature sequences are useful for differentiating these closely related enterococcal and streptococcal species/subspecies.

Identification of reference strains by ITS sequence analysis

To validate ITS sequencing for species identification, a total of 84 reference strains was analysed (Table 1). Nine strains produced discrepant identification and one strain (Streptococcus dysgalactiae subsp. equisimilis CCUG 27483) was not identified by ITS sequencing (Table 2). Strain CCUG 27483 also was not identified to subspecies level by 16S rRNA gene sequencing (Table 2). Identification of six of the nine discrepant strains, as obtained by ITS sequencing, was confirmed by 16S rRNA gene sequencing (Table 2). However, unambiguous identification of the remaining three strains (S. cristatus CCUG 35233, and S. mitis ATCC 15910 and ATCC 15914) was not obtained by 16S rRNA gene sequencing. The identification of S. cristatus CCUG 35233, and S. mitis ATCC 15910 and ATCC 15914 as S. oralis, S. vestibularis and S. oralis, respectively, by ITS sequencing was further confirmed by their signature sequences shown in Fig. 2(d, f). In brief, nine of the ten discrepant reference strains were considered to be given incorrect species names but were correctly identified by ITS sequence analysis, as confirmed by 16S rRNA gene sequencing (Table 2). Therefore, the identification rate of reference strains by ITS sequencing was 98.8 % (83/84). The mislabelling of up to 10.7 % (9/84) of the reference strains reflects the difficulties in identifying some streptococcal and enterococcal species by phenotypic methods.

Three clinical isolates (5106, 9429 and 8903-2) were not identified by ITS sequencing (Table 2). Isolate 5106 was identified as S. lutetiensis by ITS sequence analysis, but was identified as S. gallolyticus subsp. pasteurianus by 16S rRNA gene sequencing and as S. infantarius subsp. infantarius (99 % identity) by sequence analysis of the sodA gene (Poyart et al., 1998). Isolate 5106 did not display the signature sequence of S. lutetiensis (Fig. 2b). Therefore, the identity of isolate 5106 was not resolved. Isolate 9429 was not identified by ITS sequence analysis (<95 % identity to all known sequences), but was identified as S. mitis by 16S rRNA gene sequencing. Isolate 8903-2 (optochin resistant) was identified as S. pneumoniae by ITS sequencing and was identified as Streptococcus pseudopneumoniae or as S. pneumoniae by 16S RNA gene sequencing. Isolate 8903-2 did not display the ITS signature sequence typical of S. pneumoniae (Fig. 2h) and therefore the identity of the strain was not resolved. Although isolate 2000 (S. parasanguinis) was identified as Streptococcus infantis by ITS sequencing (99.4 % identity), a BLAST search of its 16S rRNA gene sequence revealed that this strain might be either S. infantis or S. oralis (Table 2). In brief, 97.7 % (130/133) of the clinical isolates were identified correctly by ITS sequencing. If reference strains and clinical isolates were taken together, the identification rate was 98.2 % (213/217) by ITS sequencing.

Sequence analysis of the 16S rRNA gene (approx. 1.5 kb) has been widely used for bacterial identification (Clarridge, 2004; Relman, 1993). As the ITS region is relatively short (from 189 to 601 bp) (see Supplementary Table S1 available in JMM Online), sequencing of the ITS region would be more efficient and accurate than sequencing of the 16S rRNA gene. This study shows an interesting approach towards identifying this group of pathogens on a molecular platform alongside 16S rRNA gene sequencing. It should be noted that species other than Streptococcus contained multiple ITS fragments and only the shortest fragment was analysed in this study (Fig. 1). A limitation of using the ITS sequence for bacterial identification is the relatively limited database compared with that of 16S rRNA genes. However, ITS sequences in the public database have increased substantially in recent years (D’Auria et al., 2006).

Phylogenetic analysis

The phylogenetic tree derived from the ITS sequences of Streptococcus (35 species) is presented in Supplementary Fig. S1 available in JMM Online. The tree was generally in agreement with the tree constructed from the 16S rRNA gene sequences (see Supplementary Fig. S2 available in JMM Online). Species of each of the following groups (S. bovis group, S. salivarius group, S. mitis group, S. sanguinis group, S. anginosus group and S. mutans group) were clustered together in the ITS sequence-based tree, as was the case in the tree derived from the 16S rRNA genes. Compared with the tree based on 16S rRNA gene sequences, two streptococcal species (S. parasanguinis and S. anginosus) branched differently or formed different clusters in the ITS sequence-based tree. It was interesting to find that the phylogenetic tree derived from ITS sequences of streptococci generally displayed a better resolution for basal branches compared with that constructed from the sequences of the 16S rRNA genes. For several major deep branches, the bootstrap values were too low in the 16S rRNA gene sequence-based tree to permit reliable interpretations.

	ACKNOWLEDGEMENTS

 
This project was supported by grants (NSC 94-2323-B006-007, NSC 94-2320-B006-078) from the National Science Council.

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