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PCR-RFLP assay for species and subspecies differentiation of the Streptococcus bovis group based on groESL sequences

¹ Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan

² Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine, Taipei, Taiwan

³ Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

⁴ Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, Tainan, Taiwan

⁵ Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan

Correspondence
Lee-Jene Teng
ljteng{at}ntu.edu.tw

Received 13 September 2007
Accepted 5 December 2007

Abbreviations: ITS, intergenic spacer.

The GenBank/EMBL/DDBJ accession numbers for the groESL gene sequences reported in this study are given in Supplementary Table S1 in JMM Online.

Tables showing the GenBank/EMBL/DDBJ accession numbers for the groES and groEL gene sequences of strains used in this study and the nucleotide lengths and sequences of groES–groEL spacer regions are available as supplementary material with the online version of this paper.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The ‘Streptococcus bovis’ group is a large bacterial complex that includes various species and subspecies isolated from humans or animals. In humans, members of the group can cause bacteraemia and endocarditis in elderly people and may sometimes cause septicaemia and meningitis in newborns (Bochud et al., 1994; Gerber et al., 2006; Kupferwasser et al., 1998; Tripodi et al., 2004). The group is also known to be associated with underlying gastrointestinal malignancy (Ellmerich et al., 2000). In previous studies, the authors found that biotype I (Streptococcus gallolyticus) was highly associated with infective endocarditis and colonic cancer, whereas biotype II was associated with primary bacteraemia (Jean et al., 2004; Ruoff et al., 1989). However, the association of species or subspecies and clinical significance is still not clear and sometimes even controversial. To better understand the relationship between pathogens and disease, it is important to identify the species or subspecies correctly.

The taxonomy/nomenclature of members of the Streptococcus bovis group is still evolving. Farrow et al. (1984) identified six different DNA groups in the Streptococcus bovis group using total DNA–DNA hybridization assays. Other investigators further separated the Streptococcus bovis group into five species (S. bovis–S. equinus, S. gallolyticus, S. infantarius, S. pasteurianus and S. lutetiensis) (Facklam, 2002; Osawa et al., 1995; Poyart et al., 2002; Schlegel et al., 2000). According to their biochemical characteristics, Streptococcus bovis strains isolated from humans were divided into three biotypes, biotype I (mannitol-positive), II/1 (mannitol-negative and β-glucuronidase-negative) and II/2 (mannitol-negative and β-glucuronidase-positive) (Clarridge et al., 2001; Coykendall, 1989). The human isolates of the Streptococcus bovis group have been reclassified as S. gallolyticus, S. infantarius and S. pasteurianus for biotypes I, II/1 and II/2, respectively (Poyart et al., 2002; Schlegel et al., 2000). Schlegel et al. (2003) suggested that biotypes I and II/2 and Streptococcus gallolyticus represent a single species and proposed the name Streptococcus gallolyticus subsp. pasteurianus for Streptococcus bovis biotype II/2. Streptococcus bovis biotype II/1 was described as Streptococcus infantarius, with two subspecies, Streptococcus infantarius subsp. infantarius (starch hydrolysis-positive, aesculin hydrolysis-variable and β-glucosidase-variable) and Streptococcus infantarius subsp. coli (reclassified as Streptococcus lutetiensis) (starch hydrolysis-variable, aesculin hydrolysis-positive and β-glucosidase-positive) (Facklam, 2002; Schlegel et al., 2000; Poyart et al., 2002). Although these new names have been proposed, most clinical laboratories have not yet accepted the nomenclature. Currently, the identification of the Streptococcus bovis group in clinical laboratories depends mostly on conventional methods and/or rapid identification systems. Misidentification has been reported due to the diversity of the biochemical characteristics of members of the group (Gavin et al., 2002). Molecular-based identification using the sodA gene, encoding manganese-dependent superoxide dismutase, has also been used recently for species identification and to distinguish the Streptococcus bovis group (Poyart et al., 1998, 2002; Sasaki et al., 2004). The sequence of the 16S rRNA gene has also been used for phylogenetic analysis (Schlegel et al., 2000, 2003), but it is difficult to differentiate species or subspecies using PCR only.

The groESL genes (also known as cpn10/60 or hsp10/60), which encode 10 kDa (GroES) and 60 kDa (GroEL) heat-shock proteins, are ubiquitous and evolutionarily highly conserved among bacteria (Hemmingsen et al., 1988). Amplification of the partial cpn60 (or groEL) gene segment has been used in the identification of bacteria such as Staphylococcus (Goh et al., 1996), Enterococcus (Teng et al., 2001b; Tsai et al., 2005), Streptococcus (Goh et al., 1998; Hung et al., 2005; Teng et al., 2002), Ehrlichia species (Sumner et al., 2000), Bartonella (Marston et al., 1999), Mycobacterium (Rastogi et al., 1999) and rickettsiae (Lee et al., 2003). We have previously determined the groESL sequence of Streptococcus bovis biotype I (Teng et al., 2002). In this study, we determined the groESL sequences of other species and subspecies of the Streptococcus bovis group and analysed the differences between them. Based on these sequences, we have developed a Streptococcus bovis group-specific PCR. Restriction digestion of the amplicon with AclI further differentiated the species and subspecies.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bacterial strains. Five reference strains and 36 clinical isolates were used in this study. The reference strains were obtained from the American Type Culture Collection (ATCC) and included Streptococcus gallolyticus ATCC 9809 (biotype I), Streptococcus gallolyticus subsp. gallolyticus ATCC 43143 (biotype I), Streptococcus infantarius subsp. infantarius ATCC BAA-102^T (biotype II/1), Streptococcus infantarius subsp. coli ATCC BAA-103 (biotype II/1) and Streptococcus gallolyticus subsp. pasteurianus ATCC 43144 (biotype II/2). Twenty-nine of the 36 clinical isolates were collected between 2000 and 2003 in the Bacteriology Laboratory, National Taiwan University Hospital (NTUH), a 2000-bed teaching hospital in northern Taiwan, and the other seven were obtained from the National Cheng Kung University Hospital (NCKUH), a teaching hospital in southern Taiwan. All strains were recovered from blood cultures. The clinical strains were identified to species level and distinguished as Streptococcus gallolyticus (n=12), Streptococcus infantarius II/1 (n=11) and Streptococcus gallolyticus subsp. pasteurianus II/2 (n=13), by using commercial identification systems [API 20 Strep system (bioMérieux Vitek), Phoenix System or Rapid ID 32 STREP system (bioMérieux Vitek)]. Strains from NCKUH were also identified by using the ribosomal 16S–23S intergenic spacer (ITS) region sequence (Tung et al., 2007).

PCR amplification and sequencing. For the determination of groESL sequences, two sets of PCR were performed. The primers Strep-ES-UP (5'-GACTATTTCTGACCAAGTGAT-3', located upstream of groES) and Strep-EL-120-100 (5'-CTCAAGAACAACRTTRCGDCC-3', where R is A or G and D is A or T or G), located in the 5'-region of groEL, and which have been described previously (Teng et al., 2002), were used to amplify a fragment containing the entire groES gene, ITS region and the short 5'-end region of groEL. The primers bovis-1F (5'-TCAGGACATTTTGCCACTTCTT-3', corresponding to positions 684–705 of the groEL gene) and Gor1907-1927 (5'-YTACATCATNCCNCCCATCAT-3', where Y is C or T and N is A, T, C or G), corresponding to positions 1623–1603 of the groEL gene, were used to amplify a partial groEL gene. PCR was carried out using a DNA thermal cycler (MJ Research) with 30 cycles of denaturation (94 °C, 30 s), annealing (50 °C, 30 s) and extension (72 °C, 1 min), followed by a final extension step (72 °C, 10 min). The PCR products were purified and subsequently sequenced using a sequencing system (model ABI PRISM 3100; Applied Biosystems) with a Taq BigDye-Deoxy Terminator cycle sequencing kit (Applied Biosystems), according to the instructions of the manufacturer.

Phylogenetic analysis. The phylogenetic relationships among species were analysed by using the neighbour-joining method in the MEGA2 (molecular evolutionary genetic analysis) analytical package (Kumar et al., 2001). For the neighbour-joining analysis, the distance between the sequences was calculated using Kimura's two-parameter model. Levels of similarity among species were determined. Bootstrap values were obtained for 500 randomly generated trees.

Intraspecies and interspecies variation. The intraspecies variation of groES genes, ITS region and partial groEL genes and the interspecies variation of groES and groEL genes were evaluated among the clinical isolates. The strains were subjected to PCR amplification of groES and ITS region with primers Strep-ES-UP and Strep-EL-120-100. PCR with primers bovis-1F and Gor1907-1927 was performed to amplify partial groEL genes. The amplified PCR products were subsequently sequenced. DNA and deduced amino acid sequences were aligned using Gene-Works software (IntelliGenetics).

Streptococcus bovis group-specific PCR. Based on the sequence obtained, we developed a Streptococcus bovis group-specific PCR. The forward primer ES5-29F (5'-TAAAACCHTTAGGHGAHCGWRTBGT-3'), together with the reverse primer, Streptococcus bovis EL1265R (5'-CAAGTTCAAGTTCAGCAACTTTTG-3'), were derived to amplify a target region with an amplicon size that ranged from 1635 to 1910 bp, depending on the species. PCR was performed with 35 cycles of denaturation (94 °C, 30 s), annealing (54 °C, 30 s) and extension (72 °C, 2 min), followed by a final extension step (72 °C, 10 min). The PCR products were analysed by using agarose gel electrophoresis (1.5 % agarose; FMC BioProducts), and then stained with ethidium bromide and photographed under UV light. A visible band with a size of 1.6–1.9 kb was considered to be a positive reaction.

PCR-RFLP for species and subspecies differentiation. After PCR, a restriction analysis was performed to further differentiate between species and subspecies. The amplification product was digested with the restriction enzyme AclI (New England BioLabs). After incubation, the DNA fragments were subjected to gel electrophoresis (FMC BioProducts), stained with ethidium bromide and photographed under UV light.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Nucleotide sequences of groESL genes in reference strains

The nucleotide sequences of full-length groES and partial groEL genes were determined for five reference strains. The sequences showed that the groES genes were all 285 bp in length. The similarities between the groES sequences ranged from 84.2 % (between Streptococcus infantarius subsp. infantarius ATCC BAA-102^T and both Streptococcus gallolyticus ATCC 9809 and Streptococcus gallolyticus subsp. pasteurianus ATCC 43144) to 99.0 % (between Streptococcus gallolyticus subsp. pasteurianus ATCC 43144 and Streptococcus gallolyticus ATCC 9809) at the nucleotide sequence level (Table 1). Pairwise sequence similarity of partial groEL (nucleotide positions 883–1572) among species and subspecies tested with reference strains ranged from 88.0 % (between Streptococcus gallolyticus ATCC 9809 and Streptococcus infantarius subsp. infantarius ATCC BAA-102^T) to 98.8 % (between Streptococcus gallolyticus ATCC 9809 and Streptococcus gallolyticus subsp. pasteurianus ATCC 43144) (Table 1).

Intraspecies similarities in clinical isolates

To evaluate the general applicability of the groESL sequence to species and subspecies identification, the groES and partial groEL sequences and the ITS regions of 36 clinical isolates were tested to identify intraspecies similarities. Comparison of these sequences from clinical isolates with reference strains for each species or subspecies are shown in Table 2. The intraspecies identity of groES was slightly higher than that for groEL sequences (Table 2). The ITS sequences from the Streptococcus gallolyticus clinical isolates were the most divergent, ranging from 95.5 to 100 %.

We found that the groES or groEL sequences provided more discrimination than the 16S rRNA gene for the differentiation of species and subspecies within the Streptococcus bovis group. The difference in the sequences of the 16S rRNA gene between Streptococcus gallolyticus and Streptococcus gallolyticus subsp. pasteurianus is only 0.3 %, whereas the differences in groES and groEL were more than 1.0 %. The higher interspecies divergence will make the differentiation easier. In addition to the low intraspecies variation of groESL sequences among the Streptococcus bovis group, these findings are attractive for species identification among this group.

Phylogenetic relationships

Unrooted phylogenetic trees constructed from full-length groES and partial groEL (nucleotide positions 883–1572) from the 36 clinical isolates and five reference strains are presented in Fig. 1. The phylogenetic analysis revealed that the nucleotide sequences of groES from 41 tested strains were divided into three major clusters, which were consistent with species and subspecies. The results of the phylogenetic analysis of the groEL gene were similar to those for the groES gene. The clinical isolates of biotype II/1 all clustered with Streptococcus infantarius subsp. coli ATCC BAA-103. In agreement with the 16S rRNA gene analysis, phylogenetic analysis of groES or groEL revealed that Streptococcus gallolyticus subsp. pasteurianus was more closely related to Streptococcus gallolyticus than to Streptococcus infantarius.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Phylogenetic relationships among Streptococcus bovis group isolates (including reference strains and clinical isolates) based on the nucleotide sequences of (a) groES and (b) groEL genes. The phylogenetic trees were generated by using the neighbour-joining method in the MEGA2 package. Numbers at nodes are confidence levels expressed as percentages of occurrence in 500 bootstrapped resamplings. Scale bars indicate the evolutionary distance between sequences determined by measuring the lengths of the horizontal lines connecting two organisms. GenBank accession numbers for the gene sequences used are available in Supplementary Table S1 in JMM Online.

View larger version (78K): [in this window] [in a new window]   Fig. 2. Streptococcus bovis group-specific PCR and RFLP analysis of the groESL genes among species and subspecies of the Streptococcus bovis group. (a) Streptococcus bovis group-specific PCR. (b) Restriction fragments of amplified products after AclI digestion. Lanes: M, 100 bp DNA ladder; 1–4, Streptococcus gallolyticus (biotype I) (strains ATCC 9809, NTUH 7051, NTUH 6222 and NTUH 8995); 5, Streptococcus infantarius subsp. infantarius ATCC BAA-102T (biotype II/1); 6, Streptococcus infantarius subsp. coli ATCC BAA-103 (biotype II/1); 7–9, clinical isolates of Streptococcus infantarius (biotypes II/1) (strains NTUH 4936, NTUH 195 and NTUH 2639); 10–13, Streptococcus gallolyticus subsp. pasteurianus (biotype II/2) (strains ATCC 43144, NTUH 3004, NTUH 1443 and NTUH 7499); 14, negative control.

PCR-RFLP for differentiation of species and subspecies

To further differentiate between species and subspecies, an AclI restriction enzyme analysis was performed (Fig. 2b). The PCR-RFLP patterns from different species and subspecies were distinguishable (Fig. 2b). Using this assay, Streptococcus gallolyticus and Streptococcus gallolyticus subsp. pasteurianus were clearly separated. All clinical isolates of Streptococcus gallolyticus and Streptococcus gallolyticus subsp. pasteurianus tested also yielded identical patterns to those of the corresponding reference strains. Two reference strains representing two subspecies of Streptococcus infantarius, Streptococcus infantarius subsp. infantarius ATCC BAA-102^T and Streptococcus infantarius subsp. coli ATCC BAA-103, generated similar but different PCR-RFLP patterns (Fig. 2b).

We checked the restriction patterns shown in the gel in Fig. 2b with the sizes estimated from the sequence data. According to the sequence data, the PCR products of Streptococcus gallolyticus were expected to be digested into five fragments (495, 363, 299, 286 and 171 bp) using the restriction enzyme AclI. Among these fragments, the 299 and 286 bp fragments are too close in size and could not be discriminated by gel electrophoresis; therefore, only four bands can be seen in the gel. The restriction fragments of the amplicon from Streptococcus gallolyticus subsp. pasteurianus showed the expected sizes (667, 495, 286 and 171 bp). Five restriction fragments of the amplicon were expected (733, 702, 286, 161 and 10 bp) for Streptococcus infantarius subsp. infantarius and four fragments (733, 702, 447 and 10 bp) for Streptococcus infantarius subsp. coli. As the 10 bp fragment was too small to be seen using electrophoresis, only three fragments were shown in Streptococcus infantarius subsp. coli. The PCR-RFLP patterns obtained for all clinical isolates of biotype II/1 were identical to that of Streptococcus infantarius subsp. coli ATCC BAA-103.

Differentiation of species and subspecies within the Streptococcus bovis group is important clinically. The association of species and subspecies with types of infection and underlying diseases has been reported previously (Jean et al., 2004; Ruoff et al., 1989). In many reports, Streptococcus gallolyticus was more commonly associated with colorectal carcinoma than other species and subspecies (Ruoff et al., 1989), whereas Streptococcus gallolyticus subsp. pasteurianus was more frequently associated with neonatal meningitis. Clarridge et al. (2001) reported that biotype II/2 (Streptococcus gallolyticus subsp. pasteurianus) formed a separate genospecies and was the most common in adult males. This hypothesis has been confirmed by additional taxonomic studies conducted by other teams (Poyart et al. 2002; Schlegel et al. 2003). In Taiwan, Streptococcus bovis biotype I has been correlated with infective endocarditis (Jean et al., 2004). In addition to the pathogenesis, different species and subspecies may display distinct antimicrobial patterns. We have reported that the prevalence of inducible erythromycin resistance among Streptococcus gallolyticus subsp. pasteurianus was higher than that of other species and subspecies (Teng et al., 2001a).

One limitation of the present study is that only human isolates were included. Avian isolates may show different geno- or phenotypes (Chadfield et al., 2007). In summary, based on the groESL sequences determined, we have developed a Streptococcus bovis group-specific PCR assay that may provide an alternative way of distinguishing the Streptococcus bovis group from other viridans streptococci. Restriction digestion of the amplicon with AclI improved the differentiation between the predominant human species or subspecies of the Streptococcus bovis group.

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