ABSTRACT
Proper identification of Streptococcus pneumoniae by conventional methods remains problematic. The discriminatory power of the 16S rRNA gene, which can be considered the “gold standard” for molecular identification, is too low to differentiate S. pneumoniae from closely related species such as Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis in the routine clinical laboratory. A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing <95.8% interspecies homology. In addition, 6 signature nucleotides specific for S. pneumoniae were identified within the 313-bp recA fragment. We show that recA analysis is a useful tool for proper identification to species level within the S. mitis group, in particular, for pneumococci.
INTRODUCTION
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is also associated with bacteremia, meningitis, otitis media, and sinusitis (34). S. pneumoniae is a member of the Streptococcus mitis group, which currently includes Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus oralis, Streptococcus infantis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus peroris, Streptococcus australis, Streptococcus oligofermentans, and Streptococcus sinensis (21, 44).
Clinical laboratories must be able to accurately differentiate S. pneumoniae from other viridans streptococci commonly found in clinical samples to facilitate appropriate antimicrobial therapy. Discrimination of S. pneumoniae from closely related species such as S. pseudopneumoniae, S. oralis, and S. mitis remains problematic since conventional phenotypic methods like colony morphology, bile solubility, and optochin susceptibility testing, as well as commercial systems (API 20 Strep and Vitek 2; bioMérieux, Marcy l'Etoile, France), do not always provide accurate identification (3, 5, 15, 18, 34) and often lead to misidentification. Moreover, sequence analysis of the 16S rRNA gene, a method widely used for bacterial identification to species level (4–8, 37), is not sufficiently discriminative (23). Several studies proposed the analysis of additional, more discriminative target genes like sodA (3, 24, 36), rpoB (12, 21), gdh (21, 35), and groEl (16) to differentiate species within the S. mitis group. However, an accurate differentiation from the more recently described S. pseudopneumoniae, which is closely related to S. pneumoniae, was either not demonstrated (3) or not investigated in detail (16, 35).
Other PCR-based approaches for accurate identification of S. pneumoniae rely on the detection of pneumococcal toxins or virulence factors, such as the pneumolysin (ply gene) and autolysin (lytA gene) (28, 33, 40), which are usually not present in other alpha-hemolytic streptococci. The usefulness of such assays is questionable, as false-positive results due to cross-reactivity among S. mitis, S. oralis, or S. pseudopneumoniae strains were generated (1, 3, 14, 17, 45).
Phylogenetic analysis of recA, encoding the highly conserved subunit of the bacterial recombinase, proved to be a valuable tool for bacterial species assignment (13, 30, 31, 42, 46) but has not been investigated in-depth for species differentiation of the S. mitis group.
The aim of this study was to assess recA as a gene target for proper identification of streptococci, particularly S. pneumoniae. We identified a 313-bp recA fragment that differentiates members of the S. mitis group and enables accurate assignment to species level.
MATERIALS AND METHODS
Bacterial strains.Type strains of S. pneumoniae (NCTC 7465), S. mitis (NCTC 12261), and S. oralis (NCTC 11427) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany). Type strains of S. pseudopneumoniae (ATCC BAA-960), S. infantis (ATCC 700779), and S. oligofermentans (LMG 21535) were obtained from the Institut Pasteur (Paris, France). Other streptococcal strains used in this study were isolated from clinical samples (blood cultures or other normally sterile body sites) in our laboratory: (i) 11 isolates collected from January to April 2009 and (ii) 20 isolates previously analyzed (5). Streptococcal strains were routinely cultured on sheep blood agar. Phenotypic characterization included colony morphology, susceptibility to optochin, bile solubility, and capsular serotyping (National Centre for Invasive Pneumococci, Institute for Infectious Diseases, University Bern, Bern, Switzerland).
recA sequence analysis.DNA was extracted from the cultures as follows. A loopful of bacteria was suspended in 500 μl 0.9% NaCl and incubated by shaking at 80°C for 10 min. After centrifugation, the pellet was resuspended in 200 μl of InstaGene matrix (Bio-Rad Laboratories, Hercules, CA) and incubated at 56°C for 2 h and subsequently at 95°C for 10 min. The mixture was centrifuged and the supernatant was used as the template for PCR.
For amplification, primers recA 2F [5′-GCCTT(T/C)ATCGATGC(C/T/G)GA(G/A)CA-3′] and recA 5R [5′-GTTTCCGG(G/A)TT(A/T/G)CC(G/A)AACAT-3′] were used. PCR cycling parameters included an initial denaturation for 5 min at 95°C; 40 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C; and a final extension for 10 min at 72°C. Five microliters of the DNA extract was used for amplification in a total volume of 50 μl containing 1.25 U of FastStart Taq DNA polymerase (Roche Diagnostics, Rotkreuz, Switzerland) and the appropriate buffer. Amplicons were purified with a QIAquick PCR purification kit (Qiagen AG, Hombrechtikon, Switzerland) and were sequenced with forward primer recA 2F and reverse primer recA 5R by use of a BigDye kit and an automatic DNA sequencer (ABI Prism 3100 genetic analyzer; Applied Biosystems, Zug, Switzerland). The sequences were edited using the software program Megalign Lasergene (version 7; DNAStar Inc., Madison, WI). Distances of the recA sequences were calculated by using Smartgene software (Zug, Switzerland). Multiple alignment of the sequences was performed with the Clustal V program (20) (Megalign Lasergene, version 7), and construction of a phylogenetic tree was performed with the neighbor-joining method (38).
Nucleotide sequence accession numbers.The recA sequences of strains S. pneumoniae NCTC 7465T, S. mitis NCTC 12261T, S. oralis NCTC 11427T, S. pseudopneumoniae ATCC BAA-960T, S. infantis ATCC 700779T, S. oligofermentans LMG 21535T, and clinical isolates of S. pneumoniae have been group deposited in GenBank under BankIt 1363616 (with individual numbers HM572273, HM572274, HM572275, HM572276, HM572277, HM572278, HM572279, HM572280, HM572281, HM572282, HM572283, HM572284, HM572285, HM572286, HM572287, HM572288, and HM572289).
RESULTS
Selection of a hypervariable recA fragment for differentiation of Streptococcus mitis group members.To identify a small hypervariable region suitable for differentiation and efficient PCR amplification, we aligned complete Streptococcus recA sequences available in GenBank (accession numbers are given in parentheses), e.g., Streptococcus pyogenes (NC_009332), S. pneumoniae (NC_008533), S. gordonii (CP000725), S. parasanguinis (AF069745), S. sanguinis (CP000387), Streptococcus mutans (NC_004350), and Streptococcus agalactiae (NC_004116). recA genes of Pseudomonas aeruginosa (NC_002516) and Escherichia coli (NC_002695) were used as outlier sequences. An internal 313-bp recA fragment including hypervariable regions was selected for amplification with consensus recA PCR primers 2F and 5R (see Materials and Methods). This region corresponds to Escherichia coli recA positions 294 to 606 (NC_002695 [19]).
Homology analysis of recA within Streptococcus mitis group.Phylogenetic analysis of the 313-bp recA fragment of the S. mitis group strains was made to determine its differentiating ability (Fig. 1A). recA sequences were generated from S. mitis group type strains (i.e., S. pneumoniae NCTC 7465T, S. mitis NCTC 12261T, S. oralis NCTC 11427T, S. pseudopneumoniae ATCC BAA-960T, S. infantis ATCC 700779T, and S. oligofermentans LMG 21535T) or were obtained from published sequences from GenBank (S. parasanguinis, accession number AF069745; S. gordonii, accession number CP000725, S. sanguinis, accession number CP000387). Partial recA sequence analysis revealed homologies of <95.8% between species (Table 1).
Homology analysis of recA (313-bp fragment) within the Streptococcus mitis group. (A) Phylogenetic tree of 313-bp recA sequences of Streptococcus mitis group strains. Validated recA sequences of S. peroris, S. australis, S. cristatus, and S. sinensis were not represented in GenBank. Bootstrap values were calculated from 1,000 trees; only values exceeding 50% are shown. (B) Phylogenetic tree of 313-bp recA sequences of Streptococcus type strains and Streptococcus clinical isolates (n = 20). Bootstrap values were calculated from 1,000 trees; only values exceeding 50% are shown.
Homology analysis of partial recA of Streptococcus sp. type strains
Intraspecies variability of Streptococcus pneumoniae recA.A set of 11 published recA sequences of S. pneumoniae strains available from GenBank (CP000410 [29], NC_008533 [29], AE005672 [41], CP001015 [11], NC_011072 [11], AE007317 [22], NC_003098 [22], CP001033 [10], NC_010582 [10], NC_003028 [41], FM211187 [9]) and 11 recA sequences obtained from accurately assigned S. pneumoniae clinical isolates from our laboratory were analyzed for homology to determine the intraspecies variability in the recA fragment sequence. The 22 sequences showed >99.7% identity.
Proof of principle: identification of viridans streptococci on the basis of sequence analysis of partial recA fragment.From 20 clinical isolates of viridans streptococci, previously unidentifiable to species level by 16S rRNA gene sequencing and with the API 20 Strep system (bioMérieux) (5), the 313-bp recA fragment was amplified and analyzed for sequence homology. For each isolate, a sequence homology of ≤95.5% to the recA sequence of the S. pneumoniae type strain was observed (Table 2). For 12 isolates (numbers 5, 6, 7, 8, 12, 13, 14, 15, 16, 18, 19, and 20), recA analysis yielded an assignment to species level (Table 2). recA sequence similarities to the best taxon ranged from 95.5% to 99.0%, with a difference of ≥1.0% to the next best taxon. For the other eight isolates (numbers 1, 2, 3, 4, 9, 10, 11, and 17), best matches were observed with S. oralis/S. oligofermentans and S. mitis/S. pseudopneumoniae, respectively.
recA sequence-based identification of viridans streptococcal clinical isolatesa
A combined phylogenetic analysis of the recA sequences of the clinical isolates and type strains showed two major clusters: one containing the type strains of S. mitis, S. pneumoniae, and S. pseudopneumoniae and the other containing the type strains of S. oralis and S. oligofermentans (Fig. 1B). Within the S. mitis/S. pneumoniae/S. pseudopneumoniae cluster, the lineage containing the S. pneumoniae type strain branched off into a tight subcluster.
Signature nucleotides in Streptococcus pneumoniae recA.The recA sequences of type strains S. pneumoniae NCTC 7465, S. pseudopneumoniae ATCC BAA-960, S. mitis NCTC 12261, S. oralis NCTC 11427, and all clinical isolates used in this study (n = 31) and published recA sequences of S. pneumoniae from GenBank (n = 11) were aligned to detect specific positions within the 313-bp recA fragment that distinguished S. pneumoniae (molecular signatures). The alignment showed 6 bp specific for S. pneumoniae at positions 97, 160, 199, 247, 250, and 280 (Table 3).
Signature nucleotides specific for Streptococcus pneumoniae observed in 313-bp recA fragmenta
DISCUSSION
Accurate identification to species level of S. mitis group members is of clinical importance, since this group contains pathogens, e.g., S. pneumoniae, and commensals of the human oral cavity, such as S. mitis and S. oralis (34). In view of increasing resistance to penicillin and macrolide antibiotics (2, 34), proper identification of viridans streptococci is important for antimicrobial therapy. Differentiation between S. pneumoniae and S. pseudopneumoniae is of relevance, as those isolates are assumed to be involved in the exacerbation of chronic obstructive pulmonary disease (25).
We have selected a 313-bp recA fragment that shows a significant variability among the S. mitis group members (Fig. 1; Table 1) and identified molecular signatures confirming accurate identification of pneumococci (Table 3). Implementation of the presented recA PCR assay in routine laboratory diagnostics is facilitated by the fact that a small part of a single gene is sufficient for accurate assignment of S. pneumoniae.
For assignment to species level using analysis of housekeeping genes, e.g., recA, approved criteria such as those for the 16S rRNA gene (5) are not available. Sequence homologies of more than 94% to 95% with a reference sequence was proposed to be appropriate for identification to species level for housekeeping genes such as rpoB (26, 32) or recA (43). This is in agreement with our data: most species showed sequence identity of more than 95.5% to the best taxon match, with a demarcation of ≥1.0% to the next homologous taxon (Table 2).
As proof of principle, we retrospectively analyzed a number of clinical isolates (n = 20) which remained unidentified to species level by phenotypic methods and 16S rRNA gene sequencing in routine diagnostics (5). For all isolates investigated, differentiation from S. pneumoniae was achieved by partial recA sequence analysis (≤95.5% sequence similarity) and from nucleotide signatures (Table 3). Twelve strains were identified to species level and eight strains were assigned to S. oralis/S. oligofermentans (n = 6) and S. mitis/S. pseudopneumoniae (n = 2), respectively. A close relation of S. oligofermentans to S. oralis was previously observed by analysis of the groEL gene (16). However, 16S rRNA gene analysis can accurately differentiate S. oligofermentans and S. oralis (data not shown). Despite a recA sequence similarity of 95.8% between the type strains of S. mitis and S. pseudopneumoniae, accurate differentiation between these species was not shown for two clinical isolates. Thus, in routine diagnostics, recA analysis is a valuable tool for identification of pneumococci, but limitations on discrimination of other members of the S. mitis group were observed.
As described earlier by analysis of other targets (24, 27, 35, 36), we observed heterogeneity within the S. mitis and S. oralis cluster (Fig. 1B) but a tight homogeneity of the S. pneumoniae strains. The hypothesis of a common ancestor of S. mitis, S. pneumoniae, and S. pseudopneumoniae proposed previously (27) is supported by our data. Our results show that the heterogeneity of S. oralis and S. mitis strains (Fig. 1B; Table 2) as well as the homogeneity of S. pneumoniae strains occurs not only in reference strains (35) but also in clinical isolates.
ACKNOWLEDGMENTS
We thank the laboratory technicians for their dedicated help. We thank Erik C. Boettger for constructive discussions and continuous support.
The study was supported by the University of Zurich.
FOOTNOTES
- Received 16 July 2010.
- Returned for modification 31 August 2010.
- Accepted 30 November 2010.
- Accepted manuscript posted online 8 December 2011.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.
