Development of 16S Rrna-Based Probes for the Identification of Gram

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ORIGINAL ARTICLE 10.1111/j.1469-0691.2007.01803.x

Development of 16S rRNA-based probes for the identiﬁcation of Gram-positive anaerobic cocci isolated from human clinical specimens A. C. M. Wildeboer-Veloo, H. J. M. Harmsen, G. W. Welling and J. E. Degener

Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

ABSTRACT Fluorescent probes targeted at 16S rRNA were designed for Peptostreptococcus anaerobius and Peptostreptococcus stomatis (Pana134), Parvimonas micra (Pamic1435), Finegoldia magna (Fmag1250), Peptoniphilus asaccharolyticus (Pnasa1254), Peptoniphilus ivorii (Pnivo731), Peptoniphilus harei (Pnhar1466), Anaerococcus vaginalis (Avag1280) and Anaerococcus lactolyticus (Alac1438), based on the 16S rRNA sequences of reference strains and 88 randomly chosen clinical isolates. These strains were also used for validation of the probes. Application of the probes to an additional group of 100 clinical isolates revealed that 87% of Gram-positive anaerobic cocci (GPAC) could be identified with this set of probes. The 16S rRNAs of 13 clinical isolates that could not be identified were sequenced. Most of these isolates were GPAC that were not targeted by the probes. No clinical isolates of Pn. asaccharolyticus were encountered. Near full-length sequences were obtained from 71 of 101 (n = 88 + 13) sequenced clinical isolates. Of these, 25 showed <98% similarity with the homologues of the closest established species. The Fmag1250, Pamic1435, Pnhar1466, Pana134, Pnasa1254 and Pnivo731 probes allowed reliable identification and hybridised with all corresponding isolates. The Avag1280 and Alac1438 probes failed to hybridise with two isolates and one isolate, respectively, because of intra-species variation. However, overall, the set of probes yielded fast and reliable identification for the majority of clinical isolates. Keywords Anaerobes, Gram-positive anaerobic cocci, fluorescent probes, hybridisation, identification, 16S rRNA Original Submission: 16 November 2006; Revised Submission: 2 May 2007; Accepted: 14 June 2007 Clin Microbiol Infect 2007; 13: 985–992

Micromonas and Finegoldia, respectively, with each INTRODUCTION being the only species present in their respective Gram-positive anaerobic cocci (GPAC) are part of genus. The genus Micromonas has now been the commensal microbiota of humans at different replaced by Parvimonas, with Parvimonas micra sites of the body, and are also known to be (formerly Micromonas micros) being the only important in human disease. They account for species in the genus [8]. Ezaki et al. [6] divided about one-third of the anaerobic isolates recov- the remaining peptostreptococci into three phy- ered from clinical material [1]. During the last logenetic groups, Peptoniphilus gen. nov., Anaero- decade, the taxonomy of GPAC has changed coccus gen. nov. and Gallicola gen. nov., with substantially [2–4]. Peptostreptococcus productus Gallicola barnesae being the only species present in was transferred to the genus Ruminococcus [5], the latter genus. The type species of the two other and the genus Peptostreptococcus was divided into new genera are Peptoniphilus asaccharolyticus and six new groups [6,7]. Murdoch and Shah [7] Anaerococcus prevotii, respectively. The only spe- transferred the species Peptostreptococcus micros cies remaining in the genus Peptostreptococcus are and Peptostreptococcus magnus to two new genera, Peptostreptococcus anaerobius and a recently described species, Peptostreptococcus stomatis, isolated from the human oral cavity [9]. Corresponding author and reprint requests: A. C. M. Wildeboer- Infections involving GPAC are often poly- Veloo, Department of Medical Microbiology, UMCG, Univer- sity of Groningen, PO Box 30001, 9700 RB Groningen, The microbial [10]. The GPAC isolated most com- Netherlands monly from infections are P. anaerobius, Pa. micra, E-mail: [email protected] Finegoldia magna and Pn. asaccharolyticus [11].

2007 The Authors Journal Compilation 2007 European Society of Clinical Microbiology and Infectious Diseases 986 Clinical Microbiology and Infection, Volume 13 Number 10, October 2007

However, although these anaerobic cocci are GPAC have also been detected using PCR in isolated commonly from infections, little attention faecal samples [23], and have been quantified is paid to their detection and identification in using fluorescent in-situ hybridisation [24]. The diagnostic laboratories because of cumbersome latter technique makes it possible to detect and and inadequate classification systems. The identify GPAC in pure culture, and perhaps phenotypic identification of GPAC is based on also directly in clinical specimens. This tech- morphological appearance, carbohydrate forma- nique is relatively inexpensive to perform, and tion and the use of gas–liquid chromatography can be implemented easily in routine diagnostic [12]. The availability of proteolytic enzyme pro- laboratories. The present report describes the files made identification of the acknowledged design and validation of 16S rRNA-based GPAC easier and reproducible [13]. This contrib- probes for the detection and identification of uted to the development of several commercial clinically relevant GPAC. Probes were designed identification kits, including the RapID ANA for a selection of GPAC isolated in our labora- (Innovative Diagnostic Systems, Atlanta, GA, tory, namely, P. anaerobius ⁄ stomatis, Pa. micra, USA) and Rapid ID 32A (bioMe´rieux, Basing- F. magna, Pn. asaccharolyticus, Anaerococcus vagi- stoke, UK) systems. However, the newly nalis, Anaerococcus lactolyticus, Pn. ivorii and described species have not yet been included in Pn. harei. the databases of these kits. In addition, several new species, assigned originally to the genus Peptostreptococcus, have been described, e.g., MATERIALS AND METHODS Peptoniphilus harei, Peptoniphilus ivorii and Anaero- Strains coccus octavius [14]. The clinical relevance of these Reference strains and clinical isolates were cultured on Brucella new species has not yet been assessed. blood agar anaerobic plates and incubated in an anaerobic The availability of genotypic data now makes atmosphere for 48 h at 37C. Cells were harvested and fixed it possible to develop molecular techniques for for fluorescent in-situ hybridisation analyses in 1:1 phosphate- buffered saline (8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 the detection and identification of GPAC. Song ⁄ % ⁄ and 0.24 g of KH2PO4 L) and ethanol 96 v v. Fixed cells et al. [15] evaluated the use of 16S rRNA were stored at )20C. In total, 188 random clinical isolates of sequences to identify GPAC. These authors were GPAC were obtained from the diagnostic laboratory of the able to identify 84% of clinical isolates of GPAC University Medical Center, Groningen, and the Regional by comparison with their own sequence database Public Health Laboratory, Enschede, The Netherlands. The of type strains. These sequences revealed ambig- original clinical samples were obtained from a variety of anatomical sites, e.g., abdomen, head ⁄ neck area, leg, arm and uous data in public databases. Song et al. [16] groin. Only one isolate of each species from a single patient developed a multiplex PCR assay for the rapid was included in the study. The isolates were identified identification of GPAC, using genus- and species- phenotypically using the Rapid ID 32A system and the specific primers. Riggio et al. [17–19] developed Wadsworth manual [12]. Isolates that could not be identified, or only ambiguously, were designated as GPAC. Pheno- additional molecular detection assays for GPAC, typically, Pn. asaccharolyticus can be distinguished from including a PCR method for the detection of Pn. harei only by colony form and cell morphology [12]. Since F. magna and Pa. micra in oral clinical specimens, this is difficult to achieve, these organisms were designated and a PCR–restriction fragment length polymor- Pn. asaccharolyticus ⁄ harei. phism assay of 16S rRNA genes for the identification of oral Peptostreptococcus isolates. DNA 16S rRNA gene sequencing probes have also been used for the identification The 16S rRNA genes of 88 randomly chosen clinical isolates of Pa. micra [20] and P. anaerobius [21]. Amplifi- were sequenced. DNA was isolated as described previously cation of the 16S)23S intergenic spacer region [25] and the 16S rRNA genes were amplified and sequenced using universal 16S rRNA-specific primers [26]. The resulting was used by Hill et al. [22] to differentiate among sequences were aligned with sequences of reference strains different species of the former peptostreptococci. derived from the EMBL database and the Ribosomal Data- When the banding patterns of 38 test strains were base Project [27], using ARB software [28]. Similarities of the compared with those of reference strains, fewer sequences were calculated using a DNA distance matrix embedded in BioEdit (http://www.mbio.ncsu.edu/BioEdit/ than half of the strains were identified. The bioedit.html). The Escherichia coli sequence for positions remaining strains could not be identified, either 82–1371 was included to ensure accurate determination of because of intra-species variation or because they species similarity. A phylogenetic tree of sequences that had differed significantly from the type strain. <98% similarity with their closest relative was constructed

2007 The Authors Journal Compilation 2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 985–992 Wildeboer-Veloo et al. Identification of Gram-positive anaerobic cocci 987 using the same alignment by the neighbour-joining method A. vaginalis and Pn. ivorii required permeabilisa- with Jukes Cantor correction, based on a distance matrix that tion with proteinase K before hybridisation. All only included positions with >50% conservation and parsimony, implemented in the ARB software. As a control, the other strains hybridised successfully without phylogenetic tree was also calculated using the maximum- additional treatment. The probes were also vali- parsimony and maximum-likelihood methods. The topology dated against a further set of 88 sequenced clinical of the tree was calculated following bootstrap analyses of isolates (Table 2) and were shown to be 100% % 1000 replicate trees. Sequences with <98 similarity were specific. deposited in the EMBL database and assigned accession numbers. The set of probes was then applied to an additional group of 100 clinical isolates that had Probe design been identified phenotypically (Table 3). The majority (29%) of the isolates was identified as Probes (Table S1, see Supplementary material) were designed using the sequences of clinical isolates and reference strains F. magna on the basis of both genotypic and present in the Ribosomal Database Project [27] and EMBL phenotypic characteristics. The Pamic1435 probe database. ARB software was used for alignment and probe hybridised with 28% of the clinical isolates. design [28]. When necessary, unlabelled helper nucleotides Three of these isolates were misidentified phe- (Table S2, see Supplementary material) were designed to notypically as F. magna; one could not be iden- increase the in-situ accessibility of 16S rRNA, as described by Fuchs et al. [29]. For practical purposes, probes were tified. designed to have a similar hybridisation temperature. All The Pana134 probe hybridised with 4% of the probes were labelled with fluorescein-5-isothiocyanate at the 3¢ clinical isolates, all identified phenotypically as ¢ and 5 ends and were synthesised by Eurogentec (Seraing, P. anaerobius. The weak hybridisation signal with Belgium). this probe was increased by adding the unlabelled helper nucleotides H115 and H155 (Table S2). The Validation Pnhar1466 probe hybridised with 17% of the Ethanol-fixed cells were spotted on a slide and fixed for a clinical isolates, four of which were misidentified further 10 min using ethanol 96% v ⁄ v. Hybridisations were performed at 50C in hybridisation buffer (0.9 M NaCl, phenotypically, one as Peptoniphilus indolicus, and 20 mM Tris-HCl, pH 7.2, SDS 0.1% w ⁄ v) containing 10 ng of three that could not be identified according to the probe, as described previously [30]. For the Pana134, current identification scheme. Of all isolates, 6% Pivo731 and Avag1280 probes (Table S1), 5 ng of the were identified genotypically as Pn. ivorii,with appropriate helper nucleotides (Table S2) were added to four isolates phenotyped correctly and two incor- the hybridisation mixture. Hybridisations were performed overnight. The bacterial probe EUB338 [31] served as a rectly. The Avag1280 probe hybridised with two positive control, and its complement non-EUB338 [32] as the (2%) clinical isolates, which was in agreement negative control. The specificities of the Pana134 and with the phenotypic identification. The hybridi- Pnasa1254 probes were increased by adding formamide sation signal of the Pnivo731 and Avag1280 20% v ⁄ v, which is a duplex-destabilising agent, to the hybridisation buffer. The hybridisation signal obtained with probes was increased using the helper nucleotides the species-specific probes was compared visually with the H716 + H750 and H1263 + H1299, respectively EUB338 and non-EUB338 signals, using an Epifluorescence (Table S2). The Alac1438 probe hybridised with BH2 microscope (Olympus, Hamburg, Germany), and scored one of the clinical isolates (1%) that could not be negative or positive. Strains of F. magna, A. vaginalis and identified phenotypically. Pn. ivorii were permeabilised with proteinase K (500 mg ⁄ L; 50 mM Tris-HCl, pH 7.6). The cell spots were covered with Overall, the set of probes allowed identification the proteinase K solution and incubated for 10 min at room of 87% of the GPAC isolates (Table 3). The 16S temperature. The enzymic reaction was stopped by incubat- rRNA genes of the remaining 13 isolates were % ⁄ ing the slides in ethanol 96 v v for 2 min. All probes were sequenced to determine whether the new probes validated against the reference strains and sequenced clinical isolates listed in Table 1. In order to assess the proportion of had hybridised correctly. The sequence results GPAC that could be identified, the set of species-specific revealed two isolates of Peptococcus niger, two of probes was also applied to an additional group of 100 A. vaginalis, one of A. lactolyticus and one of clinical isolates. Sarcina ventriculi. Of these six isolates, one of the A. vaginalis isolates was misidentified phenotyp- RESULTS ically as Peptoniphilus lacrimalis, and the A. lactolyticus isolate was misidentified as F. magna. The The set of probes used in this study demonstrated four other isolates were identified correctly using % 100 specificity when validated against the the phenotypic system. Seven remaining isolates reference strains listed in Table 1. F. magna,

2007 The Authors Journal Compilation 2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 985–992 988 Clinical Microbiology and Infection, Volume 13 Number 10, October 2007

Table 1. Reference strains and Probe sequenced clinical isolates used for Fmag Mmic Pnhar Pana Avag Alac Pnivo Pnasa validation of the species-specific Strain 1250 1435 1466 134a,b 1280b 1438 731b 1254a probes for Gram-positive anaerobic cocci Finegoldia magna DSM 20470Tc +– – – –––– Parvimonas micra DSM 20468T –+– ––––– Peptoniphilus harei DSM 10020T –– + ––––– Peptostreptococcus anaerobius DSM 2949T –– – +–––– Anaerococcus vaginalis DSM 7457Tc –– – –+––– Anaerococcus lactolyticus DSM 7456T –– – ––+–– Peptoniphilus ivorii DSM 10022Tc –– – –––+– Peptoniphilus asaccharolyticus DSM 20463T –– – ––––+ Anaerococcus prevotii DSM 20548T –– – ––––– Anaerococcus tetradius MMBd –– – ––––– Peptoniphilus indolicus DSM 20464T –– – ––––– gpac218e –– – ––––– gpac053 – – – – + – – – gpac032 + – – – – – – – gpac127 –––––––– Clostridium sporosphaeroides DSM 1294T ND ND – ND ND ND ND ND Dermabacter hominis DSM 7083T ND ND – ND ND ND ND ND Clostridium colinum DSM 6011T ND ND ND – ND ND ND ND Eubacterium tenue DSM 20695T ND ND ND – ND ND ND ND Clostridium glycolicum DSM 1288T ND ND ND – ND ND ND ND Clostridium difficile DSM 1296T ND ND ND – ND ND ND ND Proteus mirabilis MMB ND ND ND ND – ND ND ND Enterococcus moraviensis DSM 15919T ND ND ND ND ND – ND ND Clostridium beijerinckii MMB ND ND ND ND ND ND – ND aTo increase specificity of the hybridisation, formamide 20% v ⁄ v was added to the hybridisation buffer. bUnlabelled helper nucleotides were added to the hybridisation mixture to increase the in-situ accessibility of the 16S rRNA. cBefore hybridisation, cells were permeabilised with proteinase K 500 mg ⁄ L for 10 min at room temperature. dAll MMB strains are clinical isolates identified by routine procedures. eAll gpac strains are sequenced clinical isolates used in this study. DSM, Deutsche Sammlung von Mikrooganismen und Zellkulturen (Braunschweig, Germany); ND, not determined.

Table 2. Validation of the newly designed probes against Table 3. Application of the species-specific probes to 100 88 clinical isolates, the 16S rRNA gene sequences of which clinical isolates identified phenotypically were used for the design of the probes Phenotypic Sequence n n n Identification (n) Probe identification ( ) identification ( ) identification ( )

Species name Sequence Probe Finegoldia magna (29) F. magna (29) Parvimonas micra (28) Pa. micra (24) F. magna (3) Finegoldia magna 26 26 GPAC (1) Parvimonas micra 13 13 Peptoniphilus harei (17) Peptoniphilus Peptoniphilus harei 16 16 asaccharolyticus ⁄ Anaerococcus lactolyticus 77 harei (13) Anaerococcus vaginalis 65 Peptoniphilus Peptoniphilus ivorii 55 indolicus (1) Peptostreptococcus anaerobius 55 GPAC (3) Peptoniphilus lacrimalis 3 Anaerococcus lactolyticus (1) GPAC (1) Anaerococcus tetradius 1 Anaerococcus vaginalis (2) A. vaginalis (2) Anaerococcus hydrogenalis 1 Peptoniphilus ivorii (6) Pn. ivorii (4) Peptococcus niger 1 GPAC (2) Atopobium parvulum 2 Peptostreptococcus P. anaerobius (4) Ruminococcus gnavus 1 anaerobius (4) Peptostreptococcus sp. E3_32 1 Not identified (13) Peptococcus niger (2) Pc. niger (2) A. vaginalis (1) A. vaginalis (1) Pn. lacrimalis (1) A. vaginalis (1) F. magna (1) A. lactolyticus (1) Sarcina ventriculi (1) S. ventriculi (1) could not be identified phenotypically; sequence GPAC (7) Atopobium parvulum (3) analysis revealed three Atopobium parvulum, one Anaerococcus tetradius (1) Peptoniphilus octavius (1) Anaerococcus tetradius, one Pn. octavius and one Not identified (1) Bacterium N14-24. One isolate could not be iden- Bacterium N14-24 (1) tified because amplification of the 16S rRNA gene GPAC, Gram-positive anaerobic cocci. was unsuccessful. Sequence analysis of the 16S rRNA genes of the A. vaginalis and A. lactolyticus isolates revealed that the corresponding probes mismatch with the Alac1438 probe and the two failed to hybridise with these isolates because of A. vaginalis isolates had one and three mis- mismatches. The A. lactolyticus isolates had one matches, respectively.

Table 4. Similarity of the 16S rRNA sequences of clinical gpac003, AM176516 94.8 % gpac007, AM176517 94.5 % isolates to those of reference strains, calculated using the 100 92 AM176519 94.1 % a gpac018A, DNA distance matrix Pn. ivorii, Y07840 100 Peptostreptococcus sp. E3_32, AF481225 Similarity gpac148 , AM176535 95.9 % 100 Pn. asaccharolyticus, AF542228 , AY153431 ‡ % n % n n Pn. indolicus Species 98 ( ) <98 ( ) Total ( ) gpac018B, AM176520 96.5 % 96 gpac055, AM176525 96.9 % Finegoldia magna 19 0 19 100 gpac121, AM176529 95.3 % Parvimonas micra 37 10 Pn. harei, Y07839 100 Peptoniphilus harei 74 11 gpac063, AM176526 97.7 % 100 , AF542230 Peptoniphilus ivorii 03 3 Pn. lacrimalis , AM176527 96.2 % Peptostreptococcus anaerobius 30 3 gpac077 gpac137, AM176534 96.8 % Anaerococcus lactolyticus 07 7 gpac199, AM176539 96.4 % Anaerococcus vaginalis 41 5 gpac155, AM176536 97.1 % Peptoniphilus lacrimalis 11 2 100 gpac165, AM176537 95.9 % Anaerococcus tetradius 20 2 92 gpac028, AM176522 96.8 % Atopobium parvulum 40 4 gpac104, AM176528 97.3 % Peptococcus niger 10 1 98 gpac126, AM176530 96.5 % Ruminococcus gnavus 10 1 A. lactolyticus, AF542233 Bacterium N14-24 1 0 1 A. octavius, Y07841 Peptostreptococcus sp. E3_32 0 1 1 A. prevotii, AF542232 100 , AF542234 Anaerococcus hydrogenalis 01 1 A. tetradius A. vaginalis, AF542229 100 , AM176540 97.5 % aOnly sequencing experiments that yielded a near full-length sequence were gpac215 A. hydrogenalis, D14140 included in this analysis. gpac047, AM176523 97.0 % 100 Pa. micra, AF542231 gpac134, AM176532 97.6 % A distance matrix was calculated from all 96 gpac049n, AM176524 97.5 % gpac015, AM176518 97.8 % sequence results with a near complete full-length gpac022, AM176521 97.8 % gpac135, AM176533 97.8 % sequence. Partially determined sequences were gpac131, AM176531 97.8 % gpac170, AM176538 97.8 % suitable for probe design, but not for phylogenetic F. magna, AF542227 analysis. Near full-length sequences were ob- G. barnesae, AB038361 100 P. anaerobius, L04168 tained for 71 isolates, of which 25 showed <98% P. stomatis, DQ160208 R. gnavus, X94967 similarity with their closest established species 100 At. parvulum, AF292372 Pe. niger, X55797 (Table 4). Using these sequences, a phylogenetic 100 bacterium N14-24, AY880043 tree was constructed. The calculation was per- 10 % formed using three different tree construction Fig. 1. Phylogenetic tree showing the relationship between methods, each of which gave the same result for Gram-positive anaerobic cocci and clinical isolates (shown the significant branchings. The phylogenetic tree in bold) with <98% similarity to their closest relative. The obtained using the neighbour-joining method is percentages of similarity are indicated. The neighbour- joining tree was constructed using an alignment corre- shown in Fig. 1. All sequences of F. magna, P. an- sponding to Escherichia coli base-pair positions 82–1371. aerobius, A. tetradius, At. parvulum, Pc. niger, Ru- Only bootstrap values >90% are shown. The bar indicates minococcus gnavus and Bacterium N14-24 showed 10% sequence divergence. Note: Peptostreptococcus sp. ‡98% similarity to the corresponding reference E3_32 should be renamed Peptoniphilus sp. E3_32. strains, while more than half of the sequences of Pa. micra showed <98% similarity to the reference fication of GPAC; however, the results are not strain. In addition, some sequences of Pn. harei, always unambiguous, as illustrated by the A. vaginalis and Pn. lacrimalis showed <98% sim- sequence results (Tables 2 and 3), which revealed ilarity to their respective closest established spe- the presence of At. parvulum, which is actually a cies. All sequences of Pn. ivorii, A. lactolyticus, coccobacillus similar to Slackia heliotrinreducens. Anaerococcus hydrogenalis and Peptostreptococcus The GPAC encountered most frequently were sp. E3_32 showed <98% similarity to the corre- F. magna (29%), Pa. micra (22%), Pn. harei (18%), sponding reference strains. Pn. ivorii (6%), A. vaginalis (5%), A. lactolyticus (5%) and P. anaerobius ⁄ stomatis (5%). Pn. asaccharolyticus was not observed, although previous DISCUSSION studies have reported that this is one of the most In total, 188 isolates of GPAC were analysed in frequently encountered GPAC in infections [11]. this study, using 16S rRNA-targeted probes This is probably because Pn. asaccharolyticus can and ⁄ or sequencing of the 16S rRNA gene to be phenotypically distinguished from Pn. harei obtain a genotypic identification. Cell morphol- only by its colony form and cell morphology ogy is used as an initial first step in the identi- [12]. The present data suggest that Pn. harei has

2007 The Authors Journal Compilation 2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 985–992 990 Clinical Microbiology and Infection, Volume 13 Number 10, October 2007 probably been misidentified as Pn. asaccharo- members of Pn. ivorii produced identical banding lyticus in the past. Of all the GPAC analysed, patterns. Analysis of the sequence data from the 18% were identified genotypically as Pn. harei, present study confirmed the intra-species varia- indicating that this is clearly a clinically relevant tion demonstrated by Hill et al. [22], and revealed, species that can be isolated from a variety of sites, in particular, that the A. lactolyticus group showed as also suggested by Song et al. [16], who identi- extensive heterogeneity in 16S rRNA sequences. fied 48 (25%) of 190 isolates as Pn. harei. In the When designing species-specific probes for spe- present study, 17 isolates were identified as cies with such heterogeneity, the probability Pn. harei by phenotypic methods, most of them exists that probes will be restricted in specificity as Pn. asaccharolyticus ⁄ harei. One isolate was mis- and may have mismatches with the correspond- identified as Pn. indolicus and three isolates could ing isolates. Attempts were made to overcome not be identified. The four isolates identified with this by including the sequences of clinical isolates the Pana134 probe were identified phenotypically in the probe design, and this was successful for as P. anaerobius. All strains of F. magna were most of the newly designed species-specific correctly identified on the basis of phenotypic probes. Only the Avag1280 and Alac1438 probes characteristics. In contrast, A. lactolyticus strains showed mismatches with corresponding isolates. were either misidentified as F. magna, or could Among the 71 isolates for which a nearly full- not be identified. Most of the Pn. ivorii, Pa. micra length sequence was obtained, 25 had <98% and A. vaginalis strains were also correctly iden- similarity to the closest established species. This tified phenotypically. is probably because of the intra-species variation Several technical problems required resolution. within GPAC, but such isolates might also be First, the Pana134, Pnivo731 and Avag1280 probes considered as belonging to new species. initially gave a weak hybridisation signal. Fuchs Since the newly designed probes were vali- et al. [29] reported that the inclusion of unlabelled dated and directed against Gram-positive bacte- helper oligonucleotides during hybridisation ria, all hybridisations were performed overnight could increase the in-situ accessibility of 16S to obtain an optimal hybridisation signal. A rRNA. The use of unlabelled helper oligonucleo- shorter hybridisation time (2 h) is also possible, tides that bind adjacent to the probe target site although the hybridisation signal for some (Table S2) was successful in increasing the hy- GPAC will be less detectable than that after bridisation signal. Second, the initial overnight hybridisation. With the current probe hybridisation results with F. magna, A. vaginalis set, 87% of all GPAC isolates were identified and Pn. ivorii showed no signal for EUB338 and quickly and reliably. These probes make it the specific probes. This was resolved by treating possible to differentiate Pn. asaccharolyticus from these large cocci with proteinase K to increase Pn. harei, which is difficult with phenotypic permeabilisation before hybridisation. Thus, con- methods. The probe-based identification method sideration of cell morphology may faci- is more reliable than phenotypic methods, espe- litate the selection of probes for hybridisation: cially for Pn. harei, Pn. ivorii and A. lactolyticus. for large cocci, the Fmag1250, Avag1280 and Moreover, the cost of phenotypic methods is Pnivo731 probes should be used; for middle-sized higher than that of fluorescent in-situ hybridisa- cocci, the probes Pnasa1254, Alac1438 and tion. The probes also have the potential for direct Pnhar1466; for small cocci, the Pamic1435 probe; application to clinical material in order to pro- and for cocci in chains, the Pana134 probe. vide fast and direct detection without costly However, it is also possible to use the complete anaerobic cultivation. set of probes. The Avag1280 and Alac1438 probes failed ACKNOWLEDGEMENTS to hybridise with some of the corresponding isolates, and analysis of the sequence data revealed We would like to thank the technicians of the anaerobic mismatches of the probes with the clinical workgroup and the PCR group for their contribution to the practical work. We are indebted to E. Roelofsen for providing isolates. Hill et al. [22] revealed the heterogeneity clinical isolates. We thank S. Smith for critical reading of the of GPAC by analysis of 16S)23S intergenic rRNA manuscript. None of the authors has commercial relationships. polymorphisms, demonstrating a considerable This study was presented, in part, at the 8th Biennial Congress intra-species variation for A. vaginalis, while all of the Anaerobe Society of the Americas (Boise, ID, USA, 2006).

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2007 The Authors Journal Compilation 2007 European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 985–992