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1 Research Service, VA Medical Center West Los Angeles, LA, USA
2 Clinical Microbiology Laboratory, VA Medical Center West Los Angeles, LA, USA
3 Infectious Diseases Section, VA Medical Center West Los Angeles, LA, USA
4 Department of Medicine, UCLA School of Medicine, USA
5 Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, USA
Correspondence
Yuli Song
yulis1{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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; Murdoch, 1998). Most infections involving GPAC are polymicrobial (Finegold, 1995) and arise from mucocutaneous surfaces. However, there are many instances of the isolation of GPAC in pure culture; mostly, these involve Finegoldia magna, but they also occur with Peptostreptococcus (P.) anaerobius, Peptoniphilus (Pn.) asaccharolyticus, Peptoniphilus indolicus, Micromonas micros, Anaerococcus vaginalis and Peptoniphilus harei (Murdoch, 1998). Although GPAC are isolated from approximately one-quarter of all infections involving anaerobic bacteria, studies of the significance of isolates of GPAC have been hindered by an inadequate classification system and the lack of a valid identification scheme.
The taxonomy of the genus Peptostreptococcus is currently under revision. Recent 16S rRNA sequence data have shown that the genus Peptostreptococcus is very heterogeneous (Li et al., 1994; Conrads et al., 1997). Two proposals have restricted the genus Peptostreptococcus to P. anaerobius (Murdoch et al., 2000) and transferred Peptostreptococcus magnus and Peptostreptococcus micros to two new genera, Finegoldia and Micromonas, respectively (Murdoch & Shah, 1999). More recently, Ezaki et al. (2001) proposed three new genera to accommodate former Peptostreptococcus spp.: Anaerococcus, which includes the saccharolytic, butyrate-producing species A. hydrogenalis, A. lactolyticus, A. octavius, A. prevotii, A. tetradius and A. vaginalis, Peptoniphilus, which contains the non-saccharolytic, butyrate-producing species Pn. asaccharolyticus, Pn. harei, Pn. lacrimalis, Pn. indolicus and Pn. ivorii, and Gallicola, which contains a single species, G. barnesae. Furthermore, several clinically important GPAC species still await formal description (Murdoch & Mitchelmore, 1991; Murdoch & Magee, 1995). The continual changes in nomenclature are confusing, so it is difficult to escape the conclusion that the lack of a sound and stable classification scheme has substantially contributed to the neglect of GPAC.
Identification of GPAC in most laboratories is still based on phenotypic methods such as microscopic and colonial morphology, carbohydrate fermentation reactions, proteolytic enzyme profile analysis, and gas-liquid chromatography for the detection of volatile fatty acids. However, these conventional identification protocols are not only laborious and time-consuming, but also often result in inconclusive identification (Karachewski et al., 1985; Murray et al., 1985; Murdoch & Mitchelmore, 1991; Wilson et al., 2000). In the past decade, genotypic-based techniques have emerged as alternative or complementary approaches to established phenotypic-based methods of identification. 16S rDNA sequence analysis has contributed greatly to the recognition of novel species of GPAC (Ezaki et al., 1990; Li et al., 1992; Murdoch et al., 1997). DNA probes targeting the 16S rRNA gene have been used to detect P. anaerobius and M. micros (Yasui, 1989; Yasui et al., 1989; Gunaratnam et al., 1992). However, for other GPAC species also reported to be clinically important bacteria, there has been little work on molecular identification. Multiplex PCR is a variant method of PCR in which more than one locus is simultaneously amplified in the same reaction. It has the potential to yield considerable savings of time and effort in the laboratory and to identify bacteria more accurately. Since its introduction, multiplex PCR has been successfully applied to the identification of many bacteria (Tran & Rudney, 1999; Song et al., 2000; Yeboah-Manu et al., 2001; Wisselink et al., 2002). In the present study, based on accurate 16S rDNA sequence data of GPAC species obtained in a previous study (Song et al., 2003), a two-step multiplex PCR identification scheme was established to rapidly and accurately identify 14 valid GPAC species originally classified in the genus Peptostreptococcus.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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). The clinical isolates chosen represented seven GPAC species identified in our laboratory over a 5-year period using 16S rDNA sequence analysis. In addition, 23 well-characterized strains phylogenetically related to GPAC species were used to verify the specificity of the multiplex PCR assay (Table 2). All strains were cultured anaerobically overnight on Brucella blood agar (Anaerobe Systems) at 37 °C and were characterized phenotypically by conventional tests as described in the Anaerobic Bacteriology Manual (Jousimies-Somer et al., 2002) and the BD BBL Crystal Identification System (Becton Dickinson Microbiology Systems); they were characterized genetically by 16S rDNA sequence analysis.
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) and present study, as well as the related sequences retrieved from GenBank, were analysed by multialignment using CLUSTAL W (http://www.cmbi.kun.nl/bioinf/tools/clustalw.shtml).
Based on the multialignment analysis data, five potentially genus-specific primers, ANAC for the genus Anaerococcus, PEPN for the genus Peptoniphilus, PEPA for the genus Peptostreptococcus (which is also the P. anaerobius species-specific primer), MICR for the genus Micromonas (which is also the M. micros-specific primer) and FIGD for the genus Finegoldia (which is also the F. magna-specific primer), were selected from the 16S rRNA gene. In addition, six species-specific primers targeted to the six species of the genus Anaerococcus, and five species-specific primers for the five species of the genus Peptoniphilus were also designed from the 16S rDNA sequence (sequences of primers are shown in Table 3). The primer sequences were analysed for secondary structure formation, G+C content and primer-dimer formation with the NETPRIMER analysis software (http://www.premierbiosoft.com/netprimer). The specificities of these primers were predicted by comparison to the aligned SSU_rRNA database of the RDP using the CHECK_PROBE utility (Maidak et al., 2001), and were further tested by running PCRs with DNA samples from 23 well-characterized strains that are phylogenetically related to GPAC species (Table 2
). These primers were designed with minimal differences in their annealing temperature within each primer set, and to yield amplification products that ranged between 120 and 1350 bp in size and differed by at least 100 bp. The relative locations of the primers in the Escherichia coli rRNA gene sequence are indicated in Fig. 1.
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). These primers, which amplify an approximately 330 bp fragment, were used in independent amplification (i.e. not as part of the multiplex protocol) to ensure that the lack of an amplification product from species other than our targeted species reflected the specificity of our protocol rather than the lack of suitable template DNA.
Identification of GPAC species by two-step multiplex PCR assays.
Fourteen GPAC species belonging to five genera (Anaerococcus, Finegoldia, Micromonas, Peptoniphilus and Peptostreptococcus) were first identified to the genus level by multiplex PCR (designated multiplex PCR-G). Since the genera Finegoldia, Micromonas and Peptostreptococcus each have only one species, F. magna, M. micros and P. anaerobius, respectively, they were identified to species level by multiplex PCR-G. Then six species within the genus Anaerococcus were further identified to the species level by multiplex PCR-Ia and multiplex PCR-Ib. Similarly, five species of the genus Peptoniphilus were identified to the species level by multiplex PCR-IIa and multiplex PCR-IIb (Fig. 1)
PCR amplification was performed as follows: one or two colonies of the bacterial strains were suspended in 50 µl of Tris/HCl/EDTA/saline (pH 8·0), incubated for 10 min at 95 °C and centrifuged at 18 600 g for 2 min to obtain the DNA as the PCR template. PCR amplification was carried out in a volume of 50 µl containing 1·25 U Taq polymerase (Promega), 50 mM KCl, 10 mM Tris/HCl (pH 9·0), 0·1 % Triton, 2·5 mM MgCl2, 0·2 mM dNTPs and 5 µl of bacterial lysate as the template DNA. Primer concentration was 0·25 µM (each) except for MICR and PEPA (0·125 µM each) and FIGD (0·5 µM) in multiplex PCR-G. PCR was carried out for 35 cycles. Each cycle consisted of 95 °C for 20 s for denaturation; annealing was performed for 1 min at 50 °C for multiplex PCR-G, 62 °C for multiplex PCR-Ia and multiplex PCR-Ib, and 53 °C for multiplex PCR-IIa and multiplex PCR-IIb; extension was performed at 72 °C for 30 s for all. A cycle of 72 °C for 5 min was added to the final extension. PCR products were analysed by electrophoresis on a 2 % agarose gel followed by ethidium-bromide staining.
Sensitivity of multiplex PCR assays.
The sensitivities of the multiplex PCR assays were evaluated by titrating cultures of the 14 reference strains of GPAC species. The strains were suspended in Tris/HCl (pH 7·5) and the density was adjusted to approximately 108 c.f.u. ml-1 using McFarland standards. Thereafter, serial 10-fold dilutions of cultures were made in Tris/HCl. Equal volumes (100 µl) of dilutions were plated onto Brucella blood agar plates, and were taken for DNA preparation for subsequent multiplex PCR assays. Colonies were counted after 3 days incubation under anaerobic conditions. The DNA templates were obtained by heating cells at 95 °C for 10 min. Detection limits of multiplex PCR assays were determined with known numbers of bacteria diluted in Tris/HCl (pH 7·5).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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). The specificity of the multiplex PCR-G was evaluated by running PCRs with DNA from 23 well-characterized strains that are phylogenetically related to the 14 targeted GPAC species; this resulted in amplification only with the DNA from the corresponding GPAC reference strain(s).
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). The specificities of these second-step multiplex PCR assays were also verified by PCR amplification with DNA samples from the 23 reference strains (Table 2). All these multiplex PCR assays generated no amplicon with DNA other than that of the target organisms, except for multiplex PCR-IIb, which generated an amplicon from Pn. asaccharolyticus corresponding in size to that from Pn. indolicus. The quality of all the DNA samples that gave negative multiplex PCR results was checked by running PCRs with the 16S rRNA gene universal primer pair 8UA/341B. All DNA samples gave an expected band of about 300 bp in size, indicating that the lack of an amplification product from species other than our targeted organisms reflected the specificity of our protocol rather than the lack of suitable template DNA (data not shown).
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). All strains of A. tetradius (n=3), F. magna (n=60), P. anaerobius (n=19) and 40/47 strains of M. micros were consistently identified correctly by both methods. However, both multiplex PCR and 16S rRNA sequencing identified all 27 strains of Pn. asaccharolyticus and 21/22 strains of A. prevotii that had been identified by phenotypic tests as Pn. harei.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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) and was shown to be a reliable method for the identification of P. anaerobius (Holdeman et al., 1977; Murdoch & Magee, 1995), A. octavius and Pn. ivorii (Murdoch et al., 1997). However, most GPAC species produce a very limited range of VFAs. In addition, GLC is not only time-consuming, but also the capital equipment is costly. In the 1980s, Ezaki & Yabuuchi (1985) found that proteolytic enzyme profiles could distinguish clearly and reproducibly among recognized species of GPAC, which contributed to the development of several commercially available enzyme kits. These commercial kits represented a considerable advance in identification methods for GPAC in terms of speed and simplicity. However, databases accompanying the kits are often incomplete or inaccurate, and with a rapid increase in the number of newly described GPAC species, this becomes more and more of a problem. In addition, the interpretation of test results involves substantial subjective judgement. For example, in the present study, 11 strains of A. vaginalis were misidentified as A. tetradius or A. prevotii, and 48 strains of Pn. harei were misidentified as Pn. asaccharolyticus or A. prevotii using phenotypic traits. This is basically because the database for the biochemical kit used (the BBL Crystal Identification System) has not been updated since the recent taxonomic changes in the GPAC: A. vaginalis and Pn. harei were not in the database. The uncertainty associated with the classification of GPAC make their clinical importance difficult to assess. In recent years, the 16S rRNA gene has been the most widely accepted gene used for bacterial classification and identification due to its universal distribution among bacteria and the presence of species-specific variable regions within its sequence. Hybridization assays using DNA probes targeted at this gene have been developed for the clinically important GPAC species P. anaerobius and M. micros (Yasui, 1989; Yasui et al., 1989; Gunaratnam et al., 1992). However, using these techniques is often an involved task that makes their application impractical for routine laboratory use, e.g. blotting with probes is labour-intensive. More recently, Riggio et al. (2001) and Riggio & Lennon (2002) have developed PCR assays that specifically detect P. anaerobius and M. micros in clinical samples. In the present study, based on a 16S rDNA sequence analysis, we developed a multiplex PCR protocol which allows the rapid identification of 14 GPAC species, including eight clinically relevant species. Because of the complexity of multiplex PCR, such as the need for the selection of specific primers that generate a distinctive PCR product for each species under the same PCR conditions, we used a strategy of establishing a two-step multiplex PCR system. First, GPAC species were identified to the genus level by multiplex PCR-G [since the genera Finegoldia, Micromonas and Peptostreptococcus each contain only a single species (F. magna, M. micros and P. anaerobius, respectively), they were identified to the species level by multiplex PCR-G]. Then six species of the genus Anaerococcus and five species of the genus Peptoniphilus were further identified to the species level by multiplex PCR-I (including multiplex PCR-Ia and Ib) and multiplex PCR-II (including multiplex PCR-IIa and IIb).
As we were aware of the inaccuracy of the 16S rDNA sequence data of GPAC species presented in GenBank, we used the 16S rDNA sequences of 14 type strains representing 14 valid GPAC species determined in a previous study (Song et al., 2003) and this study as targets for specific primer selection. The bacterial 16S rRNA gene sequence is a good target choice for multiplex PCR since it contains conserved as well as variable domains; these can be exploited to generate genus- or species-specific amplification and, furthermore, the gene exists in large copy numbers in a single bacterial cell, exceeding by far the number of other chromosomally encoded genes. The sequence analysis showed that the sequence differences among the 14 GPAC species were clustered in three regions of the 16S rDNA. These three hypervariable regions were also observed in other bacterial species and were therefore used for species-specific primer selection. Two other regions that are unique to the genera Anaerococcus and Peptoniphilus as well as common within the species of each genus were used to design genus-specific primers. The specificity of these primers was verified by multiplex PCR amplification with DNA from the 14 GPAC reference strains and from the 23 well-characterized strains (Table 2), resulting in amplification only with the DNA from strains corresponding to the target genus or species, except for the Pn. indolicus species-specific primer (INDO) which also produced the same size band from Pn. asaccharolyticus. However, Pn. indolicus and Pn. asaccharolyticus can be distinguished by using multiplex PCR-IIa, which only yielded a specific band from Pn. asaccharolyticus.
To develop multiplex PCR assays that will identify multiple GPAC species in one reaction, the PCR conditions should be the same in the individual reactions. All the primers used in one multiplex PCR were designed to have similar PCR kinetics. Initially, equimolar primer concentrations (0·25 µM) were used in each multiplex PCR. However, multiplex PCR-G showed uneven amplification, with a weaker amplicon of F. magna even after the reaction was optimized for the cycling conditions. Therefore, the proportions of various primers used in this reaction were altered to yield approximately equal amplification products from each of the target organisms, with an increase of F. magna species-specific primer to 0·5 µM, and a decrease of M. micros- and P. anaerobius-specific primers to 0·125 µM. To reduce the complexity of the multiplex PCR, we also adopted the strategy that each multiplex setting comprised one common reverse primer, except for multiplex PCR-II, in which the forward species-specific primer for Pn. harei cross-reacted with DNA from Pn. lacrimalis; therefore, a reverse specific primer was used for Pn. harei.
The usefulness and reliability of the established scheme for the identification of clinical isolates was evaluated with 190 clinical GPAC isolates that had been previously identified to the species level by 16S rRNA sequencing and phenotypic tests. Comparison of the identification results obtained by these three methods showed that the identification obtained from multiplex PCR assays agreed 100 % with 16S rDNA sequencing identification, but only 65 % (123/190) agreed with the identification obtained by phenotypic tests. The multiplex PCR-based identification scheme always gave clear-cut results. Since we did not have clinical isolates of A. hydrogenalis, A. lactolyticus, A. octavius, Pn. asaccharolyticus, Pn. indolicus, Pn. ivorii and Pn. lacrimalis available to us, only the reference strains of these species were tested by the established multiplex PCR assays.
In conclusion, a reliable, relatively rapid and cost-effective multiplex PCR-based method for the identification of clinical isolates of GPAC species has been established. Identification of GPAC by conventional methods usually requires 
48 h after a discrete colony has been isolated. One or two weeks are required for the final species identification of the difficult GPAC species, such as some butyrate-producing members of the group. In some circumstances, no identification can be made after weeks of analysis, even by an experienced technician. Although commercially available biochemical kits shorten turnaround time, they possess the limitations inherent to all biochemical identification schemes. Determination of the 16S rRNA gene sequence represents a highly accurate and versatile method for the identification of bacteria to the species level, even when the species in question is notoriously difficult to identify by biochemical means; however, cost is a critical issue in the evaluation of 16S rDNA sequence analysis as a diagnostic tool. The multiplex PCR-based identification scheme described here can be considered, in terms of its reduction of labour time (<24 h), relatively low cost (
$5 per strain), easy application, and reliable and repeatable PCR results, a powerful potential tool for the routine clinical identification of GPAC species. Further experiments will be necessary to determine the conditions needed to detect GPAC species directly from clinical specimens.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Received 9 January 2003;
revised 25 March 2003;
accepted 14 April 2003.
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X174 RF DNA/HaeIII); 1–6, species of the genus Anaerococcus with an amplicon of 
980 bp in size (A. hydrogenalis ATCC 49630T, A. lactolyticus CCUG 31351T, A. octavius CCUG 38493T, A. prevotii CCUG 41932T, A. tetradius CCUG 46590T and A. vaginalis CCUG 31349T, respectively); 7–11, species of the genus Peptoniphilus with an amplicon of 