Development of 16S rRNA-based probes for the identification of Gram-positive anaerobic cocci isolated from human clinical specimens

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.