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Background  Members of the genera  Prevotella ,  Veillonella  and  Fusobacterium  are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus  Veillonella , the high intra-chromosomal heterogeneity of  rrs  genes has not been reported.    Methods  The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the  rrs-ITS  genes of 9  Prevotella intermedia  isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes,  hsp60 ,  recA ,  dnaJ ,  gyrB1  and  rpoB  from 89 clinical isolates of  Prevotella  were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18  Prevotella  reference strains.    Results  Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera  Prevotella  and  Veillonella . One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera  Prevotella  and  Veillonella , 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the  rrs-ITS  genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed.    Conclusion  Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera  Prevotella , ranging from 94.3–100%. However, the inter-species similarities were relatively low, ranging from 68.7–97.9%. The housekeeping genes  rpoB  and  gyrB1  were demonstrated to be alternative classification markers to the species level based on intra- and inter-species comparisons, whereas based on phylogenetic tree  rpoB  proved to be reliable phylogenetic marker for the genus  Prevotella .

Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses