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

Abstract

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.

Fig 1. 16S rRNA sequence trace data for the isolates Prevotella denticola HJX050 and Prevotella nigrescens HJX046, showing comparison sequence data for heterogeneous clones and direct sequencing with single ambiguous bases (A) or continuous ambiguous bases (B).

Prevotella nigrescens HJX046 clone A had a deletion at nucleotide position 229.

Fig 2. Phylogenetic trees based on the concatenated sequences of the gyrB1 gene.

Detailed phylogenetic trees of P. melaninogenica strains are shown in Figure (B). The trees were constructed by the neighbour-joining (NJ) method. The numbers at nodes indicate the percentage bootstrap values of 1000 replicates (>70%). Bars indicate the expected nucleotide substitutions per site. * represents reference strains.

Fig 3. Phylogenetic trees based on the concatenated sequences of the 16S rRNA gene.

Detailed phylogenetic trees of P. melaninogenica strains are shown in Figure (B). The trees were constructed by the neighbour-joining (NJ) method. The numbers at nodes indicate the percentage bootstrap values of 1000 replicates (>70%). Bars indicate the expected nucleotide substitutions per site. * represents reference strains.

Fig 4. Phylogenetic trees based on the concatenated sequences of the rpoB gene.

Detailed phylogenetic trees of P. melaninogenica strains are shown in Figure (B). The trees were constructed by the neighbour-joining (NJ) method. The numbers at nodes indicate the percentage bootstrap values of 1000 replicates (>70%). Bars indicate the expected nucleotide substitutions per site. * represents reference strains.