Species richness of gram-positive coccoid morphotypes isolated from untreated and treated root canals of teeth associated with periapical disease
Aims To estimate the species richness of gram-positive coccoid morphotypes (GPCM) from untreated and treated root canals of teeth with periapical disease, using partial 16S rRNA gene sequences and biochemical properties for identification. To further characterise the isolates by antibiotic sensitivity profiles and a subset of isolates by 16S-23S intergenic amplicon patterns. Methodology 195 isolates from 20 teeth (GPCM: 117 - untreated teeth; 78 - treated teeth) were investigated. 16S rDNA obtained from all isolates using universal primers was partially sequenced and the sequences aligned with those in databases using RDP and BLAST searches. The process of identity determination was analysed. Biochemical, commercial enzyme, and MIC tests by agar dilution and E-test (8 antibiotics) were also performed. Streptococcus, Staphylococcus, Enterococcus and Lactobacillus strains were evaluated by 16S-23S intergenic amplicon profile. The relatedness of like-strains was compared by: phylogenetic tree reconstruction using sequence data; and dendrograms using biochemical data. The putative identities from different approaches were compared. Results DNA concentration, PCR and sequencing primers, PCR protocol and origin of isolates ("untreated" or "treated") influenced sequence acquisition. The 16S rRNA sequence search method (RDP/BLAST), together with treated/untreated tooth origin, influenced putative strain identity. Thirty-eight percent of "untreated" isolates and half the "treated" isolates were identified with ? 98% sequence homology; the majority of the rest varied between 93%-97%. The 16S-23S intergenic amplicon patterns helped confirm genus designation and strain variation. Biochemical profiles were graded "unacceptable" in 38% of all isolates; a higher proportion was from treated teeth (46%:32%). Biochemical identities matched with 16S rRNA identities to genus level in 72% of isolates, to species level in 45%, and the rest did not match. MICs from agar dilution and E-test were within 2 dilutions in 90% of tests. Frequency distributions of MICs showed NCCLS (2003) breakpoint values to give reliable interpretive categories. Thirty-eight percent of isolates showed resistance, which was more common amongst "treated" isolates (53%:26%). Antibiotic resistance helped confirm or query identities and inform strain variation. Conclusions The 16S rRNA gene sequence, 16S-23S intergenic amplicon patterns and biochemical and MIC profiles gave different perspectives on isolates. Each approach provided accumulative evidence of subspecies strain variation and that strains colonising treated teeth may be influenced by treatment history.