Anaerobic culture to detect periodontal and caries pathogens

Abstract

Background: Anaerobic culture has been critical in our understanding of the oral microbiotas.

Highlight: Studies in advanced periodontitis in the 1970's revealed microbial complexes that associated with different clinical presentations. Taxonomy studies identified species newly-observed in periodontitis as Aggregatibacter (Actinobacillus) actinomycetemcomitans, Campylobacter (Wolinella) rectus and other Campylobacter species, and Tannerella (Bacteroides) forsythia. Anaerobic culture of initial periodontitis showed overlap in the microbiota with gingivitis, and added Selenomonas noxia and Filifactor alocis as putative periodontal pathogens. Porphyromonas gingivalis and T. forsythia were found to be associated with initial periodontitis in adults. The dominant microbiota of dental caries differs from that of periodontitis. The major cariogenic species are acidogenic and acid tolerant species particularly Streptococcus mutans, and Lactobacillus and Bifidobacterium species. Anaerobic culture of severe early childhood caries revealed a widely diverse microbiota, comparable to that observed using cloning and sequencing. The PCR-based cloning approach, however, underestimated Actinobacteria compared with culture. Only a subset of the caries-associated microbiota was acid tolerant, with different segments of the microbiota cultured on blood agar compared to a low pH acid agar. While the major caries-associated species was S. mutans, a new species, Scardovia wiggsiae, was significantly associated with early childhood caries. Higher counts of S. wiggsiae were also observed in initial white spot carious lesions in adolescents.

Conclusion: In periodontitis and dental caries, anaerobic culture studies of advanced disease provided a comprehensive analysis of the microbiota of these infections. Anaerobic culture highlighted the limitation of PCR with standard primers that underestimate detection of Actinobacteria.

Keywords: 16S rRNA; Anaerobic culture; Caries; Microbiome; Periodontitis.

Figure 1. Gram negative species associated with progressing advanced periodontitis

Samples from deep periodontal pockets were cultured anaerobically and species identified using biochemical tests. Three microbial complexes were observed in different clinical categories. Samples from “Young Adults” or aggressive periodontitis were dominated by A. actinomycetemcomitans and P. gingivalis; from “minimal inflammation” or refractory periodontitis by P. intermedia, T. forsythia and E. corrodens; and from “Moderate inflammation”, chronic periodontitis by P. gingivalis, F. nucleatum and T. forsythia. Data modified from Tanner et al. [4].

Figure 2. P. gingivalis and T. forsythia in (a) early periodontitis and (b) active initial periodontitis in adults

Samples from periodontitis and healthy control sites were analyzed by PCR. a. In the cross sectional analysis of early periodontitis, increased detection frequencies of P. gingivalis (p<0.001) and T. forsythia (p<0.03) were observed in subjects with over 1 site with at least 2 mm periodontal attachment loss. b. In longitudinal analyses, detection frequency of P. gingivalis (p<0.01) was associated with progressing/active periodontitis whereas the association with T. forsythia did not reach significance (p=0.075). Data modified from Tanner et al. [35, 36].

Figure 3. Bacterial phyla from clonal and cultural analyses of early childhood caries and caries-free children

Samples from 80 children were processed by cloning and sequencing [58] and by anaerobic culture [57]. All clones and isolates were identified from 16S rRNA sequences in the Human Oral Microbiome Database (HOMD) [61]. Higher proportions of Actinobacteria were observed in cultural analysis on blood or acid agars compared with the cloning/sequencing analysis. Total bacterial counts on blood agar were 10 times higher than acid agar, and the greatest microbial diversity was observed from anaerobic blood agar isolation. Sequence based analyses by Natalia I Chalmers.

Figure 4. Major acid-tolerant species cultured from early childhood caries and caries-free children

Samples were processed by anaerobic culture and isolates were identified from 16S rRNA sequences in HOMD. The detection frequencies of the major acid tolerant species associated with childhood caries were S. mutans, S. wiggsiae, Parascardovia denticolens and Streptococcus sobrinus. Other acid resistance species were detected more frequently from caries-free children including several non-mutans streptococci, and unnamed Actinomyces species as denoted by the Human Oral Taxon (HOT) number. Data modified from Tanner et al. [57].