Microbiota in dentine caries cultivable on pH-selective agars

Objective: To investigate the acid tolerant microflora at different levels in established dentine caries lesions using solid pH-selective media, as acid stress might be a major selective determinant in dentine caries ecosystems.

Methods: Primary dentine caries lesions (vital teeth, no symptoms) in five patients were sampled aseptically with a rose-bur at three levels: superficially, in the centre and the bottom of the lesion, when it was considered clinically caries free using visual and tactile criteria. Samples were incubated on neutral (blood agar) and pH-selective (Todd-Hewitt agar buffered to pH 4.0, 4.5, 5.0, 5.5) agar. Numbers of colony-forming units (cfu) were determined and colonies were characterized morphologically and with enzymatic- and sugar fermentation tests.

Results: The total numbers of bacteria recovered from the pH-neutral agars did not decrease significantly with lesion depth (median blood agar: 6.3×10³ superficially; 2.2×10³ bottom) whereas cfu recovery from low pH agars decreased with increasing agar acidity. The composition of the aciduric microflora varied both between subjects and between sample sites within the lesions. Gram-positive cocci were most abundant, but with lower pH and deeper sampling sites, the numbers of lactobacilli and other Gram-positive rods increased. Mutans streptococci were found at all sampling depths. S. anginosus, S. constellatus, S. crista, S. gordonii, S. intermedius and S. sanguis were found less frequently. 

Conclusions: The study clearly indicates that many different microorganisms can be recovered on pH 5.5 agars and thus survive in low pH environments. pH 5.5 is quite sufficient to moderately demineralize dentine, and aciduric microorganisms should thus have the potential to contribute to the dentine caries process. Approved by the ethical committee at Lund University. Funded by Faculty grants.