Population diversity and dynamics of Streptococcus mitis, Streptococcus oralis, and Streptococcus infantis in the upper respiratory tracts of adults, determined by a nonculture strategy

Abstract

We reinvestigated the clonal diversity and dynamics of Streptococcus mitis and two other abundant members of the commensal microbiota of the upper respiratory tract, Streptococcus oralis and Streptococcus infantis, to obtain information about the origin of frequently emerging clones in this habitat. A culture-independent method was used, based on cloning and sequencing of PCR amplicons of the housekeeping gene gdh, which shows remarkable, yet species-specific, genetic polymorphism. Samples were collected from all potential ecological niches in the oral cavity and pharynx of two adults on two occasions separated by 2 years. Based on analysis of close to 10,000 sequences, significant diversity was observed in populations of all three species. Fluctuations in the relative proportions of individual clones and species were observed over time. While a few clones dominated, the proportions of most clones were very small. The results show that the frequent turnover of S. mitis, S. oralis, and S. infantis clones observed by cultivation can be explained by fluctuations in the relative proportions of clones, most of which are below the level of detection by the traditional culture technique, possibly combined with loss and acquisition from contacts. These findings provide a platform for understanding the mechanisms that govern the balance within the complex microbiota at mucosal sites and between the microbiota and the mucosal immune system of the host.

FIG. 1.

Distribution of pairwise sequence distances for a fragment of the gdh gene in collections of independent isolates of S. mitis (n = 62), S. oralis (n = 47), and S. infantis (n = 12). By comparison, the range of genetic distances among alleles of housekeeping genes of S. pneumoniae is 0 to 0.012. The lower cutoff distance used to define distinct alleles in this study was 0.01.

FIG. 2.

Consensus Minimum Evolution tree showing phylogenetic associations and genetic diversity of gdh alleles amplified from samples from two individuals. Bootstrap values, based on 500 replicates, are shown for the major branches. Alleles from subjects A and B are designated A and B, respectively. The boldface values that follow the allele designations are the numbers of identical sequences in the sequenced samples of the gene libraries.

FIG. 3.

Rarefaction analysis based on the gdh clone libraries generated from samples collected from subjects A and B. The curves reflecting the observed [Mao Tau (obs.)] and estimated [Chao1 (Estim.)] richness values plotted as a function of the number of sampled clones demonstrate that virtually all genotypes were detected.

FIG. 4.

Dynamics of clones of S. mitis (black circles and lines), S. oralis (red circles and lines), and S. infantis (green circles and lines) detected on two occasions separated by 2 years in samples from all niches within the oral cavity and pharynx of two adult subjects. Circles connected by a line represent a clone detected on both occasions. Other clones were detected on only one occasion.