Interbacterial Adhesion Networks within Early Oral Biofilms of Single Human Hosts

Abstract

Specific interbacterial adhesion, termed coaggregation, is well established for three early colonizers of the plaque biofilm: streptococci, actinomyces, and veillonellae. However, little is known about interactions of other early colonizers and about the extent of interactions within the bacterial community from a single host. To address these gaps, subject-specific culture collections from two individuals were established using an intraoral biofilm retrieval device. Molecular taxonomy (Human Oral Microbe Identification Microarray [HOMIM]) analysis of biofilm samples confirmed the integrity and completeness of the collections. HOMIM analysis verified the isolation of Streptococcus gordonii and S. anginosus from only one subject, as well as isolation of a previously uncultivated streptococcal phylotype from the other subject. Strains representative of clonal diversity within each collection were further characterized. Greater than 70% of these streptococcal strains from each subject coaggregated with at least one other coisolate. One-third of the strains carry a known coaggregation mediator: receptor polysaccharide (RPS). Almost all nonstreptococcal isolates coaggregated with other coisolates. Importantly, certain Rothia strains demonstrated more coaggregations with their coisolated bacteria than did any Streptococcus or Actinomyces strain, and certain Haemophilus isolates participated in twice as many. Confocal microscopy of undisturbed biofilms showed that Rothia and Haemophilus each occur in small multispecies microcolonies. However, in confluent high-biomass regions, Rothia occurred in islands whereas Haemophilus was distributed throughout. Together, the data demonstrate that coaggregation networks within an individual's oral microflora are extensive and that Rothia and Haemophilus can be important initiators of cell-cell interactions in the early biofilm.IMPORTANCE Extensive involvement of specific interbacterial adhesion in dental plaque biofilm formation has been postulated based on in vitro coaggregation between oral bacteria from culture collections that are not subject specific. In the present study, subject-specific culture collections were obtained from early plaque biofilm of two volunteers, and coaggregations within each culture collection were assayed. Coaggregations, several of which involved a coaggregation-mediating cell surface molecule known from well-studied streptococci, were widespread. Unexpectedly, the little-studied organisms Haemophilus and Rothia participated in the greatest numbers of interactions with community members; these two organisms showed different distributions within the undisturbed biofilm. The data show that coaggregation networks encompass most organisms within the biofilm community of each individual, and they indicate prominent participation of organisms such as Haemophilus and Rothia in early plaque biofilm formation.

Keywords: Haemophilus; Rothia; biofilms; coaggregation; oral microbiome; streptococci.

Copyright © 2017 American Society for Microbiology.

Figures

FIG 1

HOMIM analysis of biofilm DNA samples (heat map) and existence of isolates corresponding to particular HOMIM probes (check marks). Red text: probes with a score of ≥2 on every chip in both subjects. Blue text: probes with a score of ≥2 on at least one chip in every visit for both subjects. Green text: probes with a score of ≥2 on at least one chip in every visit for subject 1 only. Brown text: probes with a score of ≥2 on at least one chip in every visit for subject 2 only. The probe identifier (ID) presents the species name followed by the HOT number_probe number.

FIG 2

(a) Immunofluorescence localization of Haemophilus and associated cells in undisturbed biofilms from subject 1. Top panels, 4-h biofilm. Bottom panels, 8-h biofilm. Right panels, zoom of central region in left panels. Scale bar = 10 μm. (b) Immunofluorescence localization of Rothia and associated cells in undisturbed biofilms from subject 1. All images represent the same field of view. Grayscale images show cells stained with DAPI, anti-Rm antibodies, or anti-RPS4 antibodies. The lower right panel shows a red-green-blue (RGB) overlay of grayscale images. Circles mark cells in intimate interaction. (c) Rothia cells had reduced antibody reactivity in 8-h biofilms. All images represent the same field of view. Grayscale images show cells stained with DAPI, anti-Rm antibodies, or anti-RPS4 antibodies, plus an image with the anti-Rm signal enhanced. RGB overlays include an image showing the enhanced anti-Rm channel. Scale bar = 10 μm.

FIG 3

FISH localization of genera in undisturbed 8-h biofilms of subject 1. Asterisks in low-magnification images (upper panels) mark regions shown in high-magnification fields of view (bottom panels). The vast majority of cells were STR-stained streptococci. PAS-stained cells (Haemophilus) were distributed throughout the biofilm (upper panels), as well as in small multispecies clusters (a2). Rothia (ROT probe) and Neisseria (NEI probe) occurred in distinctive packets. GEM-labeled cells (Gemella) were distributed throughout the biofilm (b, b1, and b2) and as single cells (upper right of panel b). Few cells labeled solely with EUB were seen (b2).