Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol· L -1 glycine. Genomic DNA was extracted, subjected to 16S rRNA PCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F. nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

This study aimed to study whether the Sortase A (srtA) gene helps mediate coaggregation and co-adherence between Streptococcus mutans (S. mutans) and other salivary bacteria. S. mutans UA159 and srtA-deficient mutant served as "bait" in classical co-aggregation assays and membrane-based co-adherence assays were used to examine interactions of S. mutans with Fusobacterium nucleatum (F. nucleatum), Streptococcus mitis (S. mitis), Streptococcus gordonii (S. gordonii), Streptococcus sanguis (S. sanguis), Actinomyces naeslundii (A. naeslundii) and Lactobacillus. Co-adherence assays were also performed using unfractionated saliva from healthy individuals. Co-adhering partners of S. mutans were sensitively detected using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Both UA159 and its srtA-deficient mutant bound to F. nucleatum but not to any of the other five salivary bacteria. The srtA-deficient mutant showed lower co-adherence with F.nucleatum. The two S. mutans strains also showed similar co-adherence profiles against unfractionated salivary bacteria, except that UA159 S. mutans but not the srtA-deficient bound to a Neisseria sp. under the same conditions. Deleting srtA reduces the ability of S. mutans to bind to F.nucleatum, but it does not appear to significantly affect the binding profile of S. mutans to bulk salivary bacteria.

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