4.6 Article

Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Journal

INTERNATIONAL JOURNAL OF ORAL SCIENCE
Volume 3, Issue 2, Pages 90-97

Publisher

NATURE PUBLISHING GROUP
DOI: 10.4248/IJOS11033

Keywords

membrane binding assay; polymerase chain reaction-denaturing gradient gel electrophoresis; coaggregation; Fusobacterium nucleatum; Streptococcus mutans

Funding

  1. Chinese State Scholarship
  2. US National Institutes of Health (NIH) [DE020102, GM95373]

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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 (E 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 E 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.

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