title: Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries.
abstract: Culturing methods are the primary approach for microbiological analysis of plaque biofilms in rodent models of dental caries. In this study, we developed strategies for the isolation of DNA and RNA from plaque biofilms formed in vivo to analyse the viable bacterial population and gene expression. Plaque biofilm samples from rats were treated with propidium monoazide to isolate DNA from viable cells, and the purified DNA was used to quantify total bacteria and the Streptococcus mutans population via quantitative polymerase chain reaction (qPCR) and specific primers; the same samples were also analysed by counting colony-forming units (CFU). In parallel, RNA was isolated from plaque-biofilm samples (from the same animals) and used for transcriptional analyses via reverse transcription-qPCR. The viable populations of both S. mutans and total bacteria assessed by qPCR were positively correlated with the CFU data (P < 0.001; r > 0.8). However, the qPCR data showed higher bacterial cell counts, particularly for total bacteria (vs. CFU). Moreover, S. mutans proportion in the plaque biofilm determined by qPCR analysis showed strong correlation with incidence of smooth-surface caries (P = 0.0022, r = 0.71). The purified RNAs presented high RNA integrity numbers (> 7), which allowed measurement of the expression of genes that are critical for S. mutans virulence (e.g. gtfB and gtfC). Our data show that the viable microbial population and the gene expression can be analysed simultaneously, providing a global assessment of the infectious aspect of dental caries. Our approach could enhance the value of the current rodent model in further understanding the pathophysiology of this disease and facilitating the exploration of novel anti-caries therapies.
authors.affiliation: Eastman Department of Dentistry and Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14642, USA. marlise_klein@urmc.rochester.edu
authors.forename: M I
authors.initials: MI
authors.lastname: Klein
authors.forename: K M
authors.initials: KM
authors.lastname: Scott-Anne
authors.forename: S
authors.initials: S
authors.lastname: Gregoire
authors.forename: P L
authors.initials: PL
authors.lastname: Rosalen
authors.forename: H
authors.initials: H
authors.lastname: Koo
mesh_terms: D000818:Animals; D058491:Bacterial Load; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms; D004269:DNA, Bacterial / Q000032:analysis; D003731:Dental Caries / Q000382:microbiology*; D003743:Dental Enamel / Q000382:microbiology; D003773:Dental Plaque / Q000382:microbiology; D004195:Disease Models, Animal; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005964:Glucosyltransferases / Q000235:genetics; D050296:Microbial Viability / Q000235:genetics*; D012329:RNA, Bacterial / Q000032:analysis; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013295:Streptococcus mutans / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D005964:Glucosyltransferases