Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries

Abstract

SUMMARY 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 isolation of DNA and RNA from in vivo formed plaque-biofilms to analyze 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 S. mutans population via qPCR and specific primers; the same samples were also analyzed by colony forming unit (CFU) counting. In parallel, RNA was isolated from plaque-biofilm samples (from same animals) and used for transcriptional analyses via RT-qPCR. The viable population 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 analyzed simultaneously, providing a global assessment of the infectious aspect of the disease 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.

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

“…At the end of 2 weeks, the animals were sacrificed. The jaws were aseptically dissected and were processed for microbiological analysis of each animal's plaque biofilms as described by Klein et al (52). For microbiological analysis, the left jaws were sonicated in 5 ml of 154 mM sterile NaCl solution for plaque biofilm removal.…”

Novel Antibiofilm Chemotherapy Targets Exopolysaccharide Synthesis and Stress Tolerance in Streptococcus mutans To Modulate Virulence Expression In Vivo

“…We also evaluated the expression of selected genes from in vivo RNA samples collected during our animal study. To our knowledge, this is the first time that the transcriptional profiles of S. mutans have been examined in rodent plaque samples (10,25). RNA yields from in vivo samples are typically lower than those obtained from in vitro samples.…”

“…At the end of the experimental period, the animals were sacrificed on the day following the final day of treatment. The jaws were aseptically dissected and processed for RNA extraction (see below) and microbiological analysis of the animal's plaque as detailed in Klein et al (25). For microbiological analysis, the left jaws were sonicated in 5 ml of 154 mM sterile NaCl solution for plaque removal.…”

“…RNA was extracted and purified using protocols optimized for biofilms formed in vitro (10) and in vivo (25). Briefly, disc sets and rodent jaws were incubated in RNALater (Applied Biosystems/Ambion, Austin, TX), and the biofilm material was removed from the discs and teeth.…”

“…We performed RT-qPCR to validate and further characterize specific GOIs selected from microarray data analysis (32). Using methods described previously, the RNAs purified from both in vitro (32) and in vivo (25) samples were used as templates for cDNA synthesis. For a list of the primers used in this study, see Table S1 in the supplemental material.…”