Use of Multiple-Displacement Amplification and Checkerboard DNA-DNA Hybridization To Examine the Microbiota of Endodontic Infections

Abstract

ABSTRACT Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) μg after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10 4 in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10 4 in amplified samples was 51.2 ± 2.2 and in nonamplified samples was 14.5 ± 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris , and A. baumannii .