Molecular microbial diagnosis

Abstract

Based on culture-independent molecular methods using sequence analysis of 16S rRNA genes, most of the predominant bacterial species in the oral cavity have been identified (1,2,3,6,14,17,20,26,28,30,36,39,40,46,48). Collectively speaking, there are about 620 predominant oral bacterial species, of which about 35% have not yet been cultivated in vitro. Whereas there is considerable debate as to what defines a bacterial species (53,54), the 16S rRNA approach defines, in general, a species (or more precisely a phylotype) as 16S rRNA gene sequences of strains or cloned 16S rRNA inserts with >98.5% similarity. Consequently, strain or clone sequences with <98.5% similarity to previously defined phylotypes are considered representatives of new phylotypes (for details, see the Human Oral Microbiome Database [HOMD, http://www.homd.org/]). More recently, based on evaluations of 35,000 16S rRNA gene sequences from about 400 patients, it is estimated that there are approximately 1,200 predominant species (unpublished data, HOMD). In contrast, investigators in a recent study utilizing pyrosequencing analysis of 197,600 sequences derived from the oral cavity have suggested that the microbial diversity of the human oral microbiome is much greater with approximately 19,000 phylotypes (27). However, most of these phylotypes are those found at very low densities. Bacterial species colonizing the surfaces of the human oral cavity are known to play an important role in oral health and disease and thus a rapid and accurate means of identification is crucial. Traditionally, identification has been based on phenotypic and biochemical criteria, including microscopy, biochemical reactivity, growth conditions, dye and immunofluorescence staining, bacterial end product analysis, cell membrane composition, and antibiotic sensitivity. However, these tests are labor-intensive and costly, providing sometimes inconsistent results that make identification rather tentative. This can be due to strain variation within a species. More recently, molecular DNA-based techniques have been used to identify bacteria directly from clinical samples circumventing the need for in vitro cultivation. Results from these studies implicate specific bacterial species or complexes of species that are associated with oral health and disease. In general, there are 3 main categories of molecular microbial analyses to consider, namely 1) PCR-based methods, including single target PCR, multiplex PCR and quantitative PCR; 2) DNA-DNA hybridization methods such as in situ hybridization, checkerboard hybridization, and 16S rRNA-based microarrays; and 3) sequencing methods including the latest, next-generation sequencing (NGS) techniques, such as pyrosequencing, real-time single-molecule DNA sequencing, and nanopore-based sequencing. The focus of this review is to describe the current status of these DNA-based, culture-independent methods for use or potential use in molecular microbial diagnosis.