A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria

Abstract

Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (V1–V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. No single region can differentiate among all bacteria; therefore, systematic studies that compare the relative advantage of each region for specific diagnostic goals are needed. We characterized V1–V8 in 110 different bacterial species including common blood borne pathogens, CDC-defined select agents and environmental microflora. Sequence similarity dendrograms were created for hypervariable regions V1–V8, and for selected combinations of regions or short segments within individual hypervariable regions that might be appropriate for DNA probing and real-time PCR. We determined that V1 best differentiated among Staphylococcus aureus and coagulase negative Staphylococcus sp. V2 and V3 were most suitable for distinguishing all bacterial species to the genus level except for closely related enterobacteriaceae. V2 best distinguished among Mycobacterium species and V3 among Haemophilus species. The 58 nucleotides-long V6 could distinguish among most bacterial species except enterobacteriaceae. V6 was also noteworthy for being able to differentiate among all CDC-defined select agents including Bacillus anthracis, which differed from B. cereus by a single polymorphism. V4, V5, V7 and V8 were less useful targets for genus or species-specific probes. The hypervariable sequence-specific dendrograms and the “MEGALIGN” files provided online will be highly useful tools for designing specific probes and primers for molecular assays to detect pathogenic bacteria, including select agents.

Introduction

Sequence analysis of the 16S ribosomal RNA (rRNA) gene has been widely used to identify bacterial species and perform taxonomic studies (Choi et al., 1996, Clarridge, 2004, Munson et al., 2004, Petti et al., 2005, Schmalenberger et al., 2001). Bacterial 16S rRNA genes generally contain nine “hypervariable regions” that demonstrate considerable sequence diversity among different bacterial species and can be used for species identification (Van de Peer et al., 1996). Hypervariable regions are flanked by conserved stretches in most bacteria, enabling PCR amplification of target sequences using universal primers (Baker et al., 2003, Lu et al., 2000, McCabe et al., 1999, Munson et al., 2004). Numerous studies have identified 16S rRNA hypervariable region sequences that identify a single bacterial species or differentiate among a limited number of different species or genera (Becker et al., 2004, Bertilsson et al., 2002, Choi et al., 1996, Clarridge, 2004, Kataoka et al., 1997, Lu et al., 2000, Marchesi et al., 1998, Maynard et al., 2005, Rothman et al., 2002, Yang et al., 2002). Rapid approaches that detect certain species-specific sequences within a single hypervariable region are also in common use (Bertilsson et al., 2002, Stohr et al., 2005, Varma-Basil et al., 2004, Yang et al., 2002).

Unfortunately, 16S rRNA hypervariable regions exhibit different degrees of sequence diversity, and no single hypervariable region is able to distinguish among all bacteria. Molecular diagnostic methods, such as real-time PCR (Selim et al., 2005, Varma-Basil et al., 2004, Yang et al., 2002) or melting temperature analysis (Skow et al., 2005) generally use fluorescent probes that hybridize to relatively short amplicons. This places additional limits on the size of the DNA sequence that can be used for bacterial species identification in these assay formats. Given the increasing importance of real-time PCR to medical diagnostics, it is surprising that few studies have focused on matching short segments of hypervariable 16S rRNA gene regions with the common pathogenic or environmental bacteria that can be differentiated by each segment. Furthermore, no investigations to date have applied this analysis to determine the 16S rRNA gene sequences best suited for identifying both common human pathogens and “select agents” of bioterrorism.

The purpose of the current study was to simplify the development of specific probe or primer-based PCR assays for detecting common bacterial pathogens and select agents. Our aim was to identify the most appropriate 16S rRNA hypervariable region targets for genus and species-specific probes or primers and to experimentally confirm a portion of these results. We used the neighbor joining method to create dendrograms of each 16S rRNA hypervariable segment in 113 available sequences of 110 different bacterial species, including common blood borne pathogens and select agents. Our results demonstrate that it is possible to distinguish among almost all of these pathogens using a small number of hypervariable gene segments. We also provide criteria for selection of specific target sequences based on the desired purposes of the assays and provide recommendations for probes and primer pairs that would be useful in these assays.