Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Characterization of abscesses from liver, pancreas and kidney using deep sequencing of the 16S rRNA gene.

To characterize the microbial communities in abscess material from liver, pancreas, and kidneys, we performed deep sequencing of the 16S rRNA gene, in addition to cultivation and Sanger based 16S rRNA gene sequencing directly from the samples. Fifty-nine abscess samples were investigated, 38 from liver, 11 from pancreas, 10 from kidney. Using deep sequencing we made 227 bacterial identifications in 52 specimens, as compared to 69 identifications from the 44 specimens positive by culture. Escherichia coli, Enterococcus sp., Klebsiella sp. and Streptococcus sp. were the most common findings, but various anaerobe bacteria also constituted a large part of the microflora and those were frequently not detected by culture. Culture-independent methods like 16S deep sequencing can significantly improve microbiological diagnostics of clinical specimens. They are particularly valuable for complex purulent infections like abdominal abscesses. Therefore, deep sequencing approaches should be considered as a part of the available repertoire in diagnostic hospital laboratories.