Improved Species-Level Clinical Identification of Enterobacteriaceae through Broad-Range PCR and Sequencing.

represent a diverse and medically important family of bacteria that are difficult to identify to the species level using the standard molecular method of 16S rRNA gene sequencing. Prior work has demonstrated the value of gene sequence analysis in resolving different members of the family. However, existing protocols are not optimized for clinical use and exhibit several limitations in practice. Here, we describe an improved assay for -based identification of which boasts increased broad-range specificity across genera, shorter amplicon sizes that are suitable for use with formalin-fixed or direct patient specimens, and enhanced amplification efficiency and assay sensitivity through the incorporation of locked nucleic acid chemistries. Sequence analysis of public databases indicates that the partial sequence interrogated by this design retains high discriminatory power among genera and species, with only particular lineages of sp. and proving unresolvable. Limits of detection studies using 8 disparate species indicated that amplification was consistently achievable across organisms and allowed robust dideoxynucleotide chain terminator sequencing from as little as 10 genome equivalents of template, depending on the species interrogated. Retrospective application of the assay to patient specimens enabled unambiguous classification of to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related and species. We expect that this assay will facilitate the accurate molecular identification of species from the family in a variety of clinical specimens and diagnostic contexts.