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

Abstract

Enterobacteriaceae 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 dnaJ 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 dnaJ-based identification of Enterobacteriaceae 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 dnaJ sequence interrogated by this design retains high discriminatory power among Enterobacteriaceae genera and species, with only particular lineages of Shigella sp. and Escherichia coli 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 dnaJ assay to patient specimens enabled unambiguous classification of Enterobacteriaceae to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related Escherichia coli and Shigella species. We expect that this assay will facilitate the accurate molecular identification of species from the Enterobacteriaceae family in a variety of clinical specimens and diagnostic contexts.

The Role of Enterobacteriaceae in Gut Microbiota Dysbiosis in Inflammatory Bowel Diseases

“…The dnaJ gene encodes the heat shock protein 40 and contains regions highly conserved in the Enterobacteriaceae family, suitable for broad-specificity primer design [ 143 ]. Despite this target demonstrating high performance in the unambiguous identification of Enterobacteriaceae species, it remains suboptimal for Enterobacteriaceae discrimination from direct specimens.…”

“…Despite this target demonstrating high performance in the unambiguous identification of Enterobacteriaceae species, it remains suboptimal for Enterobacteriaceae discrimination from direct specimens. In fact, the amplicon generated by polymerase chain reaction (PCR) is relatively long (758 base pair (bp)) for metagenomic-based approaches, which require short-length fragments [ 143 ].…”

Advantages and Limitations of 16S rRNA Next-Generation Sequencing for Pathogen Identification in the Diagnostic Microbiology Laboratory: Perspectives from a Middle-Income Country

“…For these bacteria, additional sequencing of other genes will lead to more accurate species identification. The dnaJ sequence shows superior species identification for Enterobacteriaceae compared to 16S rRNA [ 84 ]. Burkholderia sp.…”

“…As the medical sector moves towards digitization and becomes data-driven, new diagnostic microbiology laboratories, including those in middle-income countries, that use 16SNGS for bacterial identification will have higher technology transition readiness [ 36 , 41 , 96 ]. This happens when diagnostic microbiology ultimately moves towards usage of pathogen whole genome sequence not just for diagnostics, but also as stored information for periodical surveillance, molecular epidemiological studies and public health interventions [ 84 ].…”

Case Report: Comparison of Plasma Metagenomics to Bacterial PCR in a Case of Prosthetic Valve Endocarditis

“…The uncertainties surrounding mNGS assay utility leave it unclear if and when clinicians treating infectious diseases should prioritize mNGS testing over conventional molecular diagnostic tools. While a single class of broad-range PCR (e.g., bacterial) or organism-specific PCRs detect fewer pathogens than mNGS, such assays are reported to be at least as sensitive ( 4 ), less expensive ( 15 , 16 ), and can be performed directly on material with a preponderance of human cells, including infected body fluids, tissues, explanted devices, and, in many cases, formalin-fixed paraffin embedded tissue blocks ( 16 , 17 ).…”