Development and Application of a Rapid 23S rRNA‐Targeted Reverse Transcription Quantitative PCR System for the Detection and Quantification of Enterobacterales

ABSTRACT Bacteria belonging to the family Enterobacteriaceae occur as commensals or pathogens in humans. The increasing prevalence of multidrug‐resistant bacteria has raised global concern. Following the reclassification of Enterobacteriaceae species into the order Enterobacterales in 2016. However, no quantification method has been reported that is designed and validated in the context of the current order‐level taxonomy of Enterobacterales . Therefore, we aimed to develop a novel reverse transcription quantitative PCR (RT‐qPCR) system for the quantification of Enterobacterales based on 23S rRNA. The system was used to quantify Enterobacterales species, and its applicability was evaluated using fecal samples from 99 patients with gastrointestinal cancer and 10 healthy volunteers. Furthermore, we performed a proof‐of‐concept evaluation of its utility for rapid assessment of antimicrobial susceptibility in carbapenem‐resistant Enterobacterales (CRE). The system exhibited high analytical sensitivity and enabled quantification of diverse Enterobacterales spp., including newly classified species, with theoretical detection limits of 10 3 cells/g feces and 1 cell/mL blood. In healthy volunteers, intestinal Enterobacterales were dominated by Escherichia coli and Enterobacter , whereas preoperative patients with gastrointestinal cancer exhibited greater diversity. In vitro, viable CRE cell counts determined by the RT‐qPCR system 2 h after antibiotic treatment correlated well with culture‐based susceptibility results obtained after 24 h, suggesting the feasibility of rapid antimicrobial susceptibility assessment within 6 h. The developed 23S rRNA RT‐qPCR system provides a sensitive approach for quantifying Enterobacterales within the current taxonomic framework and may facilitate the rapid assessment of antimicrobial activity against CRE. Clinical Trial Registration There is no clinical trial registration.