Relative quantification of the recA gene for antimicrobial susceptibility testing in response to ciprofloxacin for pathogens of concern.

Antimicrobial resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antimicrobial susceptibility tests (AST) rely on organism growth rates that result in prolonged time-to-answer for slow growing organisms. Changes in the cellular transcriptome can be rapid in the presence of stressors such as antibiotic pressure, providing the opportunity to develop AST towards transcriptomic signatures. Here, we show that relative quantification of the recA gene is an indicator of pathogen susceptibly when select species are challenged with relevant concentrations of ciprofloxacin. We demonstrate that ciprofloxacin susceptible strains of Y. pestis and B. anthracis have significant increases in relative recA gene expression after 15 min of exposure while resistant strains show no significant differences. Building upon this data, we designed and optimized seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, response and housekeeping genes, respectively, for multiple biothreat and ESKAPE pathogens. Final evaluation of all seven duplex assays tested against 124 ciprofloxacin susceptible and resistant strains, including Tier 1 pathogens, demonstrated an overall categorical agreement compared to microbroth dilution of 97% using a defined cutoff. Testing pathogen strains commonly associated with urinary tract infections in contrived mock sample sets demonstrated an overall categorical agreement of 96%. These data indicate relative quantification of a single highly conserved gene accurately determines susceptibility for multiple bacterial species in response to ciprofloxacin.