Relative quantification of the recA gene for antibiotic susceptibility testing in response to ciprofloxacin for pathogens of concern
Antibiotic 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 antibiotic susceptibility tests (AST) are dependent on organism growth rates resulting in prolonged diagnostic answers for slow growing organisms. Changes in the cellular transcriptome can be instantaneous in the presence of stressors such as antibiotic pressure. Here, we demonstrate that relative quantification of the recA gene in response to breakpoint concentrations of ciprofloxacin is an indicator of pathogen susceptibly. For this purpose, we developed seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, as response and housekeeping genes respectively, for biothreat and ESKAPE pathogens. Surrogate biothreat agents Y. pestis and B. anthracis saw increases in relative recA gene expression, independent of growth rate after 15 minutes of exposure to ciprofloxacin with maximal expression seen after 60 minutes. Treatment with doxycycline also demonstrated an increase in relative recA fold changes compared to no treatment controls. Final evaluation of all seven duplex assays tested across 125 strains, including Tier 1 pathogens, from broth culture demonstrated an overall categorical agreement compared to gold standard microbroth dilution of 97.56% with major error (ME) rates of 1.59% and very major error (VME) rates of 3.23%. Testing on pathogen strains commonly associated with urinary tract infections in contrived clinical sample sets demonstrated an overall categorical agreement of 95.8% with a ME rate of 0.0% and VME rate of 7.69%. These data indicate that relative quantification of a single highly conserved gene accurately predicts susceptibility for multiple bacterial species in response to ciprofloxacin. Overall design: RNA sequencing was performed on bacterial samples exposed to breakpoint concentrations of ciprofloxacin across time. RNA sequencing analysis was compared between resistant and suceptable strains as well as compared across multple organisms including B. anthracis and Y. pestis.