RNA markers enable phenotypic test of antibiotic susceptibility in Neisseria gonorrhoeae after 10 minutes of ciprofloxacin exposure.

Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1-2 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. Rapid phenotypic approaches using quantification of DNA have been demonstrated for fast-growing organisms (e.g. E. coli) but are challenging for slower-growing pathogens such as N. gonorrhoeae. Here, we investigate the potential of RNA signatures to provide phenotypic responses to antibiotics in N. gonorrhoeae that are faster and greater in magnitude compared with DNA. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 min of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB) we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10 min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.