Cell Envelope Damage of N. gonorrhoeae after 15-min Beta-Lactam Exposure Enables Rapid Antimicrobial Susceptibility Testing

Abstract

Background: Designing diagnostic tools to perform phenotypic antimicrobial susceptibility testing (AST) at the point-of-care (POC) is a vital step in tackling the global threat of antimicrobial resistance. Gonorrhea infections with resistance to the first-line dual therapy have already emerged, highlighting the impending threat of untreatable gonorrhea. A rapid, phenotypic AST could enable evidence-based (instead of empirical) therapy and improve surveillance. The focus of this work is to develop innovative strategies to measure the phenotypic antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates after just 15–30 min of exposure with an antibiotic. We focused on the duration of the exposure step because it remains the bottleneck for phenotypic AST with fastidious and slow-growing microorganisms. Methods: We selected nucleic acid readout because our longterm goals include building fully integrated POC devices that determine the phenotypic response to antibiotic of a specific pathogen rapidly. We have been developing rapid phenotypic ASTs based on quantification of nucleic-acid concentrations in antibiotic-exposed samples. We describe a new phenotypic AST that does not depend on the speed of DNA replication and applies to beta-lactams penicillin, ceftriaxone, and cefixime acting on clinical isolates of N. gonorrhoeae very rapidly. Results: Our assay had 100% categorical agreement with the gold-standard agar dilution AST when N. gonorrhoeae isolates were incubated for 15-min with penicillin, and 100% categorical agreement when incubated for 30 min with ceftriaxone and cefixime, and steps can be performed within 35 min measured from contrived urine samples exposed to penicillin. Conclusion: By designing techniques which allow us to rapidly determine the antibiotic phenotype, evidence-based prescription of antibiotics will become possible.