Rapid Molecular Phenotypic Antimicrobial Susceptibility Test for Neisseria gonorrhoeae Based on Propidium Monoazide Viability PCR

Abstract

Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments, leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof-of-concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for the susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90 and 75% susceptible and resistant strains, respectively, to ceftriaxone and 66.7 and 83.3% susceptible and resistant strains, respectively, to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy within 30 min, a significant improvement compared to the conventional method which could take multiple days.

Rapid and Accurate Quantification of ViableLactobacillusCells in Infant Formula by Flow Cytometry Combined with Propidium Monoazide and Signal-Enhanced Fluorescence In Situ Hybridization

“…PMA is a DNA-binding red dye that enters only membrane-compromised cells. It can be photolyzed by bright visible light to produce a nitrene molecule that can form a covalent cross-link with the PMA-bound DNA. , Excessive PMA can be inactivated by exposure to bright light, thus ensuring that the results of the subsequent experiments are not affected. The staining of the fluorescently labeled oligonucleotide probe using the SEFISH technique was divided into two steps: fixation and hybridization.…”