1438 - Dissecting the Multifaceted Nature of Antibiotic Resistance in Clinical Isolates of Neisseria gonorrhoeae by Natural Transformation

Background: Neisseria gonorrhoeae (NG) causes the sexually transmitted disease gonorrhea. It has developed resistance to every antibiotic introduced for gonorrhea treatment such that NG clinical isolates with multidrug resistance (MDR) are increasingly common. We hypothesize that natural transformation could be used to transfer genetic determinants of antibiotic resistance from drug-resistant NG clinical isolates without pre-knowledge of the genetic determinants to a new background under antibiotic selection to generate isogenic transformants for further characterization.

Methods: Natural transformation, PCR amplification and DNA sequencing, and antibiotic susceptibility testing were used in the studies.

Results: We have validated the hypothesis using genomic DNA from an MDR including ciprofloxacin-resistant NG clinical isolate as a donor and a ciprofloxacin-susceptible NG isolate as a recipient under the selective pressure of ciprofloxacin. This led to a series of transformants that contain single or multiple genetic resistance determinants being generated depending on the resistance levels and transformation frequencies. Antibiotic susceptibility testing and genetic characterization of the transformants allowed us to (i) identify ciprofloxacin resistance determinants including efflux mutation mtrR_-79 and target mutations GyrA S91F D95G and ParC D86N, (ii) quantify the contribution of each genetic determinant responsible for the ciprofloxacin resistance and (iii) regenerate the ciprofloxacin resistance phenotype of the donor isolate to detect multiple paths of possible resistance development for ciprofloxacin. Furthermore, we also validated the hypothesis with a novel antibiotic gepotidacin to identify pre-existing genetic determinants contributing to varying susceptibility to this antibiotic. Finally, we envision that, along with whole genome sequencing, natural transformation could be used to identify and quantify novel genetic resistance determinants to current or novel antibiotics in drug-resistant NG clinical isolates.

Conclusion: We demonstrated the utility of natural transformation in dissecting the multifaceted nature of antibiotic resistance in NG clinical isolates.