1440 - The Genetic Basis for a Neisseria gonorrhoeae Clinical Isolate That Contains mtrR_-79 Mutation But Is Highly Susceptible to Antibiotics Effluxed by the Mtr Pump System

Background: Neisseria gonorrhoeae (NG) possesses multiple drug efflux systems that play an important role in evading antibiotics in the treatment for gonorrhea and in helping this pathogen to evade innate antimicrobial defenses during infection. The mtrR_-79 and mtr₁₂₀ mutations in the promoter region between mtrR and mtrCDE are common mutations contributing to overexpression of the MtrCDE efflux pump resulting in increased efflux to multiple antibiotics including macrolides, β-lactams and tetracycline. However, we found a NG clinical isolate that contains the mtrR_-79 mutation but is highly susceptible to antibiotics effluxed by the MtrCDE pump system.

Methods: PCR amplification, DNA sequencing and natural transformation were used to investigate the genetic basis responsible for the increased susceptibility by this isolate.

Results: We amplified by PCR the individual genes of mtrCDE, respectively, from this susceptible isolate as well as a NG isolate that contains the mtrR_-79 mutation with increased efflux; there was no difference in the size of PCR products between the susceptible isolate and the isolate with increased efflux, indicating there was no large deletion/insertion in these genes. DNA sequence analysis of mtrCDE revealed the susceptible isolate also contained a loss-of-function mutation ΔGC from a 6 GC repeat GCGCGCGCGCGC in mtrC resulting in MtrC A117 frameshift predicted to produce a truncated MtrC protein that results in a low efflux phenotype. Natural transformation of the susceptible isolate with a wild type mtrC and selection with ciprofloxacin generated transformants that corrected the ΔGC mutation and restored the increased efflux phenotype.

Conclusion: Our results indicate that genotyping of mtrR and the promoter region between mtrR and mtrCDE is insufficient to predict increased efflux phenotype and provide direct evidence that NG isolates with elevated efflux is able to genetically revert to low efflux via loss-of-function mutations in the coding region of the efflux pump genes.