Kaitlyn Jackson Tiffany Ayoub Julia Pearson Julie Richmond


Introduction: Antimicrobial resistance (AMR) is a global emergency that involves the process of microbes gaining resistance to antimicrobials. A specific example of this is Neisseria (N.) gonorrhoeae gaining resistance to azithromycin, which can be caused by mutations at ribosomal sites. New research suggests that manipulating the bacteria to create a drug efflux pump deficiency will “mask” the effects of AMR mutations when introduced to antibiotic drugs, compared to that of a drug efflux pump proficient environment. This proposal aims to investigate the effects of a drug efflux pump deficient environment compared to a proficient environment on azithromycin drug efficacy in N. gonorrhoeae with an AMR ribosomal mutation. It is hypothesized that AMR mutations can be masked through gene knockout causing MtrCDE efflux pump deficient environments of N. gonorrhoeae.

Methods: Aim 1 consists of inducing a ribosomal mutation that incurs resistance to azithromycin in N. gonorrhoeae strains using CRISPR genome editing in groups A and B. Aim 2 will utilize CRISPR-Cas9 to delete the mtrE gene in N. gonorrhoeae, rendering the MtrCDE efflux pump deficient, creating an efflux pump deficient environment in the bacteria of groups B and D. Aim 3 will determine the difference in susceptibility of N. gonorrhoeae towards azithromycin between efflux pump deficient and proficient environments, using disk diffusion and zone of inhibition.

Results: We expect Group A will have a smaller diameter zone of inhibition exhibiting azithromycin resistance, while Groups B, C and D will result in a larger diameter exhibiting azithromycin susceptibility.

Discussion: We anticipate that Group A will have the smallest diameter zone of inhibition due to the azithromycin resistant mutation and proficient efflux pump environment. We expect Group B will have the largest diameter, demonstrating that N. gonorrhoeae became susceptible to azithromycin with the deficient efflux pump environment masking the azithromycin resistant mutation. Group C (proficient pump) and D (deficient pump) are both wild-type strains therefore we predict they will have larger diameters due to their natural susceptibility to azithromycin.

Conclusion: The proposed experiment could help uncover the molecular mechanisms behind masking, which could consequently provide a gateway towards effective strategies to combat AMR.

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Research Protocol