PROJECT SUMMARY Antibiotic resistance in Neisseria gonorrhoeae is an urgent threat to public health, with the emergence of strains resistant to current clinically-approved antibiotics, and infection rates simultaneously rising (up 92% since 2009). Substantial effort has been spent on identifying resistance mechanisms to antibiotic therapies currently circulating within the gonococcal population. However, these surveys often overlook a known source of resistance for gonococci – the commensal Neisseria. Neisseria promiscuously share DNA with one another, which has been demonstrated to facilitate rapid adaptation and evolutionary change. For example, the majority of reduced susceptibility to both azithromycin and ceftriaxone in N. gonorrhoeae populations has been acquired though horizontal transfer and subsequent spread of commensal alleles. As resistance has been shown to be directly selected for in commensals after antibiotic usage in patients, these species will always be a persistent threat for resistance donation to N. gonorrhoeae. Thus, the overall goal of this project is to profile the Neisseria resistome, or the total collection of antibiotic resistance mechanisms available to members within the genus. The knowledge gained from this proposal may directly support the development of improved surveillance methods that may aid future resistance outbreak prevention as a result of DNA donation. In Aim 1, we will use experimental evolution of 10 commensal isolates to 6 antibiotics to nominate novel mechanisms of resistance that may be available to pathogenic members of the genus. This aim builds on our published work demonstrating the feasibility of rapid resistance evolution (< 20 days) in commensals, and the ease of identifying the underlying casual mutations via whole genome sequencing. In Aim 2, we will use population genomics to determine the genomic regions that demonstrate evidence of horizontal transfer from commensals to gonococci. This aim will also provide evidence supporting the possibility of novel derived mutations uncovered in Aim 1 and those that have previously been described, in being maintained in natural populations. In Aim 3, we will validate novel resistance mechanisms and assess their likelihood of transfer to N. gonorrhoeae. Transformations of candidate resistance mutations into ancestral commensal strains (not evolved) and a piliated N. gonorrhoeae will together 1) prove causality of novel mutations, and 2) provide experimental evidence of the likelihood of transfer into a Neisseria pathogen. The PI has demonstrated that these experiments are feasible at the undergraduate and graduate student levels, in both the research laboratory and in the classroom, and will provide exceptional educational and training opportunities for RIT students.

PROJECT NARRATIVE Neisseria gonorrhoeae has repeatedly been demonstrated to acquire antibiotic resistance from close commensal relatives within the genus yet the mutations encoding resistance that are harbored by commensals have been infrequently described. This limits our ability to predict novel resistance mechanisms that can be readily accessed across species boundaries and rapidly disseminated within pathogenic Neisseria populations. This project aims to nominate commensal resistance mechanisms that are direct threats to public health by testing the alleles that are at risk of transfer from commensals to pathogen using a combination of computational and experimental approaches.