Project Summary/Abstract Gonorrhea is one of the most common sexually transmitted infections (STIs) globally, and prior to the COVID- 19 pandemic, the second most common reportable infection in the U.S. In the past decade, there have been significant increases in gonorrhea among men who have sex with men (MSM) in the US, with a 375% increase observed from 2010–2018. At the same time, the percentages of antimicrobial-resistant Neisseria gonorrhoeae (AMR-NG) continues to increase markedly in MSM. Despite the significant increase in the prevalence of AMR- NG, the drivers and reservoirs of AMR-NG especially for oropharyngeal gonorrhea commonly diagnosed in MSM and responsible for all the reported cases of failed ceftriaxone treatment are yet to be fully elucidated. The oropharynx is reservoir for AMR-NG because it harbors a large microbiota and repertoire of AMR genes. The overall goal of this proposal is to describe comprehensively the oropharyngeal resistome of men who are a priority population for STI and HIV control and prevention. In Aim 1, we will use culturomics to identify NG and commensal bacteria, determine their AMR profiles, and use the phenotypic AMR data for stringent quality control of our oropharyngeal resistome profiling (Aim 2). In Aim 2, we will use an unbiased culture-independent metagenomic sequencing and comprehensive bioinformatic analysis to determine the oropharyngeal resistome of MSM and MSW. In Aim 3, we will sequence 150 commensal Neisseria spp. paired with NG isolates obtained from the same patient when isolated (Aim 1) to demonstrate that they share the same genetic markers of AMR. We will define the genetic mechanisms of AMR among multidrug-resistant (MDR) commensal Neisseria spp., and investigate the relative frequency of in vitro transfer of AMR from 10 different species of commensal Neisseria to fully susceptible and genetically diverse recipient strains of NG. We expect that the results from these studies will provide urgently needed data on the repertoire of AMR genes and genetic determinants facilitating the evolution of NG resistance to antibiotics recommended for gonorrhea treatment including ceftriaxone, the last remaining highly effective treatment option for gonorrhea, and paving the way for improved proactive identification and mitigation of emerging AMR threats. Our data will immediately contribute to the surveillance of AMR threats in MSM disproportionately impacted by gonorrhea and HIV epidemics, and guide efforts to develop novel antimicrobials for combatting MDR-NG. Public Health Significance. This study will define the magnitude and diversity AMR in the human oropharynx, an anatomic site thought to play a central role in the emergence of AMR-NG, and will provide new insights into which bacteria and under what circumstances NG may acquire AMR. This knowledge will provide important insights into how AMR-NG develops, critically important information in developing strategies to contain the threat of AMR-NG...