PROJECT SUMMARY Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is in the top tier of AMR threats as defined by WHO. NG is responsible for the second most prevalent sexually transmitted infection worldwide, which can cause long-term health consequences. Unfortunately, NG has rapidly developed resistance to all first-line antimicrobials previously recommended for the treatment of gonorrhea, including the last viable antimicrobial, ceftriaxone. As AMR continues to evolve, there is an urgent need for personalized treatment approaches that can spare the use of ceftriaxone to reduce the chances of NG becoming resistant to this antimicrobial. However, this requires clinicians to know drug resistance or susceptibility quickly enough to inform prescription decisions. The Centers for Disease Control and Prevention (CDC) periodically publishes STD treatment guidelines to help clinicians, which are informed by susceptibility data generated by the national CDC Gonococcal Isolate Surveillance Project (GISP). However, determining AMR requires prolonged (24-48 hours) microbiological cultivation in sophisticated laboratory facilities, which has been supplanted by nucleic acid amplification tests (NAAT) for diagnosis of NG infections. The widespread adoption of NAAT has created a critical void in AMR testing, leading to a loss of capability to perform culture of NG in most testing clinics. We propose to develop a rapid AST platform for NG, capable of testing against multiple antimicrobial conditions at scale directly from clinical specimens. Our proposed method involves measuring the earliest transcriptional responses to antimicrobials for rapid AST. We and others have shown that measuring mRNA expression levels following a 10-minute antimicrobial exposure can predict susceptibility well before phenotypic changes in growth are observable. This forms the basis of our proposed single-cell mRNA-enabled AST (sc-mRNA-AST) for NG. To achieve sc-mRNA-AST for NG, our platform will use droplet microfluidics to isolate and analyze individual NG cells from clinical specimens with heterogeneous bacterial flora. We will first identify the most susceptibility- informative transcripts agnostic to mechanisms of resistance against each of 3 NG-relevant antimicrobials. We will use ultrasensitive single-molecule fluorescence spectroscopy to quantify mRNA molecules from individual cells without the need for amplification. Additionally, we will incorporate an assembly-line-like, cascaded microfluidic design into our platform, allowing for numerous sc-mRNA-AST assays against clinically relevant antimicrobial conditions. With an additional 2 minutes per condition, our platform can test 15 antimicrobial conditions to determine susceptibility and minimal inhibition concentrations (MICs) for 3 antimicrobials within 1 hour, directly from clinical urogenital samples. To evaluate the performance of our platform, we will use contrived samples with NG isolates and prospectively co...