Project Summary Carbapenem-resistant Pseudomonas aeruginosa (CR-P. aeruginosa) contributes to significant mortality in hospitalized patients. Only four β-lactam agents with activity against CR-P. aeruginosa are available in the US, of which ceftolozane-tazobactam (C-T) is the most frequently used to treat CR-P. aeruginosa. Pre-clinical investigations indicate C-T had activity against >90% of P. aeruginosa isolates; after clinical use, reports of P. aeruginosa that initially tested susceptible to C-T but became resistant during therapy emerged. Our preliminary data indicate that amongst 28 consecutive patients infected with CR-P. aeruginosa with isolates initially susceptible to C-T, 50% of patients had subsequent P. aeruginosa isolates with > 4-fold increase in C-T minimum inhibitory concentrations (MICs) up to 30 days after C-T exposure. We identified genetic markers contributing to resistance through whole genome sequencing (WGS) – notably mutations in the ampC-ampR region and in PBP3. Alarmingly, 86% of P. aeruginosa isolates initially susceptible to another novel antibiotic, ceftazidime-avibactam (C-A), exhibited resistance to C-A after C-T exposure - in the absence of C-A exposure. This is concerning as the emergence of resistance to one novel agent could eliminate the few remaining treatment options. We explored modifiable risk factors that could reduce the frequency of C-T resistance and found that 30% of patients who received C-T over 1 hour had subsequent C-T resistant isolates, whereas no patients who received the drug over 3 hours developed resistant isolates. We would like to repeat this exploratory work and include experimental validation studies in a cohort of 260 patients across seven hospitals who have paired P. aeruginosa clinical isolates stored at -80°C and were prescribed C-T. In Aim 1, WGS will identify the genetic loci associated with C-T resistance comparing paired CR-P. aeruginosa isolates from the same patient before and up to 30 days after C-T exposure. Validation will occur through cloning and site-directed mutagenesis work. Broth microdilution will determine susceptibility testing results for the three other β-lactams with activity against CR-P. aeruginosa to quantify the emergence of cross-resistance. In Aim 2, we will capitalize on diverse C-T administration strategies at participating sites and develop a machine learning derived decision tree to inform clinicians how to prescribe C-T most effectively to reduce the likelihood of acquired resistance. The following are some risk factors that will be investigated for potential inclusion: (1) infusion of C-T over 3 hours, (2) high-dose C-T, (3) combination antibiotic therapy, (4) dialysis dependency, (5) source of infection and source control, (6) number of days of C-T exposure. This work will (1) inform future drug development by identifying areas of the bacterial chromosome highly prone to resistance and in need of additional reinforcement and (2) identify modifiable r...