Serial measurement of nitrite and xanthine oxidase (XO) to assess antimicrobial pharmacodynamics (PD) and treatment efficacy in urinary tract infections (UTIs): A proof-of-concept for personalized antimicrobial therapy

Introduction: UTI is a common indication for antimicrobial therapy. Biomarkers specific to the causative organism, such as nitrite, are often employed in UTI diagnosis through qualitative detection methods such as dipsticks. XO was found to be a highly sensitive predictor for UTIs, but limited data exist on the utility of these markers as an indicator of organism response to antimicrobial treatment. Here, we explore the feasibility of monitoring nitrite levels in UTIs as a means of assessing the response to antimicrobial therapy, thereby advancing in vivo measures of exposure-response. Methods: A 0.5*103 colony forming units (CFU/mL) solution of E. coli strain 25922 (susceptible to amoxicillin) and E. coli strain 11954 (beta-lactamase producing) was prepared in artificial urine (AU) containing 20mM nitrate. Before incubating at 37°C, a plastic lid was placed on the wells to keep oxygen from reaching the samples. Absorbance at 600nm (OD600) was measured in 30min intervals to monitor bacterial growth, and in 12-hour intervals at 548nm after adding Griess reagent to assess nitrite. XO activity was assessed with UV absorption using a Sigma-Kit. To explore the effect of bacterial killing on nitrite, 164mg/L amoxicillin was added to AU containing E. coli after 8 hours of growth. Nitrite and XO were assessed at 0h, 12, 24, and 48h. Results: Nitrite concentrations in NO3-containing gradually increase until E. coli reaches the stationary phase as seen in a plateau of OD600 and CFUs for both strains. For the susceptible E.Coli strain, a simultaneous decline in both colony-forming units (CFUs) as seen when plated on McHinton plates and nitrite levels was observed after adding supra-MIC doses of amoxicillin (164mg/L). CFU in the resistant E. coli strain stays high after the addition of amoxicillin, and nitrite stays significantly higher than in the susceptible strain. No differences in XO levels was observed over time and between the different strains. Discussion: Growth of E. coli in urine triggers increased nitrate-reducing activity and the formation of nitrite. Consequently, the measurement of nitrite in urine has the potential to serve as a means of monitoring bacterial growth. High concentrations of amoxicillin result in a significant reduction in nitrite levels for a susceptible E. coli strain only. This leads us to the conclusion that longitudinal monitoring of nitrite could provide an in-vivo measure of antimicrobial PD in the context of urinary tract infections (UTIs). Elevated XO levels in UTI patients could be due to elevated immune response rather than increased number of bacteria in urine alone. Conclusion: Serial measurement of nitrite during UTIs could enable real-time monitoring of pharmacodynamics and treatment success. The origin of elevated XO in UTI samples needs to be further explored.