(M-498) Efficacy of Nitric Oxide-Releasing Antibiotic for Treatment of Polymicrobial Bacterial Infections

Introduction: Polymicrobial infections adversely affect indwelling medical devices like vascular catheters. For catheter-induced bloodstream infections, gram-positive S. aureus and gram-negative P. aeruginosa are two of the most common bacterial species. The bacteria adhere to the device surface and are immersed in an extracellular polymeric substance that antimicrobial drugs cannot penetrate. Furthermore, the species interact via horizontal gene transfer, quorum sensing, and extracellular virulence factors, leading to superior drug-evading properties. Polymicrobial biofilm infections are responsible for higher rates of antibiotic resistance (AR), increased hospitalization time, higher healthcare costs, and 2-fold higher patient mortality. An emerging method to fight AR, especially for bacteria within biofilms, is nitric oxide (NO). This work builds off previously published work from our research lab in which Ampicillin, an extended spectrum penicillin, was functionalized with the RSNO donor S-nitroso-N-acetylpenicillamine (SNAP) to form SNAPicillin.1 Herein, SNAPicillin is tested against polymicrobial biofilms, specifically S. aureus and P. aeruginosa. We believe NO-releasing Ampicillin will provide a novel platform for preventing and treating polymicrobial infections in clinical settings.



Materials and Methods: First, the NO release behavior of SNAPicillin was characterized and compared to equimolar SNAP concentrations. NO release was measured via a Sievers Chemiluminescence Nitric Oxide Analyzer 280i (NOA) at physiological conditions (dark and 37 ℃) in the presence of Mueller Hinton broth (MHB) media. NO release values were taken at 0, 2, 8, 12, and 24 h time points. Next, the 24 h antimicrobial efficacy of dual-action SNAPicillin was thoroughly characterized against mono- and polymicrobial S. aureus and P. aeruginosa. For all three culture conditions, the MICs were determined, and time-kill studies at antimicrobial-relevant concentrations were evaluated. SNAPicillin efficacy was compared to equimolar solutions of unbound Ampicillin, unbound NO-donor SNAP, and SNAP mixed with Ampicillin. Bacteria solutions were combined with different drug concentrations ranging from 0.95 nM to 8 mM for all compounds except SNAP, ranging from 0.95 nM to 16 mM. Prepared plates were incubated at 37 ℃ in the dark for 24 h. The in vitro biocompatibility was determined against 3T3 mouse fibroblast cells for the range of concentrations used throughout the antibacterial studies. Lastly, biofilm-dispersing capabilities were characterized against 48 h mono- and polymicrobial biofilms grown on medical-grade polymeric surfaces.



Results, Conclusions, and Discussion: Overall, the conjugated NO-releasing antibiotic exhibits significantly superior antibacterial capabilities as well as improved biocompatibility. Regarding NO release, SNAPicillin demonstrates a dose-dependent profile and, at low concentrations, has higher release than unmodified SNAP and SNAP mixed with Ampicillin. Next, minimum inhibitory concentration (MIC) testing was performed. Standard in vitro MIC testing is completed using a single species of bacteria rather than multiple often fails to predict drug concentrations needed in clinical settings. Therefore, the effect of different concentrations was evaluated against mono- and polymicrobial S. aureus and P. aeruginosa. When investigated in polymicrobial conditions, SNAPicillin (with an MIC₅₀ of 125 µM) was more effective than unbound Ampicillin, unbound NO-donor SNAP, and SNAP mixed with Ampicillin. Time-kill studies were performed to investigate the treatment concentrations further. High concentrations (greater than 2 mM) of SNAP and SNAP mixed with Ampicillin were needed to have bactericidal effects, which were cytotoxic. Biocompatibility testing also revealed that SNAPicillin was less cytotoxic than unbound NO donor SNAP. Binding the NO donor to Ampicillin increased its cytocompatibility. SNAPicillin is biocompatible through 500 µM whereas SNAP and SNAP with Ampicillin reach 70% viability near 10 µM and 100 µM, respectively. Therefore, SNAPicillin emerges as the dominant choice over unbound NO donor. Lastly, mature biofilms were treated with 500 µM SNAPicillin to investigate antibiofilm capabilities while preserving cytocompatibility. SNAPicillin resulted in greater than a 1-log reduction of viable S. aureus and P. aeruginosa in all culture conditions. Ampicillin, NO-donor SNAP, and SNAP mixed with Ampicillin did not result in meaningful reductions. The superior antibacterial activity of SNAPicillin can be attributed to the combined, centralized effects of NO and Ampicillin. NO produces reactive oxygen and nitrogen species that easily diffuse through the bacterial membrane to initiate dispersal, and then NO and Ampicillin can target and kill planktonic bacteria together. In conclusion, this work presents NO-releasing Ampicillin as a platform for preventing and treating polymicrobial infections in clinical settings.



Acknowledgements (Optional): Funding for this work was supported by the National Institutes of Health, USA, grants R01HL134899 and R01HL151473.



References (Optional): 1. Estes Bright, L. M. et al., ACS Appl. Mater. Interfaces 2023, 15 (12), 15185-15194.