The antibacterial role of exogenous nitric oxide gas

Experimental evidence indicates that nitric oxide (NO) has a role in the host defense mechanism, and may have antibacterial characteristics. The goal of this thesis is to explore the feasibility of exogenous or gaseous NO (gNO) as a potential therapeutic antibacterial agent by confirming that gNO is bacteriocidal to bacteria and to elucidate the mechanism by which it is bacteriocidal. A successful bacteriocidal effect is defined as a decrease in bacteria of greater than 3 loglO cfu/ml. In order to test the bacteriocidal or bacteriostatic effects of gNO, we built and validated a closed in vitro environment for delivery of a variety of gases including NO. Under controlled in vitro conditions, we determined the time required to effectively induce a bacteriocidal effect with 200 parts per million gNO on a representative collection of Gram-positive and Gram-negative strains of bacteria associated with clinical infection. Further, Candida albicans, Methicillin Resistant Staphylococcus aureus (MRSA), a particularly resistant strain of Pseudomonas aeruginosa from a cystic fibrosis patient, Group B Streptococcus, and Mycobacterium smegmatis were also included to determine if yeasts, a multi-drug resistant strain of bacteria and actinomycetes had a similar response. These bacteria represented a variety of bacterial pathogens that contribute to both respiratory and wound infections. A series of in vitro studies was performed using wild-type M. smegmatis, isogenic mycothiol (MSH) mutants, and complemented mutants to show that MSH was essential for the protection of M. smegmatis against oxidative and nitrosative stressors. The results showed that 200 ppm gNO was completely bacteriocidal for all bacteria tested. Without exception, every bacterial strain challenged with 200 ppm gNO had at least a 3 loglO reduction in cfu/ml. The bacterial death curve was characterized by a latent period when it appeared that the bacteria were unaffected. This latent period was followed by an abrupt death of all cells. Mycobacteria typically had a longer latent period compared to other organisms, suggesting that mycobacteria have a highly active mechanism that protects the cell from gNO cytotoxicity. Eukaryotic cell and Gram negative bacteria synthesize glutathione (GSH), which detoxifies NO and reactive oxygen species. Some bacteria that do not make GSH produce other low-molecular weight thiols to protect themselves from oxidative damage. M smegmatis, which does not make GSH, produces MSH, which may act analogously to GSH in protecting the cell from NO and other electirophilic molecules. Mycothiol was shown to protect M. smegmatis against gNO damage and, to achieve this, an intact MSH biosynthesis pathway was required. The studies described here also determined that continuous exposure to a high level of gNO (200 ppm) was not required for the observed bacteriocidal effect. A cyclic regimen of 160 ppm gNO for 30 minutes, followed by 3.5 hours of 20 ppm gNO also resulted in a complete bacteriocidal effect. These studies begin to lay the foundation for potential use of gNO as a therapeutic agent against bacterial infections. This thesis will advance the current body of knowledge and confirm that NO is a cytocidal, non-organism specific, broad-spectrum antibacterial agent. Data from these studies will add insight into the detoxification pathways that drug-resistant bacteria use to fight antibiotic therapies. This new validated exposure chamber methodology may provide opportunities for further research on agents that block these pathways. Ultimately, the knowledge derived from this thesis might provide a rationale for further exploration of gNO as a non-organism specific, non-antibiotic based, treatment for pulmonary and wound infections.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat