Comparison of toxicogenomic effects of sodium hypochlorite, hydrogen peroxide, and peracetic acid on Pseudomonas aeruginosa

Title of Dissertation: Comparison of toxicogenomic effects of sodium hypochlorite, hydrogen peroxide, and peracetic acid on Pseudomonas aeruginosa Degree Candidate: David Andrew Small Degree and year: Doctor of Philosophy, 2007 Dissertation directed by: Professor William E. Bentley Department of Chemical Engineering Oxidative compounds are routinely used as disinfectants on pathogenic bacteria such as Pseudomonas aeruginosa. Previous studies have not been able to reveal the mechanisms by which sodium hypochlorite, hydrogen peroxide, or peracetic acid result in antimicrobial activity. However, determining the transcriptional responses of the entire genome to these oxidative stressors may lead to a better understanding the mechanisms by which these antimicrobial compounds kill. The goal of this study is to determine the global gene expression changes in P. aeruginosa after exposure to a sub-lethal dose of oxidative stress disinfectants (sodium hypochlorite, peracetic acid, and hydrogen peroxide) by means of Affymetrix Pseudomonas GeneChip arrays; to hypothesize some mechanisms for further study; and to develop predictive technologies that could be used to monitor toxicogenomic responses based on microarray research. This is the first simultaneous comparison of microarray data on the P. aeruginosa genome after treatment with sodium hypochlorite, peracetic acid, or hydrogen peroxide to elucidate the mode of action by which these oxidative stressors kill through a genomics perspective. A 20 min exposure time to 4.4 mM sodium hypochlorite, 1 mM hydrogen peroxide, and 0.5 mM peracetic acid sufficiently inhibited cellular growth of P. aeruginosa without resulting in significant cell death allowing for the proper extraction of RNA to be assayed on the microarray. These concentrations and the 20 min exposure time were confirmed using viability testing that included culture plating, cell flow cytometry, and bioluminescence (adenosine triphosphate, ATP) measurements. The microarray data was analyzed using statistical techniques that included scatter plots, analysis of variance (ANOVA), hierarchical clustering, metabolic pathway analysis, and principal component analysis (PCA). The ANOVA and successive metabolic pathway analysis and PCA suggested that the mechanism of action for all of the oxidative stressors was different, except for the increase in cellular protection mechanisms. Sodium hypochlorite exposure resulted in more gene expression changes (upregulation and downregulation) than either peracetic acid exposure or hydrogen peroxide exposure. Sodium hypochlorite exposure revealed a downregulation of oxidative phosphorylation and electron transport genes and the upregulation of organic sulfur transport and metabolism genes. Peracetic acid exposure resulted in an upregulation of glycerol metabolism genes. Hydrogen peroxide exposure resulted in an upregulation of the pyocin synthesis genes. The resulting global transcription profile of the oxidative stressors in this comparison can help identify similarly activated genes and differentially expressed genes on a genome-wide scale to help researchers better understand the mechanisms by which oxidative disinfectants kill bacterial pathogens, such as P. aeruginosa. COMPARISON OF TOXICOGENOMIC EFFECTS OF SODIUM HYPOCHLORITE, HYDROGEN PEROXIDE, AND PERACETIC ACID ON PSEUDOMONAS AERUGINOSA