Use of bioluminescent Escherichia coli O157:H7 to investigate chlorine dioxide mechanism of bacterial inactivation
Abstract
Chlorine dioxide (ClO2) is a common oxidizing broad-spectrum antimicrobial agent that does not have a well-defined mechanism of bacterial inactivation. Previous reports attributed the ClO2 mechanism of inactivation to oxidation of proteins or inhibition of protein synthesis. This work investigated the bacterial mechanism of inactivation of ClO 2 by using a lux based bioluminescent derivative of E. coli O157:H7 and an online biosensor. E. coli O157:H7-lux bioluminescence responded to changes in metabolic activity, cellular physiology and environmental conditions, allowing cellular activity to be monitored in real-time in situ. The bioluminescent response of E. coli O157:H7-lux was monitored during different environmental conditions and exposures to ClO2. When E. coli O157:H7-lux was treated with a 0.2 mg/L sublethal dose of ClO2, the bioluminescence decreased rapidly within the first six seconds, from 1.5 x 107 RLU/sec to 4.5 x 10 6 RLU/sec. After the quick decrease, bioluminescence rapidly recovered to approximately initial light levels. This recovery indicated the initial ClO2 reaction with the bacterial cell was reversible. Protein oxidation as the inactivation mechanism was disproven when E. coli O157:H7-lux recovered bioluminescence post-ClO2 treatment in the presence of chloramphenicol, an antibiotic that inhibits protein synthesis. To confirm this finding, ClO2 bioluminescent response patterns were compared to sodium hypochlorite, which has a known mechanism of protein oxidation, and found to be markedly different. An E. coli firefly luciferase (luc) bioluminescent strain was constructed in order to study the effects of ClO2 on cellular ATP levels. ATP concentration was not affected by sublethal exposures to ClO2. E. coli O157:H7-lux was then treated with 2,4-dinitrophenol and ClO2 in the presence of trimethoprim. These results indicated ClO2 reacted with reducing agents within the cell such as NADH and NADPH. This reaction was studied in vitro and found to be highly reactive with a second-order rate constant of 3.9 x 106 M-1 s-1. The NAD+ generated from this reaction was found to be biologically active using the enzyme glucose dehydrogenase. Collectively, these results show the initial step in the bacterial mechanism of inactivation of ClO 2 is oxidation of reducing agents.
Degree
Ph.D.
Advisors
Applegate, Purdue University.
Subject Area
Microbiology
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