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Title: DETECTION OF VIABLE BACTERIA CELLS BY BIOLUMINESCENCE: A BIOENERGETIC APPROACH

Author
item Tu, Shu I
item Wijey, Chandi
item Paoli, George
item Gehring, Andrew
item Irwin, Peter

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/15/2004
Publication Date: 11/15/2004
Citation: Tu, S., Wijey, C., Paoli, G., Gehring, A.G., Irwin, P.L. 2004. Detection of viable bacteria cells by bioluminescence: a bioenergetic approach. Journal of Rapid Methods and Automation in Microbiology. V.12, #3, p. 207-219.

Interpretive Summary: Contamination of viable pathogenic bacteria, e.g., E coli O157:H7 in foods may lead to serious public health concerns. To minimize possible outbreak of food poisoning by pathogenic bacteria, sensitive and rapid detection techniques are needed to call for proper treatments to intervene further distribution of contaminated foods. Current available method for detecting viable pathogenic bacteria is time consuming and has low sensitivity. In this work, we developed a new process to detect viable bacteria. The method involved the applications of chemical-induced physiological responses of live bacteria and a very sensitive bioluminescence detection process. Since the responses are related to cellular metabolic functions, the developed process may be used to ascertain the presence of viable bacteria. The information is useful for researcher and/or engineers to design a process to detect viable and specific pathogens in foods.

Technical Abstract: The cellular ATP content of fourteen freshly harvested bacteria including Bacillus, Campylobacter, Citrobacter, Escherichia, Lactobacillus, Listeria, Pediococcus, Pseudomonas, Salmonella, Streptococcus and Yersinia, was determined using a luciferin-luciferase bioluminescence approach. Incubation of bacteria with carbonyl cyanide meta-chlorophenyl hydrazone (CCCP), a membrane protonophore, prior to cell breakage substantially lowered the bioluminescence signals indicating a decrease of cellular ATP content. The addition of CCCP after cell breakage had no detectable effect on the ATP levels. This differential effect of CCCP was not observed using heat-killed bacteria, i.e., the ATP content was not affected by CCCP incubation. The CCCP effects on cellular ATP level were detectable in bacterial suspensions with 10(3) to 10(6) CFU/mL. Upon cold storage, the ATP content, but not the population of viable bacteria, decreased. The ATP content was partially restored by the addition of glucose. The restoration of ATP content by the addition of glucose was also sensitive to CCCP treatment. These results demonstrated that viable bacterial cells can be differentiated from dead cells by their responses to membrane protonophores.