Detection of Viable Bacteria Cells by Bioluminescence: a Bioenergetic Approach 1

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-Iuciferase 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 afrer 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 egects on cellular ATP level were detectable in bacterial suspensions with concentrations ranging from 106 to Id CFU/mL. Upon cold storage, the ATP content, but not the population of viable bacteria, decreased. The ATP content could be paaially restored by the addition of glucose. The ATP content restored 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. Mention of brand or firm names does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. Journal of Rapid Methods and Automation in Microbiology 12 (2004) 207-219. All Righrs Reserved. OCopyright 2004 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 207 208 S.-I. TU, C. WUEY, G. PAOLI, P. IRWIN and A. GEHRING