EFFECTS OF MEMBRANE PROTONOPHORES ON THE ATP CONTENT OF IMMUNOMAGNETIC BEADS CAPTURED ESCHERICHIA COLI O157:H7

An approach to rapidly detect the presence of viable Escherichia coli O157:H7 is described. Specific immunomagnetic beads were applied to capture the bacteria in different solutions. The captured bacteria were then lysed by commercially available reagents to release cellular ATP which was detected by firefly luciferin-luciferase induced chemiluminescence. The bioenergetic status of the bacteria was adjusted by the addition of glucose, a carbon nutrient source, and carbonyl cyanide meta-chlorophenyl hydrazone (CCCP), a membrane protonophore. The addition of glucose restored oxygen consumption and medium acidification activities and increased the ATP content of harvested bacteria after storage. On the other hand, CCCP enhanced the oxygen consumption and medium acidification but significantly decreased the ATP content. None of the glucose and CCCP effects could be detected with heat-killed and γ-ray irradiated E. coli O157:H7. Thus, immunomagnetic capture of the E. coli followed by testing the bioenergetic responses of captured bacteria would qualitatively determine the presence of viable E. coli O157:H7. When applied, the developed procedure could easily detect the presence of less than one CFU of the E. coli per gram of beef hamburg after a 6-h enrichment at 37C.