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

Abstract

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.

DETECTION OF VIABLE BACTERIA CELLS BY BIOLUMINESCENCE: A BIOENERGETIC APPROACH 1

“…Indeed, the predicted CCCP effects on the cellular ATP level, were verified in our previous studies of E. coli 0157:H7 (Tu et al 1999) and shown to be a measure of cell viability. In the current study, we have expanded the tests on CCCP effects as an indicator of cell viability to those bacteria listed in Table 1.…”

“…To minimize the potential for outbreaks, the presence of viable pathogens in foods must be rapidly and effectively detected. To this end, we have successfully expanded our previous finding on the influence of CCCP and glucose on the ATP content of E. coli 0157:H7 (Tu et al 1999;Tu et al 2000) to a number of other bacteria. Specifically, the presence of CCCP can decrease the ATP content of viable but not heat-killed cells.…”

“…Recently, we have developed a new approach to ascertain the presence of viable E. coli 0157:H7 in ground beef by an ATP-bioluminescence method (Tu et al 1999(Tu et al , 2000. In this development, E. coli 0157 specific immunomagnetic beads were applied to capture the bacteria prior to lysis by commercially available reagents to release cellular ATP for measurement via luciferinluciferase induced luminescence.…”

“…Potentially, this method may be extended to detect the presence of other viable pathogenic bacteria if suitable antibodies are available for producing specific immunomagnetic beads and the described CCCP effects are common to other bacteria. To determine the potential applicability of the CCCP effects (Tu et al 1999) in other bacteria was the primary objective of the current study. We randomly chose 14 different strains of bacteria (1 1 species from 10 genera) and found that the CCCP treatment caused decreases in cellular ATP content of viable cells of all tested bacteria.…”

ENZYME‐LINKED IMMUNOMAGNETIC CHEMILUMINESCENCE INCORPORATING ANTI‐H7 AND ANTI‐O157 ANTIBODIES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7*

“…1 alternative, screening-only tests (Feng 1992;Hartman et al 1992;Fung 1995;Benoit and Donahue 2003). Many rapid methods have been developed for the quick assessment of food samples for the presumptive presence of bacterial pathogens (Gehring et al 1998;Gehring et al 1999;Tu et al 1999;Crawford et al 2000;Tu et al 2002). However, most rapid methods that presumptively detect the presence of pathogenic bacteria in foods do not confirm/identify the presence of targeted bacteria with the exception of some that rely on nucleic acid assays (Deng and Fratamico 1996;Tims and Lim 2003).…”