Redox Mediated Respiration Measurements on E. coli in the Presence of Antibiotics.

ERTL P; ROBELLO E; BATTAGLINI F; MIKKELSEN SR

Orlando

Congreso; 49th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.; 1999

Bacterial resistance against a variety of common and new antibiotics remains a well-known problem in applied medicine. Rapid detection of antimicrobial susceptibility of administered antibiotics would provide a practical diagnostic tool. Such an assay would also aid in preventing widespread growth of resistant bacterial strains. We have investigated a new type of antimicrobial susceptibility assay that is based on measuring respiration rather than reproduction. The respiratory rate of a model microorganism, E. coli, JM 105, is measured elecrochemically using potassium hexacyanoferrate (III) and other electro-tranfer mediators, as described by others (e.g. Anal. Chem. 1996, 68 ,192).  The artificial electron acceptor is reduced at an appropriate site of the bacterial electron transport chain and hence transports electrons from the cell to an amperometric electrode such as platinum. The current measured is directly related to the respiration rate of the microorganism. Kinetic measurements show that this strain of E. coli has an apparent Km 10.6 mM  (stationary phase) for ferricyanide. Different electrode materials such as gold, carbon and platinum as well as temperature effects are discussed in this study. Cells are preincubated in the absence or presence of antibiotics having different mechanisms of action, and their respiratory behavior is monitored. Several different antibiotics effective against cell wall synthesis, protein synthesis, DNA replication and cytoplasmatic membrane are investigated. Changes in respiratory activity can be correlated with results of control experiments involving the growth of cells on agar media, made selective by the inclusion of antibiotics. In all cases in which decreased respiratory activity is measured, growth inhibition is observed on the agar plate. Validation of the assay using to 14 antibiotics shows 100% specificity, 93% efficiency and 88% sensitivity. Further, amperometric monitoring of respiration offers the opportunity to obtain results within 25 min instead of 24 hours necessary with the standard agar plate method.