Rapid antibiotic susceptibility testing via electrochemical measurement offerricyanide reduction by Escherichia coli and Clostridium sporogenes

Electrochemical measurement of respiratory chain activity allows rapid and reliable screening for antibiotic susceptibility in microorganisms. Chronoa mperometry and chronocoulometry of suspensions of aerobically cultivated E. coli combined with the non-native oxidant potassium hexacyanoferrate(III) (ferricyanide) yield signals for re oxidation of the reduction product ferr ocyanide that are much smaller if the E. coli has been incubated briefly wi th an effective antibiotic compound. Chronocoulometric results, obtained fo llowing 20-min incubation with antibiotic and 2-min measurement in assay bu ffer containing 50 mM ferricyanide and 10 mM succinate, at +0.50 V vs Ag/Ag Cl at a Pt working electrode, were compared with traditional disk diffusion susceptibility testing, which requires overnight incubation on agar plates ; the results show significantly lower accumulation of ferrocyanide in all cases in which growth inhibition was observed in the disk diffusion assay. A range of antibiotic compounds (13) were examined that possess different m echanisms of action. Quantitative determination of IC50 values for penicill in G and chloramphenicol yielded values that were 100-fold higher than thos e obtained by standard turbidity methods after 10-h incubation; this is lik ely a result of the very brief (10 min) exposure time to the antibiotics, A ddition of 5 mu M 2,6-dichlorophenolindophenol, a hydrophobic electron-tran sfer mediator, to the assay mixture allowed susceptibility testing of a Gra m-positive obligate anaerobe, Clostridium sporogenes. This rapid new assay will facilitate;clinical susceptibility testing, allowing appropriate treat ment virtually as soon as a clinical isolate can be obtained.