Electrochemical Determination of Antibiotic Susceptibilities in Microorganisms
In microbiology, a surprising variety of definitions exist for the term "viability". Life and death are defined according to the technique used to monitor viability: growth (reproduction), membrane integrity, the ability to reduce a membrane-permeable dye, localized enzyme activities, and active respiration form the basis for most of the available viability estimation methods. We have chosen to study respiration because, in principle, changes in respiration can be monitored very rapidly in comparison with traditional growth-based methods of estimating microbial cell viability.
Microbial respiration can be monitored using electrochemical techniques such as chronoamperometry and chronocoulometry. In our experiments, the respiratory cycle activity of E. coli and other organisms is measured in the presence of excess respiratory substrates and ferricyanide, an oxidant that interacts with the same terminal cytochrome oxidase site that molecular oxygen reacts with in aerobic bacteria. The organisms reduce the ferricyanide to its ferro form (iron(III) to iron(II)), and the ferrocyanide is detected electrochemically through its reoxidation to the iron(III) form. The current generated during this process is measured and is related to the concentration of ferrocyanide produced by the bacterial reduction of ferricyanide in the cell suspension.
We have recently discovered that the succinate metabolic pathway, one branch of the microbial respiratory chain, is extremely sensitive to the presence of antibiotic and cytotoxic compounds. Cell culture samples incubated briefly (10-20 min) with effective antibiotic compounds demonstrate markedly lower succinate-dependent respiratory cycle activities than samples incubated in antibiotic-free buffers. A range of organisms can be examined in this manner for their antibiotic susceptibility patterns, including aerobic, anaerobic, Gram-positive and Gram-negative species. We are developing this assay into a clinical diagnostics kit, to replace the much more time-consuming growth-based methods currently employed by clinical testing laboratories.
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