Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

The synchrotron at CERN might be close to revealing the Higgs boson but the one at SOLEIL seems to have revealed the concentration of antibiotics that accumulate within drug treated bacterial cells. Perhaps not as exciting but nevertheless important.

One way in which cells acquire resistance to antibiotics is by lowering its intracellular concentration either by active efflux or by preventing its accumulation by altering membrane permeability. However, what was lacking was a way to directly demonstrate this due to the inability to measure levels of antibiotic within single cells. Most methods that attempted to measure intracellular antibiotic concentrations, could achieve this only for a population of cells. Also the methods were invasive- employing cell lysis to release antibiotic and then measure their levels using their natural fluorescence; or they would involve modification of the antibiotic which could affect its efficacy. A recent study published in PLoS one, employed an improvement on an existing fluorimetric method by using synchrotron radiation D(deep)UV imaging and DUV microspectroscopy to measure concentration of antibiotics within single cells.

The authors manage to measure fluorescence levels as well as spectra of certain antibiotics within single cells, taking into account the large amounts of fluorescence given by other cellular components like NADH, proteins rich in tyrosine and tryptophan, etc. Using their technique, they manage to demonstrate that a multi-drug resistant(MDR) strain of Enterobacter aerogenes does not accumulate fluoroquinolone antibiotics within cells, while an antibiotic sensitive derivative of this strain does. Since a non specific drug efflux pump is responsible for the MDR in the particular strain, they thus demonstrate directly that the efflux pump was responsible for decreasing the levels of antibiotic within the cell, a fact hitherto taken for granted (the sensitive strain lacks the gene encoding this pump).

The authors argue, that by being able to reduce the level of antibiotic within cells, such populations of cells (which would otherwise have been killed) can now achieve increased resistance by mutating. Hence it is essential to dissect the molecular mechanisms required to reduce antibiotic levels in bacterial cells. In turn, to study these mechanisms it would be useful to have a direct assay that measures intracellular antibiotic concentration in a non invasive manner.

The possibility of detecting antibiotics within cells, opens avenues for addressing more difficult questions (like what is the subcellular localization of the antibiotic in the cell?) and is bound to make significant impact on the research on clinical antimicrobials.

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