Abstract
A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.
Highlights
Since the use of antimicrobial agents to combat bacterial infections, the emergence of bacteria that are resistant to antibiotics was observed [1,2,3,4,5,6,7,8]
To investigate the mechanism that controls the intracellular concentration of antibiotics in single resistant isolate and to define the ways to increase normal concentration, we have selected a trifluoroquinolone, flerofloxacin and an E. aerogenes resistant strain from a MDR isolate which overexpresses the broad spectrum AcrAB-TolC efflux pump and its efflux derivative
With the continuing emergence of MDR bacteria, methods have been developed to follow the uptake of antibiotics in bacteria, but none is presently able to quantify the quinolones uptake in single bacteria
Summary
Since the use of antimicrobial agents to combat bacterial infections, the emergence of bacteria that are resistant to antibiotics was observed [1,2,3,4,5,6,7,8]. MDR has became a major health concern worldwide Extensive research has identified three major bacterial strategies involved in the MDR development: (i) the target protection barrier (alteration/modification of the target site); (ii) the enzymatic barrier (degradation of the antibiotic molecule) and (iii) the membrane barrier (acting to limit the required intracellular dose of antibiotic) [9]. The concentration of antibiotics within bacteria decreases under the threshold required for its activity [9,10,11,12,13,14] This reduction can contribute to the development of additional mechanisms of resistance including mutation of antibiotic targets (e.g. in gyrase) or production of enzymes that cleave antibiotics (e.g. ß-lactamases), and reinforces the efficacy of these acquired mechanisms [9,12,13]. In the face of this continuously emerging threat, the development of efficient strategies to circumvent the bacterial MDR responses depends upon understanding the molecular basis of the mechanism controlling the intracellular concentration of antibacterial agents
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