Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution

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 antibiotic 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.

Figure 1. Fleroxacin uptake by Enterobacter aerogenes population. A

. Fluorescence emission spectra of Fle (λ_exc = 283 nm) detected from Glycin-HCl-induced lysis of Enterobacter aerogenes strain EA289. EA289 were incubated with Fle (2 µg/ml) for 30 min in follow conditions: (− ·− ·) Fle; (−−−) Fle + Glu (0.4%); (–) Fle + CCCP (10 µM). B. Comparison of Fle concentration uptake determined from lysated bacteria. Enterobacter aerogenes strains EA289 and EA298 were incubated with Fle at different concentrations (1, 2 or 8 µg/ml) alone or with Glu (0.4%) or CCCP (10 µM).

Figure 2. Fleroxacin uptake by individual Enterobacter aerogenes. A

. Fluorescence spectra of Fle (750 ng/ml) in PBS pH = 7: (−−−) excitation spectrum at λ_emis = 420 nm; (–) emission spectrum at λ_exc = 313 nm. B. Transmission (left), fluorescence (middle) and merge (right) images of Fle (64 µg/ml)-treated EA289 bacteria. Scale bar corresponds to 3 µm. C. Transmission (left), fluorescence (middle) and merge (right) images of Fle (64 µg/ml)- and CCCP- (25 µM) treated EA289 bacteria. Scale bar corresponds to 3 µm. D. Percentage of maximum fluorescence intensity of Fle within single bacteria from fig. 2C. E. Fluorescence intensity detected from Fle channel (λ_exc = 290 nm; DM 300 nm; BP filter 420≤ λ_emis ≤480 nm) from individual EA289 bacteria as a function of treatment conditions: (a) EA289 with no additions; (b) EA289 incubated with Fle (64 µg/ml); (c) EA289 incubated with Glu; (d) EA289 co-incubated with Glu and Fle (64 µg/ml); (e) EA289 incubated with CCCP (25 µM); (f) EA289 co-incubated with CCCP (25 µM) and Fle (64 µg/ml).

Figure 3. Individual bacteria microspectro-fluorescence measurement. A

. Transmission image of Enterobacter aerogenes EA289 bacteria. White arrow indicates on bacterium from which one of the fluorescence spectra of fig. 3B was taken. Scale bar corresponds to 3 µm. B. Fluorescence emission spectra (recorded by UV-VIS microspectrofluorimetry at λ_exc = 290 nm) from two individual Fle-untreated bacteria EA289. Fluorescence emission spectrum (− ·− ·) corresponds to bacterium marked on fig. 3A; (–) spectrum corresponds to bacterium not in the field of view.

Figure 4. Fluorescence intensity of Fleroxacin from Enterobacter aerogenes bacteria.

Mean fluorescence emission spectra (λ_exc = 290 nm) of Fle from Enterobacter aerogenes EA289 bacteria were measured by UV-VIS microspectrofluorimetry. Fle (64 µg/ml)-treated bacteria were co-incubated with follow additives: (–) CCCP (25 µM); (−−−) Glu (0.4%) and (− ·− ·) in the additives absence (EA289 incubated only with Fle).

Figure 5. Comparison of fluorescence intensity and concentration of Fleroxacin in individual bacteria and lysated bacteria. A

. Fluorescence intensity of Fle measured by UV-VIS microspectrofluorimetry from individual EA289 bacteria. EA289 were incubated with Fle (64 µg/ml) only or with Glu (0.4%) or CCCP (25 µM). B. Comparison of Fle concentration uptake determined from lysated bacteria. Enterobacter aerogenes EA289 were incubated with Fle (64 µg/ml) alone or with Glu (0.4%) or CCCP (25 µM).