Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation

Background In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

[1]  B. Cinquin,et al.  Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes , 2018, Nature Protocols.

[2]  J. Pagés,et al.  Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics. , 2017, Research in microbiology.

[3]  I. V. Leus,et al.  Synergy between Active Efflux and Outer Membrane Diffusion Defines Rules of Antibiotic Permeation into Gram-Negative Bacteria , 2017, mBio.

[4]  B. Cinquin,et al.  Fluoroquinolone structure and translocation flux across bacterial membrane , 2017, Scientific Reports.

[5]  Michelle F. Richter,et al.  Predictive compound accumulation rules yield a broad-spectrum antibiotic , 2017, Nature.

[6]  P. Ruggerone,et al.  Computational modelling of efflux pumps and their inhibitors. , 2017, Essays in biochemistry.

[7]  J. Pagés,et al.  Erratum: Unusual marine unicellular symbiosis with the nitrogen-fixing cyanobacterium UCYN-A , 2017, Nature Microbiology.

[8]  L. Piddock,et al.  How to Measure Export via Bacterial Multidrug Resistance Efflux Pumps , 2016, mBio.

[9]  R. Bonomo,et al.  Overview: Global and Local Impact of Antibiotic Resistance. , 2016, Infectious disease clinics of North America.

[10]  Hao Ge,et al.  Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells , 2016, Molecular cell.

[11]  R. Laxminarayan,et al.  Access to effective antimicrobials: a worldwide challenge , 2016, The Lancet.

[12]  B. Cinquin,et al.  Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level , 2015, Scientific Reports.

[13]  S Gnanakaran,et al.  Permeability Barrier of Gram-Negative Cell Envelopes and Approaches To Bypass It. , 2015, ACS infectious diseases.

[14]  Daniel H. Foil,et al.  A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition , 2015, PloS one.

[15]  S. Tentarelli,et al.  Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria. , 2015, Analytical chemistry.

[16]  Hiroshi Nikaido,et al.  The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria , 2015, Clinical Microbiology Reviews.

[17]  M. Webber,et al.  Molecular mechanisms of antibiotic resistance , 2014, Nature Reviews Microbiology.

[18]  Dean G. Brown,et al.  Trends and exceptions of physical properties on antibacterial activity for Gram-positive and Gram-negative pathogens. , 2014, Journal of medicinal chemistry.

[19]  M. Page,et al.  Discovery and development of new antibacterial agents targeting Gram-negative bacteria in the era of pandrug resistance: is the future promising? , 2014, Current opinion in pharmacology.

[20]  Derek S. Tan,et al.  General Platform for Systematic Quantitative Evaluation of Small-Molecule Permeability in Bacteria , 2014, ACS chemical biology.

[21]  M. Winterhalter,et al.  TRANSLOCATION Project: How to Get Good Drugs into Bad Bugs , 2014, Science Translational Medicine.

[22]  P. Ruggerone,et al.  RND efflux pumps: structural information translated into function and inhibition mechanisms. , 2013, Current topics in medicinal chemistry.

[23]  Ronald N. Jones,et al.  10 x '20 Progress--development of new drugs active against gram-negative bacilli: an update from the Infectious Diseases Society of America. , 2013, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  J. Pagés,et al.  New Peptide-based antimicrobials for tackling drug resistance in bacteria: single-cell fluorescence imaging. , 2013, ACS medicinal chemistry letters.

[25]  J. Pagés,et al.  Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution , 2012, PloS one.

[26]  H. Nikaido,et al.  Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. , 2012, FEMS microbiology reviews.

[27]  J. Handzlik,et al.  Strategies for bypassing the membrane barrier in multidrug resistant Gram‐negative bacteria , 2011, FEBS letters.

[28]  J. Pagés,et al.  Efflux pumps of gram-negative bacteria, a new target for new molecules. , 2010, Current topics in medicinal chemistry.

[29]  T. Meinnel,et al.  New Antibiotic Molecules: Bypassing the Membrane Barrier of Gram Negative Bacteria Increases the Activity of Peptide Deformylase Inhibitors , 2009, PloS one.

[30]  J. Pagés,et al.  Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates , 2009, PloS one.

[31]  M. Winterhalter,et al.  The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria , 2008, Nature Reviews Microbiology.

[32]  J. Pagés,et al.  Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens. , 2008, Current drug targets.

[33]  D. Pompliano,et al.  Drugs for bad bugs: confronting the challenges of antibacterial discovery , 2007, Nature Reviews Drug Discovery.

[34]  H. Nikaido.  Molecular Basis of Bacterial Outer Membrane Permeability Revisited , 2003, Microbiology and Molecular Biology Reviews.

[35]  H. Nikaido,et al.  Prevention of drug access to bacterial targets: permeability barriers and active efflux. , 1994, Science.