Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage

This is the final version. Available from Nature Research via the DOI in this record. All relevant data are available in this article, its Supplementary Information and Supplementary Data files (the source data behind the graphs in the paper is contained in Supplementary Data 2), except for original image files, which are available from the corresponding author upon reasonable request.Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.Biotechnology & Biological Sciences Research Council (BBSRC)Engineering and Physical Sciences Research Council (EPSRC)Wellcome TrustMedical Research Council (MRC)Gordon and Betty Moore Foundation Marine Microbiology InitiativeNHMRCNHMRCNHMRCThe Royal SocietyChina Scholarship Council (CSC)University of QueenslandInstitute for Molecular BiosciencesQUEXGW

Fast bacterial growth reduces antibiotic accumulation and efficacy

This is the final version. Available from eLife Sciences Publications via the DOI in this record. Data availability: All data acquired for this study are presented within the manuscript, the supplementary information and the source data files.Phenotypic variations between individual microbial cells play a key role in the resistance of microbial pathogens to pharmacotherapies. Nevertheless, little is known about cell individuality in antibiotic accumulation. Here, we hypothesise that phenotypic diversification can be driven by fundamental cell-to-cell differences in drug transport rates. To test this hypothesis, we employed microfluidics-based single-cell microscopy, libraries of fluorescent antibiotic probes and mathematical modelling. This approach allowed us to rapidly identify phenotypic variants that avoid antibiotic accumulation within populations of Escherichia coli, Pseudomonas aeruginosa, Burkholderia cenocepacia, and Staphylococcus aureus. Crucially, we found that fast growing phenotypic variants avoid macrolide accumulation and survive treatment without genetic mutations. These findings are in contrast with the current consensus that cellular dormancy and slow metabolism underlie bacterial survival to antibiotics. Our results also show that fast growing variants display significantly higher expression of ribosomal promoters before drug treatment compared to slow growing variants. Drug-free active ribosomes facilitate essential cellular processes in these fast-growing variants, including efflux that can reduce macrolide accumulation. We used this new knowledge to eradicate variants that displayed low antibiotic accumulation through the chemical manipulation of their outer membrane inspiring new avenues to overcome current antibiotic treatment failures.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology & Biological Sciences Research Council (BBSRC)Medical Research CouncilGordon and Betty Moore FoundationEngineering and Physical Sciences Research CouncilWellcome TrustRoyal SocietyH2020 Marie Skłodowska-Curie Action

Fluorescent Antibiotics: New Research Tools to Fight Antibiotic Resistance

Better understanding how multidrug-resistant (MDR) bacteria can evade current and novel antibiotics requires a better understanding of the chemical biology of antibiotic action. This necessitates using new tools and techniques to advance our knowledge of bacterial responses to antibiotics, ideally in live cells in real time, to selectively investigate bacterial growth, division, metabolism, and resistance in response to antibiotic challenge. In this review, we discuss the preparation and biological evaluation of fluorescent antibiotics, focussing on how these reporters and assay methods can help elucidate resistance mechanisms. We also examine the potential utility of such probes for real-time in vivo diagnosis of infections

Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (Delta tolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment

A template guided approach to generating cell permeable inhibitors of Staphylococcus aureus biotin protein ligase

Abstract not availableAshleigh S. Paparella, Jiage Feng, Beatriz Blanco-Rodriguez, Zikai Feng, Wanida Phetsang, Mark A.T. Blaskovich, Matthew A. Cooper, Grant W. Booker, Steven W. Polyak, Andrew D. Abel

An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants

An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to retain antimicrobial activity. Using facile 'click' chemistry, this versatile intermediate can be further functionalised to explore antimicrobial structure-activity relationships or conjugated to fluorophores to generate fluorescent probes. Such probes can report bacteria and their location in a sample in real time. Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bacteria using confocal microscopy

A template guided approach to generating cell permeable inhibitors of Staphylococcus aureus biotin protein ligase

Inhibitors of biotin protein ligase (BPL) are novel antimicrobial compounds with the potential to treat infections caused by bacteria resistant to current antibiotics. A novel BPL inhibitor (12, K-i; 1.4 mu M) was synthesized from biotin acetylene and an azide-functionalized analogue of fluorescent nitrobenzofurazan by Cu(I) catalysed cycloaddition and also by template guided synthesis using wild-type BPL from Staphylococcus aureus. LC/HRMS-based detection provides improved sensitivity over previous reports using a mutant BPL, with demonstrated applicability to other BPLs. Super-imaging fluorescence microscopy demonstrated the accumulation of 12 in the cytoplasm of S. aureus, but not Escherichia coli. This novel fluorescent probe can be used to gain new insights into the mechanism of uptake, efflux and metabolism of BPL inhibitors in S. aureus. (C) 2017 Elsevier Ltd. All rights reserved

Structural characterization of α-amino acid complexes of molybdates: A spectroscopic and DFT study

The reactions of L-enantiopure alpha-amino acids (aaH) with Na2MoO4 led to the high-yield isolation of Mo(VI) complexes of general formula Mo2O4(OH)(4)(aaH). A similar outcome was achieved by using (NH4)(6)Mo7O24 in the place of Na2MoO4. Solid-state IR and NMR spectroscopy indicated the presence of cis-MoO2 units linked by a zwitterionic amino acid ligand, via a bidentate bridging coordination through the carboxylate group. Thus possible dinuclear and polynuclear structures are proposed on the basis of DFT calculations