HT‐SPOTi: A Rapid Drug Susceptibility Test (DST) to Evaluate Antibiotic Resistance Profiles and Novel Chemicals for Anti‐Infective Drug Discovery

Cynthia A. Danquah1, Arundhati Maitra1, Simon Gibbons2, Jane Faull3, Sanjib Bhakta3

1 These authors contributed equally, 2 Research Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London, 3 Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, University of London, London
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.8
DOI:  10.1002/9780471729259.mc1708s40
Online Posting Date:  February, 2016
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Antibiotic resistance is one of the major threats to global health and well‐being. The past decade has seen an alarming rise in the evolution and spread of drug‐resistant strains of pathogenic microbes. The emergence of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis and antimicrobial resistance among the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species) as well as fungal pathogens (such as certain species of Candida, Aspergillus, Cryptococcus, and Trichophyton) poses a significant 21st century scientific challenge. With an extremely limited arsenal of efficacious antibiotics, techniques that can (a) identify novel antimicrobials and (b) detect antimicrobial resistance are becoming increasingly important. In this article, we illustrate the HT‐SPOTi, an assay that is principally based on the growth of an organism on agar medium containing a range of different concentrations of drugs or inhibitors. The simple methodology makes this assay ideal for evaluating novel antimicrobial compounds as well as profiling an organism's antibiotic resistance profile. © 2016 by John Wiley & Sons, Inc.

Keywords: drug susceptibility testing (DST); HT‐SPOTi; antimicrobial resistance; whole‐cell phenotypic evaluation; infectious diseases; diagnosis; drug discovery

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: HT‐SPOTi as a Drug Susceptibility Test
  • Basic Protocol 2: HT‐SPOTi for Novel Anti‐Infective Drug Discovery
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: HT‐SPOTi as a Drug Susceptibility Test

  • Sterile Middlebrook 7H10 (MB7H10) agar medium (unit 10.1; Larsen et al., ) with 0.5% (v/v) glycerol, supplemented with 10% (v/v) (OADC, BD Difco, cat. no. 212351)
  • Sterile dimethyl sulfoxide (DMSO), double‐distilled water or any other solvent
  • Working stock of drugs (2 or 5 mg/ml) dissolved in a suitable solvent (see Strategic Planning)
  • 70% ethanol
  • Clinical sample (in this case, sputum)
  • MycoDDR reagent kit (Immy)
  • Sterile Middlebrook 7H9 (MB7H9) broth medium (unit 10.1; Larsen et al., ) with 0.5% (v/v) glycerol and 0.2% (v/v) Tween 80 supplemented with 10% (v/v) OADC; BD Difco, cat. no. 212351)
  • Steam bath/water bath/microwave
  • Microcentrifuge tubes or darkened glass vials for storing drug stocks
  • PCR half‐skirted 96‐well plates
  • Standard 96‐well plates
  • Multidrop Combi Reagent Dispenser (Thermo Scientific)
  • Single‐channel (2 to 20 μl and 100 to 1000 μl) and multichannel (2 to 20 μl) pipettors with sterile tips
  • Centrifuge
  • Spectrophotometer
  • Digital camera
  • Additional reagents and equipment for culture of M. tuberculosis (Larsen et al., )

Basic Protocol 2: HT‐SPOTi for Novel Anti‐Infective Drug Discovery

  • Bacterial sample (M. aurum in this protocol)
  • Additional reagents and equipment for HT‐SPOTi ( protocol 1)
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Literature Cited

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