Genetic Screens and Biochemical Assays to Characterize Vibrio cholerae O1 Biotypes: Classical and El Tor

Mike S. Son1, Ronald K. Taylor1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6A.2
DOI:  10.1002/9780471729259.mc06a02s22
Online Posting Date:  August, 2011
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Abstract

Vibrio cholerae serogroup O1 has two biotypes, classical and El Tor, the latter of which has displaced the prior and has been the causative agent for the ongoing seventh pandemic. However, reports since 2001 have identified clinical isolates of El Tor that have classical O1 biotype genetic and phenotypic characteristics. These El Tor variants have been emerging in clinical settings with increased frequency, including the 2010 cholera outbreak in Haiti. The emergence of El Tor variants warrants the proper and timely identification of clinical (or environmental) isolates' biotype. This unit describes some quick and simple genetic screens and phenotypic assays (biochemical characterization), to be performed simultaneously, commonly used to distinguish biotype and initiate characterization of any clinical (or environmental) isolates of Vibrio cholerae O1. Curr. Protoc. Microbiol. 22:6A.2.1‐6A.2.17. © 2011 by John Wiley & Sons, Inc.

Keywords: Vibrio cholerae O1; classical biotype; El Tor biotype; genetic screens; biochemical characterization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR‐Based Genetic Screens Using tcpA and ctxB
  • Basic Protocol 2: Polymyxin B Resistance Assay
  • Basic Protocol 3: Citrate Metabolism
  • Basic Protocol 4: Casein Hydrolysis Protease Assay on Milk Agar Plates
  • Basic Protocol 5: Hemolysis Assay on Blood Agar Plates
  • Basic Protocol 6: Motility Assay
  • Basic Protocol 7: Voges‐Proskauer (VP) Assay
  • Basic Protocol 8: Biofilm Production Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR‐Based Genetic Screens Using tcpA and ctxB

  Materials
  • tcpA/ctxB forward and reverse primers
  • Overnight cultures
  • QIAprep Spin Miniprep kit (Qiagen, cat. no. 27104) containing:
    • EB buffer
  • 1.25 mM dNTP mix (NEB, cat. no. N0446S; see recipe)
  • 10× Pfu Turbo Cx reaction buffer (Stratagene, cat. no. 600410‐52)
  • Pfu Turbo Cx HotStart DNA polymerase (Agilent, cat. no. 600412‐51)
  • 1% agarose gel in 1× TBE (see reciperecipes)
  • 6× DNA gel loading dye (see recipe)
  • QIAquick PCR Purification Kit (Qiagen, cat. no. 28104)
  • NanoDrop ND100 spectrophotometer (NanoDrop, model no. ND100)
  • 200‐µl sterile Thin‐walled PCR tubes (Corning, cat. no. 6571)
  • PCR thermal cycler (to hold 200‐µl PCR tubes) with heated lid

Basic Protocol 2: Polymyxin B Resistance Assay

  Materials
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • Polymyxin B (Sigma‐Aldrich, cat. no. P1004‐100MU)
  • Double‐distilled water
  • V. cholerae strains
  • Sterile flat toothpicks
  • 37°C incubator
  • Marker

Basic Protocol 3: Citrate Metabolism

  Materials
  • 100 × 15–mm minimal citrate medium plates (see recipe)
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • 37°C incubator
  • Patching grid (see Fig. )
  • Sterile flat‐ended toothpicks

Basic Protocol 4: Casein Hydrolysis Protease Assay on Milk Agar Plates

  Materials
  • 100 × 15–mm milk agar plates (see recipe)
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • 37°C incubator
  • Marker
  • Sterile flat toothpicks

Basic Protocol 5: Hemolysis Assay on Blood Agar Plates

  Materials
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • Blood agar plates (Fisher, cat. no. R01200)
  • 37°C incubator
  • Sterile wooden applicator sticks (Fisher, cat. no. 01‐340)

Basic Protocol 6: Motility Assay

  Materials
  • 100 × 15–mm motility agar plates (see recipe)
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • Bunsen burner
  • Inoculating wire stab (Fisher, cat. no. 22‐032‐099)
  • Standard measuring ruler

Basic Protocol 7: Voges‐Proskauer (VP) Assay

  Materials
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • MR‐VP broth (see recipe)
  • 5% (w/v) α‐naphthol (see recipe)
  • 40% (w/v) potassium hydroxide (see recipe)
  • 37°C incubator
  • Vortex

Basic Protocol 8: Biofilm Production Assay

  Materials
  • V. cholerae strains
  • Luria‐Bertani (LB) agar plates (standard size of 100 mm × 15 mm)
  • 0.01% (w/v) crystal violet (see recipe)
  • 50% glacial acetic acid (see recipe)
  • Victor2 multilabel counter (PerkinElmer, cat. no. 1420‐018)
  • 37° and 30°C incubators
  • 96‐well U‐bottom flexible plate (BD, cat. no. 353911) and Lid (BD, cat. no. 353913)
  • Paper towels
  • 200‐µl 12‐channel multichannel pipettor
  • 96‐well flat‐bottom microtiter plate (Dynex Technologies, cat. no. 3455)
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Figures

Videos

Literature Cited

Literature Cited
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