Genotypic and Phenotypic Assays to Distinguish Vibrio cholerae Biotype

  • Kyle D. Brumfield
  • Bailey M. Carignan
  • Mike S. SonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1839)


Vibrio cholerae is a motile gram-negative bacterium found in brackish water and the etiological agent of the fecal-oral disease cholera. Classical and El Tor are two main biotypes that make up the V. cholerae O1 serogroup, which each display unique genotypic and phenotypic characteristics that allow for reliable biotype characterization. While treatment for cholera is much the same despite the causative strain’s biotype, such classification can be imperative for laboratory experiments and may have broader impacts in the biomedical field. In the early 2000s, clinical isolates were identified that contained genotypic and phenotypic traits from both biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to be more complicated than previous single-assay identification. Herein, we describe a series of PCR-based genetic screens (tcpA and ctxB) and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and Voges-Proskauer). Together, these assays are used for reliable biotype characterization of V. cholerae clinical (and environmental) isolates.

Key words

Vibrio cholerae Biotypes Classical El Tor El Tor variants Biochemical assays 



Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103506 and a Plymouth State University Research Advisory Council (RAC) grant for MSS.


  1. 1.
    Shimada T, Arakawa E, Itoh K et al (1994) Extended serotyping scheme for Vibrio cholerae. Curr Microbiol 28:175–178CrossRefGoogle Scholar
  2. 2.
    Yamai S, Okitsu T, Shimada T et al (1997) Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin and addition of novel serogroups. J Jpn Infect Dis 71:1037–1045Google Scholar
  3. 3.
    Karaolis D, Lan R, Reeves P et al (1995) The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-O1 Vibrio cholerae. J Bacteriol 177:3191–3198CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Albert J, Siddique A, Islam M et al (1993) Large outbreak of clinical cholera due to V. cholerae non-O1 in Bangladesh. Lancet 341:704CrossRefPubMedGoogle Scholar
  5. 5.
    Kaper J, Bradford H, Roberts N et al (1982) Molecular epidemiology of Vibrio cholerae in the U.S. Gulf Coast. J Clin Microbiol 16:129–134PubMedPubMedCentralGoogle Scholar
  6. 6.
    Karaolis D, Johnson J, Bailey C et al (1998) A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. Proc Natl Acad Sci U S A 95:31343139CrossRefGoogle Scholar
  7. 7.
    Karaolis D, Lan R, Kaper J et al (2001) Comparison of Vibrio cholerae pathogenicity islands in sixth and seventh pandemic strains. Infect Immun 69:1947–1952CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nair G, Faruque S, Bhuiyan N et al (2002) New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh. J Clin Microbiol 40:3296–3299CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nursin S, Khan G, Bhuiyan N, Ansaruzzaman M et al (2004) Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic. J Clin Microbiol 42(12):5854–5856CrossRefGoogle Scholar
  10. 10.
    Ghosh-Banerjee J, Senoh M, Takahashi T et al (2010) Cholera toxin production by the El Tor variant of Vibrio cholerae O1 compared to the prototype El Tor and classical biotypes. J Clin Microbiol 48:4283–4286CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Son M, Megli C, Kovacikova G et al (2011) Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J Clin Microbiol 49:3739–3749CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lan R, Reeves P (2002) Pandemic spread of cholera: genetic diversity and relationships within the seventh pandemic clone of Vibrio cholerae determined by amplified fragment length polymorphism. J Clin Microbiol 40:172–181CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Nair G, Safa A, Bhuyian N et al (2006) Isolation of Vibrio cholerae O1 strains similar to pre-seventh pandemic El Tor strains during an outbreak of gastrointestinal disease in an island resort in Fiji. J Med Microbiol 55:1559–1562CrossRefPubMedGoogle Scholar
  14. 14.
    Ansaruzzaman M, Bhuyian N, Nair G, et al. The Mozambique Cholera Vaccine Demonstration Project Coordination Group (2004) Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis 10:2057–2059CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ansaruzzaman M, Bhuyian N, Safa A et al (2007) Genetic diversity of El Tor strains of Vibrio cholerae O1 with hybrid traits isolated from Bangladesh and Mozambique. Int J Med Microbiol 297:443–449CrossRefPubMedGoogle Scholar
  16. 16.
    Safa A, Bhuyian N, Nursin S et al (2006) Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes. J Med Microbiol 55:1563–1569CrossRefPubMedGoogle Scholar
  17. 17.
    Barua D (1992) History of cholera. In: Barua D, Greenough W III (eds) Cholera. Plenum Publishing Corporation, New York, NYCrossRefGoogle Scholar
  18. 18.
    Morales R, Delgado G, Cravioto A (2008) Population genetics of Vibrio cholerae. In: Faruque S, Nair G (eds) Vibrio cholerae—genomics and molecular biology. Caister Academic Press, NorfolkGoogle Scholar
  19. 19.
    Sack D, Sack R, Nair G et al (2004) Cholera. Lancet 363:223–233CrossRefPubMedGoogle Scholar
  20. 20.
    Taylor R, Miller V, Furlong D et al (1987) Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera-toxin. Proc Natl Acad Sci U S A 84:2833–2837CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Herrington D, Hall R, Losonsky G et al (1988) Toxin, toxin coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med 168:1487–1492CrossRefPubMedGoogle Scholar
  22. 22.
    Kovacikova G, Skorupski K (2002) Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter. Mol Microbiol 46:1135–1147CrossRefPubMedGoogle Scholar
  23. 23.
    Jobling M, Holmes R (1997) Characterization of hapR, a positive regulator of the Vibrio cholerae HA protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol Microbiol 26:1023–1034CrossRefPubMedGoogle Scholar
  24. 24.
    Wang Y, Wang H, Cui Z et al (2011) The prevalence of functional quorum-sensing systems in recently emerged Vibrio cholerae toxigenic strains. Environ Microbiol Rep 3(2):218–222CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Chuang L, Cheng Y, Yang C (2012) URPD: a specific product primer design tool. BMC Res Notes 5:306CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Kyle D. Brumfield
    • 1
  • Bailey M. Carignan
    • 1
  • Mike S. Son
    • 1
    Email author
  1. 1.Department of Biological SciencesPlymouth State UniversityPlymouthUSA

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