Current Microbiology

, Volume 62, Issue 3, pp 758–763 | Cite as

Characterization of Novel Alleles of Toxin Co-Regulated Pilus A Gene (tcpA) from Environmental Isolates of Vibrio cholerae

  • Praveen Kumar
  • Anuja Thulaseedharan
  • Gautam Chowdhury
  • Thandavarayan Ramamurthy
  • Sabu Thomas


Vibrio cholerae is causative agent of life threatening diarrheal disease, cholera. The toxin co-regulated pilus (TCP) is a critical colonization factor of V. cholerae and it also serves as receptor for CTXФ. In this study, we describe nucleotide sequence of four novel alleles of tcpA gene from toxigenic and non-toxigenic V. cholerae isolated from environmental sources. The phylogenetic analysis of tcpA revealed that it is related to tcpA of newly emerged O1 strain and unrelated to tcpA of wild type (classical and El Tor strains). All strains showed variant tcpA and also harbored intact Vibrio Pathogenicity Island (VPI). The expression of all variant alleles was demonstrated by RT-PCR.



This study was supported by an intramural support from Rajiv Gandhi Centre for Biotechnology, Trivandrum, India. Praveen Kumar gratefully acknowledges the Council of Scientific and Industrial Research, Govt. of India for research fellowship. We are grateful to Dr. D. V. Singh, Institute of Life Sciences, Bhubaneswar, India for providing V. cholerae strains VC20 and 569B used in this study. The authors are thankful to Prof. M. Radhakrishna Pillai, Director, RGCB for the facilities provided. The helpful technical assistance of Dr. Manoj P, Technical Officer, RGCB is acknowledged.


  1. 1.
    Boyd EF, Waldor MK (2002) Evolutionary and functional analyses of variants of the toxin-coregulated pilus protein TcpA from toxigenic Vibrio cholerae non-O1/non-O139 serogroup isolates. Microbiology 148:1655–1666PubMedGoogle Scholar
  2. 2.
    Chakraborty S, Mukhopadhyay AK, Bhadra RK et al (2000) Virulence genes in environmental strains of Vibrio cholerae. Appl Environ Microbiol 66:4022–4028PubMedCrossRefGoogle Scholar
  3. 3.
    Cheng NC, Tsai JL, Kuo YS et al (2004) Bacteremic necrotizing fasciitis caused by Vibrio cholerae serogroup O56 in a patient with liver cirrhosis. J Formos Med Assoc 103:935–938PubMedGoogle Scholar
  4. 4.
    Chun J, Huq A, Colwell RR (1999) Analysis of 16S–23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl Environ Microbiol 65:2202–2208PubMedGoogle Scholar
  5. 5.
    Coelho A, Vicente AC, Baptista MA et al (1995) The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints. Res Microbiol 146:671–683PubMedCrossRefGoogle Scholar
  6. 6.
    Faruque SM, Asadulghani, Alim AR et al (1998) Induction of the lysogenic phage encoding cholera toxin in naturally occurring strains of toxigenic Vibrio cholerae O1 and O139. Infect Immun 66:3752–3757PubMedGoogle Scholar
  7. 7.
    Faruque SM, Asadulghani, Saha MN et al (1998) Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential. Infect Immun 66:5819–5825PubMedGoogle Scholar
  8. 8.
    Faruque SM, Mekalanos JJ (2003) Pathogenicity islands and phages in Vibrio cholerae evolution. Trends Microbiol 11:505–510PubMedCrossRefGoogle Scholar
  9. 9.
    Hoshino K, Yamasaki S, Mukhopadhyay AK et al (1998) Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunol Med Microbiol 20:201–207PubMedCrossRefGoogle Scholar
  10. 10.
    Karaolis DK, Lan R, Kaper JB et al (2001) Comparison of Vibrio cholerae pathogenicity islands in sixth and seventh pandemic strains. Infect Immun 69:1947–1952PubMedCrossRefGoogle Scholar
  11. 11.
    Karaolis DK, Somara S, Maneval DR et al (1999) A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria. Nature 399:375–379PubMedCrossRefGoogle Scholar
  12. 12.
    Kirn TJ, Lafferty MJ, Sandoe CM et al (2000) Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae. Mol Microbiol 35:896–910PubMedCrossRefGoogle Scholar
  13. 13.
    Ko WC, Chuang YC, Huang GC et al (1998) Infections due to non-O1 Vibrio cholerae in southern Taiwan: predominance in cirrhotic patients. Clin Infect Dis 27:774–780PubMedCrossRefGoogle Scholar
  14. 14.
    Kumar P, Peter WA, Thomas S (2008) Detection of virulence genes in Vibrio cholerae isolated from aquatic environment in Kerala, Southern India. Appl Biochem Biotechnol 151:256–262PubMedCrossRefGoogle Scholar
  15. 15.
    Kumar P, Wilson PA, Bhai R et al (2009) Characterization of an SXT variant Vibrio cholerae O1 Ogawa isolated from a patient in Trivandrum, India. FEMS Microbiol Lett 303:132–136PubMedCrossRefGoogle Scholar
  16. 16.
    Kumar P, Peter WA, Thomas S (2010) Rapid detection of virulence-associated genes in environmental strains of Vibrio cholerae by multiplex PCR. Curr Microbiol 60:199–202PubMedCrossRefGoogle Scholar
  17. 17.
    Meyer TF, Gibbs CP, Haas R (1990) Variation and control of protein expression in Neisseria. Annu Rev Microbiol 44:451–477PubMedCrossRefGoogle Scholar
  18. 18.
    Morris JG Jr (2003) Cholera and other types of vibriosis: a story of human pandemics and oysters on the half shell. Clin Infect Dis 37:272–280PubMedCrossRefGoogle Scholar
  19. 19.
    Mukhopadhyay AK, Garg S, Mitra R et al (1996) Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993 to 1995) analysis. J Clin Microbiol 34:2537–2543PubMedGoogle Scholar
  20. 20.
    Novais RC, Coelho A, Salles CA et al (1999) Toxin-co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene. FEMS Microbiol Lett 171:49–55PubMedCrossRefGoogle Scholar
  21. 21.
    Restrepo D, Huprikar SS, Van Horn K et al (2006) O1 and non-O1 Vibrio cholerae bacteremia produced by hemolytic strains. Diagn Microbiol Infect Dis 54:145–148PubMedCrossRefGoogle Scholar
  22. 22.
    Rivera IN, Chun J, Huq A, Sack RB et al (2001) Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl Environ Microbiol 67:2421–2429PubMedCrossRefGoogle Scholar
  23. 23.
    Rozemeijer W, Korswagen LA, Voskuyl AE et al (2009) Vibrio cholerae non-O1 non-O139 infection in an immunocompromised patient returning from Spain. Eurosurveillence 14:1–2Google Scholar
  24. 24.
    Sabeena F, Thirivikramji G, Radhakutty G et al (2001) In vitro susceptibility of Vibrio cholerae O1 biotype El Tor strains associated with an outbreak of cholera in Kerala, Southern India. J Antimicrob Chemother 47:361–362PubMedCrossRefGoogle Scholar
  25. 25.
    Safa A, Bhuiyan NA, Murphy D et al (2009) Multilocus genetic analysis reveals that the Australian strains of Vibrio cholerae O1 are similar to the pre-seventh pandemic strains of the El Tor biotype. J Med Microbiol 58:105–111PubMedCrossRefGoogle Scholar
  26. 26.
    Sarkar A, Nandy RK, Nair GB et al (2002) Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae. Infect Immun 70:4735–4742PubMedCrossRefGoogle Scholar
  27. 27.
    Sharma C, Thungapathra M, Ghosh A et al (1998) Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India. J Clin Microbiol 36:756–763PubMedGoogle Scholar
  28. 28.
    Stine OC, Sozhamannan S, Gou Q et al (2000) Phylogeny of Vibrio cholerae based on recA sequence. Infect Immun 68:7180PubMedCrossRefGoogle Scholar
  29. 29.
    Taylor RK, Miller VL, Furlong DB et al (1987) Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc Natl Acad Sci USA 84:2833–2837PubMedCrossRefGoogle Scholar
  30. 30.
    Tamura K, Dudley J, Nei M et al (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596PubMedCrossRefGoogle Scholar
  31. 31.
    Thomas S, Ramachandran D, Remani B et al (2008) Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India. World J Microbiol Biotechnol 24:433–434CrossRefGoogle Scholar
  32. 32.
    Waldor MK, Mekalanos JJ (1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:1910–1914PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Praveen Kumar
    • 1
  • Anuja Thulaseedharan
    • 1
  • Gautam Chowdhury
    • 2
  • Thandavarayan Ramamurthy
    • 2
  • Sabu Thomas
    • 1
  1. 1.Department of Molecular Microbiology, Cholera and Environmental Microbiology LaboratoryRajiv Gandhi Centre for BiotechnologyTrivandrumIndia
  2. 2.National Institute of Cholera and Enteric DiseasesKolkataIndia

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