Iranian Journal of Pathology

Genetic Diversity of ctxB Gene Among Classical O1 and El Tor Strains of Vibrio cholerae using High-Resolution Melting Curve Analysis

Document Type : Original Research

Authors

Mahdieh Mahboobi 1
Reza Mirnejad 2
Hamid Sedighian 1
Vahhab Piranfar 3
Abbas Ali Imani Fooladi 1

1 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Molecular Biology Research Center Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Research and Development Department, Farname Inc, Thornhill, Canada

https://doi.org/10.30699/ijp.2020.127793.2393
Abstract
Background & Objective:  Vibrio cholerae is a natural inhabitant of the environment and causes severe diarrhea ailments (cholera) that affects thousands of people each year worldwide. The most important virulence factors of this pathogen are cholera toxin (cholera toxin CT) and Type IV pili (toxin co-regulated pili TCP), which are encoded within the genome of the filamentous bacteriophage CTXφ. In the present study, according to researchers’ report on genotypic variations of cholera toxin, we evaluated the sequence of ctxB subunit obtained from 100 strains of patients infected with cholera in Iran.
Methods:  The evaluation of genotype variations of cholera toxin was made by high-resolution melting curve analysis illustrating a single nucleotide change. Then, ctxB gene sequencing was performed. Through this analysis and the sequencing process, two standard samples were studied.
Results:  Using serologic tests, all the strains analyzed in this study were identified to be in O1 serotype. However, there have been differences in sequences of ctxB as some were similar to V. cholerae O1 biovar El Tor str. N16961 while others were similar to the genotype of V. cholerae ATCC 14035. We did not observe any particular pattern within the process of mutation.
Conclusion:  The analysis of the new samples of ctxB showed that they were potentially different. It seems that these complicated species were affected by a new genetic exchange of El Tor and classic genotypes.

Keywords

ctxB
Vibrio cholerae
High-resolution melt analysis
Genotyping Techniques

Subjects

  1. Hun Yoon S, Waters CM. Vibrio cholerae. Trends Microbiol. 2019;27(9):806-7. [DOI:10.1016/j.tim.2019.03.005] [PMID]
  2. Mandal S, Mandal MD, Pal NK. Cholera: a great global concern. Asian Pac J Trop Med.. 2011;4(7):573-80. [DOI:10.1016/S1995-7645(11)60149-1]
  3. Albert M. Epidemiology & molecular biology of Vibrio cholerae O139 Bengal. Indian J Med Res. 1996;104:14-27. [PMID]
  4. Muanprasat C, Chatsudthipong V. Cholera: pathophysiology and emerging therapeutic targets. Future Med Chem. 2013;5(7):781-98. [DOI:10.4155/fmc.13.42] [PMID]
  5. Shinoda S, Furumai Y, Katayama S-I, Mizuno T, Miyoshi S-I. Ecological study of pathogenic vibrios in aquatic environments. Biocontrol Sci. 2013;18(1):53-8. [DOI:10.4265/bio.18.53] [PMID]
  6. Alam M, Nusrin S, Islam A, Bhuiyan NA, Rahim N, Delgado G, et al. Cholera between 1991 and 1997 in Mexico was associated with infection by classical, El Tor, and El Tor variants of Vibrio cholerae. J Clin Microbiol. 2010;48(10):3666-74. [DOI:10.1128/JCM.00866-10] [PMID] [PMCID]
  7. Neogi SB, Chowdhury N, Awasthi SP, Asakura M, Okuno K, Mahmud ZH, et al. Novel cholera toxin variant and ToxT regulon in environmental Vibrio mimicus isolates: potential resources for the evolution of Vibrio cholerae hybrid strains. Appl Environ Microbiol. 2019;85(3):e01977-18. [DOI:10.1128/AEM.01977-18] [PMID] [PMCID]
  8. Broeck DV, Horvath C, De Wolf MJ. Vibrio cholerae: cholera toxin. Int J Biochem Cell Biol. 2007;39(10):1771-5. [DOI:10.1016/j.biocel. 2007.07.005] [PMID]
  9. Faruque SM, Albert MJ, Mekalanos JJ. Epidemiology, Genetics, and Ecology of ToxigenicVibrio cholerae. Microbes and Environments. 1998;62(4):1301-14. [DOI:10.1128/MMBR.62.4.1301-1314.1998] [PMID] [PMCID]
  10. Reguera G, Kolter R. Virulence and the environment: a novel role for Vibrio cholerae toxin-coregulated pili in biofilm formation on chitin. J. Bacteriol. 2005;187(10):3551-5. [DOI:10.1128/JB.187.10.3551-3555.2005] [PMID] [PMCID]
  11. Boyd EF, Heilpern AJ, Waldor MK. Molecular analyses of a putative CTXφ precursor and evidence for independent acquisition of distinct CTXφs by toxigenic Vibrio cholerae. J. Bacteriol. 2000;182(19):5530-8. [DOI:10.1128/JB.182.19 .5530-5538.2000] [PMID] [PMCID]
  12. Safa A, Sultana J, Dac Cam P, Mwansa JC, KONG YCR. Vibrio cholerae O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis. 2008. [DOI:10.3201/eid1406.080129] [PMID] [PMCID]
  13. Kaper JB, Bradford HB, Roberts NC, Falkow S. Molecular epidemiology of Vibrio cholerae in the US Gulf Coast. J Clin Microbiol 1982;16(1):129-34. [DOI:10.1128/JCM.16.1.129-134.1982] [PMID] [PMCID]
  14. Nair GB, Qadri F, Holmgren J, Svennerholm A-M, Safa A, Bhuiyan NA, et al. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J of clin microbiol. 2006;44(11):4211-3. [DOI:10.1128/JCM.01304-06] [PMID] [PMCID]
  15. Son MS, Megli CJ, Kovacikova G, Qadri F, Taylor RK. 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. 2011;49(11):3739-49. [DOI:10.1128/JCM.01286-11] [PMID] [PMCID]
  16. Marashi SMA, Rajabnia R, Fooladi AAI, Hojati Z, Moghim S, Esfahani BN. Determination of ctxAB expression in Vibrio cholerae Classical and El Tor strains using Real-Time PCR. International journal of molecular and cellular medicine. 2013;2(1):9.
  17. Tehrani FHE, Moradi M, Ghorbani N. Bacterial Etiology and Antibiotic Resistance Patterns in Neonatal Sepsis in Tehran during 2006-2014. Iran J Pathol. 2017;12(4):356. [PMCID] [DOI:10.30699/IJP.2017.27992]
  18. Nusrin S, Khan GY, Bhuiyan N, Ansaruzzaman M, Hossain M, Safa A, et al. Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh. J clin microbiol. 2004;42(12):5854-6. [DOI:10.1128/JCM.42. 12.5854-5856.2004] [PMID] [PMCID]
  19. van der Stoep N, van Paridon CD, Janssens T, Krenkova P, Stambergova A, Macek M, et al. Diagnostic guidelines for high‐resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96‐well LightScanner™. Human mutation. 2009;30(6):899-909. [DOI:10.1002/humu.21004] [PMID]
  20. Giannopoulos A, Rougkala N, Loupis T, Mantzourani M, Viniou N-A, Variami E, et al. Detection of CALR mutations using high resolution melting curve analysis (HRM-A); application on a large cohort of greek et and mf patients. Mediterr J Hematol Infect Dis. 2019;11(1). [DOI:10.4084/mjhid.2019.009] [PMID] [PMCID]
  21. Cruz TD, Publow AJ, University VC, America USo. qPCR Genotype Determination and Mixture Detection Using High Resolution Melting Curve Analysis of STR Loci. 2019.
  22. Mehrabadi JF, Morsali P, Nejad HR, Fooladi AAI. Detection of toxigenic Vibrio cholerae with new multiplex PCR. J Infect Public Health. 2012;5(3):263-7. [DOI:10.1016/j.jiph.201 2.02.004] [PMID]
  23. Sedighian H, Halabian R, Amani J, Heiat M, Taheri RA, Fooladi AAI. Manufacturing of a novel double-function ssDNA aptamer for sensitive diagnosis and efficient neutralization of SEA. Analytical biochemistry. 2018;548:69-77. [DOI:10.1016/j.ab.2018.02.017] [PMID]
  24. Rad HS, Mousavi SL, Rasooli I, Amani J, Nadooshan MRJ. EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157: H7. Iran J Microbiol. 2013;5(3):244.
  25. Habibian R, Khayyat Khameneie M, Sedighian H, Daneshi F, Bagheri Moghadam M, Mahboobi M. Virulence factor diversity between imipenem resistant and imipenem susceptible strains of Escherichia coli isolated from hospitalized patients with severe urinary tract infections. Biosciences Biotech Res Asia. 2014;11(2):469-77. [DOI:10.13005/bbra/1297]
  26. Xi D, Li Y, Yan J, Li Y, Wang X, Cao B. Small RNA coaR contributes to intestinal colonization in Vibrio cholerae via the two‐component system EnvZ/OmpR. Env Microbiol. 2019.
  27. Taylor C. Mutation scanning using high-resolution melting. Biochemical Society Transactions. 2009;37(2):433. [DOI:10.1042/BST0370433] [PMID]
  28. Fazil MT, Bhanumathi R, Pandey H, Singh D. Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala, southern India. Infection, Genetics and Evolution. 2011;11(2):454-9. [DOI:10.1016/j.meegid.2010.12.008] [PMID]
  29. Choi SY, Lee JH, Jeon Y-S, Lee HR, Kim EJ, Ansaruzzaman M, et al. Multilocus variable-number tandem repeat analysis of Vibrio cholerae O1 El Tor strains harbouring classical toxin B. J med microbiol 2010;59(7):763-9. [DOI:10.1099/jmm.0.017939-0] [PMID]
  30. Le Roux WJ, Van Blerk GN. Use of a high resolution melt real-time polymerase chain reaction (PCR) assay for the environmental monitoring of Vibrio cholerae. 2011.
  31. Jin D, Luo Y, Zhang Z, Fang W, Ye J, Wu F, et al. Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis. FEMS Microbiol Lett. 2012;330(1):72-80. [DOI:10.1111/j.1574-6968.2012.02535.x] [PMID]
  32. Piranfar V, Sharif M, Hashemi M, Vahdati AR, Mirnejad R. Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP. Iran J Basic Med Sci. 2015;18(9):909.
  33. Masjedian Jazi F, Mirnejad R, Piranfar V, Amir Mozafari N, Zahraei Salehi T, Khormali M, et al. Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella. Journal of Laboratory Medicine. 2017;41(6):325-31. [DOI:10.1515/labmed-2017-0030]
Iranian Journal of Pathology
Volume 15, Issue 4
Autumn 2020
Pages 320-325

  • Receive Date 17 May 2020
  • Accept Date 07 June 2020

Home

Guide for Authors

Submit Manuscript

Contact Us