Molecular characterization of Vibrio cholerae O1 strains circulating in Assam: a north eastern state of India
,
Abstract
Background and Objectives: Information on the genetic epidemiology of cholera in Assam, a northeastern state of India is lacking despite cholera being a major public health problem. The study aimed to determine the virulence genes and genes encoding antibiotic resistance in Vibrio cholerae isolates and to determine the prevalent genotypes based on the presence or absence of the virulence genes and ctxB genotype.
Materials and Methods: Twenty-five V. cholerae strains were subjected to conventional biotyping and serotyping followed by multiplex PCR to detect ctxA, ctxB, zot, ace, O1rfb, tcpA, ompU, ompW, rtxC, hly and toxR and antibiotic resistance genes. Cholera toxin B (ctxB) gene was amplified followed by sequencing.
Results: All the V. cholerae O1 isolates were El Tor Ogawa and showed the presence of the core toxin region representing the genome of the filamentous bacteriophage CTXø. The complete cassette of virulence genes was seen in 48% of the isolates which was the predominant genotype. All the isolates possessed amino acid sequences identical to the El Tor ctxB subunit of genotype 3. sulII gene was detected in 68% of the isolates, dfrA1 in 88%, strB in 48% and SXT gene was detected in 36% of the isolates.
Conclusion: Toxigenic V. cholerae O1 El Tor Ogawa strains of ctxB genotype 3 carrying a large pool of virulence genes are prevailing in Assam. Presence of a transmissible genetic element SXT in 36% of the strains is of major concern as it indicates the emergence of multiple drug resistance among the V. cholerae isolates.
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3. Siriphap A, Leekitcharoenphon P, Kaas RS, Theethakaew C, Aarestrup FM, Sutheinkul O, et al. Characterization and genetic variation of Vibrio cholerae isolated from clinical and environmental sources in Thailand. PLoS One 2017;12(1): e0169324.
4. Chatterjee SN, Chaudhuri K. Lipopolysaccharides of Vibrio cholerae. I. physical and chemical characterization. Biochim Biophys Acta 2003; 1639:65-79.
5. Garg P, Nandy RK, Chaudhury P, Roy Chowdhury NR, De K, Ramamurthy T, et al. Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India. J Clin Microbiol 2000; 38:4249-4253.
6. Bhandari M, Jennison AV, Rathnayake IU, Huygens F. Evolution, distribution and genetics of atypical Vibrio cholerae - a review. Infect Genet Evol 2021; 89:104726.
7. Mishra A, Taneja N, Sharma RK, Kumar R, Sharma NC, Sharma M. Amplified fragment length polymorphism of clinical and environmental Vibrio cholerae from a freshwater environment in a cholera-endemic area, India. BMC Infect Dis 2011; 11:249.
8. Kumar P, Peter WA, Thomas S. Detection of virulence genes in Vibrio cholerae isolated from aquatic environment in Kerala, southern India. Appl Biochem Biotechnol 2008; 151:256-262.
9. Nandi B, Nandy RK, Mukhopadhyay S, Nair GB, Shimada T, Ghose AC. Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clin Microbiol 2000; 38:4145-4151.
10. Goel AK, Jiang SC. Association of heavy rainfall on genotypic diversity in V. cholerae isolates from an outbreak in India. Int J Microbiol 2011; 2011:230597.
11. Koelle K, Pascual M, Yunus M. Pathogen adaptation to seasonal forcing and climate change. Proc Biol Sci 2005; 272:971-977.
12. Lü H, Yuan Y, Sun N, Bi Z, Guan B, Shao K, et al. Characterization of Vibrio cholerae isolates from 1976 to 2013 in Shandong province, China. Braz J Microbiol 2017; 48:173-179.
13. Chowdhury FR, Nur Z, Hassan N, von Seidlein L, Dunachie S. Pandemics, pathogenicity and changing molecular epidemiology of cholera in the era of global warming. Ann Clin Microbiol Antimicrob 2017; 16:10.
14. Sharma A, Sarmah Dutta B, Rasul ES, Barkataki D, Saikia A, Hazarika NK. Prevalence of Vibrio cholerae O1 serogroup in Assam, India: a hospital-based study. Indian J Med Res 2017; 146:401-408.
15. Old DG (2006). Vibrio, Aeromonas, Plesiomonas, Campylobacter, Arcobacter, Helicobacter, Wolinella. In: Mackie and McCartney Practical Medical Microbiology. Ed, Collee JG, Fraser AG, Marmion BP, Simmons A. Churchill Livingstone, 14th ed. New Delhi, pp.425-448.
16. Perilla MJ, Ajello G, Bopp C, Elliott J, Facklam R, Knapp JS, et al. Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world. World Health Organization 2003.
17. Goel AK, Ponmariappan S, Kamboj DV, Singh L. Single multiplex polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and non-O1 Vibrio cholerae. Folia Microbiol (Praha) 2007; 52:81-85.
18. Keasler SP, Hall RH. Detecting and biotyping Vibrio cholerae O1 with multiplex polymerase chain reaction. Lancet 1993; 341:1661.
19. Kumar P, Jain M, Goel AK, Bhadauria S, Sharma SK, Kamboj DV, et al. A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, eastern India. J Med Microbiol 2009; 58:234-238.
20. Chow KH, Ng TK, Yuen KY, Yam WC. Detection of RTX toxin gene in Vibrio cholerae by PCR. J Clin Microbiol 2001; 39:2594-2597.
21. Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, et al. Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates. Appl Environ Microbiol 2001; 67:910-921.
22. Hochhut B, Lotfi Y, Mazel D, Faruque SM, Woodgate R, Waldor MK. Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob Agents Chemother 2001; 45:2991-3000.
23. Falbo V, Carattoli A, Tosini F, Pezzella C, Dionisi AM, Luzzi I. Antibiotic resistance conferred by a conjugative plasmid and a class I integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy. Antimicrob Agents Chemother 1999; 43: 693-696.
24. Ramachandran D, Bhanumathi R, Singh DV. Multiplex PCR for detection of antibiotic resistance genes and the SXT element: application in the characterization of Vibrio cholerae. J Med Microbiol 2007; 56:346-351.
25. Bhanumathi R, Sabeena F, Isac SR, Shukla BN, Singh DV. Molecular characterization of Vibrio cholerae O139 bengal isolated from water and the aquatic plant Eichhornia crassipes in the river Ganga, Varanasi, India. Appl Environ Microbiol 2003; 69:2389-2394.
26. Das B, Bischerour J, Barre FX. Molecular mechanism of acquisition of the cholera toxin genes. Indian J Med Res 2011; 133:195-200.
27. Abana D, Gyamfi E, Dogbe M, Opoku G, Opare D, Boateng G, et al. Investigating the virulence genes and antibiotic susceptibility patterns of Vibrio cholerae O1 in environmental and clinical isolates in Accra, Ghana. BMC Infect Dis 2019; 19:76.
28. Alishahi A, Imani Fooladi AA, Fallah Mehrabadi J, Mahmoodzadeh Hosseini H. Facile and rapid detection of Vibrio cholerae by multiplex PCR based on ompU, ctxA, and toxR genes. Jundishapur J Microbiol 2013; 6(10): e7933.
29. Zaw MT, Emran NA, Ibrahim MY, Suleiman M, Awang Mohd TA, Yusuff AS, et al. Genetic diversity of toxigenic Vibrio cholerae O1 from Sabah, Malaysia 2015. J Microbiol Immunol Infect 2019; 52:563-570.
30. Akoachere JF, Mbuntcha CK. Water sources as reservoirs of Vibrio cholerae O1 and non-O1 strains in Bepanda, Douala (Cameroon): relationship between isolation and physicochemical factors. BMC Infect Dis 2014; 14:421.
31. Borkakoty B, Biswas D, Devi U, Yadav K, Mahanta J. Emergence of classical ctxB genotype 1 and tetracycline resistant strains of Vibrio cholerae O1 El Tor in Assam, India. Trans R Soc Trop Med Hyg 2012; 106:382-386.
32. Chatterjee S, Patra T, Ghosh K, Raychoudhuri A, Pazhani GP, Das M, et al. Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant. J Med Microbiol 2009; 58:239-247.
33. Goel AK, Jain M, Kumar P, Jiang SC. Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India. World J Microbiol Biotechnol 2010; 26:281-287.
34. Faruque AS, Alam K, Malek MA, Khan MGQ, Ahmed S, Saha D, et al. Emergence of multidrug-resistant strain of Vibrio cholerae O1 in Bangladesh and reversal of their susceptibility to tetracycline after two years. J Health Popul Nutr 2007; 25:241-243.
35. Garg P, Sinha S, Chakraborty R, Bhattacharya SK, Nair GB, Ramamurthy T, et al. Emergence of fluoroquinolone-resistant strains of Vibrio cholerae O1 biotype El Tor among hospitalized patients with cholera in Calcutta, India. Antimicrob Agents Chemother 2001; 45:1605-1606.
36. Krishna BV, Patil AB, Chandrasekhar MR. Fluoroquinolone-resistant Vibrio cholerae isolated during a cholera outbreak in India. Trans R Soc Trop Med Hyg 2006; 100:224-226.
37. Bhowmick TS, Das M, Ruppitsch W, Stoeger A, Pietzka AT, Allerberger F, et al. Detection of virulence-associated and regulatory protein genes in association with phage typing of human Vibrio cholerae from several geographical regions of the world. J Med Microbiol 2009; 58:1160-1167.
38. Goel AK, Jiang SC. Genetic determinants of virulence, antibiogram and altered biotype among the Vibrio cholerae O1 isolates from different cholera outbreaks in India. Infect Genet Evol 2010; 10:815-819.
39. Jain M, Kushwah KS, Kumar P, Goel AK. Molecular characterization of Vibrio cholerae O1 reveals continuous evolution of its new variants in India. Indian J Microbiol 2013; 53:137-141.
40. Ratnam PV, Sundararaj T, Rajkumar S, Anand A, Banu S. Genotyping of Vibrio cholerae strains based on the cholera toxin and virulence associated genes. Indian J Appl Res 2015; 5:525-528.
41. Jain M, Kumar P, Goel AK. Emergence of tetracycline resistant Vibrio cholerae O1 biotype El Tor serotype Ogawa with classical ctxB gene from a cholera outbreak in Odisha, eastern India. J Pathog 2016; 2016:1695410.
42. Nayak AK, Nayak SR, Behera DR, Pal BB. Dissemination of Vibrio cholerae O1 isolated from Odisha, India. Environ Microbiol Rep 2021; 13:355-363.
2. World Health Organization (2014). Cholera surveillance and number of cases. World Health Organization. Geneva.
3. Siriphap A, Leekitcharoenphon P, Kaas RS, Theethakaew C, Aarestrup FM, Sutheinkul O, et al. Characterization and genetic variation of Vibrio cholerae isolated from clinical and environmental sources in Thailand. PLoS One 2017;12(1): e0169324.
4. Chatterjee SN, Chaudhuri K. Lipopolysaccharides of Vibrio cholerae. I. physical and chemical characterization. Biochim Biophys Acta 2003; 1639:65-79.
5. Garg P, Nandy RK, Chaudhury P, Roy Chowdhury NR, De K, Ramamurthy T, et al. Emergence of Vibrio cholerae O1 biotype El Tor serotype Inaba from the prevailing O1 Ogawa serotype strains in India. J Clin Microbiol 2000; 38:4249-4253.
6. Bhandari M, Jennison AV, Rathnayake IU, Huygens F. Evolution, distribution and genetics of atypical Vibrio cholerae - a review. Infect Genet Evol 2021; 89:104726.
7. Mishra A, Taneja N, Sharma RK, Kumar R, Sharma NC, Sharma M. Amplified fragment length polymorphism of clinical and environmental Vibrio cholerae from a freshwater environment in a cholera-endemic area, India. BMC Infect Dis 2011; 11:249.
8. Kumar P, Peter WA, Thomas S. Detection of virulence genes in Vibrio cholerae isolated from aquatic environment in Kerala, southern India. Appl Biochem Biotechnol 2008; 151:256-262.
9. Nandi B, Nandy RK, Mukhopadhyay S, Nair GB, Shimada T, Ghose AC. Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J Clin Microbiol 2000; 38:4145-4151.
10. Goel AK, Jiang SC. Association of heavy rainfall on genotypic diversity in V. cholerae isolates from an outbreak in India. Int J Microbiol 2011; 2011:230597.
11. Koelle K, Pascual M, Yunus M. Pathogen adaptation to seasonal forcing and climate change. Proc Biol Sci 2005; 272:971-977.
12. Lü H, Yuan Y, Sun N, Bi Z, Guan B, Shao K, et al. Characterization of Vibrio cholerae isolates from 1976 to 2013 in Shandong province, China. Braz J Microbiol 2017; 48:173-179.
13. Chowdhury FR, Nur Z, Hassan N, von Seidlein L, Dunachie S. Pandemics, pathogenicity and changing molecular epidemiology of cholera in the era of global warming. Ann Clin Microbiol Antimicrob 2017; 16:10.
14. Sharma A, Sarmah Dutta B, Rasul ES, Barkataki D, Saikia A, Hazarika NK. Prevalence of Vibrio cholerae O1 serogroup in Assam, India: a hospital-based study. Indian J Med Res 2017; 146:401-408.
15. Old DG (2006). Vibrio, Aeromonas, Plesiomonas, Campylobacter, Arcobacter, Helicobacter, Wolinella. In: Mackie and McCartney Practical Medical Microbiology. Ed, Collee JG, Fraser AG, Marmion BP, Simmons A. Churchill Livingstone, 14th ed. New Delhi, pp.425-448.
16. Perilla MJ, Ajello G, Bopp C, Elliott J, Facklam R, Knapp JS, et al. Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world. World Health Organization 2003.
17. Goel AK, Ponmariappan S, Kamboj DV, Singh L. Single multiplex polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and non-O1 Vibrio cholerae. Folia Microbiol (Praha) 2007; 52:81-85.
18. Keasler SP, Hall RH. Detecting and biotyping Vibrio cholerae O1 with multiplex polymerase chain reaction. Lancet 1993; 341:1661.
19. Kumar P, Jain M, Goel AK, Bhadauria S, Sharma SK, Kamboj DV, et al. A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, eastern India. J Med Microbiol 2009; 58:234-238.
20. Chow KH, Ng TK, Yuen KY, Yam WC. Detection of RTX toxin gene in Vibrio cholerae by PCR. J Clin Microbiol 2001; 39:2594-2597.
21. Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, et al. Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates. Appl Environ Microbiol 2001; 67:910-921.
22. Hochhut B, Lotfi Y, Mazel D, Faruque SM, Woodgate R, Waldor MK. Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob Agents Chemother 2001; 45:2991-3000.
23. Falbo V, Carattoli A, Tosini F, Pezzella C, Dionisi AM, Luzzi I. Antibiotic resistance conferred by a conjugative plasmid and a class I integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy. Antimicrob Agents Chemother 1999; 43: 693-696.
24. Ramachandran D, Bhanumathi R, Singh DV. Multiplex PCR for detection of antibiotic resistance genes and the SXT element: application in the characterization of Vibrio cholerae. J Med Microbiol 2007; 56:346-351.
25. Bhanumathi R, Sabeena F, Isac SR, Shukla BN, Singh DV. Molecular characterization of Vibrio cholerae O139 bengal isolated from water and the aquatic plant Eichhornia crassipes in the river Ganga, Varanasi, India. Appl Environ Microbiol 2003; 69:2389-2394.
26. Das B, Bischerour J, Barre FX. Molecular mechanism of acquisition of the cholera toxin genes. Indian J Med Res 2011; 133:195-200.
27. Abana D, Gyamfi E, Dogbe M, Opoku G, Opare D, Boateng G, et al. Investigating the virulence genes and antibiotic susceptibility patterns of Vibrio cholerae O1 in environmental and clinical isolates in Accra, Ghana. BMC Infect Dis 2019; 19:76.
28. Alishahi A, Imani Fooladi AA, Fallah Mehrabadi J, Mahmoodzadeh Hosseini H. Facile and rapid detection of Vibrio cholerae by multiplex PCR based on ompU, ctxA, and toxR genes. Jundishapur J Microbiol 2013; 6(10): e7933.
29. Zaw MT, Emran NA, Ibrahim MY, Suleiman M, Awang Mohd TA, Yusuff AS, et al. Genetic diversity of toxigenic Vibrio cholerae O1 from Sabah, Malaysia 2015. J Microbiol Immunol Infect 2019; 52:563-570.
30. Akoachere JF, Mbuntcha CK. Water sources as reservoirs of Vibrio cholerae O1 and non-O1 strains in Bepanda, Douala (Cameroon): relationship between isolation and physicochemical factors. BMC Infect Dis 2014; 14:421.
31. Borkakoty B, Biswas D, Devi U, Yadav K, Mahanta J. Emergence of classical ctxB genotype 1 and tetracycline resistant strains of Vibrio cholerae O1 El Tor in Assam, India. Trans R Soc Trop Med Hyg 2012; 106:382-386.
32. Chatterjee S, Patra T, Ghosh K, Raychoudhuri A, Pazhani GP, Das M, et al. Vibrio cholerae O1 clinical strains isolated in 1992 in Kolkata with progenitor traits of the 2004 Mozambique variant. J Med Microbiol 2009; 58:239-247.
33. Goel AK, Jain M, Kumar P, Jiang SC. Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India. World J Microbiol Biotechnol 2010; 26:281-287.
34. Faruque AS, Alam K, Malek MA, Khan MGQ, Ahmed S, Saha D, et al. Emergence of multidrug-resistant strain of Vibrio cholerae O1 in Bangladesh and reversal of their susceptibility to tetracycline after two years. J Health Popul Nutr 2007; 25:241-243.
35. Garg P, Sinha S, Chakraborty R, Bhattacharya SK, Nair GB, Ramamurthy T, et al. Emergence of fluoroquinolone-resistant strains of Vibrio cholerae O1 biotype El Tor among hospitalized patients with cholera in Calcutta, India. Antimicrob Agents Chemother 2001; 45:1605-1606.
36. Krishna BV, Patil AB, Chandrasekhar MR. Fluoroquinolone-resistant Vibrio cholerae isolated during a cholera outbreak in India. Trans R Soc Trop Med Hyg 2006; 100:224-226.
37. Bhowmick TS, Das M, Ruppitsch W, Stoeger A, Pietzka AT, Allerberger F, et al. Detection of virulence-associated and regulatory protein genes in association with phage typing of human Vibrio cholerae from several geographical regions of the world. J Med Microbiol 2009; 58:1160-1167.
38. Goel AK, Jiang SC. Genetic determinants of virulence, antibiogram and altered biotype among the Vibrio cholerae O1 isolates from different cholera outbreaks in India. Infect Genet Evol 2010; 10:815-819.
39. Jain M, Kushwah KS, Kumar P, Goel AK. Molecular characterization of Vibrio cholerae O1 reveals continuous evolution of its new variants in India. Indian J Microbiol 2013; 53:137-141.
40. Ratnam PV, Sundararaj T, Rajkumar S, Anand A, Banu S. Genotyping of Vibrio cholerae strains based on the cholera toxin and virulence associated genes. Indian J Appl Res 2015; 5:525-528.
41. Jain M, Kumar P, Goel AK. Emergence of tetracycline resistant Vibrio cholerae O1 biotype El Tor serotype Ogawa with classical ctxB gene from a cholera outbreak in Odisha, eastern India. J Pathog 2016; 2016:1695410.
42. Nayak AK, Nayak SR, Behera DR, Pal BB. Dissemination of Vibrio cholerae O1 isolated from Odisha, India. Environ Microbiol Rep 2021; 13:355-363.
| Files | ||
| Issue | Vol 13 No 5 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i5.7420 | |
| Keywords | ||
| Vibrio cholerae O1; Cholera toxin; Virulence; Genotype; Drug resistance | ||
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How to Cite
1.
Sharma A, Dutta B, Rabha D, Rasul E, Hazarika N. Molecular characterization of Vibrio cholerae O1 strains circulating in Assam: a north eastern state of India. Iran J Microbiol. 2021;13(5):583-591.
