Open Access Open Access  Restricted Access Subscription Access

Molecular Characterization and Toxin-Typing of Clostridium difficile Isolates of Dogs and Pigs from Assam and Mizoram of North East India


Affiliations
1 Department of Microbiology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
2 Department of Animal Biotechnology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
3 Department of Biotechnology, Gauhati University, Guwahati 781 014, India
4 Department of Microbiology, Central Agricultural University, Selesih, Aizwal, Mizoram 796 014, India
5 Department of Veterinary Clinical Medicine, Assam Agricultural University, Khanapara, Guwahati 781 022, India
 

Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecalsamples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection ofbinary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 fromdog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A+B+ isolates.

Keywords

Diarrhoea, NE India, Pseudomembranous Colitis, Toxin-Typing, Virulence.
User
Notifications
Font Size

  • Hall, I. C. and O’Toole, E., Intestinal flora in new-born infants with a description of a new pathogenic anaerobe, Bacillus difficilis. Am. J. Dis. Child, 1935, 49, 390–402.
  • Bartlett, J. G., Chang, T. W., Gurwith, M., Gorbach, S. L. and Onderdonk, A. B., Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N. Engl. J. Med., 1978, 298, 531–534.
  • George, R. H. et al., Identification of Clostridium difficile as a cause of pseudomembranous colitis. Br. Med. J., 1978, 1, 695.
  • Larson, H. E., Price, A. B., Honour, P. and Borriello, S. P., Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet, 1978, 1, 1063–1066.
  • Libby, J. M., Jortner, B. S. and Wilkins, T. D., Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters. Infect. Immun., 1982, 36, 822–829.
  • Rupnik, M., Avesani, V., Janc, M., Von Eichel-Streiber, C. and Delmee, M., A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J. Clin. Microbiol., 1998, 36, 2240–2247.
  • Rupnik, M., Widmer, A., Zimmermann, O., Eckert, C. and Barbut, F., Clostridium difficile toxinotype V, ribotype 078, in animals and humans. J. Clin. Microbiol., 2008, 46, 2146.
  • Niyogi, S. K. et al., Prevalence of Clostridium difficile in hospitalised patients with acute diarrhoea in Calcutta. J. Diarrhoeal. Dis. Res., 1991, 9, 16–19.
  • Dhawan, B., Chaudhry, R. and Sharma, N., Incidence of Clostridium difficile infection: a prospective study in an Indian hospital. J. Hosp. Infect., 1999, 43, 275–280.
  • Chaudhry, R., Joshy, L., Kumar, L. and Dhawan, B., Changing pattern of Clostridium difficile associated diarrhoea in a tertiary care hospital: a 5 year retrospective study. Indian J. Med. Res., 2008, 127, 377–382.
  • Vaishnavi, C. and Singh, M., Preliminary investigation of environmental prevalence of Clostridium difficile affecting inpatients in a north Indian hospital. Indian J. Med. Microbiol., 2012, 30, 89–92.
  • Delmee, M., Van, B. J., Simon, A., Janssens, M. and Avesani, V., Laboratory diagnosis of Clostridium difficile-associated diarhoea; a plea for culture. J. Med. Microbiol., 2005, 54, 187–191.
  • Cohen, Stuart, H., Tang, Y. J. and Silva Joseph Jr, Analysis of the Pathogenecity locus of Clostridium difficile strains. J. Infect. Dis., 2000, 18, 659–663.
  • Zheng, L. et al., Multicenter evaluation of a new screening test that detects Clostridium difficile in fecal specimens. J. Clin. Microbiol., 2004, 42, 3837–3840.
  • Lemee, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J. F. and Pons, J. L., Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J. Clin. Microbiol., 2004, 42, 2609–2617.
  • Stubbs, S., Rupnik, M., Gibert, M., Brazier, J., Duerden, B. and Popoff, M., Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol. Lett., 2000, 186, 307–312.
  • Rupnik, M., Brazier, J. S., Duerden, B. I., Grabnar, M. and Stubbs, S. L., Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology, 2001, 147, 439–447.
  • Porter, M. C., Reggiardo, C., Bueschel, D. M., Keel, M. K. and Songer, J. G., Association of Clostridium difficile with bovine neonatal diarrhea. In Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO, USA, 2002.
  • Songer, J. G. and Anderson, M. A., Clostridium difficile: an important pathogen of food animals. Anaerobe, 2006, 12, 1–4.
  • Hammitt, M. C. et al., A possible role for Clostridium difficile in the etiology of calf enteritis. Vet. Microbiol., 2008, 127, 343–352.
  • Schneeberg, A., Rupnik, M., Neubauer, H. and Seyboldt, C., Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia, Germany. Anaerobe, 2012, 18, 484–488.
  • Struble, A. L., Tang, Y. J., Kass, P. H., Gumerlock, P. H., Madewell, B. R. and Silva Jr, J., Fecal shedding of Clostridium difficile in dogs: a period prevalence survey in a veterinary medical teaching hospital. J. Vet. Diagn. Invest., 1994, 6, 342–347.
  • Weese, J. S., Reid-Smith, R. J., Avery, B. P. and Rousseau, J., Detection and characterization of Clostridium difficile in retail chicken. Lett. Appl. Microbiol., 2010, 50, 362–365.
  • Marks, S. L., Kather, E. J., Kass, P. H. and Melli, A. C., Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J. Vet. Intern. Med., 2002, 15, 533–540.
  • Koene, M. G. J. et al., Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates. Clin. Microbiol. Infect., 2012, 18, 778–784.
  • Silva, R. O. S. et al., Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil. Brazilian J. Microbiol., 2013, 44, 133–137.
  • Lefebvre, S. L., Waltner-Toews, D., Peregrine, A. S., Reid-Smith, R., Hodge, L., Arroyo, L. G. and Weese, J. S., Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control. J. Hosp. Infect., 2006, 62, 458–466.
  • Fry, P. R., Thakur, S., Abley, M. and Gebreyesa, W. A., Antimicrobial resistance, toxinotype, and genotypic profiling of Clostridium difficile isolates of swine origin. J. Clin. Microbiol., 2012, 50, 2366–2372.
  • Schwan, C. et al., Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog., 2009, 5, e1000626.
  • Alvarez-Perez, S., Blanco, J. L., Bouza, E., Alba, P., Gibert, X., Maldonado, J. and Garcia, M. E., Prevalence of Clostridium difficile in diarrhoeic and non-diarrhoeic piglets. Vet. Microbiol., 2009, 137, 302–305.
  • Baker, A. A., Davis, E., Rehberger, T. and Rosener, D., Prevalence and diversity of toxigenic Clostridium perfringens and Clostridium difficile among swine herds in the midwest. Appl. Environ. Microbiol., 2010, 76, 2961–2967.
  • Janezic, S. et al., International Clostridium difficile animal strain collection and large diversity of animal associated strains. BMC Microbiol., 2014, 14, 173.

Abstract Views: 364

PDF Views: 131




  • Molecular Characterization and Toxin-Typing of Clostridium difficile Isolates of Dogs and Pigs from Assam and Mizoram of North East India

Abstract Views: 364  |  PDF Views: 131

Authors

B. Das
Department of Microbiology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
R. K. Sharma
Department of Microbiology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
P. Borah
Department of Animal Biotechnology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
S. Das
Department of Biotechnology, Gauhati University, Guwahati 781 014, India
L. M. Barkalita
Department of Animal Biotechnology, Assam Agricultural University, Khanapara, Guwahati 781 022, India
R. K. Mandakini Devi
Department of Microbiology, Central Agricultural University, Selesih, Aizwal, Mizoram 796 014, India
B. C. Baishya
Department of Veterinary Clinical Medicine, Assam Agricultural University, Khanapara, Guwahati 781 022, India

Abstract


Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecalsamples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection ofbinary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 fromdog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A+B+ isolates.

Keywords


Diarrhoea, NE India, Pseudomembranous Colitis, Toxin-Typing, Virulence.

References





DOI: https://doi.org/10.18520/cs%2Fv113%2Fi06%2F1099-1106