Mismatch Amplification Mutation Assay (MAMA) PCR Reveals Altered El Tor Vibrio Cholerae O1 Circulating in Delhi, India

Abstract

The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. V. cholerae has more than 200 known serotypes but not all the strains are pathogenic. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. 30 V. cholerae O1 isolates (Table 1) were revived from stock cultures maintained at Laboratory Department in Maharishi Valmiki Infectious Diseases Hospital (MVIDH), Delhi during 2012-2014. Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. All isolates were biochemically identified as V. cholerae biotype El Tor and serologically O1 Ogawa. All isolates were amplified with classical specific primers. Cholera continues to be endemic in large number of states in the eastern, western, northern and southern parts of India and there were 38 cholera outbreak reports published in India during 2007 to 2013. These Indian outbreaks have been associated with new El Tor variant. Key-wordsV. cholerae O1, El Tor, MAMA PCR, ctxB -------------------------------------------------IJLSSR----------------------------------------------INTRODUCTION The acute diarrheal disease cholera is caused by Vibrio cholerae, a major public health problem in Asia, Africa and Latin America. According to the World Health Organisation (WHO) estimates there were 3-5 million cholera cases and 1,00,0001,20,000 deaths each year throughout the world. V. cholerae has more than 200 known serogroups but not all the strains are pathogenic. Among them, O1 and O139 antigens are highly pathogenic and acknowledged to cause epidemic and pandemic disease. The symptom of the acute cholera is rice water stools. Access this article online Quick Response Code: Website: www.ijlssr.com DOI: 10.21276/ijlssr.2016.2.6.6 Clinical manifestations of the disease range from mild symptoms such as abdominal cramps, nausea and vomiting, to more severe symptoms such as dehydration, shock and death. A devastating cholera outbreak, that had occurred in Haiti since October 2010, involved 6, 98, 893 cases and 8,540 deaths. V. cholerae serogroup O1 is classified into two biotypes, which are termed ‘classical’ and ‘El Tor’. The current seventh pandemic of cholera is caused by the El Tor biotype was originated in 1961 from Celebes Islands in Indonesia. Spread of this biotype was recorded for the first time during 1964 in India and it made its first appearance in Delhi during June, 1965. In recent years, it has been seen that the emergence of new variants of V. cholerae O1 have carried characteristics of both classical and El Tor biotypes and these variants of V. cholerae O1 are called as ‘atypical El Tor’. These strains might have evolved from El Tor variants that acquired certain characteristics from classical genome. In this study, we focused on type of CT genotype existing among V. cholerae Research Article (Open access) Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 6 http://ijlssr.com Copyright © 2015-2016 International Journal of Life-Sciences Scientific Research Page 679 O1 isolated from the patients who were admitted in MVIDH, Delhi during 2012-2014. MATERIALS AND METHODS Collection and processing of samples 30 V. cholerae O1 isolates were revived from stock cultures were maintained at Laboratory Department in MVIDH, Delhi during 2012-2014. These samples were revived and transferred to alkaline peptone water (APW, pH-8.6) and incubated at 37°C for 4-6 hrs. After incubation, the enriched cultures were further inoculated to the thiosulphate-citrate-bile-salts-sucrose (TCBS) agar and bile salt agar (BSA, pH-8.6) (Hi-Media, Mumbai). Confirmation of V. cholerae Typical colonies appearing on TCBS/BSA were confirmed by standard biochemical tests. These isolates were further tested serologically with commercially available V. cholerae O1 polyvalent and monovalent and O139 antiserum (BD, USA and Denka Seiken, Ltd. Japan). Genomic DNA preparation Genomic DNA from each isolate was extracted using protocol described previously. PCR assays for detection of ctxB genotype Mismatch amplification mutation assay was used for classifying the strains into prototype El Tor, hybrid, or El Tor variant biotype based on their ctxB gene. In this PCR, the common forward primer for both classical and El Tor alleles was used. Two allele specific primers Re-Cla and Re-Elt were used respectively. The PCR conditions and cycles were described previously. V. cholerae O1 strains 569B (classical) and N16961 (El Tor) were used as control strains. The primers used were synthesized by Invitrogen, India and dNTPs, Taq polymerase, 10X Taq buffer used in this study were obtained from Genei, Bangalore. The primer sequences were used in this PCR is given in Table 2.