kes them especially suited as bacterial strains discriminator. Since the late 1960s when molecular and modern methods were not widely available, lytic phages of V. cholerae have been used for typing of V. cholerae O1 which remained primary choice for classification of V. cholerae strains. Basu and Mukherjee initially proposed a phage typing scheme for V. cholerae O1 biotype ElTor in 1968, which was considered as a primary choice and a gold standard approach for differentiating V. cholerae biotype ElTor strains in the past. Six phage types were identified by Basu and Mukherjee to characterize V. cholerae O1 biotype ElTor strains, among them phage type 2 and 4 are predominant whereas types 1, 3, 5, and 6 are not in circulation due to some unknown reason, investigation of which may require a separate study. A fundamental feature of the phage typing of V. cholerae was its usefulness in differentiating strains into several types as well as for differentiating classical and ElTor biotypes. Phage typing schemes for V. cholerae O1 and O139 were developed form NICED over the last few decades.,,,
Apart from the importance of cholera bacteriophages to discriminate cholera strains, phages have been used as an alternative to antibiotic therapy to circumvent the burden of antibiotic resistance., An earlier study has found that V. cholerae isolates from Kolkata, India are strongly correlated with resistance to some antibiotics such as streptomycin, tetracycline, and trimethoprim–sulfamethoxazole. Other studies conducted in Nepal on V. cholerae strains isolated from cholera patients have found that 6.45% of the strains were multidrug resistant., These studies suggest that antibiotic-resistant strains of V. cholerae in the Indian subcontinent are on the rise.,, Horizontal gene transfer is an widely accepted methods of antibiotics resistance spreads.
To use bacteriophages for therapeutic application in cholera, phage candidate or phage cocktal must have a broad host range and minimum effect on commensal organisms. A recent epidemiological study had identified the cholera hot spot region in India, but data on the phenotypic nature of presently circulating vibrio strains are missing in this part of the world. In this study, we have not only characterized the V. cholerae strains but did a comparative analysis on the host range of these phages for future therapeutic applications. Phenotypic characteristics including their susceptibility to vibrio phages described in this study are beneficial to keep the scientific community better equipped with the knowledge about the nature of circulating strains and the capability of phages to kill circulating V.cholerae strains to subvert future epidemic.
| Materials and Methods|| |
V. cholerae O1 biotype ElTor strain MAK 757 (ATCC 51352) was used as the standard propagating strain or as the host for the propagation of V. cholerae O1 phages. We received clinical and environmental samples used in this study from different parts of the country. We included a total of 1882 V. cholerae O1 strains isolated from different cholera endemic areas in India between 2012 and 2017 in this study. We also included V. cholerae O139 strain NPR-4, classical biotype strains V. cholerae 154 and 569B which were used as negative control. Bacterial strains used in the recent study were grown in selected media (TCBS) using the standard methodology.
The panel of five phages of Basu and Mukherjee phage typing scheme (Gr 1 to Gr V) and five phages from the new phage typing scheme (i.e., N4, S5, S20, M4, and D10] were used for phage typing study. These phages are in regular use in our laboratory which were reported earlier as different from each other and biotype specific., All the phages were purified from the stock by successive single-plaque isolation against the respective standard propagating strain.
Propagation of bacteriophages
Standard broth lysis procedure was used to prepare phage lysates. Nutrient broth inoculated with 1% (vol/vol) cell suspension from the overnight grown culture of MAK757 and grown at 37°C for 4–6 h in a 