ological 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 shaker incubator. Mid-logarithmic-phase cultures of V. cholerae MAK757 were infected with respective phages at a multiplicity of infection of 0.1, and the infected cultures were incubated with shaking at 37°C until complete lysis occurred. After complete lysis of the culture solution, few drops of chloroform was added in the lysate, mixed well and centrifuged at 10,000 rpm for 20 min at 4°C. Phage lysate was collected and stored at 40 C followed by centrifugation. For concentrating, phage lysate was centrifuged at 30,000 rpm for 2 h at 4°C using a Beckman 50.2 Ti rotor to pellet down the phage particles, resuspended in 0.05 M Tris [pH 7.5] with 0.02 M MgCl2 and kept at 4°C. Broth lysis was used for preparation of larger stock. However, to get small volume high titer stock of phages, alternate to the above lysis method, plate lysis procedure was used for phage propagation following the standard methodology. Bacteriophages used in the present study were purified using cesium chloride density (1.3–1.7 g/ml) gradient as per the standard protocol.,
Serological identification was carried out with each strain by using polyvalent O1 and consequently monospecific Inaba and Ogawa antisera (Difco, Detroit, Mich., USA) according to the WHO guideline (World Health Organization (1993): Guidelines for Cholera Control. World Health Organization, Geneva).
Determination of routine test dilution and Phage typing
Phage typing was carried out based on the standard technique regularly performed in our laboratory., A panel of the typing phages were used to type all the 1882 V. cholerae O1 biotype ElTor strains used in this study. Briefly, a single colony of V. cholerae strain grown in nutrient agar media was inoculated in nutrient broth and incubated under static conditions for 4–5 h at 37°C. An aliquot of the fresh culture (0.1 ml) was mixed with molten 0.8% soft agar (3.5 ml, maintained at 42°C) and poured onto a nutrient agar plate to prepare a uniform lawn of bacteria. Routine test dilutions (RTDs) of the phages were determined by 