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 the method as described by Chattopadhyay et al. In brief, bacteriophage stock was serially diluted and the dilutions were spotted onto the lawn of the standard propagating host strain. Lowest dilution which was capable to lyse or react positively (appearance of at least five plaques) was considered as RTD and used for phage typing.,
Phage typing was performed as per the standard protocol of our laboratory. Mid logarithmic phase young culture of V. cholerae (0.1 ml.) was mixed with molten soft agar and poured onto a nutrient agar plate. The plates were then kept to dry at room temperature (20–30 min) followed by the application of small drops of all the typing phages at the respective RTD dilutions. The drops on the plates were allowed to dry at room temperature for around 10 min and incubated at 37°C for 16–18 h. V. cholerae MAK757 was used in each set of an experiment as a positive control due to the ability of all the typing phages to lyse MAK757. Presence of a clear lytic zone or appearance of at least five or more plaques for individual phage drop was considered as positive reaction.
We have calculated individual phage sensitivity by dividing the total number of strains sensitive for specific phage with the total number strain studied.,
| Results|| |
Bacteriophage and host range
A total of ten bacteriophages which are in routine use for phage typing in Vibrio phage reference laboratory were used in this study to determine phenotypic characterization of V. cholerae strains circulating in India. V. cholerae O1 biotype ElTor strain MAK757 (ATCC 51352) was found sensitive to all the ten bacteriophages but V. cholerae O1 classical biotype strain 569B and V. cholerae 154 were found resistant to these ten ElTor phages. Strains of common enteropathogens such as Salmonella, Shigella, and enteropathogenic Escherichia coli were not sensitive to these phages.
Biotyping and serotyping
All the 1882 strains used in this study were characterized and confirmed as V. cholerae. Our result showed that all the strains included in