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 this study belonged to V. cholerae O1 biotype ElTor [Table 1]. This result is indicative of complete replacement of classical biotypes by ElTor and revealed that V. cholerae O1 biotype ElTor is the only circulating pathogenic strain in India.
|Table 1: Distribution of phenotypes of Vibrio cholerae O1 strain isolated from different states in India|
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Based on cholera strains O1 antigen seroreactivity V. cholerae O1 strains can be divided into three groups, Inaba, Ogawa, and Hikojama. 1882 strains of V. cholerae O1 biotype ElTor were serotyped in our laboratory. Results indicated 1754 (93.2%) strains belonged to serotype Ogawa. Only 128 (6.8%) strains were found as serotype Inaba. We did not found any Hikojima serotype in our analysis [Table 1].
A total of 1882 V. cholerae O1 biotype ElTor strains isolated from different cholera endemic areas in India were used for phage typing with the set of typing phages available in the Vibrio Phage Reference Laboratory. Out of the ten phages, five phages were from Basu and Mukherjee scheme and five were from the new phage typing scheme.
Basu and Mukherjee phage typing
Five phages of the Basu and Mukherjee phage typing scheme was used to discriminate the ElTor strains and our results indicated that the strains were typed into two different phage types, phage type 2 (T-2) and phage type 4 (T-4). It was found that 1529 (81.25%) strains were grouped as T-2 and 334 (17.75%) were grouped as T-4. Almost 1% strains were found untypeable (UT) [Table 1].
New phage typing
Along with the Basu and Mukherjee typing phages, new phage typing scheme was simultaneously used to classify the 1882 V. cholerae O1 isolates. In contrast to Basu and Mukherjee's scheme, new phage typing scheme, developed in our institute has a better discriminatory power and is capable of differentiating V.cholerae O1 biotype ElTor strains into 27 different types. Phage typing analysis based on the lytic reaction with the phages from the new phage typing scheme [Table 1] indicated 100% type ability of