 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 V. cholerae O1 biotype El Tor strains. Analysis revealed that V. cholerae O1 biotype ElTor strains were distributed into several different phage tapes of which (Types 27, 26, 23.24, 13, 21 and 25) were the major types that represented almost 81% of the total typed strains in this study [Table 2]. Majority of the 1882 strains belonged to phage type 27 (60.41 %). Phage type 26 (8.66%) was the next major phage type followed by type 23 (4.94%), type 24 (4.41%), type 13 (3.82%), type 21 (3.08%) and type 25 (2.92%) [Table 1]. It was found that the V. cholerae O1 biotype ElTor strains belonging to phage type 27 were widely and almost equally distributed in all the nine states from where strains were selected for the present study.
|Table 2: Major Phage type classification based upon sensitivity to phages N4, S5, S20, M4 and D10. (+) indicates sensitive to specific phage, (-) indicates resistance to specific phage|
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Identification of candidate phage for an alternative therapy
Sensitivity of the circulating V. cholerae strains against the five phages of the new phage typing scheme was calculated and it was found that all the phages have the ability to lyse at least 80% of the circulating strains in India. As shown in [Table 3], phage N4 can lyse 91.17% strains, phage S20 can lyse 83.95%, phage M4 can lyse 86.29% of strains and phage D10 can lyse 80.49%. However, phage S5 has shown highest lethality which can kill or lyse 94% of the circulating V. cholerae O1 biotype El Tor strains.
|Table 3: Comparative analysis of vibriophage susceptibility. All the 1882 strains used in the present study were analyzed for sensitivity against the individual phages. Maximum number of strains were found sensitive to phage S5 followed by N4, M4, S20 and D10|
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| Discussion|| |
We are using phage typing as one of the primary methods in monitoring the V. cholerae strains responsible for cholera infection in India at Vibrio Phage Typing Reference Laboratory. Besides potential application of phage typing schem