Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India

Abstract

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years.

Fig 1. Genetic organization of VSP-II in V. cholerae O1 isolated from Kolkata, India.

(A) Intact VSP-II of reference strain N16961, (B) VSP-IIB, (C) VSP-IIC, (D) VSP-IID of VSP-II negative strain. Arrows represent ORFs according to the annotation of V. cholerae N16961. Deduced sequence and location of att site are also shown.

Fig 2. Maximum likelihood tree generated from SNPs in the core genome.

(A) Phylogenetic tree of the 7th pandemic El Tor strains with the non-O1, non-O139 and pre-seventh pandemic strains. 100,600 SNPs were identified in 276 strains. Gray triangle show collapsed clade containing the 7th pandemic El Tor strains. (B) Phylogenetic tree of the 7th pandemic clade containing all Kolkata isolates. 1,772 SNPs were identified in 243 strains. Branches colored blue and red indicate lineage 1 and 2, respectively. Gray thin circle show wave 3 lineage. Positions of the Kolkata isolates are indicated with color rings representing their characteristics on the outside tree.

Fig 3. Phylogenetic sub-tree of strains in lineage 2.

Magenta, yellow, orange, and red branches represent sublineage 2-I, 2-II and 2-III, respectively. Other characteristics of strains are indicated by the three colored rings surrounding the tree. Information of VSP-II type was obtained only from Kolkata isolates.

Fig 4. Maximum clade credibility tree of V. cholerae O1 isolates in Kolkata.

Branches colored blue and red indicate lineage 1 and 2, respectively. Node bars represent the 95% highest posterior density (HPD) range for the estimated divergence time at each node. Background of strain is colored according to the respective sublineage in Fig 3.