Genomic evidence of two-staged transmission of the early seventh cholera pandemic

Abstract

The seventh cholera pandemic started in 1961 in Indonesia and spread across the world in three waves in the decades that followed. Here, we utilised genomic evidence to detail the first wave of the seventh pandemic. Genomes of 22 seventh pandemic Vibrio cholerae isolates from 1961 to 1979 were completely sequenced. Together with 152 publicly available genomes from the same period, they fell into seven phylogenetic clusters (CL1-CL7). By multilevel genome typing (MGT), all were assigned to MGT2 ST1 (Wave 1) except three isolates in CL7 which were typed as MGT2 ST2 (Wave 2). The Wave 1 seventh pandemic expanded in two stages, with Stage 1 (CL1-CL5) spread across Asia and Stage 2 (CL6 and CL7) spread to the Middle East and Africa. Three non-synonymous mutations, one each, in three regulatory genes, csrD (global regulator), acfB (chemotaxis), and luxO (quorum sensing) may have critically contributed to its pandemicity. The three MGT2 ST2 isolates in CL7 were the progenitors of Wave 2 and evolved from within Wave 1 with acquisition of a novel IncA/C plasmid. Our findings provide new insight into the evolution and transmission of the early seventh pandemic, which may aid future cholera prevention and control.

Fig. 1. Maximum-likelihood phylogenetic tree of the complete genomes of the early seventh pandemic (1961–1979).

The tree was rooted using V. cholerae strain C5 as an outgroup. The metadata is noted on the right side of the tree. Genome rearrangement structures of chromosome 1 are marked as GS1-GS4 (see Supplementary Fig S5 for details). Filled and empty squares represent gene presence and absence of virulence factors or genes shown respectively: the cholera toxin (CTXφ), the Vibrio pathogenicity island (VPI), Vibrio seventh pandemic islands (VSP-I and VSP-II), type VI secretion system (T6SS) and the repeats-in-toxin (MARTX) toxin (RTX). Filled circles represent the presence of an antibiotic resistance gene as listed. catB9 is annotated in grey since it has not been associated with phenotypic resistance. Red stars denote the two new IncA/C plasmids. The colours of the branches correspond to the clusters identified in Fig. 2 (see Fig. 2 for details). Other colours in different columns were for visual separation purposes.

Fig. 2. Maximum-likelihood phylogenetic tree of the early seventh pandemic (1961–1979).

The tree was constructed using 29 complete genomes and 152 Illumina sequenced genomes from the seventh pandemic isolates between 1961 to 1979 and rooted using strain C5 as an outgroup. Stage 1 and Stage 2 were demarcated with light and dark grey shading. Cluster 1 (CL1) to Cluster 7 (CL7) are demarcated with different colour branches and labelled at each node. Dashed branches were shortened for clarity. Year of isolation is shown using a heatmap with reds for 1960s and blues for 1970s. Continent/Subcontinent of origin for each isolate is displayed with a colour bar and country of origin is displayed when that country was mentioned specifically in the text. All other countries were marked in grey colour with country details in Supplementary Fig S3. Only complete genomes are named for clarity. See Supplementary Fig. S3 for full details of isolate names and complete metadata.

Fig. 3. Two new IncA/C plasmids compared with the most closely related plasmid pRMH760 of Klebsiella pneumoniae.

From the inner circle to the outer circle are the three plasmids pRMH760 (Klebsiella pneumoniae), pM646 (V. cholerae M646) and pM714 (V. cholerae M714). The genes are annotated based on pRMH760 (NCBI Reference Sequence: NC_023898.1). Antibiotic resistance genes are highlighted in red while antiseptic and metal resistance genes are marked in blue. The inner rings are size scale, G + C content and G + C skews per colour legend.

Fig. 4. Alignments of the superintegrons from complete genomes.

Superintegrons (right panel) were extracted from chromosome 2 of each complete genome and aligned against strain C5. The number under the tick marks of the integron size is in base pairs (bp). The empty line segments represent deletions. The red triangles on the top of the segments represent large insertions. The genome sequences of the chromosome 2 of the strains marked in red had not been closed and thus the superintegrons were not presented. The arrows denote the orientation of each aligned sequence. The white arrow denotes the missing bases. Colours for year and country are for visual separation purpose. The tree was the same as that of Fig. 1.