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Sequencing, Phylogeny Pinpoint Bay of Bengal as Source of Pandemic Cholera

By Andrea Anderson

NEW YORK (GenomeWeb News) – An international research team reported online in Nature today that it has used a combination of genome sequencing and phylogenomics to trace the bacteria behind recent cholera outbreaks, including the outbreak in Haiti, back to a source in the Bay of Bengal.

The researchers compared sequence patterns in Vibrio cholerae from more than 150 samples collected around the world over decades to get insights into the source of the El Tor strain of V. cholerae involved in the Haitian cholera outbreak and other outbreaks from the current cholera pandemic, the seventh such pandemic documented so far. They found that the strains share an ancestor going back to the Bay of Bengal in the 1950s. And, they say, at least three waves of cholera transmission have stemmed from this ancestral cholera strain.

"It's quite clear that there have been multiple eruptions from that region of the same related strains, but slight different genetic variants over time," senior author Gordon Dougan, head of the microbial pathogenesis group at the Wellcome Trust Sanger Institute, told GenomeWeb Daily News.

Along with these gradual waves of Vibrio cholerae movement out of the Bay of Bengal, Dougan added, the findings point to smaller subsequent transmission events outside, along with a few larger jumps from one part of the world to another following Vibrio cholerae migration from the Bay of Bengal.

Cholera, an intestinal infection characterized by diarrhea, vomiting, and dehydration, is blamed for between three and five million deaths annually. The disease typically becomes a problem in places with poor sanitation or hygiene — circumstances that can arise in the wake of natural disasters, such as last year's earthquake in Haiti, or when access to safe water becomes limited for other reasons.

Most documented cholera outbreaks have been linked to V. cholerae in the O1 serogroup, which includes both the so-called classical and El Tor biotypes, or the related O139 serogroup, with the the El Tor biotype turning up in the most recent outbreaks.

Even so, it's been difficult to discern relationships between cholera-causing El Tor isolates from different parts of the world, the researchers explained, since most analyses have relied on microbiological methods or relatively few genetic markers.

"There are a few tests of phenotypic variation between variants, but they're fairly limited and they're also limited to particular hotspots of variation in the genome, like the cholera toxin genes," Dougan explained, noting that these approaches were "basically a method of discrimination rather than trying to identify relationships between strains."

To look at these relationships in more detail, the researchers used Illumina GAIIx paired-end sequencing to sequence the genomes of 136 V. cholerae isolates collected in more than 20 countries since the 1950s.

By comparing newly sequenced genomes, as well as sequences for another 18 isolates characterized previously, with the N16961 El Tor reference sequence, the team was able to track down high-resolution SNP markers for doing phylogenetic analyses of the isolates.

In the process, they found eight V. cholerae lineages, with classical isolates clustering far from the seventh pandemic El Tor isolates. Based on the phylogenetic relationships, the researchers concluded that this latest pandemic El Tor strain descended from an ancestral V. cholerae strain in the Bay of Bengal.

"Once we'd identified the rare, recently inherited SNPs, we were able to build a family tree or a phylogenetic tree based upon those SNPs," Dougan said. "We actually found a number of clades. They're all interrelated and every time we looked at a clade it always led us back to the Bay of Bengal."

And, they reported, their findings suggest that an antibiotic resistance mechanism based on the so-called SXT/R391 integrative and conjugative element first showed up in the V. cholerae genome between about 1978 and 1984, about 10 years before this resistance was recognized in a V. cholerae O139 strain.

"It's an element that's clearly come several times into cholera starting around 1981 and it's almost certainly come from other environmental bacteria or other organisms that [cholera] meets in the intestine," Dougan said. "It really gives the Vibrio an advantage, generally, against exposure to antibiotics — not necessarily antibiotics used in the treatment of cholera."

In the past 40 years or so, the researchers estimate that three overlapping waves of V. cholerae emanated from the Bay of Bengal region, coinciding with three clades of recent El Tor pandemic or "L2" isolates within the tree. These strains appear to have spread through human migration and travel and then continued to evolve locally in some regions.

"The clonal clustering of L2 isolates, the constant rate of SNP accumulation, and the temporal and geographical distribution support the concept that the seventh pandemic has spread by periodic radiation from a single source population located in the Bay of Bengal," the researchers wrote, "followed by local evolution and ultimately local extinction in non-endemic areas."

The Haitian outbreak strain, for instance, appears to have stemmed from L2 isolates in South Asia within the past six years or so.

Indeed, an independent team of researchers from Denmark, the US, and Nepal reported in mBio earlier this week that their sequencing, sequence typing, antimicrobial susceptibility, and other analyses point to a close relationship between the Haitian cholera outbreak isolates and V. cholerae found in South Asia, specifically Nepal — a pattern that appears to be consistent with the proposed introduction of the bug by peacekeepers from Nepal after the Haitian earthquake.

Such genetic analyses are useful for tracking cholera movement, though Dougan noted that a combination of genetics and classical epidemiology is important for tracing outbreak events, since some Vibrio cholerae strains, including those found on either side of the Nepalese border, are highly related to one another.

Nevertheless, he said, having a phylogenetic framework for the cholera-causing microbe is expected to prove useful to track, manage, and determine the source of cholera outbreaks.

"As the bacteria replicates in a particular city or in a chain of infection, they accumulate SNPs that can be used to track the evolution and the spread locally," Dougan said. "The frame that we provide really will be useful to people because it will be able to tell you if you've got an outbreak, whether it's a local strain causing the outbreak or whether it's a strain that's been brought in from the outside."

Dougan and his colleagues have now looked at strains from a cholera outbreak in Zimbabwe, for instance, to determine how they fit into the phylogenetic tree. They are also in the process of assessing isolates collected in Calcutta before and after vaccination in that region and doing genetic analyses to look at the local evolution of Vibrio cholerae isolates from Mexico, Kenya, and Somalia. The group ultimately plans to set up an international, SNP typing-based system for tracing future cholera outbreaks.

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