Cholera was recognised in accounts from ancient India and China 
, but it was only in 1817 that an epidemic in India spread westwards to become the first of 7 pandemics. The etiological agent of pandemic cholera was the bacterium Vibrio cholerae
serotype O1, which was later divided into two biotypes: classical biotype and El Tor biotype. The first six pandemics were caused by the classical biotype, but in the early 1900s a new form of the species emerged that caused mild or no symptoms, and was later put into the El Tor biotype, to distinguish it from the form causing cholera (classical biotype). The El Tor biotype differed by being haemolytic, and positive in the Voges Proskaur reaction that detects a specific fermentation pathway. The 6th
pandemic continued until 1923, but from then on was largely confined to Asia (and thus not pandemic). The 7th
pandemic, which erupted in 1961, was El Tor in biotype, which was quite unexpected. However there had been several outbreaks of cholera, now known as pre-7th
pandemic outbreaks, in the Makassar area, Sulawesi, Indonesia between 1937 and 1957. The organism was El Tor but the disease resembled true cholera in severity and mortality. It differed in not spreading rapidly but remained localised as reported in a review soon after the events 
. The 7th
pandemic (also El Tor) arose in the same area, and is generally thought to have arisen from the Makassar outbreak strain. It spread rapidly and eventually fully replaced the classical biotype 6th
pandemic strain, which was present in India throughout the period between the 6th
pandemics and for many years after the 7th
pandemic arrived (see also Supporting Introduction in Text S1
V. cholerae has also been found in water, particularly brackish water, and associated sediments, and also on invertebrates in these habitats. These environmental isolates are phenotypically very diverse with over 200 serotypes reported. They resemble the El Tor biotype, but the term is not used as the biotyping is confined to serotype 1. The fact that the 6th and 7th pandemics are of different biotype, with the 7th pandemic strain more similar to environmental isolates than the 6th, suggests that the 7th pandemic strain did not arise from the 6th pandemic but arose independently.
We have already looked at the relationships of the pathogenic strains using sequence of 26 house keeping genes 
. This confirmed earlier conclusions from several groups, based on MLEE and sequence data, that the 6th
pandemic clones, the pre 7th
pandemic outbreak isolates, and some other pathogenic strains are related. However with 26 genes and 12 isolates an unambiguous tree was obtained, with no reverse or parallel events. It was also clear that the 6th
pandemic clone stands apart from the other pathogenic strains (). Single base change mutational and multi bases change recombinational events could be distinguished, and this showed that there has been extensive recombination, with 8 of the 26 genes having undergone recombination in either 6th
pandemic lineage since divergence. A 1937 Makassar outbreak isolate (strain M66-2) appeared to be on the direct line to the 7th
pandemic. The picture that we have for V. cholerae
is of a species that is diverse with as many O antigen forms as E. coli
, with one lineage that has adapted to a new niche as an intestinal pathogen of humans. On at least 2 occasions this pathogenic form developed a high level of transmission to become pandemic 
Relationships of M66-2, 6th and 7th pandemic clones, and other closely related toxigenic strains based on 26 house keeping genes (Salim et al. 2005).
These circumstances provide an ideal opportunity to study at DNA level the events that enabled the Makassar outbreak strain to become a pandemic strain and enabled the 7th pandemic clone to fully replace the 6th clone. We sequenced the genomes of strain M66-2, and 6th pandemic isolate O395, and compared them with the published genome of 7th pandemic isolate N16961. We describe a method to distinguish mutation from recombination-based changes over a full genome, and this provided the data to assess the rate of mutational change, as the 7th pandemic strain N16961 was isolated in 1971, and the 6th pandemic strain, O395, was isolated in 1965. Comparison of the genomes enables us to document the changes that had occurred during divergence of the three clones. Significant findings include an estimate for the mutation rate that is 100 times higher than generally assumed, a model for change in the major integron, and assessment of mutational, recombination, integron and other evolutionary events to the separate lineages.