Abstract
A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.
Highlights
From ancient civilizations to the recent Haiti epidemic [1], cholera continues to remain a public health concern in developing countries where a large fraction of the population may not have access to safe drinking water and adequate sanitation
Similar to strain N16961, the El Tor strains C6709 and E7946 and the serogroup O139 strain SG-24 (El Tor derivative) [5] were able to outcompete the classical O395 strain in cocultures within 30 to 40 hours of mixing (Fig. S1). Another classical strain 569B, was eliminated when cocultured with the El Tor strain N16961. These results suggested that the loss of colony forming units (CFU) of the classical biotype when cocultured with the El Tor biotype may not be strain specific
The El Tor biotype of V. cholerae is a highly adapted organism that within a decade displaced the classical biotype as the predominant cause of epidemic cholera [3]
Summary
From ancient civilizations to the recent Haiti epidemic [1], cholera continues to remain a public health concern in developing countries where a large fraction of the population may not have access to safe drinking water and adequate sanitation. The O1 serogroup can be classified into two biotypes, classical and El Tor. Strains of the classical biotype, that had probably been responsible for most of the cholera pandemics between 1817 and 1961, were in general extremely virulent and caused devastating epidemics. The classical and El Tor biotypes are closely related, several biochemical and genetic differences have been reported between the two biotypes. These include striking differences in carbohydrate metabolism [7], regulation of virulence gene expression [8,9,10,11], virulence gene content [12], primary sequence of virulence genes [13] and susceptibility to lytic bacteriophages [14,15,16]. In spite of the accumulation of substantial data on the differences between the classical and El Tor biotypes, the molecular and genetic mechanisms responsible for the competitive exclusion of the classical biotype following the emergence of the El Tor biotype are unknown
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