Comparative Genomics Study of Vibrio cholerae

The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. Many infectious agents that cause emerging and re-emerging diseases appear to evolve from non-virulent forms. We still lack a clear understanding about the natural history of various microbial agents that cause human infectious diseases and the events leading to acquisition of their pathogenic potential. There have been seven pandemics of V. cholerae throughout the history of the mankind. To date, the world population is still experiencing the seventh one which started in the early 1960s. From almost 200 recognized V. cholerae serotypes, the majority of these epidemics are associated with primarily O1 serotype. However there is evidence that this species is undergoing some phenotypic changes during the last decades. Such examples include shifts in some metabolic pathways used for biotyping, phage sensitivity profiling and the acquisition of plasmids that carry multiple genes conferring antimicrobial resistance. Furthermore, the recent emergence of a non-O1 serotype (‘Bengal strain’, classified serologically as O139) has prompted the experts to think that perhaps this genotype will be the predominant one in the upcoming (eighth) pandemic. Besides the O1 and O139, the non-O1 and non-O139 V. cholerae stains are occasionally associated with other severe forms of gastrointestinal disease in humans. Interestingly, many of these non-canonical strains lack the genes encoding the typical virulence factors for this species such as the Cholera-toxin (ctx) and toxin co-regulated pilus (tcpA). Therefore it has been hypothesized that this group of non-canonical V. cholerae pathogens consist of several sub-clones that elicit disease via unknown virulence determinants and underlying mechanisms. The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a "species" DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 10 diverse V. cholerae and one V. mimicus were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for V. cholerae group members. The availability of the V. cholerae species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of V. cholerae group. One hundred thirteen query strains were investigated in this study, with each query strain hybridized against the reference strain, El Tor. Each strain has a single dye experiment. Each oligo is spotted on the V. cholerae species microarray once. Positive controls on the array consist of oligos designed from the sequenced reference genome, El Tor, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana. The microarrays also had Agilent internal controls.