טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentCohen Lyora
SubjectDevelopment of a Method for the Identification and Typing of
Vibrio cholerae Based on SSR Variation
DepartmentDepartment of Biotechnology
Supervisor Professor Yechezkel Kashi


Abstract

Vibrio cholerae, an aquatic bacterium, is the etiological agent of cholera. The clinical disease is characterized by profuse diarrhea and vomiting that rapidly leads to dehydration. Death can ensue if prompt and appropriate treatment is not initiated.

V.cholerae isolates are divided to serogroups of which only O1 and O139 are associated with cholera pandemics. Virulent isolates carry the cholera-toxin gene (ctx). Practical typing of strains is mainly serological and requires about 200 antisera. Various DNA-based methods were also applied for typing.

Our objective was to develop a Simple Sequence Repeats (SSR) based method for V.cholerae typing. SSRs are a class of short DNA motifs tandemly repeated at a specific locus. A subgroup of SSRs is the mononucleotide repeats (MNR). Large-SSR (L-SSR) consists of three to nine bp motifs locus. Being highly mutable, SSR provide a source of genomic polymorphism used in bacterial typing.

A computer-based screen of the V.cholerae genome revealed thousands of SSR tracts. The longest 17 SSR loci were chosen for analysis. A panel of 32 V.cholerae isolates was tested. Variation was evaluated by capillary sizing of fluorescent-labeled amplified L-SSR loci, and by sequencing for the MNR loci. High genetic diversity was found among the V.cholerae isolates in the SSR loci. Also, sequence analysis of 8 MNR loci provided information of both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences, presenting high levels of polymorphism.

Phylogenetic analysis of the combined SSR data showed clear discrimination between the clinical strains and the environmental isolates. Furthermore, discrimination between most of the strains was achieved. Results suggest that SSR-based typing methods, which combine both L-SSR length polymorphism and MNR multi-locus sequence typing (MNR-MLST) could be efficiently applied for V.cholerae typing.

In order to rapidly identify pathogenic V.cholerae strains a SYBR Green based Real-Time PCR assay was developed. A range of Vibrio and non-Vibrio strains was tested for amplification of ompW and ctxA V.cholerae  genes, and of two L-SSR loci, that were found to be correlated with all tested ctxA positive strains or with O1 and O139 (ctxA+) strains. Specific detection of V.cholerae and identification of pathogenic strains was achieved by either pure DNA or direct colony amplifications, within only 30 minutes. Detection sensitivity from pure culture was 103CFU, higher in two orders of magnitude than conventional PCR method. This rapid and sensitive detection of V.cholerae should facilitate the diagnosis, help the management of disease and control the outbreaks of this waterborne pathogen.