טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYoav Broza
SubjectGenetic Variation, Environmental and Epidemiology Study
of the Invasive Human Pathogen Vibrio vulnificus
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Kashi Yechezkel
Full Thesis textFull thesis text - English Version


Abstract

Vibrio vulnificus is a highly invasive human pathogen with worldwide distribution. It is commonly divided into three biochemical groups ('Biotypes') most of which are pathogenic. The emergence of V.vulnificus biotype 3 occurred over a decade ago most likely as the result of genome-hybridization of two bacterial populations. Since then, it has been the cause of dozens of clinical cases each year in Israel. However, except for three cases, no environmental isolates of this group have been retrieved. Simple Sequence Repeats (SSR) or variable number of tandem repeats (VNTR) loci provides a source of high genomic polymorphism used in bacterial typing. Here we describe; the use of variation in SSR loci for accurate and rapid genotyping of V.vulnificus; Environmental and clinical survey of V.vulnificus conducted from 2004 to 2006 using SSR for typing and epidemiology; Studying the evolutionary aspects of environmental and clinical isolates retrieved from the "melting pot" of this new group, aquaculture fish environments in Israel. In-silico screen of the genome of two V.vulnificus strains revealed thousands of SSR tracts. Twelve SSR (≥5 bp), were tested for polymorphism in a 32 characterized V.vulnificus reference panel, representing clinical and environmental isolates from all biotypes. All tested SSR were polymorphic revealing high discrimination among isolates. Genetic analysis of SSR data resulted in clear distinction of biotype 3 isolates, furthermore revealed high discriminatory power within biotype 3 group, opposed to other molecular methods. We successfully isolated dozens of environmental biotype 3 isolates. The isolation method applied suggests that biotype 3 constitutes ~21% of the V.vulnificus population in the tested aquaculture-ponds, in contrast to ~86% of the clinical cases, suggesting this groups' high virulence. Genetic relations of 189 isolates were determined based on the 12 SSR loci. Analysis revealed an intermediate cluster between the main groups of biotype 1&2 and biotype 3. Further examination of representative strains at 15 additional MLST (multi locus sequence types) loci reveled as well the presence of a new sub-group. Thus, SSR are suitable for rapid typing and classification of V.vulnificus using high throughput capillary electrophoresis methods. The use of SSR as an epidemiological tool proved to be highly efficient and we were able to show connection between specific clinical and environmental cases. The study results suggest the possibility of a new inter-specific hybrid of V.vulnificus and stress the aquaculture fish environment and the influence of man-made ecological intervention on the emergence of possible new pathogens.