טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLilach Somer
SubjectVariation of Microsatellites Sequences in Listeria
monocytogenes Genome
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Kashi Yechezkel


Abstract

The genus Listeria comprises six closely related species of which only Listeria monocytogenes is a human pathogen. Detection, identification and typing of pathogenic microorganisms have become major objectives over the past decade in microbiology. We have developed a PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and L. monocytogenes in food products. The method detects 1-5 CFU in a 25 g food sample in 24 hours. 

Two approaches were developed for the identification of genetic variation among 28 L. monocytogenes strain and six strains of the other Listeria spp. The first approach is based on presence, absence and size variation of the products at Selected Amplified Intergenic Locus (SAIL). Phylogenetic analysis based of data from 25 loci, revealed clear distinction between L. monocytogenes and other Listeria species. L. monocytogenes was further separated into the three known lineages. The SAIL method enables very simple initial bacterial typing that would serve as a basis for efficient epidemiological investigation. The second approach is based on sequence variations at SSR loci. Simple Sequence Repeats (SSR, or Microsatellites) are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. A computer-based screen of the genome of L. monocytogenes, revealed 32,000 SSR tracts, with core motifs ranging from one to six nucleotides and average appearance of one SSR every 90bp. Nineteen evenly distributed SSR loci were selected to evaluate polymorphism among strains of L. monocytogenes and the other Listeria spp. All loci contain few polymorphic SSR tracts indicating the high information content of these loci. Phylogenetic analyses discriminate among 19 of the 28 L. monocytogenes strains and among all Listeria species. Results show that Multi-Locus Sequence Typing of SSR loci, termed SSR-MLST, is an efficient tool for bacterial phylogenetic and epidemiological studies.