Towards the definition of pathogenic microbe ☆ Yael Danin-Poleg a , Lilach Somer a , Lyora A. Cohen a , Eran Diamant a , Yniv Palti b , Yechezkel Kashi a, ⁎ a Department of Biotechnology and Food Engineering, Grand Water Research Institute, Technion, Israel Institute of Technology, Haifa 32000, Israel b National Center for Cool and Cold Water Aquaculture, USDA-ARS, 11861 Leetown Rd., Kearneysville, WV 25430, USA Accepted 26 April 2006 Abstract Identification and typing of spoilage and pathogenic microorganisms have become major objectives over the past decade in microbiology. In food, strain typing is necessary to ensure food safety and for linking cases of foodborne infections to suspected items. Recent advances in molecular biology have resulted in the development of numerous DNA-based methods for discrimination among bacterial strains. Here, we present the use of Simple Sequence Repeats (SSR, or Microsatellites) for bacterial typing. SSRs are a class of short DNA sequence motifs that are tandemly repeated at a specific locus. Computer-based screen of the complete genomic DNA sequences of various prokaryotes showed the existence of tens of thousands well distributed SSR tracts. Mono Nucleotides Repeats (MNRs) are the majority of SSR tracts in bacteria, therefore selected MNR loci were analyzed for variation among strains belonging to three bacterial species: Escherichia coli, Listeria monocytogenes and Vibrio cholerae. High levels of polymorphism in the number of repeats was observed. The finding that most of the MNR tracts are variable in bacterial genomes, but stable at the strain level, allows the use of MNRs for bacterial strains identification. The variation in MNR tracts enables the separation between virulent and non-virulent strain groups and further discriminates among bacterial isolates, in the three tested bacterial species. The uncovered MNR polymorphism is important as a genome-wide source of variation, both in practical applications (e.g. rapid strain identification) and in evolutionary studies. This multi-locus MNR strategy could be applied for high throughput bacterial typing by assigning an “identity number” for each strain based on MNR variations. The developed typing technology should include the fingerprint database for large bacterial strain collections and a high throughput scanner. This accurate and rapid tool can have a major role in decreasing the incidences of food-related outbreaks and will contribute to limit epidemics. © 2006 Elsevier B.V. All rights reserved. Keywords: Bacterial typing; Simple sequence repeats; Microsatellite; Mononucleotide repeats; Listeria monocytogenes; Escherichia coli; Vibrio cholerae 1. Introduction Foodborne pathogens are a major cause of human illness with estimated number of 76 million cases, 325,000 hospitalizations, and 5200 deaths in the United States each year and much higher proportion in the developed countries. In addition to the effect on human health, large percentages of the food products are lost by microbial spoilage. The modern food industry, based on inter- national market, long shelf-life products and ready to eat food, raises new microbial safety problems and demands. Consequently pathogenic microorganism detection and identification have become major objectives over the past decade in food and water microbiology. One specific problem is the discrimination among species and strains that may vary from normal pro-biotic micro-flora to virulent strains. Bacterial typing allows distinguishing among strains within a species, to follow epidemics and route of contamination or to identify the source of spoilage. The ability to rapidly type an organism to the specific strain, particularly one that can pose a more serious threat, can lead to faster crisis response, limiting infection, aiding in treatment, and preventing potential spread of the organism. In addition we are witnessing the development of a new field of forensic microbiology. It requires accurate and reproducible technology that produces DNA typing signature or International Journal of Food Microbiology 112 (2006) 236 – 243 www.elsevier.com/locate/ijfoodmicro ☆ This paper was presented at the 19th International ICFMH Symposium, Food Micro 2004, Potorož Slovenia, 12-16 September 2004. ⁎ Corresponding author. Tel.: +972 4 8293074; fax: +972 4 8293399. E-mail address: kashi@tx.technion.ac.il (Y. Kashi). URL: http://www2.technion.ac.il/biotech/ (Y. Kashi). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.04.010