Development of a Genome Fingerprint Database to Identify Genetically Engineered Microbes

The development of recombinant DNA technology and the current state of bioscience has for the first time made the laboratory creation of new biological weapons a real possibility. A critical part of any countermeasures is the ability to identify strains that are unlike previous strains that have been isolated, i.e. are “outside the box” of known genetic variability for the organism. A database of this type will help medical care providers plan an appropriate course of treatment and aid strategic decisions as to the possible origin of the new isolate. Such a database should be capable of including many types of data including source characterization and be capable of determining the phylogenetic relationship of any new isolate to the data contained in the database. We are using the Bionumerics software package and Yersinia pestis as our test organism. We examined 37 isolates from the United States (CONUS) and 20 isolates obtained from four different continents (OCONUS) that span a time period of 100 years. We examined our group of strains using a variety of techniques selective housekeeping gene sequencing and pulsed field gel electrophoresis (PFGE). We compared these methods with the established plague typing technique of ribotyping with ribosomal RNA gene probes. All of the CONUS strains belonged to a single ribotype. Chromosomal gene sequencing revealed that Y. pestis coding regions are highly stable and have not varied in any of the OCONUS strains. In contrast, PFGE was able to distinguish 46 strains (CONUS and OCONUS) that were derived from different parents. All CONUS strains were at least 70% similar to each other and could be divided into four groups that were greater than 90% similar. Furthermore, PFGE could distinguish one of two Y. pestis strains that were isogenic except for a single uncharacterized 14 kilobase pair insertion. Taken together, our results demonstrate that the Y. pestis genome is variable at the macromolecular level and that this instability is useful for determining the phylogenetic relationship between isolates and identification of potentially engineered BW strains.