Microarray Bactericidal Testing of Natural Products Against Yersinia intermedia and Bacillus anthracis

To address the Defense Department's need for safe, effective protection against biological warfare agents (BWA's), natural products with a historical record of bactericidal efficacy such as bacteriocins, biosurfactants, lytic enzymes and essential oils or their active components were tested against non-pathogenic Bacillus anthracis and the plague pathogen surrogate Yersinia intermedia. Unlike the traditional well diffusion or spot lawn tests, the microplate/microarray test method used in these studies greatly simplified screening and interpretations of dose-response testing of the natural compounds as it relates to decontamination applications. Growth of treated cultures was measured turbidometrically with a microplate reader. Dose-reponse plots were made from the resulting turbidometric data to assess bactericidal efficacy of the test compounds. Several natural compounds were found effective against Yersinia and B. anthracis cells and spores using this method. These results show the promise of natural compounds in the development of environmentally responsible BWA decontamination preparations. INTRODUCTION The US Department of Defense recognizes the threat of biological warfare agents (BWA’s) as a weapon of mass destruction from foreign military powers and terrorists. Although the chemical warfare agent (CWA) decontamination solution 2 (DS2) is also an effective bactericide, it is corrosive and it’s use produces hazardous waste. As the DoD is concerned with the environmental impact of decontaminants for fixed site and large area applications, decontamination solutions with an adverse effect on the environment are less desirable so alternatives were sought. Microbial chemical agent decontaminating enzymes such as Organophosphorus Acid Anhydrolase (OPAA) and Organophosphorus Hydrolase (OPH) are good alternatives to DS2 for nerve agent decontamination, but they lack bactericidal activity. Inclusion of a bactericide to these enzymes for an antiCWA/BWA preparation would require chemicals with little effect on enzyme activity or the environment. Several natural products are effective bactericides and have low environmental impact, making them good candidates as biological decontaminants. Among these are the bacteriocins, essential oils and biosurfactants. Bacteriocins are anti-microbial polypeptides that are bactericidal to non-host bacteria. Some bacteriocins have chacteristics that make them desirable biological decontaminants, such as resistance to proteolytic degradation, heat stability and a wide target range. Commercially available Nisin, from Lactococcus lactis, has these characteristics and in addition is a FDA GRAS food additive. Plant essential oils (e.g. oil of cloves, eucalyptus, etc) are attractive as BWA bactericides because of their historical use in the preservation of food, embalming and medicine. These oils and their active components (AC’s) are also commercially available. As the bacteriocins from the lactic acid bacteria were more noted for their effectiveness with gram positive bacteria than gram negative bacteria , we also included the active components of plant essential oils in our decontamination studies for the Yersinia in addition to Bacillus anthracis. Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 00 JAN 2002 2. REPORT TYPE N/A 3. DATES COVERED 4. TITLE AND SUBTITLE Microarray Bactericidal Testing of Natural Products Against Yersinia intermedia and Bacillus anthracis 5a. CONTRACT NUMBER