Anthrax Environmental Decontamination Network Neli Chakvetadze1, Lile Malania1, Ketevan Sidamonidze1, Nino Modebadze1, Ekaterine Khmaladze1, Mariam Zakalashvili1, Paata Imnadze1, James Blaxland2, Ashley Otter2, Les Baillie2 1National Center for Disease Control and Public Health (NCDC), Tbilisi, Georgia 2School of Pharmacy and Pharmaceutical Sciences, Cardiff University Table 1. Bacteriophage plaque characteristics produced by phage amplified on B. anthracis Sterne. Introduction Anthrax is a disease of animals caused by the spore forming bacterium Bacillus anthracis, which can infect humans either directly through contact with infected animals or indirectly as a consequence of bioterrorism. Anthrax is spread in Georgia and remains a permanent threat to humans due to the presence of active, resistant soil foci. Historically, anthrax has been registered across the entire country of Georgia (66 rayons); approximately 10% of the county is recognized as ‘active foci’ with a significant risk of animal and human infection. In this study, we present results from the training program, “Anthrax Environmental Decontamination Network”. This training program sought to develop an approach to decontaminate soil containing B. anthracis, with minimal impact on the environment. It is thought that a combination of germinant chemicals and B. anthracis specific bacteriophage will be an effective and ecologically friendly means of reducing viable B. anthracis spores in the soil. Phage Average plaque size (mm) Range of plaque size (mm) Number of plaques measured Plaque description Typical phage titre (PFU/mL) RW 1.30 1.0 – 1.4 45 Clear and small with cloudy edge. 105 - 109 AB1 1.00 0.9 – 1.1 32 Turbid, small, and circular. 103 – 105 Figure 1. Bacteriophage (RW, Gamma, and AB1) plaques on the surface of Bacillus anthracis Sterne, Figure 2. Scanning electron micrographs of bacteriophage A) RW and B) AB1; isolated from Welsh soil. A B Materials & Methods Two specific trainings were conducted as part of the program. A training on the outdoor decontamination of B. anthracis spores was conducted. During this training, defined combinations of germinant chemicals were used to trigger germination of B. anthracis spores. Also, general procedures for bacteriophage isolation were used. Individual phage were purified before proliferation to ensure singular phage types were used. The phage were then characterized on the basis of their host range using a panel of B. cereus and B. anthracis isolates.The plaque morphology of each phage was determined using an agar overlay method. The phage preparation (at a range of dilutions) was added to overnight cultures of the propagation strain of bacteria in 5 mL of molten TSA agar. The agar was poured over the surface of a TSA plate and allowed to dry. The plate was then incubated overnight at 37°C for 18 to 24 hours. After incubation, the plates were examined for the presence of plaques. To investigate the phage morphology, the phage were subjected to scanning electron microscopy. Table 3: The various number of Bacillus isolates each phage can infect. Gamma phage is included as a reference. Phage Number of Bacillus isolates infected Number of Bacillus species infected Bacillus species phage is capable of infecting RW 7 4 B. anthracis, B. cereus, B. mycoides ,& B. thuringiensis. AB1 9 6 B. anthracis, B. cereus, B. subtilis, B. megaterium, B. pseudomycoides ,& B. thuringiensis. Gamma 12 Conclusions Using a simple laboratory based method, bacteriophage capable of lysing B. anthracis were isolated. These methods could be used to isolate B. anthracis specific bacteriophage from B. anthracis contaminated sites in Georgia. This training program formed the basis of a research model that will allow new technological approaches for anthrax decontamination to be developed. These new technologies will have the potential to impact a broad number of areas. Techniques to be developed will include the development of environmentally friendly phage based and chemical germinant based approaches of decontaminating B. anthracis from agricultural land to return it to productive use. In addition to the agricultural and economic benefits, such techniques would lead to a reduction in the number of cases of human infection (which is largely acquired through the handling of contaminated animal products) as the environmental spore burden would be reduced. Results B. anthracis failed to persist in a vegetative form after it had been induced to germinate. The addition of L-alanine and L-inosine to a suspension of B. anthracis spores stimulated germination and resulted in a four log reduction of the spore count in the soil. To date, two novel bacteriophage, RW and AB1, have been isolated from soil collected in Wales near Cardiff, UK. The characteristics of the plaques produced by these bacteriophage on B. anthracis Sterne are shown in Table 1 and Figure 1. All descriptions are noted from growth on B. anthracis overlay plates. Throughout the study, plaque titres (after overnight incubation with B. anthracis) were recorded, typical phage titres after overnight incubation are reported in the final column of the table under ‘Phage titre’. The phage titres are recorded in Plaque Forming Units per Millilitre (PFU/mL). The morphology/structure of the phage as revealed by scanning electron microscopy can been seen in Figure 2. Results from the specificity testing of each bacteriophage on the collection of previously characterized bacillus strains is recorded in Table 3. Acknowledgements This research was supported by the European Union’s Seventh Framework Programme (FP7) under grant #PIRSES-GA-2013-612309 (website: https://aednetproject.wordpress.com). We are grateful to the U.S. Department of Defense (DoD), Defense Threat Reduction Agency (DTRA), for the financial support to attend this conference. The findings, opinions, and views expressed herein belong to the authors and do not reflect any official position of the U.S. DoD, the U.S. Government, or any other organization listed.