GENTAMICIN AS A BIOCIDAL AGENT IN CONTROLLING ACANTHAMOEBA A POTENTIAL BIOLOGICAL WEAPON Katherine J. Barter, B.S., MT., Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine James R. Palmieri, Ph.D., Department of Microbiology, Division of Biomedical Sciences, Virginia College of Osteopathic Medicine and Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia. INTRODUCTION OBJECTIVE REFERENCES ABSTRACT Acanthamoeba are free living protozoa that inhabit both terrestrial and aquatic environments. Most species of Acanthamoeba are nonpathogenic; however, several species are pathogenic and cause both acute and chronic illnesses within humans. Previous publications indicate that many lethal human pathogens, including: Francisella, Yersinia, and Mycobacterium have been experimentally cultivated within Acanthamoeba trophozoites. While inside the protective cysts of Acanthamoeba, these pathogenic bacteria are not only able to withstand harsh and potentially lethal environmental conditions, but also replicate faster than when grown alone. It has been hypothesized that Acanthamoeba may have enabled some intracellular bacteria to survive and adapt their existence within macrophages. Due to the extremely tough cyst of Acanthamoeba, some species are able to endure harsh conditions, including extreme temperatures, chemical and radiological treatment. As a result, Acanthamoeba could potentially be manipulated as a vehicle for transporting virulent, environmentally fragile organisms through the public water supply. Our data indicates that pathogenic Acanthamoeba species are susceptible to treatment with the amino- glycoside Gentamicin in vitro. Further studies may clarify the dosing requirements needed for Gentamicin prophylaxis in vertebrates. DISCUSSIONRESULTS Our objective was to determine whether or not increasing concentrations of Gentamicin would have amoebicidal effects on the presence of Acanthamoeba Culbertsoni, Acanthamoeba Castellani or Acanthamoeba Astronyxis. METHODS This work was supported in part by funds from the Virginia College of Osteopathic Medicine Interdisciplinary Research Program and by the facilities provided by Thomas J. Inzana Ph.D., Virginia Maryland Regional College of Veterinary Medicine Department of Biomedical Sciences and Pathobiology in Blacksburg, Virginia. It has previously been reported that Acanthamoeba castellanii growth dropped 25% when co-cultured with Francisella tularensis compared to when grown alone. In the indicated study, 250µg/mL of Gentamicin was added to a centrifuged Acanthamoeba- Francisella pellet. The researchers attributed their decrease in Acanthamoeba growth to the extreme virulence of F. tularensis. Numerous publications indicate the extreme conditions under which Acanthamoeba are able to survive, including exposure to high doses of multiple antibiotics, and the addition of chlorine to drinking water. As a result, our laboratory hypothesized the decrease in growth of Acanthamoeba in Abd et al’s reports was due to the addition of Gentamicin, not to the presence of Francisella. We sought to determine the concentrations at which Gentamicin exhibited any significant amoebicidal effects on the growth of three Acanthamoeba species. We used Corning 275mL tissue culture flasks to grow Acanthamoeba in freshly prepared Oxoid media with varying concentrations of Gentamicin Sulfate (concentrations varied from 0 micrograms/milliliter to 300 micrograms/milliliter). We started the culture flasks with approximately 1,000,000 Acanthamoeba per milliliter in a total volume of 30 milliliters Oxoid Culture Media, than removed 20 microliter samples for analysis at designated time periods using aseptic technique. The individual Acanthamoeba samples were each loaded onto a hemacytometer and counted via phase contrast microscopy. The total number of Acanthamoeba per flask was calculated using the average of the eight corner hemacytometer grids and then converting this quantity to number of Acanthamoeba per milliliter Oxoid Culture Media supplemented with Gentamicin. Our results indicated there is a significant decrease in growth of Acanthamoeba species in response to increasing concentrations of Gentamicin. The most pathogenic Acanthamoeba species we manipulated, A.culbertsoni, showed a very significant drop in growth in response to increasing concentrations of Gentamicin. A.culbertsoni also demonstrated significantly increased growth over the other two less pathogenic species over the same period of time. This rapidity of growth, combined with the resistance to extreme changes in environmental conditions, may contribute to the increased virulence associated with A.culbertsoni. In the current time of increased bioterrorism risk, further research is necessary to identify agents that exhibit both bactericidal and amoebicidal characteristics. This data would help increase the protection of American citizens against future bioterrorist threats. Abd et al “Survival and Growth of Francisella tularensis in Acanthamoeba castellanii”. Applied and Environmental Microbiology. Vol. 69 No. 1 p Francine Marciano-Cabral and Guy Cabral “Acanthamoeba spp. as Agents of Disease in Humans”. Clinical Microbiology Reviews. Vol.16 No.2, p. 273–307. Khan et al “Pathogenesis of Acanthamoeba infections”. Microbial Pathogenesis. 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