GENTAMICIN AS A BIOCIDAL AGENT IN CONTROLLING ACANTHAMOEBA

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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
ABSTRACT
DISCUSSION
RESULTS
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.

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 als 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.
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.




METHODS
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/millilite
r 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.
REFERENCES
Abd et al. 2003. Survival and Growth of
Francisella tularensis in Acanthamoeba
castellanii. Applied and Environmental
Microbiology. Vol. 69 No. 1 p. 600-606. Francine
Marciano-Cabral and Guy Cabral. 2003.
Acanthamoeba spp. as Agents of Disease in
Humans. Clinical Microbiology Reviews. Vol.16
No.2, p. 273307. Khan et al. 2003.
Pathogenesis of Acanthamoeba infections.
Microbial Pathogenesis. Vol. 34 No.6 p.
277-285. Tomov et al. 1999. Persistence and
Multiplication of Obligate Anaerobe Bacteria in
Amebae Under Aerobic Conditions Anaerobe. Vol. 5
p.19-23.
OBJECTIVE
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.
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.