Transcript Document

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.
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 aminoglycoside Gentamicin in vitro. Further studies may
clarify the dosing requirements needed for
Gentamicin prophylaxis in vertebrates.
INTRODUCTION
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 AcanthamoebaFrancisella 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.
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/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.
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.
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.
RESULTS
DISCUSSION
Our results indicated there is a significant
decrease in growth of Acanthamoeba
species in response to increasing
concentrations of Gentamicin.
Effects of Gentamicin on A.astronyxis Growth
2.50E+06
2.00E+06
1.50E+06
003μg
002μg
001μg
Control
1.00E+06
5.00E+05
0.00E+00
0 Hour
12 Hour
24 Hour
48 Hour
Elapsed Time of Growth
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.
Effects of Gentamicin on A.castellanii Growth
2.00E+07
1.50E+07
003μg
002μg
001μg
Control
1.00E+07
5.00E+06
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.
0.00E+00
0 Hour
12 Hour
24 Hour
48 Hour
Elapsed Time of Growth
Effects of Gentamicin on A.culbertsoni Growth
REFERENCES
1.20E+08
Abd et al. 2003. “Survival and Growth of Francisella tularensis in
Acanthamoeba castellanii”. Applied and Environmental Microbiology. Vol.
69 No. 1 p. 600-606.
1.00E+08
8.00E+07
003μg
002μg
001μg
Control
6.00E+07
4.00E+07
2.00E+07
0.00E+00
0 Hour
12 Hour
24 Hour
48 Hour
Elapsed Time of Growth
Francine Marciano-Cabral and Guy Cabral. 2003. “Acanthamoeba spp. as
Agents of Disease in Humans”. Clinical Microbiology Reviews. Vol.16 No.2,
p. 273–307.
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.
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.