Ocular Microbiology and Immunology Group
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2012 OMIG Abstract 23

Susceptibility of Acanthamoebae to Amikacin
J.D. Keenan, M.C. Chen, V. Cevallos, T.M. Lietman
Francis I. Proctor Foundation, University of California, San Francisco, San Francisco CA

Purpose: Acanthamoebae are the causative agents for both keratitis and granulomatous amoebic encephalitis. One of the treatments used for granulomatous amoebic encephalitis consists of intrathecal and intravenous amikacin. Although other aminoglycocides have been used for acanthamoeba keratitis, we are unaware of reports of amikacin for this blinding corneal infection. Therefore, we performed susceptibility testing of several acanthamoeba specimens to determine the cysticidal activity of amikacin against acanthamoeba.

Methods: We performed a cysticidal susceptibility assay reported by Elder that relies on the observation that acanthamoeba cysts adhere to the walls of a 96-well microtiter plate. Briefly, we added E. coli to stored acanthamoeba specimens to promote excystment, then incubated samples at 30°C for 1 week, after which most trophozoites had re-encysted. We added 50μl of a 10⁴ cysts/ml acanthamoeba suspension to each well of a 96-well microtiter plate, then added 50μl of serial dilutions of amikacin. We incubated plates at 30°C for 48 hours. We aspirated the drug and triple rinsed the wells with Page saline to remove residual drug. We added 100μl of a 0.5 MacFarland standard concentration of E. coli to each well, and observed every other day for growth of trophozoites. The well with the lowest concentration of drug at which no trophozoites grew at 1 week was classified as the minimum cysticidal concentration (MCC). We performed each run of the assay in duplicate and used the higher MCC when reporting results. We performed positive and negative controls with each plate by adding Page saline to 1 well with acanthamoeba and to another well without acanthamoeba. An identical assay was performed for chlorhexidine as a control.

Results: We performed the assay on 1 ATCC acanthamoeba strain and 9 keratitis specimens. The clinical specimens had been obtained by physicians at the Proctor Foundation from 2008-2011. The MCCs for chlorhexidine ranged from 3.13 to 25 μg/ml, with a median of 6.25 μg/ml (IQR 3.13 to 10.94). The MCCs for amikacin ranged from 781.25 to 12,500 μg/ml, with a median of 6,250 μg/ml (IQR 6,250 to 6,250).

Conclusion: We demonstrated that amikacin has cysticidal activity against acanthamoeba obtained from clinical keratitis specimens. We also found chlorhexidine to be cysticidal at concentrations reported previously, which suggests our assay was valid. Amikacin is usually administered topically at concentrations of 3-5%; the 3% solution would be nearly 5-fold higher than the median MCC for amikacin found in this study. Amikacin may be considered as an adjunctive therapy for acanthamoeba keratitis.

Disclosure: S     Research to Prevent Blindness

2012 Agenda and Abstracts | < Previous | Next >