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Christopher Kovacs, Shawn Lynch, Joseph Carr, Christine Sanfilippo, Wolfgang Haas, Jenilee Kilbury, Kimberly Millard, Timothy Morris; Novel Minimum Inhibitory Concentration (MIC) Assay to Measure the Effectiveness of Antimicrobial Treatments Against Acanthamoeba Trophozoites and Cysts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2921.
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© ARVO (1962-2015); The Authors (2016-present)
Acanthamoeba protozoa cause rare, sight-threatening contact lens associated infections (AK, or Acanthamoeba Keratitis). Therapy is typically empiric and not well documented, requiring treatments administered over weeks or months, thus highlighting the need for standardized methods to assess efficacy of commonly used biocidal agents. This study describes a novel method of minimum inhibitory concentration (MIC) testing to determine the effectiveness of a variety of antimicrobial agents against both trophozoites and cysts of Acanthamoeba.
A. castellanii ATCC 50370 trophozoites were cultured axenically in antibiotic-free AC6 medium and adjusted to 5 x 104 trophozoites/ml for testing. Cysts were prepared with Neff’s encystment medium and adjusted to 5 x 104 cysts/ml. Test agents used were benzalkonium chloride (BAK), polyaminopropyl biguanide (PAPB), polyquaternium (PQ), chlorhexidine, and alexidine. Two-fold serial dilutions of biocides were done in 100 µl AC6 medium across 96-well plates to yield a test range of 256-0.125 µg/ml, then 100 µl of amoeba were added to all wells and incubated at 28°C. After 48 hr of outgrowth, resazurin dye, which changes from blue to pink in presence of respiring cells, was added to all wells and incubated for an additional 24 hr at 28°C.
MIC values were scored as the lowest dilution of test agent where the well remained blue 24 hr after dye was added. Similar to already standardized MIC testing of bacteria, MIC values against amoeba never varied more than one serial two-fold dilution across multiple assays. Consensus results for BAK, PAPB, PQ, chlorhexidine and alexidine against trophozoites/cysts were 16.0/16.0, 4.0/4.0, 1.0/1.0, 2.0/0.5 and 0.5/0.5 µg/ml, respectively.
The methods described here provide a simple, reproducible MIC assay for testing antimicrobial agents against Acanthamoeba trophozoites and cysts. Further, an entirely axenic assay that relies on colorimetric readout eliminates the need for bacterized co-culture or indirect statistical analysis (i.e. most probable number methods) to generate results. Finally, while these results showed minimal potency differences against trophozoites or cysts with the tested agents, this MIC method can also be used for testing against other amoeba strains as well as other potential therapeutic agents.
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