Development of Colorimetric Microtiter Plate Assay for Assessment of Antimicrobials against Acanthamoeba

Abstract

We have developed and optimized a 96-well microtiter plate assay, based on the reduction of alamarBlue, to assess the efficacies of much needed new antimicrobials against Acanthamoeba species. This assay has been optimized for determination of drug efficacy against two potentially pathogenic species, Acanthamoeba castellanii and Acanthamoeba polyphaga, and has been validated by comparison of their relative susceptibilities to chlorhexidine, a drug widely used to treat Acanthamoeba keratitis. The results demonstrate that the assay is comparable to a manual counting assay and that A. polyphaga is more resistant to chlorhexidine than A. castellanii. Thus, by use of the manual counting assay, 3.125 M chlorohexidine was almost completely effective against A. castellanii, whereas this concentration was less than 20% effective against A. polyphaga. Similar results were obtained by the alamarBlue assay. The new assay was used to determine the relative susceptibilities of A. castellanii and A. polyphaga to the alkylphosphocholines (APCs) hexadecylphosphocholine (hexadecyl-PC; miltefosine) and octadecylphosphocholine (octadecyl-PC) as well as an alkylgycerolphosphocholine, edelfosine. Both APCs studied were equally effective against A. castellanii, but octadecyl-PC was less effective than hexadecyl-PC against A. polyphaga. Both APCs were more effective than edelfosine against both Acanthamoeba species. A. polyphaga was found to be significantly less susceptible to each of the phosphocholine analogues. The newly described assay offers a number of advantages over those described previously. It is less labor-intensive than previously described assays and is sensitive and rapid, and the results can be read in a nonsubjective manner. As it is based on a standard 96-well, microtiter plate, it is amenable to automation and high throughput.

Resistance of Acanthamoeba Cysts to Disinfection in Multiple Contact Lens Solutions

“…Studies demonstrate that nearly all rigid and soft contact lens solutions sold in the United States have inadequate Acanthamoeba disinfection efficacy (1,3,4,5,12,13,14,16,19,20,25,26,37,39).…”

Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

“…To study the effect on growth of A. castellanii and alveolar epithelial cell cultures, the Alamar blue assay was used (3,54). The concentrations of A. castellanii and alveolar epithelial cell suspensions were adjusted to 1 ϫ 10 5 and 6 ϫ 10 4 cells per ml, respectively.…”

Differential Modulation of TLR3- and TLR4-Mediated Dendritic Cell Maturation and Function by Progesterone

“…Wells containing medium only were used as a blank. After 48 h, well contents were assayed for alamarBlue reduction by measuring the absorbance of the wells at 570 and 600 nm on a SpectroMax 190 plate reader (Molecular Devices) and the percentage reduction of alamarBlue calculated as previously described (28). ; catalog number 3031L), were purchased from Cell Signaling Technology (Beverly, MA).…”