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Journal of Clinical Microbiology, February 2005, p. 629-634, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.629-634.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

James McBride,{dagger} Paul R. Ingram,{dagger} Fiona L. Henriquez, and Craig W. Roberts*

Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, United Kingdom

Received 29 July 2004/ Returned for modification 13 September 2004/ Accepted 24 September 2004

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 µM 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.

* Corresponding author. Mailing address: Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor St., Glasgow, Scotland, United Kingdom. Phone: (0)141 548 4823. Fax: (0)141 548 4823. E-mail: c.w.roberts{at}strath.ac.uk.

{dagger} J.M. and P.R.I. contributed equally to this work.

Journal of Clinical Microbiology, February 2005, p. 629-634, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.629-634.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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