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

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...

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Veröffentlicht in:Journal of Clinical Microbiology 2005-02, Vol.43 (2), p.629-634
Hauptverfasser: McBride, James, Ingram, Paul R, Henriquez, Fiona L, Roberts, Craig W
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container_title Journal of Clinical Microbiology
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creator McBride, James
Ingram, Paul R
Henriquez, Fiona L
Roberts, Craig W
description 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 [micro]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.
doi_str_mv 10.1128/JCM.43.2.629-634.2005
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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 [micro]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. 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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. 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Psychology</subject><subject>Indicators and Reagents - metabolism</subject><subject>Infectious diseases</subject><subject>Medical sciences</subject><subject>Microbiology</subject><subject>Oxazines - metabolism</subject><subject>Parasitic Sensitivity Tests</subject><subject>Parasitology</subject><subject>Phospholipid Ethers</subject><subject>Phosphorylcholine - analogs &amp; derivatives</subject><subject>Xanthenes - metabolism</subject><issn>0095-1137</issn><issn>1098-660X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkktv1DAUhSMEotPCTwCyobsEv2MvWIyGt1qBBJXYWdeuM3GVxIPtKeq_x9EMLaxs6X7n3Ht9XFUvMGoxJvLNl81ly2hLWkFUIyhrCUL8UbXCSMlGCPTzcbVCSPEGY9qdVKcp3SCEGeP8aXWCuVBccLGqhnfu1o1hN7k516GvN2EM0U8uR2_rS29jyD67WH8bIbt6nRLc1X2Iy82l9Fe1nrOfFth4GFMNW_BzyvXawpwHmIIz8Kx60peae348z6qrD-9_bD41F18_ft6sLxrLZJcbB4YhZhTFknHrqDW96jrSS0pxJxUzjncKGWl6aq4BE6sIl0xayQQGKhE9q94efHd7M7lrWyaMMOpdWQrinQ7g9f-V2Q96G241ZxKprujPj_oYfu1dynryybpxhNmFfdKiY-X92dKIH8CydkrR9fc9MNJLRLpEpBnVRJeIdIlILxEV3ct_B3xQHTMpwOsjAMnC2EeYrU8PXGFw8SlcfeAGvx1---g0pEnf2Om-aUFeHZAegoZtLDZX3wnCtPwMJaQi9A84eLCk</recordid><startdate>20050201</startdate><enddate>20050201</enddate><creator>McBride, James</creator><creator>Ingram, Paul R</creator><creator>Henriquez, Fiona L</creator><creator>Roberts, Craig W</creator><general>American Society for Microbiology</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20050201</creationdate><title>Development of Colorimetric Microtiter Plate Assay for Assessment of Antimicrobials against Acanthamoeba</title><author>McBride, James ; Ingram, Paul R ; Henriquez, Fiona L ; Roberts, Craig W</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c487t-eab404b931845ce3cbf9772f83317894be5790b8bf3bda12c925848c8461a3803</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Acanthamoeba - drug effects</topic><topic>Acanthamoeba - growth &amp; development</topic><topic>Acanthamoeba castellanii - drug effects</topic><topic>Acanthamoeba castellanii - growth &amp; development</topic><topic>Amebicides - pharmacology</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Chlorhexidine - pharmacology</topic><topic>Colony Count, Microbial</topic><topic>Colorimetry</topic><topic>Culture Media</topic><topic>Fundamental and applied biological sciences. 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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. 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source American Society for Microbiology; MEDLINE; EZB-FREE-00999 freely available EZB journals; PubMed Central
subjects Acanthamoeba - drug effects
Acanthamoeba - growth & development
Acanthamoeba castellanii - drug effects
Acanthamoeba castellanii - growth & development
Amebicides - pharmacology
Animals
Biological and medical sciences
Chlorhexidine - pharmacology
Colony Count, Microbial
Colorimetry
Culture Media
Fundamental and applied biological sciences. Psychology
Indicators and Reagents - metabolism
Infectious diseases
Medical sciences
Microbiology
Oxazines - metabolism
Parasitic Sensitivity Tests
Parasitology
Phospholipid Ethers
Phosphorylcholine - analogs & derivatives
Xanthenes - metabolism
title Development of Colorimetric Microtiter Plate Assay for Assessment of Antimicrobials against Acanthamoeba
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