title: Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.
abstract: A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism.
authors.affiliation: Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain. Electronic address: irene.heredero@edu.uah.es.
authors.forename: I
authors.initials: I
authors.lastname: Heredero-Bermejo
authors.affiliation: Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Criado-Fornelio
authors.affiliation: Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Soliveri
authors.affiliation: Oxoprobics Biosciences S.L., Francisco Giralte 2, Desp.12, 28002 Madrid, Spain.
authors.forename: J A
authors.initials: JA
authors.lastname: Díaz-Martín
authors.affiliation: Oxoprobics Biosciences S.L., Francisco Giralte 2, Desp.12, 28002 Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Matilla-Fuentes
authors.affiliation: Oxoprobics Biosciences S.L., Francisco Giralte 2, Desp.12, 28002 Madrid, Spain.
authors.forename: J A
authors.initials: JA
authors.lastname: Sánchez-Arias
authors.affiliation: Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: J L
authors.initials: JL
authors.lastname: Copa-Patiño
authors.affiliation: Departamento de Biomedicina y Biotecnología, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Pérez-Serrano
mesh_terms: D000048:Acanthamoeba / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D000563:Amebicides / Q000494:pharmacology*; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D002138:Calibration; D002710:Chlorhexidine / Q000031:analogs & derivatives / Q000494:pharmacology; D050091:Dendrimers / Q000494:pharmacology; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D020128:Inhibitory Concentration 50; D010101:Oxygen Consumption
chemical_list: D000563:Amebicides; D000891:Anti-Infective Agents, Local; D050091:Dendrimers; C010882:chlorhexidine gluconate; D002710:Chlorhexidine
keywords: Acanthamoeba castellanii; Acanthamoeba polyphaga; Drug screening; Oxygen consumption rate; Viability assay