title: Berberine Antifungal Activity in Fluconazole-Resistant Pathogenic Yeasts: Action Mechanism Evaluated by Flow Cytometry and Biofilm Growth Inhibition in Candida spp.
abstract: The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 μg/ml and 16 μg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001).
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Anderson Ramos
authors.initials: AR
authors.lastname: da Silva
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: João Batista
authors.initials: JB
authors.lastname: de Andrade Neto
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Cecília Rocha
authors.initials: CR
authors.lastname: da Silva
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Rosana de Sousa
authors.initials: Rde S
authors.lastname: Campos
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Rose Anny
authors.initials: RA
authors.lastname: Costa Silva
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Daniel Domingues
authors.initials: DD
authors.lastname: Freitas
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Francisca Bruna Stefany Aires
authors.initials: FB
authors.lastname: do Nascimento
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Larissa Nara Dantas
authors.initials: LN
authors.lastname: de Andrade
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Letícia Serpa
authors.initials: LS
authors.lastname: Sampaio
authors.affiliation: Department of Biology, Science Center, Molecular Genetics Laboratory, Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Thalles Barbosa
authors.initials: TB
authors.lastname: Grangeiro
authors.affiliation: School of Pharmacy, Federal University of Paraiba, João Pessoa, PB, Brazil.
authors.forename: Hemerson Iury Ferreira
authors.initials: HI
authors.lastname: Magalhães
authors.affiliation: Department of Physiology and Pharmacology, Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Bruno Coêlho
authors.initials: BC
authors.lastname: Cavalcanti
authors.affiliation: Department of Physiology and Pharmacology, Federal University of Ceara, Fortaleza, CE, Brazil.
authors.forename: Manoel Odorico
authors.initials: MO
authors.lastname: de Moraes
authors.affiliation: School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil label_ufc@yahoo.com.br.
authors.forename: Hélio Vitoriano
authors.initials: HV
authors.lastname: Nobre Júnior
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001599:Berberine / Q000009:adverse effects / Q000494:pharmacology*; D018441:Biofilms / Q000254:growth & development; D002175:Candida / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D002177:Candidiasis / Q000188:drug therapy* / Q000382:microbiology; D002460:Cell Line; D049109:Cell Proliferation; D003453:Cryptococcosis / Q000188:drug therapy* / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000009:adverse effects / Q000494:pharmacology*; D006801:Humans; D007739:L Cells; D051379:Mice; D008826:Microbial Sensitivity Tests; D051336:Mitochondrial Membranes / Q000187:drug effects; D058889:Molecular Typing; D016533:Mycological Typing Techniques
chemical_list: D000935:Antifungal Agents; D004271:DNA, Fungal; D001599:Berberine; D015725:Fluconazole