RESUMO
Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).
Assuntos
Antifúngicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida/efeitos dos fármacos , Candida/fisiologia , Anfotericina B/farmacologia , Fluconazol/farmacologia , Violeta Genciana/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Coloração e Rotulagem/métodos , Sais de Tetrazólio/metabolismoRESUMO
The paradoxical growth (PG) of Candida sp. biofilms in the presence of high caspofungin (CAS) concentrations was previously unknown. We sought to characterize the PG at supra-MICs of CAS among clinical Candida sp. isolates grown as biofilms in 96-well polystyrene microtiter plates. The MICs of CAS were determined for 30 clinical Candida sp. isolates (4 Candida albicans, 6 C. tropicalis, 7 C. parapsilosis, 8 C. orthopsilosis, and 5 C. metapsilosis isolates) when they were grown as planktonic cells and biofilms and were defined as the lowest drug concentrations that resulted in a prominent decrease in growth and a 50% reduction in metabolic activity, respectively. PG was defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above the MIC. With the exception of C. tropicalis, all isolates displayed PG more frequently when they were grown as biofilms than when they grown as planktonic cells. PG was undetectable among C. metapsilosis isolates in planktonic cell MIC tests but was present in 100% of the isolates in biofilm MIC tests. The drug concentration and the number of drug dilutions supporting PG were higher for biofilms than for planktonic cells. Microscopic changes in cell morphology were observed among both planktonic and biofilm cells with PG. Specifically, the accumulation of enlarged, globose cells was associated with PG, and we hypothesize that CAS-induced changes in the cell wall composition may be the explanation.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/fisiologia , Equinocandinas/farmacologia , Candida/crescimento & desenvolvimento , Candidíase/microbiologia , Caspofungina , Lipopeptídeos , Testes de Sensibilidade Microbiana , Risco , Sais de TetrazólioRESUMO
We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.