Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans

Abstract

There is concern about the rise of antifungal drug resistance, but little is known about comparative biological properties and pathogenicity of drug-resistant strains. We generated fluconazole (FLC; CO23 RFLC)- or micafungin (FK; CO23 RFK)-resistant strains of Candida albicans by treating a FLC- and FK-susceptible strain of this fungus (CO23 S) with stepwise-increasing concentrations of either drug. Molecular analyses showed that CO23 RFLC had acquired markedly increased expression of the drug-resistance efflux pump encoded by the MDR1 gene, whereas CO23 RFK had a homozygous mutation in the FSK1 gene. These genetic modifications did not alter to any extent the growth capacity of the drug-resistant strains in vitro, either at 28 degrees C or at 37 degrees C, but markedly increased their experimental pathogenicity in a systemic mouse infection model, as assessed by the overall mortality and target organ invasion. Interestingly, no apparent increase in the vaginopathic potential of the strains was observed with an estrogen-dependent rat vaginal infection. The increased pathogenicity of drug-resistant strains for systemic infection was associated with a number of biochemical and physiological changes, including (i) marked cellular alterations associated with a different expression and content of major cell wall polysaccharides, (ii) more rapid and extensive hypha formation in both liquid and solid media, and (iii) increased adherence to plastic and a propensity for biofilm formation. Overall, our data demonstrate that experimentally induced resistance to antifungal drugs, irrespective of drug family, can substantially divert C. albicans biology, affecting in particular biological properties of potential relevance for deep-seated candidiasis.

FIG. 1.

Growth curves (A), scanning electron micrographs (EM) (B), and cell volume (C) of CO23_S (wild-type strain) and CO23_RFK and CO23_RFLC (FK- and FLC-resistant strains, respectively) of C. albicans. O.D. 560, optical density at 560 nm. The growth curves were obtained by growing the strains at 37°C in liquid YNB medium. The cell volumes were calculated from SEM observations, as described in Materials and Methods. In panel B, parts A, B, and C indicate the strains CO23_S, CO23_RFK, and CO23_RFLC, respectively, with corresponding cell volumes.

FIG. 2.

EM localization of MP constituents in freeze-substituted yeast cells of C. albicans strains CO23_S (A), CO23_RFK (B), and CO23_RFLC (C) following postembedding labeling with the MAb AF1 followed by gold-labeled secondary antibody. For technical details, see the text.

FIG. 3.

Experimental pathogenicity of C. albicans strains in systemic (A) and mucosal (B) infection models. (A) Male BALB/c mice were infected intravenously with the indicated C. albicans strains (inoculum size, 10⁷ cells). Survival was determined over the time indicated. There was a statistically significant difference (P < 0.01) between the parental and each of the two drug-resistant strains. For further details, see Materials and Methods. (B) Oophorectomized, estrogen-treated rats were infected intravaginally with the indicated C. albicans strains (inoculum size, 10⁷ cells). At the indicated time intervals, the intravaginal burden of fungal cells was measured as described in Materials and Methods. No statistically significant differences were found among the strains.

FIG. 4.

Hyphae and invasive hyphal growth of C. albicans strains CO23_S, CO23_RFK, and CO23_RFLC. (A) Cells were induced to form hyphae by 24-h incubation at 37°C in YNB medium containing 0.1% (wt/vol) N-acetyl-d-glucosamine (line 1) or RPMI 1640 medium containing 10% fetal calf medium (line 2). Photomicrographs were taken with a phase contrast microscope using a 40× objective and are representative of 50% random fields observed. (B) Cells (10⁴) of CO23_S, CO23_RFK, and CO23_RFLC strains were suspended in 1 μl of YPD broth and spotted onto the surface of 4% serum plates containing 0.8 to 3% agar, and the plates were incubated at 37°C. Line 1, top view of spot colonies at 7 days of incubation on 0.8% agar using a 10× objective. Line 2, images of spot colony edges at 7 days of incubation on 3% agar using a 40× objective. The photographs are representative of the whole-mounted microscopic plates.

FIG. 5.

Adherence, biofilm, and XTT assay of C. albicans strains CO23_S, CO23_RFK, and CO23_RFLC. (A) Percentage of plastic adherent cells of the strains CO23_S, CO23_RFK, and CO23_RFLC. The cells were allowed to adhere to the polystyrene surface, and then 1 ml of Sabouraud dextrose agar, which was allowed to solidify, was poured onto the cells, and the mixture was incubated at 37°C for 24 h. (B) Production of biofilm on polystyrene surfaces. Biofilms were stained with crystal violet and photographed at 10× and 40× magnifications using an inverted microscope. (C) Equal numbers of cells from CO23_S, CO23_RFK, and CO23_RFLC were suspended in 1 ml of RPMI medium and incubated in 12-well plates for the times indicated. Nonadherent cells were then removed by washing, and adherent cells were XTT assayed. Experiments were repeated three times with similar results.