Patients with long-term oral carriage harbor high-persister mutants of Candida albicans - PubMed (original) (raw)

Patients with long-term oral carriage harbor high-persister mutants of Candida albicans

Michael D Lafleur et al. Antimicrob Agents Chemother. 2010 Jan.

Abstract

Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.

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Figures

FIG. 1.

FIG. 1.

Oral Candida carriage in patients undergoing chemotherapy. Cancer patients were sampled weekly for oral Candida carriage, and the first week in which Candida was no longer isolated was plotted. Carets above the bars indicate patients who left the study after chemotherapy concluded. The patients were divided into two groups. In group A, Candida was isolated for less than 8 consecutive weeks, and in group B, Candida was isolated for more than 8 weeks.

FIG. 2.

FIG. 2.

Levels of C. albicans persisters in clinical isolates. Persisters were determined by exposing biofilms of a panel of clinical isolates to 100 μg/ml AMB for 48 h. The number of weeks denotes the time of C. albicans carriage in a given patient from which a particular strain was isolated. The biofilms were washed, disrupted, diluted, and plated for determination of colony counts. The percentage of persisters was determined by comparing the number of cells surviving AMB treatment to the number of cells of the untreated control for each strain. Each strain was tested in triplicate.

FIG. 3.

FIG. 3.

C. albicans high-persister strains isolated from cancer patients. The level of surviving persister cells was determined by exposing the biofilms of a panel of clinical isolates to 100 μg/ml AMB or 200 μg/ml CHX for 48 h. After antifungal challenge, the biofilms were washed, disrupted, diluted, and plated for determination of colony counts. A total of 131 C. albicans strains were tested for survival in the presence of AMB, and representative strains were also tested for survival in the presence of CHX. Clinical isolates from patients A to E are representatives from group A in which Candida was isolated for less than 8 consecutive weeks (n = 39). Clinical isolates from patients F to L are hip mutants from group B patients, who had long-term Candida carriage (n = 92). The number following each patient indicates the week that each strain was isolated. The data represent the average of three independent experiments performed with biological duplicates, and the error bars are standard deviations.

FIG. 4.

FIG. 4.

Dose-dependent killing of C. albicans biofilms by AMB. Biofilms were cultured in RPMI 1640 medium for 24 h and exposed to AMB for 48 h. After the antifungal challenge, the biofilms were washed, disrupted, diluted, and plated for determination of colony counts. The data represent the average of three independent biological replicates for each strain, and the error bars are standard deviations.

FIG. 5.

FIG. 5.

Susceptibility of Candida albicans isolates to AMB and CHX. The MICs for the hip mutants and susceptible C. albicans strains isolated from cancer patients were determined by broth microdilution assay. The data for the isolates are expressed as the percentage of all strains in each group: n = 116 and n = 15 for susceptible strains and hip strains, respectively.

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