Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection-Scotland, 2012-2013

Abstract

Bloodstream infections caused by Candida species remain a significant cause of morbidity and mortality in hospitalized patients. Biofilm formation by Candida species is an important virulence factor for disease pathogenesis. A prospective analysis of patients with Candida bloodstream infection (n = 217) in Scotland (2012-2013) was performed to assess the risk factors associated with patient mortality, in particular the impact of biofilm formation. Candida bloodstream isolates (n = 280) and clinical records for 157 patients were collected through 11 different health boards across Scotland. Biofilm formation by clinical isolates was assessed in vitro with standard biomass assays. The role of biofilm phenotype on treatment efficacy was also evaluated in vitro by treating preformed biofilms with fixed concentrations of different classes of antifungal. Available mortality data for 134 patients showed that the 30-day candidaemia case mortality rate was 41%, with predisposing factors including patient age and catheter removal. Multivariate Cox regression survival analysis for 42 patients showed a significantly higher mortality rate for Candida albicans infection than for Candida glabrata infection. Biofilm-forming ability was significantly associated with C. albicans mortality (34 patients). Finally, in vitro antifungal sensitivity testing showed that low biofilm formers and high biofilm formers were differentially affected by azoles and echinocandins, but not by polyenes. This study provides further evidence that the biofilm phenotype represents a significant clinical entity, and that isolates with this phenotype differentially respond to antifungal therapy in vitro. Collectively, these findings show that greater clinical understanding is required with respect to Candida biofilm infections, and the implications of isolate heterogeneity.

Keywords: Antifungal; Candida albicans; Candida glabrata; biofilm; candidaemia; catheters; drug resistance.

Fig. 1

Biofilm formation by Candida species. Candida bloodstream isolates were evaluated for biofilm formation with standardized methods. Candida isolates standardized (1 × 10⁶ cells/mL) in RPMI-1640 were grown in flat-bottomed 96-well microtitre plates for 24 h at 37°C. Mature biofilms were carefully washed with phosphate-buffered saline and allowed to air dry, and biomass quantified by staining with 0.05% w/v crystal violet solution. The biofilms were washed and destained with 100% ethanol. Biomass was quantified spectrophotometrically by reading the optical density (OD) at 570 nm in a microtitre plate reader (FluoStar Omega BMG Labtech, Aylesbury, UK). Six replicates were used for each isolate, and the mean of each is represented. ***p <0.0001.

Fig. 2

Survival of patients with Candida albicans and Candida glabrata infection. Survival of patients infected with C. albicans (solid line) and C. glabrata (dotted line) was monitored over a period of 30 days from the first Candida-positive blood culture. Cox regression plots, adjusted only for patient age (n = 95) (a) or for age and catheter removal (n = 42) (b), in patients with C. albicans and C. glabrata infection are shown. Comparison between these curves showed a statistically significant difference in the mortality rate in (b) (p <0.05).

Fig. 3

Survival of patients with Candida albicans high biofilm formers (HBFs) and low biofilm formers (LBFs). Survival of patients infected with C. albicans HBFs (n = 17) and LBFs (n = 17) was monitored over a period of 30 days from the first Candida-positive blood culture. Cox regression plots adjusted for (a) age only (n = 34) or (b) age and parenteral nutrition (n = 28) in patients with C. albicans HBFs (solid line) and LBFs (dotted line) are shown. Comparison between these curves showed a statistically significant difference in the mortality rate in (b) (p <0.05).

Fig. 4

Impact of Candida albicans biofilm formation on antifungal susceptibility. Ten low biofilm formers (LBFs) and high biofilm formers (HBFs) were standardized to 1 × 10⁶ cells/mL in RPMI-1640, and grown as biofilms in flat-bottomed 96-well microtitre plates for 24 h. Biofilms were washed with phosphate-buffered saline before being treated with 2 mg/L (a) or 200 mg/L (b) voriconazole (VRZ), caspofungin (CAS), and amphotericin B (AMB). After incubation for 24 h, metabolic activity was measured with the XTT assay, with optical density being read at 492 nm. Percentage viability was calculated relative to untreated controls, and data are presented as mean ± standard deviation. Eight replicates were used for each isolate, and repeated on two separate occasions. *p <0.05, ***p <0.001.