Candida parapsilosis Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates

Abstract

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.

Keywords: Candida parapsilosis; biofilm; colony morphology; drug susceptibility.

Figure 1

Colony morphotypes formed by C. parapsilosis. (A) Mixed morphotypes observed on a routine diagnostic plate. (B) Cells with distinct morphological colony phenotypes can be sub-cultured, but some strains (here strain PEU582: smooth-glossy vs. crepe, see below) sometimes undergo switching with strain-dependent frequencies. Smooth colonies are composed mainly of yeast-form cells, whereas non-smooth colonies are composed of pseudohyphal cells or mixtures of both morphologies.

Figure 2

C. parapsilosis collection and selection of isolates for downstream experiments. (A) Distribution of collection isolates stratified according to site of isolation and biofilm production on polystyrol. Category “invasive” includes, e.g., blood culture, biopsies, or intraoperative swabs. Orange diamonds: six isolates used in pre-formed biofilm experiments, see text. Red and green lines: mean and two-fold mean values. (B) Biofilm formation capacity; isolates sorted by value from low to high. Intersection of black lines: approximated cut-off. Red boxes indicate strain selection of representative low (LBF, left box), intermediate (IBF, middle box) and high (HBF, right box) biofilm formers for subsequent experiments.

Figure 3

Drug susceptibility. (A) Biofilm formation-phenotype dependent susceptibility testing where inoculum was prepared from cells after 1 day of growth on Sabouraud dextrose agar (SDA) (gray boxes) and after 8 days of growth (white boxes) on the same plates, when colonies had fully developed morphotypes. For each substance tested, the values for 1 and 8-day inoculum are depicted for each group (LBF, IBF, and HBF). Red lines: EUCAST clinical breakpoint (R>); green lines, susceptible cut-off (S ≤). *: statistical significance (B,C) Effect of antifungal drugs on cell viability in pre-formed biofilms of selected biofilm-forming isolates tested in (B) RPMI (Roswell Park Memorial Institute )or (C) YEPD(yeast extract peptone dextrose) media.

Figure 4

Emergence of colony morphotype over time. (A) Development of colony morphotype and (B) agar invasiveness scoring the same plates consecutively from 4 days to 10 days after inoculation. Isolates with low biofilm formation capacity (left panels), intermediate biofilm formation capacity (middle panels), and high biofilm formation capacity (right panels) were scored for the most frequent colony morphotype visible. Of note, HBF isolates rated “smooth” still developed minor frequencies of non-smooth colonies. See Supplementary Figure S1 for scoring references.