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. 2014 Jul 5:14:182.
doi: 10.1186/1471-2180-14-182.

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

Leighann Sherry, Ranjith Rajendran, David F Lappin, Elisa Borghi, Federica Perdoni, Monica Falleni, Delfina Tosi, Karen Smith, Craig Williams, Brian Jones, Chris J Nile, Gordon Ramage  1
Affiliations

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

Leighann Sherry et al. BMC Microbiol. .

Abstract

Background: Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation.

Results: Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1.

Conclusions: Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.

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Figures

Figure 1
Figure 1
Candida albicans clinical isolates vary in their ability to form biofilms. Forty-two C. albicans bloodstream isolates were used to evaluate biofilm formation of strains derived from a clinical setting. (A) Standardised C. albicans (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 PBS, 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 absorbance at 570 nm in a microtitre plate reader (FluoStar Omega, BMG Labtech). Eight replicates were used for each isolate and was carried out on two separate occasions, with the mean of each represented. C. albicans LBF (square), HBF (triangle) and IBF (circle) were defined by the upper and lower quartiles, as shown by crystal violet stained biofilms. (B) Three C. albicans LBF and HBF were standardised (1 × 106 cells/mL) in RPMI-1640 and grown in 12 well plates for 24 h at 37°C. Biofilms were washed with PBS, biomass scraped and passed through 0.22 μm filters before the filters containing the biofilms were dried at 37°C for 24 h. Biofilm dry weight was then measured for LBF and HBF, in triplicate on three separate occasions. Data represents mean ± SD with significance **p < 0.005. (C) One C. albicans LBF (i, ii) and HBF (iii, iv) were grown on Thermanox™ coverslips for 24 h at 37°C. Biofilms were then processed and viewed on a JEOL JSM-6400 scanning electron microscope and images assembled using Photoshop software. Note the lack of biomass and hyphal cells in LBF. Scale bars represent 20 μm and 5 μm for 1000× (i, iii) and 3000× (ii, iv) magnifications, respectively.
Figure 2
Figure 2
Cell surface hydrophobicity impacts biofilm phenotype. Ten C. albicans LBF and HBF were standardised (1 × 106 cells/mL) in RPMI-1640 and grown in 75 cm2 flasks for 4 and 24 h. Biofilms were washed with PBS, biomass scraped in to YPD media and standardised to OD590nm 1.0 before xylene was added to each sample. Planktonic cells were also standardised to OD590nm 1.0. Samples were allowed to separate into two phases and the OD590nm of the lower aqueous layer was measured (i). A visual representation hydrophobicity is shown for planktonic LBF (ii) and HBF (iii), 4 h biofilms LBF (iv) and HBF (v) and 24 h biofilms LBF (vi) and HBF (vii). Note the cloudy upper layer denoted by arrows showing hydrophobic cells. Ten isolates from each group were measured on two separate occasions. Data represented mean ± SD. Significant differences between LBF and HBF were observed when 4 and 24 h biofilms were compared to their planktonic counterparts (*p < 0.05, ***p < 0.0001,###p < 0.0001). Furthermore, significant differences were found between 4 and 24 h in HBF (†††p < 0.0001) and between LBF and HBF at 24 h (§§§p < 0.0001).
Figure 3
Figure 3
Amphotericin B sensitivity is significantly impacted by biofilm formation. Ten isolates with LBF and HBF were standardised 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 PBS before treated with 2 fold serial dilutions of amphotericin B for 24 h. Biofilms were washed and metabolic activity measured using the XTT metabolic assay with absorbance read at 492 nm. Each isolate was tested in duplicate, on three separate occasions with data represented by mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
C. albicans HBF have a significant impact on morbidity and mortality rate in vivo. Larvae of G. mellonella were infected with C. albicans LBF or HBF at 1 × 105 CFU/larva and monitored over a period of 7 days (A). Kaplan-Meier plots of G. mellonella survival after injection of C. albicans demonstrated a strain dependant variation in pathogenicity in vivo. Groups of HBF and LBF clinical isolates were compared to each other and to the SC5314 type strain. The HBF isolates resulted in higher killing rate compared to LBF and SC5314. In contrast, LBF isolates exhibit a slower rate of kill and 100% mortality did not occur within 7 days. PBS injected larvae were included as a negative control. (B) Infected larvae were formalin fixed and sectioned for histology analysis. At 24 h, LBF infected larvae (i) had several melanisation spots and nodules were present mainly under the cuticle and in the peripheral fat body (Feulgen staining, 20× original magnification (o.m.); inset: 4× o.m.), whereas HBF infected larvae (iv) had larger nodules with a greater melanin deposition characterised by the recruitment in the external layers of a huge number of haemocytes (20× o.m.; inset: 10× o.m). At 48 h, LBF (ii) small nodules containing both yeast and some hyphae were observed deeper in the larval tissues, sometimes reaching the external part of the gut wall (PAS staining, 20× o.m.; inset: 10× o.m), with HBF (v) having elongated hyphae _targeting the intestinal walls (PAS staining, 40× o.m.; inset: 10× o.m.) At 72 h, LBF (iii) showed segmental invasion of the gut walls (PAS staining, 20× o.m.; inset: 10× o.m.) however, HBF (vi) displayed hyphae endoluminal invasion after breaching the intestinal wall (PAS staining, 40× o.m.; inset: 10× o.m.) with few yeast cells.
Figure 5
Figure 5
Genes associated with C. albicans biofilm development are up-regulated in HBF. Ten C. albicans isolates with LBF and HBF were standardised to 1 × 106 cells/mL in RPMI-1640 and grown as biofilms in 24 well microtitre plates for 4 (A) and 24 h (B) at 37°C. Biofilms were washed with PBS and RNA extracted using the TRIzol method, cDNA synthesised and real-time PCR used to measure the expression of genes related to C. albicans biofilm formation. Percentage of gene expression is shown as log10 mean ± SD, relative to housekeeping gene ACT1. All strains were assessed in duplicate and included appropriate no RT and non-template controls. *p < 0.05, **p < 0.005.
Figure 6
Figure 6
Clustering analysis identified the transcriptional relationship of biofilm specific genes. Percentage expression of each gene was also assessed by clustering and heat map analysis using GenEx software. Data was log transformed and mean values were used for heat map construction. Increased expression of genes is shown by red and a decrease is represented by green.
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