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

BMC Microbiology Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity Leighann Sherry 0 Ranjith Rajendran 0 David F Lappin 0 Elisa Borghi Federica Perdoni Monica Falleni Delfina Tosi Karen Smith Craig Williams Brian Jones Chris J Nile 0 Gordon Ramage 0 0 Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow , 378 Sauchiehall Street, Glasgow G2 3JZ , UK 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. Candida albicans; Biofilm; Candidaemia; Antifungal - Background Bloodstream infections (BSI) caused by Candida species remain a frequent cause of morbidity and mortality, particularly within the immunocompromised population [1,2]. Overall, Candida species have been identified as the most common fungal pathogen found in bloodstream infections in the United States, and are the fourth most common organism responsible for all BSI, and are the third most common within the intensive care unit (ICU) [2]. Candidaemia is often associated with the ability of Candida to adhere to and form biofilms on indwelling medical devices, such as central venous catheters (CVC) and prosthesis [3,4]. Biofilms are a population of microorganisms attached to one another and/or a surface, surrounded by an extracellular matrix (ECM) [5]. A defining feature of biofilms is their resistance to antimicrobial therapy, with higher drug concentrations required to kill biofilms and their dispersed cells when compared to equivalent free-floating planktonic cells [5-7]. Another feature of C. albicans biofilms is their enhanced pathogenicity. For example, cells detaching from biofilms have been shown to be more cytotoxic than their planktonic counterparts and significantly increase mortality within a murine model of infection [7]. These observations have been demonstrated clinically, where a significant association was observed between C. albicans biofilm formation and mortality rates in candidaemia patients [8]. Whilst there is growing evidence of the importance of Candida biofilms in clinical medicine, not all clinical isolates are able to form biofilms. There is therefore a fundamental gap in understanding exactly what drives biofilm formation and its clinical implications. Establishing methods to differentiate these isolates is challenging, as many studies rely on either metabolic assays or biomass, and these frequently use a variety of different substrates and media [9-12]. Therefore, comparison between these studies is not possible, and further interpretation of the data to improve clinical management both for diagnostics and antifungal therapy is limited. The purpose of this study was therefore to investigate and characterise biofilm formation by clinical isolates of C. albicans using standard methodologies and subsequently analyse biofilm subsets phenotypically and transcriptionally. Here we report that C. albicans clinical isolates form biofilms that are heterogeneous, and this is associated with altered antifungal drug sensitivity and pathogenic potential. Results Candida albicans clinical isolates exhibit heterogeneous biofilm formation C. albicans bloodstream isolates displayed heterogeneity with respect to their biofilm biomass when grown in RPMI (Figure 1A). RPMI was shown to support the optimal growth of C. albicans over 24, 48 and 72 h (Additional file 1: Figure S1). Isolates were categorised as low biofilm formers (LBF) or high biofilm formers (HBF) if their biomass absorbance were less than the first quartile (Q1 OD570 = 0.565) or greater than the third quartile (Q3 OD570 = 1.682), respectively. Those isolates in between the first and third quartile (Q1-Q3) were defined as intermediate biofilm formers. When HBF were stained with crystal violet (cv), the extent of the biofilm formation was observed macroscopically, where the bottom of the well was clearly covered with cellular biomass (Figure 1A). In contrast, minimal staining was retained on isolates classed as LBF, as demonstrated by the well remaining almost colourless. We analysed a subset of isolates from the LBF and HBF group (n = 3) using dry weight measurements and confirmed our previous observations that biofilm biomass was significantly greater in isolates termed HBF (p = 0.0023) (Figure 1B). These differences are clearly evident when viewed under a SEM at low (Figure 1C [i, iii]) and high magnification (Figure 1C [ii, iv]). LBF isolates were characterised by a predominance of yeast cells and lack of hyphal cells (Figure 1C i, ii). In contrast, C. albicans HBF were highly filamentous with a multi-dimensional structure with very few yeast cells (Figure 1C iii, iv). Biofilm phenotype is affected by cell surface hydrophobicity (CSH) The CSH of LBF and HBF isolates was quantified to det (...truncated)