Abstract
Polymicrobial biofilms consisting of fungi and bacteria are frequently formed on endotracheal tubes and may contribute to development of ventilator associated pneumonia (VAP) in critically ill patients. This study aimed to determine the role of early Candida albicans biofilms in supporting dual-species (dual-kingdom) biofilm formation with respiratory pathogens in vitro, and investigated the effect of targeted antifungal treatment on bacterial cells within the biofilms. Dual-species biofilm formation between C. albicans and three respiratory pathogens commonly associated with VAP (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was studied using quantitative PCR. It was shown that early C. albicans biofilms enhanced the numbers of E. coli and S. aureus (including methicillin resistant S. aureus; MRSA) but not P. aeruginosa within dual-species biofilms. Transwell assays demonstrated that contact with C. albicans was required for the increased bacterial cell numbers observed. Total Internal Reflection Fluorescence microscopy showed that both wild type and hyphal-deficient C. albicans provided a scaffold for initial bacterial adhesion in dual species biofilms. qPCR results suggested that further maturation of the dual-species biofilm significantly increased bacterial cell numbers, except in the case of E.coli with hyphal-deficient C. albicans (Ca_gcn5Δ/Δ). A targeted preventative approach with liposomal amphotericin (AmBisome®) resulted in significantly decreased numbers of S. aureus in dual-species biofilms, as determined by propidium monoazide-modified qPCR. Similar results were observed when dual-species biofilms consisting of clinical isolates of C. albicans and MRSA were treated with liposomal amphotericin. However, reductions in E. coli numbers were not observed following liposomal amphotericin treatment. We conclude that early C. albicans biofilms have a key supporting role in dual-species biofilms by enhancing bacterial cell numbers during biofilm maturation. In the setting of increasing antibiotic resistance, an important and unexpected consequence of antifungal treatment of dual-species biofilms, is the additional benefit of decreased growth of multi-drug resistant bacteria such as MRSA, which could represent a novel future preventive strategy.
Highlights
Biofilm formation is a phenotype of many microorganisms, linked to the ability to survive in a hostile host environment [1]
We initially studied whether the presence of early biofilms of C. albicans could enhance cell numbers of P. aeruginosa, E. coli, S. aureus or methicillin resistant S. aureus (MRSA) (S2 Fig)
To demonstrate that results obtained with laboratory strains were applicable to clinical isolates we showed that cell numbers of a clinical isolate of MRSA were significantly increased when grown in the presence of a clinical isolate of C. albicans (2.4x107) compared with axenic MRSA biofilms (1.0x106) (S2D Fig)
Summary
Biofilm formation is a phenotype of many microorganisms, linked to the ability to survive in a hostile host environment [1]. Biofilm colonisation of the endotracheal tube in VAP patients has been reported to occur rapidly after intubation [13], with biofilms acting as reservoirs for pathogenic bacteria [14]. Of particular interest is the finding that Candida albicans [17], is often present in endotracheal tubes [11], and is associated with an increased risk of VAP ( not as the pathogen causing VAP), as well as prolonged intensive care unit and hospital stays [18]. Establishment of an early C. albicans biofilm could facilitate respiratory pathogen integration, leading to subsequent polymicrobial biofilm formation and drive the development of VAP
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