Ranjith Rajendran

Fungal biofilm resistance.

Fungal biofilm infections have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. This paper aims to illustrate the importance of fungal biofilms, particularly Candida albicans, and discusses some of the key fungal biofilm resistance mechanisms that include, extracellular matrix (ECM), efflux pump activity, persisters, cell density, overexpression of drug targets, stress responses, and the general physiology of the cell. The paper demonstrates the multifaceted nature of fungal biofilm resistance, which encompasses some of the newest data and ideas in the field.

Hsp90 Governs Dispersion and Drug Resistance of Fungal Biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections.

The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus

OBJECTIVES Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations. METHODS Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates. RESULTS All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. CONCLUSIONS The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.

Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity

ABSTRACT This study investigated the phase-dependent expression and activity of efflux pumps in Aspergillus fumigatus treated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P < 0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P < 0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for the A. fumigatus germling cells by 2- to 8-fold. Quantitative expression analysis of AfuMDR4 mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P < 0.001). Finally, an in vivo mouse biofilm model was used to evaluate efflux pump expression, and it was shown that AfuMDR4 was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P < 0.01). Our results demonstrate that efflux pumps are expressed in complex A. fumigatus biofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

ABSTRACT Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.

Aspergillus biofilms: clinical and industrial significance.

The biofilm phenotype is an increasingly important concept in mycological research. Recently, there has been a developing interest in whether Aspergillus species are truly able to form biofilms or not. Industrial mycologists have long been aware of biofilms and their benefit in fermentation processes, whereas clinically their role is uncertain. This review provides an update on the impact that Aspergillus biofilms have medically and industrially, and will discuss biofilm development, and our current understanding of its molecular basis. The role of exopolymeric substance and how this substance relates to antimicrobial recalcitrance will also be discussed.

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

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.

Antifungal treatment affects the laboratory diagnosis of invasive aspergillosis

Aims The purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA), and how these tests are impacted upon by the use of different classes of antifungal agents in an in-vivo model of IA. Methods A standardised rat inhalation model of IA was used to examine the effects of an azole, posaconazole, a polyene, amphotericin B and an echinocandin caspofungin. Daily blood samples were collected for subsequent analysis using a commercially available galactomannan assay and an inhouse qPCR assay. Results No significant differences were observed in the CE/g of Aspergillus fumigatus in the lungs of each group. qPCR was statistically more sensitive than galactomannan for both the early detection of infected controls (p=0.045) and for overall detection (p=0.018). However, antifungal treatment significantly reduced the overall sensitivity of qPCR (p=0.020); these effects were due to posaconazole and caspofungin. In the latter stages of infection (days 4 and 5) there were no significant differences in the numbers of infections detected by galactomannan and qPCR; however, the antifungal class used caused significant qualitative differences (p=0.041). Galactomannan showed improved detection in posaconazole-treated animals. Conclusions Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.

Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro.

OBJECTIVE The aim of this study was to evaluate and compare the activity of prescription and over-the-counter antimicrobial compounds against planktonic and biofilm forms of Candida albicans isolated from cases of oral candidiasis in vitro. STUDY DESIGN The efficacy of azoles, polyenes, an echinocandin, and 4 over-the-counter mouthwashes were tested against C. albicans-derived planktonic and biofilm cells. RESULTS Planktonic cells were shown to be highly sensitive to all of the antifungal agents tested. Sessile cells were highly resistant to azoles (≥128 mg/L) but equally sensitive to caspofungin and short treatments with Corsodyl, Listerine, and Oraldene. CONCLUSIONS Although C. albicans is sensitive to azole antifungal agents in planktonic form, it is highly resistant within the biofilm. The good efficacy of the over-the-counter mouthwashes against candidal biofilms in vitro suggests that clinical trials should now be designed to establish their role in the clinical management of oral candidal infections.