Aspergillus fumigatus biofilms in the clinical setting.

We discuss in this work the role of Aspergillus biofilms in the clinical setting by reviewing the most recent findings on this topic. Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with typical biofilm characteristics under all static conditions tested, i.e., agar media, polystyrene and bronchial epithelial cells. Under static conditions the mycelial growth is greater than in shaken, submerged conditions. The extracellular matrix (ECM) is composed of galactomannan, α-1,3-glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. Typical biofilm structures were observed in the aspergillomas from two patients and in a murine model of invasive pulmonary aspergillosis. The results indicate that α-1,3-glucans plays a predominant role in the agglutination of the hyphae together in aerial conditions, and that nutrient starvation was responsible for mycelial death in aspergilloma. Melanin was produced during the infection, suggesting that this pigment is necessary for lung tissue invasion. All antifungal drugs are significantly less effective when A. fumigatus is grown under biofilm vs. planktonic conditions. Chronic persistence of a unique genotype of A. fumigatus in the respiratory tract of CF-patients and the presence of an ECM in vivo may have some therapeutical application for aspergillosis. The most appropriate antifungal drug should not be selected only on the basis of its efficiency to kill in vitro grown fungal cells, but also on its ability to penetrate the ECM.

Characteristics of pathogenic fungi and antifungal therapy in cystic fibrosis.

A defective mucociliary clearance facilitates colonization with bacteria and fungal spores in cystic fibrosis patients. Yeasts and molds are cultured from the cystic fibrosis respiratory tract and often their clinical relevance is unknown. Candida spp. are the most commonly isolated yeasts, whereas Aspergillus spp., Scedosporium apiospermum, as well as Exophiala dermatitidis in some countries, are the most frequent molds recovered from respiratory specimens. Molecular biotyping studies have revealed that some fungal genotypes are capable of chronically colonizing the airways. Persistent Aspergillus fumigatus infection is associated with an increased risk of pulmonary exacerbations requiring hospitalization. The prevalence of non-Aspergillus molds may be underestimated due to overgrowth of Pseudomonas and Aspergillus spp. on routine media. Allergic bronchopulmonary aspergillosis is usually treated by oral steroids and an antifungal azole drug. Interactions with the co-medication have to be considered. A small number of antifungal pharmacokinetic studies indicate a high inter-subject variability for itraconazole, voriconazole and posaconazole, and therefore therapeutic drug monitoring is recommended.

Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin.

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic- and biofilm-grown A. fumigatus mycelium after 24 and 48 h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real-time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Effects of caspofungin, Candida albicans and Aspergillus fumigatus on toll-like receptor 9 of GM-CSF-stimulated PMNs.

The possible involvement of Toll-like receptors (TLRs) 1, 2, 4 and 9 in the interaction of antifungal drugs with polymorphonuclear neutrophils (PMNs) in response to Aspergillus fumigatus and Candida albicans as stimuli was investigated. Caspofungin revealed the broadest capacity to enable C. albicans and A. fumigatus to stimulate TLR upregulation, TLR 2 by A. fumigatus and TLRs 4, 9 by C. albicans. Conventional amphotericin B (cAMB) stimulated only A. fumigatus to induce TLRs 2 and 4 upregulation; voriconazole stimulated A. fumigatus and fluconazole C. albicans to induce TLR 9 upregulation. For cAMB, only TLR 9 was upregulated by A. fumigatus, whereas in the case of voriconazole, TLRs 2, 4, 9 were upregulated. Caspofungin revealed the broadest capacity: C. albicans was stimulated to upregulate TLRs at least at one of the concentrations, and A. fumigatus was stimulated to upregulate TLRs 2, 4. TLR 9 was upregulated two to three fold by all antifungal drugs on protein, except for fluconazole at the RNA level. Candida albicans preincubated with caspofungin has additional effects on CD11b expression and IL8 chemotaxis in CpG-DNA-stimulated PMNs. These results indicate a relevant upregulation with a functional relevance of TLR 9 in the presence of C. albicans strains preincubated with caspofungin at three concentrations.

Liposomal amphotericin B eradicates Candida albicans biofilm in a continuous catheter flow model.

The aim of this study was to test whether a Candida albicans biofilm can be eradicated by liposomal amphotericin B (LAMB) at the minimal inhibitory concentration in a novel catheter continuous flow model. After 24-h biofilm formation and a 24-h treatment with LAMB, the growth of the hyphal network was reduced to 20% in comparison with the untreated control, whereas fluconazole and caspofungin remained at an intermediate phase (50%). After 24-h biofilm formation and a 24-h treatment with LAMB, 20% of the surface was covered in biofilm and LAMB caused an uneven surface. For caspofungin and fluconazole, the surface covering was 80%. The extracellular matrix (ECM) of the infected, but untreated catheters had a thickness of 5-20 microm at 24 h and 10-150 microm at 48 h. After 24-h biofilm formation and a 24-h treatment with LAMB, the ECM was virtually cleared with 0 microm ECM. After 24-h biofilm formation and a 24-h treatment with fluconazole, the ECM thickness was comparable to the infected, but untreated catheter at 24 h with 10-25 microm; with caspofungin, the ECM thickness was comparable to the infected, but untreated catheter at 48 h with 10-130 microm. Comparing the blastospores, pseudohyphae and ECM, 0.5 microg mL(-1) LAMB could eradicate Candida biofilm, whereas fluconazole and caspofungin were less effective.

Persistence of Candida species in the respiratory tract of cystic fibrosis patients.

It is still controversial as to whether Candida spp. are transient or persistent colonizers of the respiratory tract of cystic fibrosis (CF) patients. We conducted a prospective study of 56 CF patients over a 30 month period to assess the distribution and persistence of different Candida spp. In vitro antifungal susceptibility testing was performed and the C. albicans isolates were typed with CARE-2 hybridization and other Candida spp. by RAPD-PCR for persistence and transmission. We found that the mean persistence of the most frequent Candida spp. was >or= 9 months. In patients from whom more than 10 isolates were recovered, we noted that at least 30% were genetically related and transmission of C. albicans in siblings was observed. The majority of all isolates were susceptible to all antifungals tested. We concluded that there was long-term persistence of Candida in the respiratory tract of CF patients and that transmission between siblings may be one possible means of acquisition. Whether long-term colonization with Candida strains can contribute to the chronic infection and inflammation in the CF lung requires further investigation.

Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells.

Aspergillus fumigatus is a leading cause of death in immunocompromised patients and a frequent colonizer of the respiratory tracts of asthma and cystic fibrosis (CF) patients. Biofilms enable bacteria and yeasts to persist in infections and can contribute to antimicrobial resistance. We investigated the ability of A. fumigatus to form biofilms on polystyrene (PS) and human bronchial epithelial (HBE) and CF bronchial epithelial (CFBE) cells. We developed a novel in vitro coculture model of A. fumigatus biofilm formation on HBE and CFBE cells. Biofilm formation was documented by dry weight, scanning electron microscopy (SEM), and confocal scanning laser microscopy (CSLM). The in vitro antifungal activities of seven antifungal drugs were tested by comparing planktonic and sessile A. fumigatus strains. A. fumigatus formed an extracellular matrix on PS and HBE and CFBE cells as evidenced by increased dry weight, SEM, and CSLM. These biofilms exhibited decreased antifungal drug susceptibility and were adherent to the epithelial cells, with fungi remaining viable throughout 3 days. These observations might have implications for treatment of A. fumigatus colonization in chronic lung diseases and for its potential impact on airway inflammation, damage, and infection.

Cross-resistance to medical and agricultural azole drugs in yeasts from the oropharynx of human immunodeficiency virus patients and from environmental Bavarian vine grapes.

Cross-resistance among Candida albicans isolates from the oropharynges of human immunodeficiency virus-infected patients (n = 16) and environmental yeast strains of various species (n = 54) to medical and agricultural azole drugs was observed. Precautions against the unnecessary widespread use of azoles in the environment and human medicine are strongly recommended to prevent patients from acquiring azole-resistant yeasts.

In vitro effects of micafungin against Candida biofilms on polystyrene and central venous catheter sections.

Long-term inserted and surgically implanted catheters can be colonised by Candida spp. Candida biofilms in vitro are often resistant to antifungal agents. The aim of this study was to investigate the in vitro activity of micafungin (MFG) against six Candida spp. biofilms on polystyrene (PS) and central venous catheter (CVC) sections. Safranin staining and differential interference contrast microscopy were used to demonstrate biofilm production. MFG activity was determined by the reduction in metabolic activity (%RMA) by tetrazolium reduction assay on both substrates. In vitro, Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida dubliniensis and Candida kefyr produced mature biofilms on PS and CVC sections. MFG was active against C. kefyr (0.5 microg/mL) and C. glabrata (<0.5 microg/mL) on PS. However, MFG displayed resistance (>16 microg/mL) against C. albicans, C. dubliniensis,C. tropicalis and C. parapsilosis. On CVC disks, MFG was active against C. glabrata (1 microg/mL) as well as C. parapsilosis and C. albicans (<0.5 microg/mL). MFG was resistant (>16 microg/mL) against C. dubliniensis, C. tropicalis and C. kefyr. MFG was active in vitro against all six Candida spp. on both substrates. However, MFG could not reduce the metabolic activity completely even at the highest concentration.

Effect of voriconazole combined with micafungin against Candida, Aspergillus, and Scedosporium spp. and Fusarium solani.

Effects of voriconazole combined with micafungin against 101 isolates of Candida spp. and 100 isolates of filamentous fungi have been evaluated by in vitro checkerboard analysis. The combination was indifferent for 97% of the Candida isolates and synergistic for 64% of the filamentous fungi (79% for Aspergillus fumigatus).

Effect of media composition and in vitro activity of posaconazole, caspofungin and voriconazole against zygomycetes.

OBJECTIVES: The effect of different media and composition on the in vitro activity of posaconazole, caspofungin and voriconazole against 59 zygomycetes species was determined. METHODS: The media tested were RPMI 1640 medium with and without 2% glucose, antibiotic medium 3 (AM3) with and without 2% glucose, and high resolution (HR) medium. RESULTS: Posaconazole was significantly more active than caspofungin and voriconazole, both in RPMI 1640 medium with 2% glucose and in HR medium. Adding glucose improved the determination of end points, but had only minor influence on the MICs. MICs evaluated in AM3 were lower than in RPMI 1640 medium or HR medium. CONCLUSIONS: The in vivo effect of posaconazole in zygomycosis needs further evaluation.

Effects of several antifungal drug combinations against clinical and environmental isolates of Cryptococcus neoformans from China.

The in vitro interactions of caspofungin (CSP) with terbinafine (TRB) and ravuconazole (RVC) with 5-fluorocytosine (5-FC) were tested against 82 clinical and environmental isolates of Cryptococcus neoformans from China. The interaction of CSP with TRB proved synergistic against those isolates with a CSP MIC < or =2 microg ml-1 (5% of the isolates), additive against 42% of the isolates and indifferent against 53%. The effects of RVC with 5-FC were synergistic, additive or indifferent against 8%, 26% and 67% of the isolates, respectively. No antagonistic effects were found among any of the drugs. The combinations of CSP with TRB and RVC with 5-FC may display beneficial effects in a strain-dependent manner, while in no case showed antagonistic effects. These data might be of use to design safer and more efficient treatments for patients with cryptococcosis and warrant further evaluation.

Micafungin enhances neutrophil fungicidal functions against Candida pseudohyphae.

We evaluated the effect of the combination of micafungin and polymorphonuclear leukocytes (PMN) against hyphae of Candida albicans and Candida dubliniensis. Micafungin enhanced the PMN oxidative burst dose dependently. The combination was synergistic (C. albicans) or additive (C. dubliniensis); when PMN were pretreated with granulocyte-macrophage colony-stimulating factor, the combination was more effective.

Clinical manifestations and diagnosis of invasive aspergillosis in immunocompromised children.

UNLABELLED: Invasive aspergillosis (IA) is a serious life-threatening complication in immunocompromised children. The commonest risk groups are children with acquired immunodeficiency syndrome, leukaemia, corticosteroid and other immunosuppressive therapy, chronic granulomatous disease and severe combined immunodeficiency as well as neonates. The clinical manifestations are heterogeneous and many organ systems can be involved. Diagnosis based on the clinical presentation alone is cumbersome. Innovative and sensitive laboratory test systems which detect fungal antigens or DNA in clinical specimens have been recently developed. Specific Aspergillus antibody detection using recombinant antigen technique has also been introduced. Although each individual technique has drawbacks, the combined use of culture with antigen and antibody ELISA as well as PCR should result in an earlier and more definitive diagnosis of IA in children presenting with clinical and/or radiological signs of aspergillosis. In high risk children these methods are valuable for serial screening and early detection of Aspergillus infection. The implementation of accurate diagnostic criteria and standardised diagnostic flow charts in children at risk will lead to a better outcome of IA in the future. CONCLUSION: definite, well-timed early diagnosis and sufficient therapy is elementary for a successful outcome of invasive aspergillosis in immunocompromised children. To date, the diagnosis of invasive aspergillosis remains a combination of clinical presentation, radiology and microbiological tests.

Effect of Micafungin (FK463) on Candida albicans adherence to epithelial cells.

BACKGROUND: Adherence is considered a major virulence trait of Candida albicans. FK463 is a new investigational intravenous antifungal of the 'candin family' with potent in vitro and in vivo activity against Candida spp. OBJECTIVE: The aim of the present study was to investigate the effect of Micafungin (FK463) on Candida adherence to epithelial cells of azole-sensitive and azole-resistant C. albicans isolates. METHODS: An in vitro assay using microtest plate technology and fluorescence measurement was developed to compare the adherence of C. albicans SC5314 and of paired C. albicans isolates to epithelial cells in the presence and in the absence of FK463. RESULTS: FK463 showed a marked inhibitory effect on the adherence of C. albicans SC5314. The addition of FK463 reduced the adherence of C. albicans SC5314 to 90% of the value of control without drug. A dose-dependent adherence inhibition was observed with FK463 in the range of 10-0.015 microg ml(-1). The comparison of paired C. albicans isolates, either a fluconazole-susceptible and a fluconazole-resistant isolate of one patient, revealed no significant difference in the adherence behavior between azole-susceptible and azole-resistant. CONCLUSION: Micafungin (FK463) has the capacity to reduce adherence of C. albicans azole-susceptible and azole-resistant strains to epithelial cells.