Gerhard Blum

Potential Basis for Amphotericin B Resistance in Aspergillus terreus

ABSTRACT This study investigated the basis for intrinsic amphotericin B (AMB) resistance in Aspergillus terreus. The ergosterol content, cell wall composition, and lipid peroxidation level had no influence on AMB resistance. The level of catalase production in A. terreus was significantly higher than that in A. fumigatus (P < 0.05). This higher-level production may contribute to AMB resistance in A. terreus since oxidative damage has been implicated in AMB action.

New Insight into Amphotericin B Resistance in Aspergillus terreus

ABSTRACT Amphotericin B (AMB) is the predominant antifungal drug, but the mechanism of resistance is not well understood. We compared the in vivo virulence of an AMB-resistant Aspergillus terreus (ATR) isolate with that of an AMB-susceptible A. terreus isolate (ATS) using a murine model for disseminated aspergillosis. Furthermore, we analyzed the molecular basis of intrinsic AMB resistance in vitro by comparing the ergosterol content, cell-associated AMB levels, AMB-induced intracellular efflux, and prooxidant effects between ATR and ATS. Infection of immunosuppressed mice with ATS or ATR showed that the ATS strain was more lethal than the ATR strain. However, AMB treatment improved the outcome in ATS-infected mice while having no positive effect on the animals infected with ATR. The in vitro data demonstrated that ergosterol content is not the molecular basis for AMB resistance. ATR absorbed less AMB, discharged more intracellular compounds, and had better protection against oxidative damage than the susceptible strain. Our experiments showed that ergosterol content plays a minor role in intrinsic AMB resistance and is not directly associated with intracellular cell-associated AMB content. AMB might exert its antifungal activity by oxidative injury rather than by an increase in membrane permeation.

Species and susceptibility distribution of 1062 clinical yeast isolates to azoles, echinocandins, flucytosine and amphotericin B from a multi-centre study.

Descriptive values were determined for eight antifungal agents within the course of a multi‐centre study encompassing 1062 German and Austrian clinical yeast isolates. Candida albicans (54%) was the predominant species isolated followed by Candida glabrata (22%), Candida parapsilosis (6%), Candida tropicalis (5.7%), Candida krusei (4.3%), as well as eleven further candidal and four non‐Candida yeast species. While 519 (48.9%) isolates were tested susceptible to all antifungals tested, no isolate was found to exhibit complete cross resistance. For C. albicans, the proportions of susceptible isolates were 93.2% (amphotericin B), 95.6% (flucytosine), 84.3% (fluconazole), 83.8% (posaconazole), 91.8% (voriconazole), 96.5% (anidulafungin), 96.2% (caspofungin) and 97.6% (micafungin). Patterns of complete parallel resistances were observed within azoles (8.8%) and echinocandins (1.7%). While a decreased susceptibility was found infrequently for echinocandins and flucytosine, it was more common for azoles with highest proportions for isolates of C. glabrata (fluconazole, 40.6%; posaconazole, 37.2%), Candida guilliermondii (fluconazole and posaconazole, each 25.0%), C. krusei (posaconazole, 28.3%; voriconazole, 60%), C. parapsilosis (fluconazole, 70.3%) and C. tropicalis (fluconazole, 62.3%). The descriptive values obtained in this study represent a valid basis for the comparison of recent and future epidemiological surveys to analyse the susceptibility of yeast isolates towards major antifungal substances.

A 1-year Aspergillus terreus surveillance study at the University Hospital of Innsbruck: molecular typing of environmental and clinical isolates

Aspergillus terreus appears to have become an increasingly frequent cause of opportunistic infections in the University Hospital of Innsbruck (UHI) and is of serious concern because of in vivo and in vitro resistance to amphotericin B. In order to determine the possible relationship between environmental contamination by A. terreus and the occurrence of invasive aspergillosis, a 1-year prospective study (2004-2005) was carried out in the UHI. Isolates obtained from air samples of various high-risk settings and those from surveillance cultures of proven and probable aspergillosis (EORTC/MSG criteria) were examined by genotyping. Within 1 year, 34 and 15 A. terreus isolates were collected from the environment and from patients, respectively. Genotypic analysis with rapid amplification of polymorphic DNA (RAPD) PCR and the combination of three different primers (R108, CII, P4) revealed 46 distinct genotypic profiles (types 1-46). No strain similarity was detected among and within the patients and environmental areas, indicating a great genomic diversity in A. terreus, which is common in the environment of Innsbruck and a source of invasive infections in immunosuppressed patients. Genotypical diversity was found in clinical and environmental A. terreus isolates.

In vitro and in vivo role of heat shock protein 90 in Amphotericin B resistance of Aspergillus terreus

Aspergillus terreus (A. terreus) is of serious concern because of a high propensity to dissemination and in vitro and in vivo resistance to Amphotericin B (AmB). The underlying molecular mechanism of AmB is not known yet and here we want to explore whether fungal heat shock protein 90 (HSP90) is involved in polyene resistance in A. terreus. AmB-susceptible (ATS) and AmB-resistant (ATR) A. terreus and AmB-susceptible Aspergillus fumigatus (AFS) were investigated in response to AmB with a special focus on HSP90. HSP90 inhibitors resulted in significant improvement of AmB activity against ATR as minimum inhibitory concentrations (MIC) decreased from 32 to 0.38 mg/L. Gene expression profiling showed a greater basal amount of HSP90 levels in ATR and ATS when compared with AFS. HSP90 blockers in combination with AmB were evaluated in a murine model of disseminated aspergillosis. HSP90 inhibitors were not beneficial for mice infected with ATR, and neither mono- nor combination treatment with AmB yielded clinical improvement. HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) was harmful. HSP90 seems to play a vital role in antifungal stress response in all aspergilli tested, whereas HSP90 does not substantiate the origin of AmB resistance in ATR.

Blocking Hsp70 Enhances the Efficiency of Amphotericin B Treatment against Resistant Aspergillus terreus Strains

ABSTRACT The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.

Virulence and thrombocyte affectation of two Aspergillus terreus isolates differing in amphotericin B susceptibility

Aspergillus terreus-induced invasive infections exhibit high lethality, partly due to the intrinsic resistance for amphotericin B (AmB). We compared the virulence and pathogenesis of an AmB-resistant isolate of A. terreus (ATR) with that of a rare variant showing enhanced sensitivity for AMB (ATS). The modifications that result in enhanced AmB sensitivity of isolates are not associated with reduced virulence in vivo; instead, the ATS-infected mice died even faster than the ATR-infected animals. Since A. terreus enters the blood stream in most patients and frequently induces thrombosis, we studied a putative correlation between virulence of the two A. terreus isolates and their effect on thrombocytes. Those mice infected with the more virulent ATS isolate had lower thrombocyte numbers and more phosphatidylserine exposure on platelets than ATR-infected mice. In vitro experiments confirmed that ATS and ATR differ in their effect on thrombocytes. Conidia, aleurioconidia and hyphae of ATS were more potent than ATR to trigger thrombocyte stimulation, and thrombocytes adhered better to ATS than to ATR fungal structures. Furthermore, ATS secreted more soluble factors that triggered platelet stimulation than ATR. Thus, it might be suggested that the capacity of a fungal isolate to modulate thrombocyte parameters contributes to its virulence in vivo.

Airborne fungus exposure prior to hospitalisation as risk factor for mould infections in immunocompromised patients.

The aim of this study was to investigate the relationship between fungal exposure prior to hospitalisation and ensuing onset of invasive mould infections (IMI) in patients at risk. Patients admitted to the Department of Haematology, Oncology and Transplant Surgery of the Medical University Innsbruck received a questionnaire regarding fungal exposure prior to hospital stay. Questions inquired heavy fungal exposures up to 5 days before hospitalisation. A total of 234 patients were enrolled in this study. Multiple fungus exposures were associated with the onset of community‐acquired IMI in patients with haematological malignancies. In univariate analysis, haematological malignancies (P = 0.013) and allergy to dust, pollen or moulds (P = 0.015) were significantly associated with fungal infections. In multivariate analysis, logistic regression showed that haematological patients (P = 0.015) and patients with allergy (P = 0.015) were significantly more frequently infected with fungi. Hospital‐independent fungal sources highlight risk‐factors for IMI in severe immunocompromised patients and the rate of community‐acquired IMI does increase.

Evaluation of a modified EUCAST fragmented mycelium inoculum method for in vitro susceptibility testing of dermatophytes and the activity of novel antifungal agents

ABSTRACT For antifungal susceptibility testing of nonsporulating or poorly sporulating dermatophytes, a fragmented-mycelium inoculum preparation method was established and compared to broth microdilution testing according to CLSI and EUCAST guidelines. Moreover, the in vitro activity of new antifungal agents against dermatophytes was evaluated. Agreement between the mycelial inoculum method and the CLSI broth microdilution method was high (93% to 100%). Echinocandins (minimal effective concentration [MEC], ≤0.5 mg/liter) and posaconazole (MIC, ≤3.00 mg/liter) showed good activity against all tested dermatophytes.

Rep-PCR and RAPD-PCR fingerprinting of Aspergillus terreus

We compared two PCR methods for molecular typing the medically important filamentous fungus Aspergillus terreus. In a set of 46 strains investigated we found 19 and 12 different fingerprinting types obtained by random amplified polymorphic DNA PCR (RAPD) and semi-automated repetitive element PCR (rep-PCR), respectively.