Clinical Isolates Of Aspergillus Fumigatus Research Articles

An electrophoretic molecular karyotype of a clinical isolate of Aspergillus fumigatus and localization of the MDR-like genes AfuMDR1 and AfuMDR2

The molecular karyotype of a clinical isolate of Aspergillus fumigatus (10AF/86/10) was determined by contour-clamped homogeneous electric field gel electrophoresis. Five chromosomal bands were resolved by this method. The resolved chromosomes ranged in size from 1.7 to 4.8 Mb, and together constituted a total genomic size of at least 15.8 Mb. Southern analysis of the separated chromosomes located the position of two MDR-like genes, AfuMDR1 and AfuMDR2, on chromosomes III and IV, respectively. The methods described herein may enable the application of molecular karyotyping of A. fumigatus in epidemiologic surveillance studies.

Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection

Given the increased choice of therapeutic agents and the rising incidence of serious invasive disease, it is important that reliable in-vitro methods for detecting antifungal drug resistance in Aspergillus spp. are developed. Six clinical isolates of Aspergillus fumigatus, obtained from patients in whom the clinical outcome was known, were selected for study. Each was used to examine a range of parameters affecting agar dilution and broth microdilution susceptibility test results. The in-vitro results were compared with outcome in a neutropenic mouse model of invasive aspergillosis. Groups of animals were treated with itraconazole at 25 mg/kg and 75 mg/kg and survival rates and organ burdens were determined. Itraconazole was efficacious against four isolates (susceptible) but failed for two (resistant) in the animal model of infection. Both the resistant isolates had been obtained from patients receiving itraconazole treatment with good serum concentrations of the drug. Conditions for the agar dilution test which produced results that correlated best with our in-vivo observations included the use of RPMI agar with L-glutamine buffered to pH 7 with MOPS, inoculated with 10(6)-10(7) conidia/mL and incubated for 48-72 h at 28 or 35 degrees C with a no-growth endpoint. Optimal conditions for the broth microdilution method included the use of RPMI medium with L-glutamine and 2% glucose buffered to pH 7 with MOPS, an inoculum of 2 x 10(5) conidia in 200 microL incubated for 48 h at 35 degrees C with a growth (or trace) endpoint. The MICs for the susceptible isolates were 0.12-1.0 mg/L and > or = 16 mg/L for the resistant isolates. With careful selection and standardization of test conditions it is possible to generate reproducible in-vitro susceptibility data for Aspergillus spp. that will predict clinical outcome.

Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuvéglise, J. Sarfati, J. P. Latgé, and S. Paris, Nucleic Acids Res. 24:1428-1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host.

In vitro antifungal activity of CAN-296: a naturally occurring complex carbohydrate.

The in vitro activity of a naturally occurring complex carbohydrate, CAN-296, was evaluated by testing 132 clinical and ATCC isolates of yeast and Aspergillus fumigatus, many of which were azole-resistant. The in vitro susceptibility tests were performed by standardized broth micro- and macrodilution methods and results were compared with those obtained for amphotericin B, fluconazole, ketoconazole, flucytosine and the pneumocandin L-733,560. All tested Candida species showed highly uniform susceptibility to CAN-296 at concentrations of 0.078 to 0.312 microgram/ml; non-albicans Candida were as susceptible to CAN-296 as the Candida albicans strains. Multi-azole-resistant Candida species were highly sensitive to CAN-296. Minimum inhibitory concentration measurements did not differ from minimum lethal concentrations by more than two-fold for all tested Candida species. Aspergillus fumigatus, on the other hand, showed only moderate susceptibility to CAN-296. The kinetics of the anti-Candida activity of CAN-296 was investigated by kill-curve experiments using C. albicans and C. glabrata and the results were compared with those obtain for amphotericin B. CAN-296 was found to be rapidly fungicidal in concentrations ranging from 4-16 fold the mean MIC value. The broad spectrum of anti-Candida activity together with the rapid fungicidal effect make this complex carbohydrate a promising agent for clinical use.

Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.

Immunologic significance of a collagen-derived culture filtrate containing proteolytic activity in Aspergillus-related diseases

Background: Despite increasing evidence implicating fungal proteases in the virulence of pulmonary fungal diseases, routine fungal culture media do not favor protease production. Hence, filtrates that serve as the source of antigen for serologic determinations are poor in proteases, and consequently, the immunologic significance of these enzymes is unknown. Methods: A clinical isolate of Aspergillus fumigatus was cultured on collagen medium, resulting in excretion of high levels of fungal proteases in the culture filtrate. This was compared with standard culture filtrates by diverse analytic techniques including immunoblotting with sera of patients with pulmonary aspergilloma (PA) and allergic bronchopulmonary aspergillosis (ABPA). Results: Protein profiles of collagen medium filtrate showed several (glyco)proteins not found in conventional culture filtrates, including a prominent 32 kd glycoprotein, which coisolated in gel filtration and ion-exchange chromatography with elastase activity, as well as 67 kd and 94 kd (glyco)proteins. Intense IgG binding was seen with the 32 kd glycoprotein when ABPA and PA sera were used. The 94 kd protein showed intense binding with PA sera but not with ABPA sera, whereas for the 67 kd glycoprotein the reverse tended to be the case. Conclusion: Fungal culture on collagen media results in the production of filtrates with high protease activity, containing unique (glyco)proteins of which at least one (32 kd) is closely associated with fungal elastase activity. These constituents are immunologically relevant, eliciting IgG production in patients with PA and ABPA, suggesting production of these (glyco)proteins during disease in vivo. The use of collagen media filtrates may enhance our serodiagnostic capacity in patients with fungal pulmonary diseases.

Use of DNA moderately repetitive sequence to type Aspergillus fumigatus isolates from aspergilloma patients.

Polymorphism of forty-seven sequential clinical isolates from 3 patients with an aspergilloma was analyzed. DNA from each isolate was digested with EcoRI and hybridized in Southern blots with a 32P-labeled nonribosomal DNA repetitive sequence. Most isolates from each patient displayed the same hybridization pattern. Southern blot patterns obtained with DNA repetitive sequences can be used to type clinical isolates of Aspergillus fumigatus and have shown that aspergilloma patients are most probably infected by a single strain.

Purification and characterization of an extracellular aspartic proteinase from Aspergillus fumigatus.

An aspartic proteinase (PEP) from the culture supernatant of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 24%. The procedure involved affinity chromatography on pepstatin agarose, the interaction requiring a chaotropic salt (sodium trifluoroacetate) for complete elution of the enzyme. Among 11 amino acids of the N-terminal region, nine were identical with the corresponding sequence of the aspartic proteinase aspergillopepsin A from Aspergillus niger var. awamori (previously called Aspergillus awamori). Thus PEP belongs to the aspergillopepsins, a family of closely related aspartic proteinases produced by aspergilli. Specific antibodies against PEP were detected by dot blot assay in sera of two patients with aspergillosis. In addition, PEP antigen was demonstrated by immunofluorescence in mycotic human lung, using specifically elicited antibodies from guinea-pigs. PEP had an estimated molecular mass of 38 kDa and the pI was determined at pH 4.2. PEP is therefore likely to be closely related to an acid proteinase of A. fumigatus which was originally described in 1981.

Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue

A serine proteinase (Alp) from the culture supernate of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 41%. The procedure involved affinity chromatography on agarose-epsilon-amino-caproyl-D-tryptophan methyl ester. Alp had an estimated mol. wt of 32 Kda and the pI was determined at pH 7.9. The enzyme was fully inhibited by phenylmethyl sulphonyl fluoride, chymostatin and alpha-1-proteinase inhibitor, and it was largely inhibited by alpha-1-anti-chymotrypsin. Partial inhibition was observed with tosyl-phenylalanine chloromethyl ketone, but tosyl-lysine chloromethyl ketone was ineffective. Thus, Alp may be identical with the major chymotryptic activity of A. fumigatus, which has already been described. The N-terminal sequence of 25 amino acids revealed an 88% homology of Alp with the subtilisin-related proteinase of A. oryzae. Alp acted on casein over a broad range from pH 5.5 to 11.5 and also acts to a lesser extent on haemoglobin and serum albumin. The enzyme degraded elastin and a synthetic elastase substrate; hence, it may be identical with the previously described elastinolytic activity of the fungus. At pH 7.3 and a concentration of 1 microgram/ml, Alp was not toxic for Vero cells, but it efficiently detached such cells from a plastic surface. Specific antibodies against Alp were detected by enzyme immunoassay in the sera of patients and Alp-antigen was demonstrated by immunofluorescence in mycotic human lung. In addition, a second proteinase (Exalp) with extremely alkaline activity, and an aspartic proteinase of A. fumigatus are described.

Identification of extracellular siderophores of pathogenic strains ofAspergillus fumigatus

Clinical and environmental isolates of Aspergillus fumigatus synthesized extracellular siderophores when grown in defined medium. Six hydroxamate siderophores were purified from culture filtrates and identified by thin layer chromatography. The most prominent siderophore was identified as N,N',N"-triacetylfusarinine C and the second most prominent siderophore was identified as ferricrocin. In addition, a hydrolytic product of N,N',N"-triacetylfusarinine C was identified. Three other siderophores were present in smaller amounts and were not identified. Since the same siderophores were produced by isolates from diseases of varying severity and from environmental material, it is unlikely that the extracellular siderophores function as virulence factors during infection. However, they may function as growth factors by mediating iron uptake by the fungus in the micro-environment of the inflammatory focus.

Development of a chromosomal DNA probe for the laboratory diagnosis of aspergillosis

Chromosomal DNA was extracted from clinical isolates of Aspergillus fumigatus of human and animal origin using the protoplast lysate method. The probe was developed by the nick translation of the chromosomal DNA genome fraction with p32 as the radiolabel. Hybridization of the probe with endonuclease-cleaved DNA of the same species resulted in a pattern of recognition sites specific for the species. The latter was not seen in other species encountered in clinical specimens. Trials were carried out on sputum experimentally inoculated with the fungus where crude DNA was directly extracted, treated with the endonuclease and hybridized with the probe. The efficacy of the probe was as good with the crude as the purified DNA. The specificity of the probe was determined by testing it against single and mixed DNA populations extracted from different species of several fungal and bacterial genera isolated from and/or known to occur in clinical specimens of respiratory infection origin. The sensitivity of the probe was assessed by detecting a DNA concentration in the specimen equivalent to 3 C.F.U.

Evaluation of the Antifungal Activity of Lemon Grass Oil

AbstractThe antifungal activity of Lemon grass Oil (LGO) has been evaluated using fungistatic (MIC and agar diffusion tests) and fungicidal (spore germination) studies. Appreciable activity was observed against various isolates of Candida and clinical isolates of Aspergillus fumigatus, Microsporum gypseum and Trichophyton mentagrophytes. The most resistant organism was A. fumigatus while M. gypseum and the Candida spp. were the most susceptible of the isolates. Comparative studies with pure samples of citral and citronellal, constituents of LGO, showed good activity against the test fungi while dipentene and myrcene showed no activity.Exposure of the spores of A. fumigatus to 0.1% LGO for 5 mins resulted in 93% of spores not germinating while lower concentrations (0.08% and 0.05%) caused 80% and 60% reduction in spore germination respectively. Challenge tests on a formulated aqueous cream containing LGO indicated that 0.25% LGO would effectively preserve the cream against fungal contamination.

Polyene sensitivity during germination of conidia of Aspergillus fumigatus.

A system for the rapid and relatively synchromous germination of conidia from a clinical isolate of Aspergillus fumigatus is described. The polyene-mediated release of K plus from germinating conidia has been determined. Ungerminated conidia were insensitive to amphotericin B methyl ester (AME) at concentrations greater than 50 mug/ml, but rapidly became sensitive to 1 to 2 mug AME/ml during the intitial stages of germination. These findings have been correlated with minimum inhibitory concentration values obtained in studies of conidial germination and hyphal outgrowth using a variety of growth tests.