Isolation and purification of chiamydospores of Candida albicans.

Two methods were investigated for their efficiency in isolating and purifying chlamydospores of Candida albicans. Chlamydospores were disconnected from pseudomycelial cells either enzymatically using beta-glucuronidase or mechanically by ultrasonic treatment. Free chlamydospores were separated from other cell material by sucrose gradient centrifugation. The resulting preparations were inspected by light-microscopy and electron-microscopy. Both methods yielded preparations with a level of over 90% chlamydospore cells. Ultrasonic treatment caused little change to the ultrastructure of the chlamydospores, whereas the enzyme treatment profoundly affected the cell wall. It is concluded that ultrasonic treatment is an efficient method for obtaining pure preparations of chlamydospores.

Influence of intestinal decontamination using metronidazole on the detection of methanogenic Archaea in bone marrow transplant recipients.

Methane-forming microbes of the phylogenetic domain Archaea are part of the strictly anaerobic microflora of the human intestine. In bone marrow transplant (BMT) recipients, the regimen of intestinal decontamination with metronidazole is targeted to anaerobic bacteria. The effect on the anaerobic methanoarchaea, however, is unknown. Therefore, the faeces of patients undergoing BMT were investigated for methane production. The anoxic Hungate technique and an archaeal growth medium were used to culture faecal specimens. Methane production was measured in the head space of the culture bottles by gas chromatography using a thermal conductivity detector. In a testing serial specimen of 100 patients, 13 patients were found to bear methanogens, and 11 of these patients received metronidazole. The methane-producing faecal specimens occurred before metronidazole use in three patients, during the first week in five patients, and after cessation in three patients. No specimen of the 11 patients that was obtained during the 2nd-5th week of gut decontamination showed methane production. It is concluded that use of metronidazole directed against faecal anaerobic bacteria also suppresses or eliminates faecal methanogenic Archaea.

Susceptibility testing of Aspergillus spp. by means of an automated blood culture system.

The BacT/Alert blood culture system was evaluated for the MIC determination of amphotericin B for 27 strains of Aspergillus from five different species. An inoculum of c. 10(8) cfu/mL and an incubation time of 48 h resulted in MICs for 26 of 27 strains that corresponded with published MICs obtained in a microdilution assay. Twelve of 13 strains with MIC(48) values >/=2 mg/L showed growth within 24 h in bottles containing 0.25 mg/L amphotericin B, indicating that results can be obtained the day following inoculation. It is concluded that the BacT/Alert blood culture system can be used for susceptibility testing of Aspergillus spp. with amphotericin B.

Isolation and antimicrobial susceptibility testing of fecal strains of the archaeon Methanobrevibacter smithii.

BACKGROUND: The archaeon Methanobrevibacter smithii is regarded as part of the indigenous microflora of the human intestine but may be connected with pathological conditions. The microbe is extremely oxygen intolerant and is not detectable by anaerobic culture techniques for bacteria. Accordingly, to date quantitative antimicrobial susceptibility data of human isolates are missing. METHODS: The anoxic Hungate technique and a three-step culture procedure using media supplemented with antibiotics were applied to isolate M. smithii from randomly selected human feces. The minimum inhibitory concentrations (MICs) of 15 isolates and the reference strain DSM 861 were determined using a broth macrodilution test resembling the procedure for testing anaerobic bacteria. RESULTS: The 16 strains were highly resistant (MICs >64 mg/l) against penicillin G, cephalothin, vancomycin, streptomycin, gentamicin, ciprofloxacin, and clindamycin. Metronidazole inhibited the strains at MICs between 0.5 and 64 mg/l. CONCLUSIONS: Multiple-antibiotic-resistant Methanoarchaea occur in the human gut. They may be selected during therapy with common antibacterial agents and may be eliminated by the application of metronidazole.

Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region.

The amplified 5.8S RNA coding DNA with the neighbouring internal transcribed spacers ITS I and ITS II (ITS I--5.8S rDNA--ITS II) of 27 culture collection strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus were investigated by single strand conformational polymorphism (SSCP) analysis. All strains showed a polymerase gel electrophoresis (PCR) product of 0.6 kb. Separation of DNA single strands of the PCR product in an acrylamide-bisacrylamide gel containing formamide SSCP resulted in individual patterns for each of the species. A minor variability within the species A. fumigatus and A. flavus did not affect the correct species identification. The results were confirmed when investigating 55 wild strains from patients and the environment. It is concluded that the analysis of the amplified ITS I--5.8S rDNA--ITS II region by SSCP allows the differentiation of the medically most relevant aspergilli. As the method does not require morphologically fully developed fungal colonies, it yields species diagnosis faster than the conventional macroscopic and microscopic identification.

Value of different methods for the characterisation of Aspergillus terreus strains.

To evaluate different methods for strain differentiation, 10 isolates of Aspergillus terreus from Germany and two epidemiologically unrelated strains were investigated. The sources of the isolates were patients with cystic fibrosis (4), immunosuppression (2), otitis externa (2), sinusitis (1) and endocarditis (1). Environmental isolates were obtained from a contaminated cell culture and from soil. The isolates did not differ in their macroscopic and microscopic morphology, in their protein patterns analysed by SDS-PAGE and in their susceptibility to amphotericin B and itraconazole. The RFLP analysis of total genomic DNA digested by EcoRI resulted in patterns that were too faint for interpretation. However, after hybridisation of the digested DNA with a short DNA probe of repetitive sequence, six different patterns were found. Based on the patterns of the randomly amplified polymorphic DNA (RAPD) with three primers, nine different genotypes were discriminate. RAPD patterns discriminated the epidemiologically unrelated reference strains (endocarditis isolate from Thailand, soil isolate from the USA) and the isolates from Germany. It is concluded that, in contrast to the phenotypic methods, the analysis of RAPD patterns is useful for strain differentiation of A. terreus.

Haemolytic activity of Helicobacter pylori against human and animal erythrocytes.

Clinical isolates and reference strains of H. pylori (n = 28) were investigated for lytic activity against red blood cells (RBC) of man, sheep, horse, rabbit, hamster and guinea pig. The test medium (HTA) consisted of agar base supplemented with newborn calf serum and washed RBC. In contrast to previous reports, haemolysis of human RBC was detected. Blood group A RBC supported the haemolytic growth of all strains studied. On HTA with blood group 0 RBC, one strain failed to grow and five strains grew nonhaemolytically. The diversity of growth and haemolysis on HTA containing the human and several animal RBC species allowed a differentiation of the strains into eight groups. Using a liquid RBC assay for quantitative measurement of haemolysis, extracts of broth-grown cells of strain ATCC 43504 showed an average haemolysis rate of 75% against sheep RBC and of 83% against human A RBC. Cell-free culture supernatants, however, showed an average haemolysis rate of 57% against sheep RBC and no activity against human A RBC. The data indicate that H. pylori expresses an intracellular haemolytic factor and a cell surface-associated or secreted haemolytic factor which have different RBC specificities. The demonstration of the sensitivity of human RBC supports the hypothesis that the haemolytic property of H. pylori is involved in the pathogenesis of human infection.

Epidemiological typing of Alcaligenes xylosoxidans subsp. xylosoxidans by antibacterial susceptibility testing, fatty acid analysis, PAGE of whole-cell protein and pulsed-field gel electrophoresis.

Antibacterial susceptibility testing, fatty acid analysis, protein analysis and DNA analysis of Alcaligenes xylosoxidans subsp. xylosoxidans were compared to determine the efficiency of the methods available for strain typing. Thirty isolates were investigated: 20 clinical isolates from a nonsocomial outbreak in Essen (Germany), 9 clinical isolates from sporadic nosocomial cases in Paris (France) and reference strain ATCC 2402. The highest microbiological discriminative power was exhibited by pulsed-field gel electrophoresis (PFGE) yielding nine types, followed by fatty acid methyl ester (FAME) analysis with six types, and antibacterial susceptibility testing and polyacrylamide gel electrophoresis with five types each. By combining the results of the four typing methods, 14 varieties could be differentiated. Protein analysis and fatty acid analysis failed to discriminate between isolates from Essen and Paris and the reference strain, while antibacterial susceptibility testing and DNA analysis clearly discriminated them. It is concluded that a combination of antibacterial susceptibility testing and PFGE typing is most suitable for epidemiological typing of Alcaligenes xylosoxidans subsp. xylosoxidans strains.

Antibacterial activity of East African medicinal plants.

In an ethnopharmacological survey, extracts of the six East African medicinal plants Entada abyssinica (stem bark), Terminalia spinosa (young branches), Harrisonia abyssinica (roots), Ximenia caffra (roots), Azadirachta indica (stem bark and leaves), and Spilanthes mauritiana (roots and flowers) were tested against 105 strains of bacteria from seven genera (Staphylococcus, Enterococcus, Pseudomonas, Escherichia, Klebsiella, Salmonella, Mycobacterium). The minimum inhibitory concentration reached by 50% (MIC50%) and 90% (MIC90) of the strains for the extracts of E. abyssinica, T. spinosa, X. caffra, and A. indica (stem bark) ranged from 0.13-8 mg/ml and from 0.5 to > 8 mg/ml, respectively. Their minimum bactericidal concentration by 50% (MBC50%) and MBC90% were all between 0.5 and > 8 mg/ml. H. abyssinica, A. indica (leaves), and S. mauritiana (roots and flowers) had MIC and MBC values > or = 8 mg/ml. Mycobacteria were not inhibited at extract concentrations of 0.5-2 mg/ml. It is concluded that plant extracts with low MIC and MBC values may serve as sources for compounds with therapeutic potency.

Characterization of the periodontal microflora by the fatty acid profile of the broth-grown microbial population.

The applicability of fatty acid analysis to the characterization of periodontal microflora was investigated using gas-liquid chromatography (GLC) and the software of the Sherlock Microbial Identification System (MIS) from MIDI Inc. Sulcus fluid was collected with paper points and anaerobically cultured in broth at 35 degrees C for four days. The broth-grown microbial population was extracted and the fatty acid methyl esters (FAME) were separated by GLC. The investigation of 67 specimens from asymptomatic sulci and of 32 specimens from inflamed sulci showed that the patterns of FAME profiles, the clustering of FAME profiles by computerized 2-D plot procedure, and the determination of the peak area index (PAI) of the FAME profiles differentiate between normal and pathological sulcus flora. Comparison of the clinical sulcus rating and the FAME data indicated that a pathological FAME profile may precede manifest periodontitis, and the normalization of the FAME profile may precede healing. It is concluded that the FAME analysis of sulcus fluid is a diagnostic aid for periodontological surveillance, for the initiation of preventive treatment of periodontitis, and for controlling the antimicrobial efficiency of therapeutic measures.

Adhesion of Helicobacter pylori to yeast cells.

In order to get information as to whether direct interaction of H. pylori and yeasts may modulate the course of H. pylori infections, the adhesion of H. pylori to C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis and S. cereviseae was investigated. H. pylori adhered significantly more frequently to C. tropicalis (adhesion ratio > 10%) than to the other yeasts (adhesion ratios < 5%). On an average, no significant difference to the adhesion ratios of E. coli and S. aureus was found. Electron microscopic examinations showed that H. pylori cells contacted the cells of C. tropicalis either by knob-like structures or by close surface-to-surface adhesion. Cholesterol-depleted H. pylori cells adhered to the yeast no more than cholesterol-carrying cells. There was no indication that a direct cooperation with yeasts plays a role in H. pylori infections.

Value of environmental sampling and molecular typing of aspergilli to assess nosocomial sources of aspergillosis.

In order to investigate the risk of hospital-acquired infections due to environmental Aspergillus, air sampling outside and inside the bone marrow transplantation (BMT) clinic of the University Hospital, Essen, Germany, was performed prospectively for one year. The spore concentration in the air was matched with meteorological data. Two BMT-patients, who were hospitalized during the sampling period, suffered from aspergillosis after discharge. The patients' isolates obtained at re-admission were compared with environmental isolates obtained during the first hospitalization. Analysis by randomly amplified polymorphic DNA showed that the two BMT-patients were infected with Aspergillus strains that were different from the environmental strains. It is concluded that it is not possible to predict the environmental Aspergillus spore concentration by analysis of meteorological data. Since the concentration of specific strains may fluctuate rapidly, a hospital-acquired Aspergillus infection cannot be excluded even if the infecting strain is not found in the hospital environment.

A comparison of methods of phenotypic and genotypic fingerprinting of Exophiala dermatitidis isolated from sputum samples of patients with cystic fibrosis.

Phenotypic and genotypic characteristics of 11 strains of Exophiala dermatitidis were investigated. Ten strains (including three reference strains) were isolated from sputum samples of six patients with cystic fibrosis (CF) in Germany, and one reference strain was isolated from a patient with phaeohyphomycosis in Japan. The strains showed differences in their ability to assimilate sorbitol, palatinose, rhamnose, gluconate and melezitose, leading to the differentiation of seven auxotypes. The IC30 of amphotericin B, and ketoconazole and itraconazole, respectively, indicated susceptibility, whereas the IC30 of fluconazole and 5-fluorocytosine indicated resistance in all strains. Protein patterns in SDS-PAGE revealed no major differences. The glycoconjugate patterns distinguished the Japanese strain from the other strains. Cluster analysis of whole-cell fatty acid methyl ester (FAME) profiles with the Microbial Identification System (MIS) revealed two major clusters separating a reference strain and the Japanese strain from the other strains. Analysis of patterns resulting from random amplification of polymorphic DNA (RAPD) with two arbitrary primers showed four genotypes. Comparison of the results revealed no agreement between the different fingerprinting methods, except the separation of the Japanese strain from the European CF strains. As the results of assimilation tests seem to vary between different laboratories, the analysis of FAME profiles and RAPD analysis are recommended for typing E. dermatitidis.

Genetic diversity among isolates of Aspergillus fumigatus in patients with cystic fibrosis.

Strains of Aspergillus fumigatus (n = 24) were isolated from the sputa of six patients with cystic fibrosis during periods from 3 to 11 months. The genetic polymorphisms of the strains were studied using the random amplified polymorphic DNA (RAPD) assay with three single oligonucleotides and pairwise combined primers. The analysis of RAPD patterns resulted in 15 different RAPD types. In four patients, the colonizing type changed, whereas in two others the same types were detected over periods between 3 and 11 months. The genetic diversity as well as the shift of the colonizing strains found in some patients might be important for the epidemiology of Aspergillus infections in patients with cystic fibrosis.

Detection of Aspergillus galactomannan antigen in foods and antibiotics.

The specificity of the Pastorex Aspergillus latex agglutination test for the diagnosis of manifest aspergillosis is hampered by the occurrence of false-positive results. In order to prove whether or not the false-positive reactions may be caused by the uptake of the soluble galactomannan antigen from the environment, the presence of the antigen was tested in foods, air samples, antibiotics for therapeutic use and faeces. Reactions of the Aspergillus latex agglutination test were found in 15 (79%) out of 19 samples of meals prepared in a hospital kitchen, in five out of six canned vegetables from a supermarket, in all of six samples of pasta and rice bought in health shops, in the faeces of four bone marrow transplant (BMT) recipients and of four healthy subjects and in one and two batches of the antibiotics co-amoxyclav and piperacillin respectively. The concentration of the antigen in faecal material was calculated to be in the range of 1.2-38.4 micrograms g-1. It is concluded that the faecal galactomannan antigen may reach the circulation in patients with dysfunction of the intestinal mucosal barrier, e.g. BMT recipients, thus leading to diagnostically false-positive antigenaemia.

Non-value of Aspergillus antigen detection in bronchoalveolar lavage fluids of patients undergoing bone marrow transplantation.

A commercially available antigen assay (Pastorex Aspergillus) was used to detect Aspergillus antigen in serial bronchoalveolar lavage (BAL) fluids and sera of patients undergoing bone marrow transplantation (six patients with autopsyproven aspergillosis and 10 control patients without evidence of fungal infection). Aspergillus antigen was not detected in 17 BAL fluids of the six patients with proven aspergillosis. In two of the six patients the assay gave positive results in serum specimens. Three of the 10 control patients showed reactive BAL fluids. It is concluded that the latex agglutination assay of BAL fluids has no value in the diagnosis of invasive (pulmonary) aspergillosis in patients undergoing bone marrow transplantation.