Göran Blomquist

18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

ABSTRACT In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.

Volatile metabolites from microorganisms grown on humid building materials and synthetic media

Growth of different microorganisms is often related to dampness in buildings. Both fungi and bacteria produce complicated mixtures of volatile organic compounds that include hydrocarbons, alcohols, ketones, sulfur- and nitrogen-containing compounds etc. Microbially produced substances are one possible explanation of odour problems and negative health effects in buildings affected by microbial growth. A mixture of five fungi, Aspergillus versicolor, Fusarium culmorum, Penicillium chrysogenum, Ulocladium botrytis and Wallemia sebi were grown on three different humid building materials (pinewood, particle board and gypsum board) and on one synthetic medium. Six different sampling methods were used, to be able to collect both non-reactive volatile organic compounds and reactive compounds such as volatile amines, aldehydes and carboxylic acids. Analysis was performed using gas chromatography, high-performance liquid chromatography and ion chromatography, mass spectrometry was used for identification of compounds. The main microbially produced metabolites found on pinewood were ketones (e.g. 2-heptanone) and alcohols (e.g. 2-methyl-1-propanol). Some of these compounds were also found on particle board, gypsum board and the synthetic medium, but there were more differences than similarities between the materials. For example, dimethoxymethane and 1,3,5-trioxepane and some nitrogen containing compounds were found only on particle board. The metabolite production on gypsum board was very low, although some terpenes (e.g. 3-carene) could be identified as fungal metabolites. On all materials, except gypsum board, the emission of aldehydes decreased during microbial growth. No low molecular weight carboxylic acids were identified.

VOLATILE METABOLITES PRODUCED BY TWO FUNGAL SPECIES CULTIVATED ON BUILDING MATERIALS

Two fungal species commonly found in indoor environments, Penicillium commune and Paecilomyces variotü, were cultivated on pine wood and on a combination of gypsum board and mineral wool. Air from the cultures was adsorbed on Tenax TA and analysed using thermal desorption-cold trap injection gas chromatography. Identification of the produced volatile metabolites was performed by mass spectrometry. The majority of the compounds produced were alcohols, ketones, ethers and terpenoid compounds. Commonly produced metabolites were 2-methyl-1-propanol, 3-methyl-1-butanol, 1-hexanol, 2-heptanone, 2-pentanone and 2,5-dimethyl-furan. The production was highly influenced by both medium and species.

Evaluation of PCR primers and PCR conditions for specific detection of common airborne fungi

We examined the selectivity of 53 sets of primers for environmental monitoring of indoor air quality. Thirty-six fungal strains, representing 26 species from 14 genera of commonly occurring fungi, and 16 different bacterial strains, representing both gram-negative and gram-positive species, were included in the experiment. We verified the specificity of 28 of the 53 sets of primers, which were classified as universal fungal, universal bacterial, group or species specific. The PCR conditions required for optimal specificity were also determined. These results can serve as a guide for the step-wise PCR-based detection and identification of airborne fungi commonly found in indoor environments.

Classification of fungi by means of pyrolysis-gas chromatography pattern recognition

Repetitive samples of three strains of the mould Penicillium were subjected to pyrolysis-gas chromatography (Py-GC). From the chromatograms, 26 peak heights were used in a subsequent SIMCA pattern recognition analysis. This data analysis gives a marked improvement in the classification of the samples (100% correct, 85% unique) in comparison with the traditional analysis based on the average chromatogram of each class (92% correct, 45% unique). The data analytical method is described in detail using the Py-GC data as an illustration.

Airborne chemical compounds on dairy farms

The chemical environment that dairy farmers are exposed to during milking was investigated. Volatile organic compounds (VOCs) were analysed and identified, and the levels of formaldehyde, ammonia and carbon dioxide were measured in eight farms in northern Sweden. Both stationary and personal samples were taken. A total of 70 VOCs were identified from the adsorbent samples, with p-cresol, 2-butanone, ethyl acetate, alpha-pinene and delta 3-carene occurring at the highest levels. All monitored levels were significantly lower for compounds having a stated highest occupational exposure level (OEL). Using multivariate techniques some differences in the composition of the workplace air between and within the farms were found. No difference was found between personal exposure and the surrounding environment in the cowshed.

Application of PCR and probe hybridization techniques in detection of airborne fungal spores in environmental samples

Specific PCR amplification and probe hybridization techniques were applied to examine the compositions of airborne fungi in samples from three different environments. The results from microscopic and CFU counting were compared to those of the molecular-based detections. The detection sensitivity for PCR amplifications was 9 to 73 spores and 1.3 to 19.3 CFUs per PCR reaction. The hybridization detection limit was 2 to 4 spores and 0.2 to 1.2 CFU. The hybridization method was more sensitive than PCR amplification and showed less variation among samples. Using specific PCR primers and probes we identified the presence of several fungal groups and species in the air samples. Specific detections through probe hybridization to PCR products amplified with universal or group-specific fungal primers have promising applications in the examination of air samples for environmental monitoring.

Data analysis of pyrolysis—chromatograms by means of simca pattern recognition

The variability between repetitive pyrolysis—chromatograms of the same type of samples is not totally random, but can in part be modelled by a principal components (PC) model. This makes it possible to use efficiently repetitive pyrolysis-chromatograms of samples of known types to obtain separate PC models for each type. Samples of unknown origin can then be classified according to which of the PC models their pyrolysis-chromatograms are most similar. The methodology is illustrated using pyrolysis—gas chromatograms of two species of the fungal genus Penicillium.

Reproducibility of pyrolysis-gas chromatographic analyses of the mould penicillim brevi-compactum

The reproducibility of multivariate measurements is discussed. Reproducibility of a second kind is defined, in which part of the variability between samples is described by a principal components model. The use of this generalized reproducibility is shown to give an improved precision in the pyrolysis-gas chromatography of a Penicillium species.

Mould exposure in museum personnel.

SummaryIn the basement archives of a local arts and crafts museum where the books and bookkeeping registers were handled, a woman on the museum staff had had ten attacks of fever, chill, nausea and cough during one year. The symptoms appeared at the end of the working day and disappeared after one to three days at home. No symptoms could be detected during the summer holidays. The books stored in the basement archives had earlier been stored in a small house with a leaky roof, which had led to the growth of mould on the books. When the books were handled at the museum, they were still covered with mould. The exposure measurements showed 106 CFU/m3 and a total of 108 microorganisms/m3.