Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques.

A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin.     

Enumeration of airborne bacteria and fungi using solid phase cytometry

Conventional methods for the enumeration of airborne micro-organisms are inaccurate and time-consuming, hence the interest in novel approaches is increasing. In the present study, the use of solid phase cytometry (SPC) was evaluated for the enumeration of airborne micro-organisms. A 4 h SPC procedure based on viability staining was applied to samples from 50 locations and compared with an optimised culture-based method. Plate counts after air sampling were repeatable but strongly dependent on sampling volume. Samples with low or high microbial load were difficult to analyse using the culture-based method, unlike with SPC. Results show that SPC can be considered superior to the culture-based method because of its much higher dynamic range, its speed and its ability to enumerate not only culturable but all viable micro-organisms.     

天梯山石窟壁画保存环境中空气细菌的季节性变化

【背景】微生物侵蚀是古代壁画常见生物病害,影响壁画的长久保存和安全陈展。空气微生物作为壁画病害菌的主要来源,近年在文物赋存环境监测和预防性保护中引起广泛重视。【目的】对天梯山石窟壁画的2处保存地,即天梯山原址和武威西夏博物馆壁画保存环境中的空气细菌浓度、群落结构及其季节变化规律进行分析。【方法】利用生物气溶胶采样器,在2016年春、夏、秋、冬4季分别采集各位点空气样品;基于传统培养方法获得空气中细菌浓度及纯培养菌株;通过提取细菌基因组DNA、扩增其16S rRNA基因、测序和系统发生关系分析等技术研究不同位点细菌群落时空动态变化规律;结合环境监测数据,分析影响文化遗产地空气细菌群落变化的主要因素。【结果】空气可培养细菌的总浓度在16.7-1 451.8 CFU/m3范围内变动。原址第18窟和第13窟,各季节细菌浓度无显著性差异,且呈明显季节性变动规律,总体特征为夏秋季低,冬春季高。西夏博物馆外空气细菌浓度在各季节均高于库房内,冬季最高。本研究共鉴定出19个细菌属,隶属于4个门;其中不动杆菌属(Acinetobacter)、节杆菌属(Arthrobacter)、芽孢杆菌属(Bacillus)、考克氏菌属(Kocuria)、短波单胞菌属(Brevundimonas)、肉食杆菌属(Carnobacterium)、 Pseudoclavibacter和薄层菌属(Hymenobacter)为优势属。【结论】天梯山石窟空气细菌群落结构具有明显的时空分布特征;相对湿度、温度及季节性降水均会影响其变化;鉴定得到部分种属具备引起壁画生物腐蚀的潜势;本研究可为当地开展遗址和馆藏环境中文物预防性保护提供本底资料。     

Relationship between sampling duration and concentration of culturable airborne mould and bacteria on selected culture media

AIMS: This study was designed to evaluate potential effects of sampling duration on observed concentrations of airborne culturable mould and bacteria on selected media. METHODS AND RESULTS: Airborne culturable mould and bacteria from lightly to moderately contaminated environments were collected on selected culture media using two co-located, concurrently operated, Andersen N-6 samplers for five sampling durations in the range of 1-10 min. Differences in mean concentrations, as well as linear relationships between sampling duration and both concentration and variability, were evaluated using nonparametric procedures. For the five sampling durations, there were no significant differences in mean concentrations of mould; for bacteria, there were significant differences, with a trend of decreasing concentrations as sampling duration increased. Data variability decreased with increasing sampling duration for both mould and bacteria. CONCLUSIONS: Airborne culturable mould concentrations were similar for sampling durations in the range of 1-10 min. Airborne bacteria concentrations tended to trend downwards with sampling durations exceeding 3 min. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that sampling durations of 1-10 min are appropriate for collection of airborne culturable mould on malt extract agar (MEA) and dichloran glycerol agar (DG-18); based on the apparent trend of decreasing bacterial sample concentrations associated with increasing sampling duration, sampling durations of 相似文献   

Detection of airborne genetically modified maize pollen by real-time PCR

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.     

Levels of airborne biological agents and related factors in indoor environments of fish toxicity laboratory

In this study, we evaluated the levels of airborne biological agents, such as bacteria, fungi, endotoxin, and (1→3)-β-d-glucan in university fish toxicity laboratory every month for one year and assessed the associated environmental factors. A single-stage viable cascade impactor connected with a pump was used for culturable bacteria and fungi. An analysis of airborne endotoxin and (1→3)-β-d-glucan was performed using kinetic Limulus amebocyte lysate assays. Levels of culturable bacteria and fungi were the highest in summer, whereas levels of endotoxin and (1→3)-β-d-glucan were the highest in winter and spring, respectively. Human activity was correlated with culturable bacteria and fungi, and culturable fungi were also associated with culturable bacteria. Although additional studies based on advanced analyzing technology are required, simultaneous sampling with biomarkers of bacteria is required to further elucidate the characteristics of biological agents.     

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

麦积山石窟赋存环境中空气细菌的时空分布特征

【目的】空气微生物沉降及污染与文化遗产的微生物退化密切相关,本文对世界文化遗产地麦积山石窟赋存环境空气中细菌浓度和群落结构的季节性变化特征进行了系统研究,为石窟环境监测预警和文物预防性保护提供依据。【方法】利用生物气溶胶采样器,在2016年春、夏、秋和冬季分别采集空气样品;基于传统培养方法获得空气中细菌浓度及纯培养菌株;通过提取基因组DNA、扩增细菌16S rRNA、测序和系统发生树等分子技术研究细菌群落时空动态变化规律;结合环境监测数据,分析影响遗产地空气细菌变化的主要因素。【结果】监测期内,空气细菌浓度在(281.20–1409.20)CFU/m3之间,最高浓度出现在MJ4处的夏季,最低浓度出现在MJO处的春季;具有明显季节性变化特征,在空间层位分布上有所差异,但不显著(P0.05)。培养的细菌菌株经鉴定属于4个门11个属;芽孢杆菌属(Bacillus)、Paenarthrobacter、节杆菌属(Arthrobacter)、薄层菌属(Hymenobacter)和考克氏菌属(Kocuria)等为优势属。【结论】麦积山石窟空气细菌群落结构具有明显的季节性和空间分布动态变化特征;在石窟不同层位,空气中细菌群落分布与相对湿度、温度与降雨量相关;部分细菌种属如芽孢杆菌属、微球菌属(Micrococcus),为壁画及彩塑生物腐蚀的潜在病害菌;麦积山石窟及周边环境空气细菌的监测可为石窟保护和旅游开放管理提供重要参考。     

Molecular vs culture methods for the detection of bacterial faecal indicators in groundwater for human use

AIMS: The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. METHODS AND RESULTS: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml(-1). Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml(-1). CONCLUSIONS: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.     

Development of an Analytical Method for the Metaproteomic Investigation of Bioaerosol from Work Environments

The metaproteomic analysis of air particulate matter provides valuable information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters is developed. Different protein extraction procedures are tested to select the best extraction protocol based on protein recovery. The optimized method is tested for the extraction of proteins from spores of ubiquitous bacteria species and used for the metaproteomic characterization of filters from three work environments. In particular, ambient aerosol samples are collected in a composting plant, in a wastewater treatment plant, and in an agricultural holding. A total of 179, 15, 205, and 444 proteins are identified in composting plant, wastewater treatment plant, and agricultural holding, (cow stable and blending plant), respectively. In agreement with the major categories of primary biological aerosol particles, all identified proteins originated primarily from fungi, bacteria, and plants. The paper is the first metaproteomic study applied to bioaerosol samples collected in occupationally relevant environmental sites and, even though not aimed at monitoring the risk exposure of workers, it provides information on the possible exposure in the working environmental sites.     

Seasonal seawater temperature as the major determinant for populations of culturable bacteria in the sediments of an intact mangrove in an arid region

Mangroves are highly productive marine ecosystems where bacteria (culturable and non-culturable) actively participate in biomineralization of organic matter and biotransformation of minerals. This study explores spatial and seasonal fluctuations of culturable heterotrophic bacteria and Vibrio spp. in the sediments of an intact mangrove ecosystem and determines the dominant environmental factors that govern these fluctuations. Sediment samples were collected monthly from three stations in the mangroves of Laguna de Balandra, Baja California Sur, Mexico, through an annual cycle. Physicochemical parameters included seawater temperature, salinity, and concentration of dissolved oxygen. Viable counts of culturable heterotrophic bacteria and Vibrio spp. were made. In one sample (March 2003), nutrient concentrations (ammonium, nitrites, nitrates, and phosphates), organic matter, pH and sediment texture were also determined. General cluster analyses, analysis of variance of specific variables, and several principal component analyses demonstrated that seawater temperature is the principal determinant of seasonal distribution of culturable heterotrophic bacteria and Vibrio spp. in mangrove sediments. Soil texture, concentration of nutrients, and organic matter concentration contribute to heterogenicity to a lesser extent. A large spatial variation in bacterial populations was observed over short distances ( approximately 1 m) in sampling areas within the same site. These analyses show that the culturable bacterial distribution in sediments of mangroves has high spatial and temporal heterogeneity.     

Exposure of workers to airborne microorganisms in open-air swine houses

This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan. We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries. Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler. Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 x 10(5) and 143.7 CFU/m(3), respectively. The concentration of airborne culturable fungi was about 10(3) CFU/m(3), with Cladosporium the predominant genus. The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units. The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria. Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration.     

Site-related and seasonal variation of bioaerosol emission in an indoor wastewater treatment station: level,characteristics of particle size,and microbial structure

The emission of the airborne bacteria and fungi from an indoor wastewater treatment station adopting an integrated oxidation ditch with a vertical circle was investigated. Microbial samples were collected by the six-stage viable Andersen cascade impactor, and the samples were collected in triplicate in each sampling site per season. Culture-based method was applied to determine the concentrations of the airborne bacteria and fungi, while the cloning/sequencing method was used to characterize the genetic structure and community diversity of airborne bacteria. The highest concentrations of airborne bacteria (4155 ± 550 CFU/m³) and fungi (883 ± 150 CFU/m³) were obtained in June (summer). The lowest concentration of bacteria (1458 ± 434 CFU/m³) was determined in January (winter), and the lowest concentration of fungi (169 ± 40 CFU/m³) was found in March (spring), respectively. The particle size distribution analysis showed that most culturable bacteria obtained in all the sampling sites were in the particle size range of 1.1–4.7 µm. Most culturable fungi had particle sizes in the range 1.1–3.3 µm. Microbial population analysis showed that Bacillus sp., Acinetobacter sp., and Lysinibacillus were the main groups obtained in S1. Enterobacter was the dominant group in sampling site S2. Both the concentrations and particle size distribution of the bioaerosols in the enclosed space presented a seasonal and site-related variation. Concentration and richness of microorganisms in bioaerosols in June were higher than in September and January. The particle size distribution varied between the sampling sites, and proportion of large particles was higher in S2 than in S1 because of the settlement of large particles. Pathogenic species, such as Acinetobacter lwoffii, Staphylococcus saprophyticus, and Enterobacter sp., were isolated from the bioaerosols, which could pose serious latent danger to sewage workers’ health.     

Exposure of Workers to Airborne Microorganisms in Open-Air Swine Houses

This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan. We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries. Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler. Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 × 10⁵ and 143.7 CFU/m³, respectively. The concentration of airborne culturable fungi was about 10³ CFU/m³, with Cladosporium the predominant genus. The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units. The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria. Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration.     

A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.     

Short-term dynamic patterns of bioaerosol generation and displacement in an indoor environment

The short-term dynamics and distribution of airborne biological and total particles have been assessed in a large university hallway by particle counting using laser particle counters and impaction air samplers. Particle numbers of four different size ranges were determined every 2 min over several hours. Bioaerosols (culturable bacteria and fungi determined as colony-forming units) were directly collected every 5 min on Petri dishes containing the appropriate growth medium. Results clearly show distinct short-term dynamics of particulate aerosols, of both biological and non-biological origin. These reproducible periodic patterns are closely related to periods when lectures are held in lecture rooms and the intermissions in between when students are present in the hallway. Peaks of airborne culturable bacteria were observed with a periodicity of 1 h. Bioaerosol concentrations follow synchronously the variation in the total number of particles. These highly reproducible temporal dynamics should be considered when monitoring indoor environments to determine air quality.     

Influence of meteorological parameters and PM2.5 on the level of culturable airborne bacteria and fungi in Abadan,Iran

In recent years, monitoring of airborne bacteria and fungi concentrations has obtained increasing universal attraction not only for influences on ecological balance but also for evaluating their public health consequences. In this study, we aimed to investigate culturable airborne bacteria and fungi levels in different sites of Abadan, and their association with meteorological parameters and PM_2.5 levels. Abadan is one of the most industrialized cities in the southwest of Iran where over the current decade has experienced lots of dust storm episodes. In total, 400 air samples were collected in 6 months (autumn and winter) using a single-stage viable Andersen cascade impactor for sampling airborne bacteria and fungi and portable DustTrak Aerosol Monitor 8520 for measuring PM_2.5 concentrations and meteorological parameters. Microbial concentrations showed a significant difference between various sites over the study period with averages of 569.57?±?312.64 and 482.73?±?242.86 CFU/M³ for bacteria and fungi, respectively. The air temperature had a significant effect on the concentration of both airborne bacteria and fungi. A significant positive correlation between relative humidity and fungi but no correlation between relative humidity and bacteria concentrations were observed. The average airborne PM_2.5 concentrations of all sites among the study period was 93.24?±?116.72 μg/m³. The atmospheric bacterial and fungal communities were strongly positively correlated with the ambient PM_2.5 level. The levels of airborne bacteria and fungi along with PM_2.5 in the air of the city were relatively higher than the recommended levels. Therefore, the best course of action is needed to control emission sources. Further studies are also needed to evaluate the clinical analysis of the health effects of exposure to these pollutants.

     

Application of rapid enzyme assay techniques for monitoring of microbial water quality

Rapid enzyme assay techniques based on direct measurement of beta-d-galactosidase (GALase) or beta-d-glucuronidase (GLUase) activity without selective cultivation are used for rapid estimation of the level of coliform bacteria and Escherichia coli in water samples. Reported detection limits using fluorogenic substrates correspond to culturable target bacteria concentrations that can be appropriate within present guidelines for recreational waters. The rapidity, that is detection within one hour, compromises the specificity of the assay; enzyme activity contributions from other than target bacteria need to be considered, particularly at low levels of target bacteria. Enzyme activities are more persistent than the culturability of target bacteria to environmental and disinfection stress, thus water samples may express enzyme activities of both culturable and viable non-culturable cells.     

Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour‐intensive methods involving cultivation and morphology‐based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt–isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean‐up step using solid‐phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples—regardless of biomass or other properties—being successfully PCR‐amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus‐associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology‐based identifications, we find a species‐rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus‐associated interaction webs and communities.