Determining the influence of environmental and patient specific factors on the polymicrobial communities of the cystic fibrosis airway

The aim of this study was to investigate the polymicrobial communities in an adult Cystic Fibrosis population stratified by gender and the most common CFTR mutation, F508del. In this pilot study, DNA was extracted from sputum samples of 29 adult patients (16 male: 13 female) with an F508del mutation in a stable clinical state. Universal primers were used to amplify DNA from bacterial and fungal communities and the resulting fragments were analysed by denaturing gradient gel electrophoresis. Bacterial profiles showed a significant effect of gender (P = 0.046) and P. aeruginosa carriage (P = 0.034) on community structure. Bacterial communities were found to be randomly assembled. Fungal community analysis found that F508del homozygous patients had a greater diversity than heterozygous patients (P = 0.007). This study indicates that the bacterial lung communities of adult CF patients are randomly assembled but have distinct gender based differences. Furthermore, the fungal communities colonising the CF lung are more diverse in F508 homozygotes. This is the first paper to identify a reduced bacterial diversity in female patients with CF and to implicate more severe CFTR genotypes with increased risk of infection with multiple fungal species.     

The airway microbiota in cystic fibrosis: a complex fungal and bacterial community--implications for therapeutic management

Background

Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways.

Methodology and Principal Findings

Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus.

Conclusions

In light of the recent concept of CF lung microbiota, we viewed the microbial community as a unique pathogenic entity. We thus interpreted our results to highlight the potential interactions between microorganisms and the role of fungi in the context of improving survival in CF.     

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log₁₀ copies/mL for M. abscessus complex and by a mean of 0.43 log₁₀ copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.     

Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies

The purpose of this study is to evaluate whether sample preservation can affect the yield of nucleic acid extracts from environmental samples. Storage of microbial samples was studied using three sediment types of varying carbon contents (10–57% carbon of dry weight). Four different storage solutions were tested at three temperatures. Freezing of samples at ?20 °C or ?80 °C, either without preservative or in phenol–chloroform solution, retained nucleic acid quantities very efficiently. Storage of samples in phenol–chloroform solution at +4 °C also gave good yields except for sediment with extremely high-carbon content. Ethanol and RNAlater® preservation decreased nucleic acid yields drastically at all temperatures. To study how sample preservation may affect the result of microbial community analysis, one type of sediment was selected for length heterogeneity-PCR analysis and PCR cloning of the 16S rRNA genes. Ethanol and RNAlater® preservation caused a slight bias towards certain microbial types in the community analyses shown by underrepresentation of Bacteroidetes, Betaproteobacteria and Gammaproteobacteria-affiliated peak sizes and overrepresentation of Actinobacteria, Chloroflexi and Alphaproteobacteria-affiliated peak sizes. Based on the results of this study, preservation in phenol–chloroform solution can be recommended as an alternative storage method when freezing is not possible such as during extended field sampling; however, ethanol and RNAlater® may cause serious problems when used as preservatives for environmental samples containing humic acids.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Chinese white truffles shape the ectomycorrhizal microbial communities of Corylus avellana

Here, we investigated the influence of Chinese white truffle (Tuber panzhihuanense) symbioses on the microbial communities associated with Corylus avellana during the early development stage of symbiosis. The microbial communities associated with ectomycorrhizae, and associated with roots without T. panzhihuanense colonization, were determined via high-throughput sequencing of bacterial 16S rRNA genes and fungal ITS genes. Microbial community diversity was higher in the communities associated with the ectomycorrhizae than in the control treatment. Further, bacterial and fungal community structures were different in samples containing T. panzhihuanense in association with C. avellana compared to the control samples. In particular, the bacterial genera Rhizobium, Pedomicrobium, and Herbiconiux were more abundant in the ectomycorrhizae, in addition to the fungal genus Monographella. Moreover, there were clear differences in some physicochemical properties among the rhizosphere soils of the two treatments. Statistical analyses indicated that soil properties including exchangeable magnesium and exchangeable calcium prominently influenced microbial community structure. Lastly, inference of bacterial metabolic functions indicated that sugar and protein metabolism functions were significantly more enriched in the communities associated with the ectomycorrhizae from C. avellana mycorrhized with T. panzhihuanense compared to communities from roots of cultivated C. avellana without T. panzhihuanense. Taken together, these results highlight the interactions among ectomycorrhizal fungi, soil properties, and microbial communities that are associated with host plants and further our understanding of the ecology and cultivation of the economically important T. panzhihuanense truffles.

     

不同树叶凋落物对人参土壤理化性质及微生物群落结构的影响

树种选择是林下山参护育成败的关键,研究树叶凋落物对人参土壤养分、微生物群落结构组成的影响,旨在为林下山参护育选择适宜林地及农田栽参土壤改良提供科学依据和理论指导。通过盆栽试验,研究添加5.0 g色木槭Acer mono.Maxim.var.mono(A)、赤松Pinus densiflora Sieb.et Zucc.(B)、胡桃楸Juglans mandshurica Maxim.(C)、紫椴Tilia amurensis Rupr.(D)、蒙古栎Quercus mongolica Fisch.ex Ledeb.(E)树叶凋落物到土壤中,种植人参(Panax ginseng C.A.meyer)后研究土壤理化性质以及微生物群落结构的变化。结果表明:添加不同树叶处理后人参土壤性质发生改变,土壤p H值显著高于对照土壤5.91(P0.05),土壤全氮、速效氮磷、微生物碳氮在所有树叶处理中显著增加(P0.05),而土壤容重、速效钾和C/N在添加树叶处理中降低。18个土壤样品基因组,经16S和ITS1测序分别得到6064和1900个OUTs。其中细菌涵盖了42门、117纲、170目、213科、225属,真菌涵盖了24门、98纲、196目、330科、435属。不同树叶处理人参土壤细菌和真菌地位发生改变,细菌Proteobacteria是树叶分解的关键微生物,添加树叶后其多样性显著高于对照(P0.05)。而细菌Bacteroidetes和真菌Basidiomycota可能是区别阔叶林和针叶林树种的关键微生物,针叶林中含量显著低于阔叶林(P0.05),而真菌Ascomycota是针叶林分解的关键微生物。进一步从不同分类水平上得到特定树叶凋落物的特异细菌和真菌。典型相关分析(CDA)表明细菌Bacteroidetes、Chloroflexi、Actinobacteria及真菌Basidiomycota、Zygomycota、Chytridiomycota及Ascomycota的位置及多样性的改变均与土壤因子SMBN、TN、AP、SOC、AK、C/N、p H有关。综上所述,添加不同树叶后不仅提高土壤微生物量碳氮、改善土壤理化性质,同时改变微生物群落结构组成,不同树叶处理土壤理化性质不同导致人参土壤微生物组成的差异,本结果对于林下参选地和农田栽参土壤微生物改良具有理论指导作用。     

A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota

Powdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored. We performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs). We found that after infection of soil-grown plants, G. orontii outcompeted numerous resident leaf-associated fungi while fungal community structure in roots remained unaltered. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-derived SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root-derived SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a twofold increase in leaf bacterial load. Our findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.     

Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis

In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.     

Contrasting primary successional trajectories of fungi and bacteria in retreating glacier soils

Early community assembly of soil microbial communities is essential for pedogenesis and development of organic legacies. We examined fungal and bacterial successions along a well‐established temperate glacier forefront chronosequence representing ~70 years of deglaciation to determine community assembly. As microbial communities may be heavily structured by establishing vegetation, we included nonvegetated soils as well as soils from underneath four plant species with differing mycorrhizal ecologies (Abies lasiocarpa, ectomycorrhizal; Luetkea pectinata, arbuscular mycorrhizal; Phyllodoce empetriformis, ericoid mycorrhizal; Saxifraga ferruginea, nonmycorrhizal). Our main objectives were to contrast fungal and bacterial successional dynamics and community assembly as well as to decouple the effects of plant establishment and time since deglaciation on microbial trajectories using high‐throughput sequencing. Our data indicate that distance from glacier terminus has large effects on biomass accumulation, community membership, and distribution for both fungi and bacteria. Surprisingly, presence of plants rather than their identity was more important in structuring bacterial communities along the chronosequence and played only a very minor role in structuring the fungal communities. Further, our analyses suggest that bacterial communities may converge during assembly supporting determinism, whereas fungal communities show no such patterns. Although fungal communities provided little evidence of convergence in community structure, many taxa were nonrandomly distributed across the glacier foreland; similar taxon‐level responses were observed in bacterial communities. Overall, our data highlight differing drivers for fungal and bacterial trajectories during early primary succession in recently deglaciated soils.     

不同生境黑果枸杞根际与非根际土壤微生物群落多样性

研究典型生境黑果枸杞根际与非根际土壤微生物群落多样性及其与土壤理化性质间的关系,为进一步研究黑果枸杞抗逆性提供理论数据。采集新疆精河县艾比湖地区(EB)盐碱地、乌苏市(WS)路旁荒地、五家渠市(WQ)人工林带的黑果枸杞根际与非根际土壤,利用Illumina-MiSeq高通量测序技术分析细菌和真菌群落组成和多样性。结果表明:根际土壤细菌多样性高于非根际土壤(WQ除外),而根际真菌多样性低于非根际土壤。WQ非根际土壤细菌和真菌多样性均高于EB和WS;根际细菌多样性排序为EBWSWQ,根际真菌多样性排序为WSEBWQ。根际土壤优势细菌门依次是变形菌门、拟杆菌门、放线菌门、酸杆菌门,真菌优势门为子囊菌门、担子菌门。根际土壤细菌变形菌门、拟杆菌门、酸杆菌门的相对丰度高于非根际土壤,而厚壁菌在根际土壤中的丰度显著降低,真菌优势门丰度在根际土和非根际土中的变化趋势因地区而异; Haliea、Gp10、Pelagibius、Microbulbifer、假单胞菌属、Thioprofundum、Deferrisoma是根际土壤细菌优势属;多孢子菌属、支顶孢属、Corollospora、Cochlonema是根际真菌优势属。细菌、真菌优势类群(门、属)的组成以及丰富度存在地区间差异,厚壁菌门在EB地区的丰富度显著高于含盐量较低的WS、WQ;盐碱生境EB中根际土壤嗜盐细菌的丰度高于非盐碱生境(WQ、WS),如盐单胞菌属、动性球菌属、Geminicoccu、Pelagibius、Gracilimonas、Salinimicrobium等。小囊菌属是EB根际真菌的最优势属,Melanoleuca是WQ和WS的最优势属,地孔菌属、Xenobotrytis、Brachyconidiellopsis、多孢子菌属等在EB根际土壤中的丰度显著高于WQ和WS。非盐碱生境(WS和WQ)的微生物群落之间的相似性较高,并且高于与盐碱环境(EB)之间的相似性,表明土壤含盐量对微生物群落组成丰度具有重要的影响。     

Vertical Distribution and Activity of Soil Microbial Population in a Sandy Desert Ecosystem

The vertical distribution of a microbial community on a temporal and spatial (vertical) scale under the Tamarix aphylla salt-resistant plant was studied. The uniqueness of this plant is in the creation of “islands of salinity” beneath the plant, due to salt excretion and leaf shedding at the plant base. Our working hypothesis was that the activity and biomass of the soil microbial population and the ratio between bacterial biomass and fungal biomass will decrease toward the driest season. Soil samples were collected beneath and between plants under the T. aphylla canopy down to the 0- to 50-cm depth at 10-cm intervals. The substrate-induced respiration method was used to estimate the total soil microbial biomass (MB), CO₂ evolution, metabolic quotient (qCO₂) index, and MB of both the fungal and bacterial communities. A significant difference was observed (p < 0.05) in MB and qCO₂ index between the different soil layers in the control samples, whereas there was no significant difference in the soil samples in the vicinity of the plant (p > 0.05) between the depths, due to the plant rhizosphere effect.     

Vertical distribution of the soil microbiota along a successional gradient in a glacier forefield

Spatial patterns of microbial communities have been extensively surveyed in well‐developed soils, but few studies investigated the vertical distribution of micro‐organisms in newly developed soils after glacier retreat. We used 454‐pyrosequencing to assess whether bacterial and fungal community structures differed between stages of soil development (SSD) characterized by an increasing vegetation cover from barren (vegetation cover: 0%/age: 10 years), sparsely vegetated (13%/60 years), transient (60%/80 years) to vegetated (95%/110 years) and depths (surface, 5 and 20 cm) along the Damma glacier forefield (Switzerland). The SSD significantly influenced the bacterial and fungal communities. Based on indicator species analyses, metabolically versatile bacteria (e.g. Geobacter) and psychrophilic yeasts (e.g. Mrakia) characterized the barren soils. Vegetated soils with higher C, N and root biomass consisted of bacteria able to degrade complex organic compounds (e.g. Candidatus Solibacter), lignocellulolytic Ascomycota (e.g. Geoglossum) and ectomycorrhizal Basidiomycota (e.g. Laccaria). Soil depth only influenced bacterial and fungal communities in barren and sparsely vegetated soils. These changes were partly due to more silt and higher soil moisture in the surface. In both soil ages, the surface was characterized by OTUs affiliated to Phormidium and Sphingobacteriales. In lower depths, however, bacterial and fungal communities differed between SSD. Lower depths of sparsely vegetated soils consisted of OTUs affiliated to Acidobacteria and Geoglossum, whereas depths of barren soils were characterized by OTUs related to Gemmatimonadetes. Overall, plant establishment drives the soil microbiota along the successional gradient but does not influence the vertical distribution of microbiota in recently deglaciated soils.     

Differential microbial communities in hot spring mats from Western Thailand

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.     

冰川生态系统固碳微生物研究进展

[目的] 海南海口含有丰富的温泉资源,对温泉微生物多样性进行研究,有助于进一步开发和利用海南温泉微生物资源。[方法] 本文采用Illumina HiSeq高通量测序技术对海口3个温泉[海甸岛荣域温泉（S1）、火山口开心农场温泉（S2）和西海岸海长流温泉（S3）] 水样中微生物ITS序列和16S rRNA基因V3-V4区进行测序及生物信息学分析,探究海口市3个不同区域的温泉真菌多样性与细菌多样性。[结果] （1）α多样性分析表明,真菌群落中,S3 > S1 > S2,而在细菌群落中,S2 > S1 > S3。β多样性分析表明,3个温泉真菌群落和细菌群落组成差异皆显著。（2）分类分析表明,温泉真菌群落优势菌门为子囊菌门（Ascomycota）和担子菌门（Basidiomycota）,细菌群落优势菌门为变形菌门（Proteobacteria）、拟杆菌门（Bacteroidetes）、Thermi、硝化螺旋菌门（Nitrospirae）、绿菌门（Chlorobi）、厚壁菌门（Firmicutes）、绿弯菌门（Chloroflexi）、放线菌门（Actinobacteria）。（3）CCA（Canonical correspondence analysis）分析表明,3个温泉的真菌群落主要影响因子是温度,细菌群落主要影响因子是总磷。[结论] 海南省海口市温泉中含有丰富的微生物资源,其微生物群落组成受多种环境因子影响,且影响真菌和细菌的主要环境因子不同。     

Analysis of Lung Microbiota in Bronchoalveolar Lavage,Protected Brush and Sputum Samples from Subjects with Mild-To-Moderate Cystic Fibrosis Lung Disease

Individuals with cystic fibrosis (CF) often acquire chronic lung infections that lead to irreversible damage. We sought to examine regional variation in the microbial communities in the lungs of individuals with mild-to-moderate CF lung disease, to examine the relationship between the local microbiota and local damage, and to determine the relationships between microbiota in samples taken directly from the lung and the microbiota in spontaneously expectorated sputum. In this initial study, nine stable, adult CF patients with an FEV₁>50% underwent regional sampling of different lobes of the right lung by bronchoalveolar lavage (BAL) and protected brush (PB) sampling of mucus plugs. Sputum samples were obtained from six of the nine subjects immediately prior to the procedure. Microbial community analysis was performed on DNA extracted from these samples and the extent of damage in each lobe was quantified from a recent CT scan. The extent of damage observed in regions of the right lung did not correlate with specific microbial genera, levels of community diversity or composition, or bacterial genome copies per ml of BAL fluid. In all subjects, BAL fluid from different regions of the lung contained similar microbial communities. In eight out of nine subjects, PB samples from different regions of the lung were also similar in microbial community composition, and were similar to microbial communities in BAL fluid from the same lobe. Microbial communities in PB samples were more diverse than those in BAL samples, suggesting enrichment of some taxa in mucus plugs. To our knowledge, this study is the first to examine the microbiota in different regions of the CF lung in clinically stable individuals with mild-to-moderate CF-related lung disease.