Influences of environmental bacteria and their metabolites on allergies, asthma, and host microbiota.

The prevalence of allergic diseases and asthma has dramatically increased over the last decades, resulting in a high burden for patients and healthcare systems. Thus, there is an unmet need to develop preventative strategies for these diseases. Epidemiological studies show that reduced exposure to environmental bacteria in early life (eg, birth by cesarean section, being formula-fed, growing up in an urban environment or with less contact to various persons) is associated with an increased risk to develop allergies and asthma later in life. Conversely, a reduced risk for asthma is consistently found in children growing up on traditional farms, thereby being exposed to a wide spectrum of microbes. However, clinical studies with bacteria to prevent allergic diseases are still rare and to some extent contradicting. A detailed mechanistic understanding of how environmental microbes influence the development of the human microbiome and the immune system is important to enable the development of novel preventative approaches that are based on the early modulation of the host microbiota and immunity. In this mini-review, we summarize current knowledge and experimental evidence for the potential of bacteria and their metabolites to be used for the prevention of asthma and allergic diseases.

Environmental and mucosal microbiota and their role in childhood asthma.

BACKGROUND: High microbial diversity in the environment has been associated with lower asthma risk, particularly in children exposed to farming. It remains unclear whether this effect operates through an altered microbiome of the mucosal surfaces of the airways. METHODS: DNA from mattress dust and nasal samples of 86 school age children was analyzed by 454 pyrosequencing of the 16S rRNA gene fragments. Based on operational taxonomic units (OTUs), bacterial diversity and composition were related to farm exposure and asthma status. RESULTS: Farm exposure was positively associated with bacterial diversity in mattress dust samples as determined by richness (P = 8.1 × 10-6 ) and Shannon index (P = 1.3 × 10-5 ). Despite considerable agreement of richness between mattress and nasal samples, the association of richness with farming in nasal samples was restricted to a high gradient of farm exposure, that is, exposure to cows and straw vs no exposure at all. In mattress dust, the genera Clostridium, Facklamia, an unclassified genus within the family of Ruminococcaceae, and six OTUs were positively associated with farming. Asthma was inversely associated with richness [aOR = 0.48 (0.22-1.02)] and Shannon index [aOR = 0.41 (0.21-0.83)] in mattress dust and to a lower extent in nasal samples [richness aOR 0.63 = (0.38-1.06), Shannon index aOR = 0.66 (0.39-1.12)]. CONCLUSION: The stronger inverse association of asthma with bacterial diversity in mattress dust as compared to nasal samples suggests microbial involvement beyond mere colonization of the upper airways. Whether inhalation of metabolites of environmental bacteria contributes to this phenomenon should be the focus of future research.

Contribution of above- and below-ground plant traits to the structure and function of grassland soil microbial communities.

BACKGROUND AND AIMS: Abiotic properties of soil are known to be major drivers of the microbial community within it. Our understanding of how soil microbial properties are related to the functional structure and diversity of plant communities, however, is limited and largely restricted to above-ground plant traits, with the role of below-ground traits being poorly understood. This study investigated the relative contributions of soil abiotic properties and plant traits, both above-ground and below-ground, to variations in microbial processes involved in grassland nitrogen turnover. METHODS: In mountain grasslands distributed across three European sites, a correlative approach was used to examine the role of a large range of plant functional traits and soil abiotic factors on microbial variables, including gene abundance of nitrifiers and denitrifiers and their potential activities. KEY RESULTS: Direct effects of soil abiotic parameters were found to have the most significant influence on the microbial groups investigated. Indirect pathways via plant functional traits contributed substantially to explaining the relative abundance of fungi and bacteria and gene abundances of the investigated microbial communities, while they explained little of the variance in microbial activities. Gene abundances of nitrifiers and denitrifiers were most strongly related to below-ground plant traits, suggesting that they were the most relevant traits for explaining variation in community structure and abundances of soil microbes involved in nitrification and denitrification. CONCLUSIONS: The results suggest that consideration of plant traits, and especially below-ground traits, increases our ability to describe variation in the abundances and the functional characteristics of microbial communities in grassland soils.

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

Denitrifying pathways dominate nitrous oxide emissions from managed grassland during drought and rewetting.

Nitrous oxide is a powerful greenhouse gas whose atmospheric growth rate has accelerated over the past decade. Most anthropogenic N2O emissions result from soil N fertilization, which is converted to N2O via oxic nitrification and anoxic denitrification pathways. Drought-affected soils are expected to be well oxygenated; however, using high-resolution isotopic measurements, we found that denitrifying pathways dominated N2O emissions during a severe drought applied to managed grassland. This was due to a reversible, drought-induced enrichment in nitrogen-bearing organic matter on soil microaggregates and suggested a strong role for chemo- or codenitrification. Throughout rewetting, denitrification dominated emissions, despite high variability in fluxes. Total N2O flux and denitrification contribution were significantly higher during rewetting than for control plots at the same soil moisture range. The observed feedbacks between precipitation changes induced by climate change and N2O emission pathways are sufficient to account for the accelerating N2O growth rate observed over the past decade.

Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

Changes of diversity pattern of proteolytic bacteria over time and space in an agricultural soil.

The genetic heterogeneity of neutral metalloprotease (npr) gene fragments from soil proteolytic bacteria was investigated at a cultivated field site with four different soil types and at three different depths in April, July, and October. Terminal restriction fragment length polymorphism (T-RFLP) analyses of polymerase chain reaction-amplified npr gene fragments were applied to study the dynamic of the npr gene pool with regard to environmental conditions. The aim of this study was to relate differences in npr community structure and richness to the vertical, site, and seasonal variations naturally occurring at the field site under investigation. T-RFLP analysis revealed a noticeable seasonal variability in the community structure of npr-containing bacteria. The data suggest that the composition of the npr proteolytic bacterial population in July differed from those at the other dates. Additionally, the diversity of npr genes decreased with increasing soil depth revealing the highest values in upper layers. The reasons behind the observed patterns in the community structure might be mainly seasonal and vertical variation of the quantity and heterogeneity of available substrates as well as spatial isolation caused by a varying water amount and the connectivity of soil particles among the soil profile. Sequencing and phylogenetical analysis of 120 npr clones from the top soils collected in July revealed that most of the clones exhibit only poor homology to npr genes of isolates previously obtained from various environments, indicating the presence of until now uncharacterized npr coding proteolytic bacteria at the study site.

Ecology and evolution of bacterial microdiversity.

Using high resolution molecular fingerprinting techniques like random amplification of polymorphic DNA, repetitive extragenic palindromic PCR and multilocus enzyme electrophoresis, a high bacterial diversity below the species and subspecies level (microdiversity) is revealed. It became apparent that bacteria of a certain species living in close association with different plants either as associated rhizosphere bacteria or as plant pathogens or symbiotic organisms, typically reflect this relationship in their genetic relatedness. The strain composition within a population of soil bacterial species at a given field site, which can be identified by these high resolution fingerprinting techniques, was markedly influenced by soil management and soil features. The observed bacterial microdiversity reflected the conditions of the habitat, which select for better adapted forms. In addition, influences of spatial separation on specific groupings of bacteria were found, which argue for the occurrence of isolated microevolution. In this review, examples are presented of bacterial microdiversity as influenced by different ecological factors, with the main emphasis on bacteria from the natural environment. In addition, information available from some of the first complete genome sequences of bacteria (Helicobacter pylori and Escherichia coli) was used to highlight possible mechanisms of molecular evolution through which mutations are created; these include mutator enzymes. Definitions of bacterial species and subspecies ranks are discussed in the light of detailed information from whole genome typing approaches.

Molecular analysis of ammonia oxidation and denitrification in natural environments.

This review summarizes aspects of the current knowledge about the ecology of ammonia-oxidizing and denitrifying bacteria. The development of molecular techniques has contributed enormously to the rapid recent progress in the field. Different techniques for doing so are discussed. The characterization of ammonia-oxidizing and -denitrifying bacteria by sequencing the genes encoding 16S rRNA and functional proteins opened the possibility of constructing specific probes. It is now possible to monitor the occurrence of a particular species of these bacteria in any habitat and to get an estimate of the relative abundance of different types, even if they are not culturable as yet. These data indicate that the composition of nitrifying and denitrifying communities is complex and apparently subject to large fluctuations, both in time and in space. More attempts are needed to enrich and isolate those bacteria which dominate the processes, and to characterize them by a combination of physiological, biochemical and molecular techniques. While PCR and probing with nucleotides or antibodies are primarily used to study the structure of nitrifying and denitrifying communities, studies of their function in natural habitats, which require quantification at the transcriptional level, are currently not possible.

Mycorrhizosphere responsiveness to atmospheric ozone and inoculation with Phytophthora citricola in a phytotron experiment with spruce/beech mixed cultures.

The aim was to analyze functional changes in the mycorrhizosphere (MR) of juvenile spruce and beech grown in a mixture under ambient and twice ambient ozone and inoculated with the root pathogen Phytophthora citricola. The phytotron experiment was performed over two vegetation periods, adding the pathogen at the end of the first growing season. Root biomass data suggest that the combined treatment affected spruce more than beech and that the negative influence of ozone on stress tolerance against the root pathogen P. citricola was greater for spruce than for beech. In contrast, beech was more affected when the pathogen was the sole stressor. The functional soil parameter chosen for studies of MR soil samples was activity of extracellular enzymes. After the first year of ozone exposure, MR soil samples of both species showed increased activity of almost all measured enzymes (acid phosphatase, chitinase, beta-glucosidase, cellobiohydrolase) in the O3 treatment. Species-specific differences were observed, with a stronger effect of P. citricola on beech MR and a stronger ozone effect on spruce MR. In the second year, the effects of the combined treatment (ozone and P. citricola) were a significant increase in the activity of most enzymes (except cellobiohydrolase) for both tree species. The results indicated that responsiveness of MR soils towards ozone and P. citricola was related to the severity of infection of the plants and the reduction of belowground biomass, suggesting a strong, direct influence of plant stress on MR soil enzyme activity. Additional research is needed using different species and combined stresses to determine the broader ecological relevance of shifts in rhizosphere enzymes.

Short term effects of ozone on the plant-rhizosphere-bulk soil system of young beech trees.

Plant growth largely depends on microbial community structure and function in the rhizosphere. In turn, microbial communities in the rhizosphere rely on carbohydrates provided by the host plant. This paper presents the first study on ozone effects in the plant-rhizosphere-bulk soil system of 4-year-old beech trees using outdoor lysimeters as a research platform. The lysimeters were filled with homogenized soil from the corresponding horizons of a forest site, thus minimizing field heterogeneity. Four lysimeters were treated with ambient ozone (1 x O3) and four with double ambient ozone concentrations (2 x O3; restricted to 150 ppb). In contrast to senescence, which was almost unaffected by ozone treatment, both the photochemical quantum yield of photosystem II (PSII) and leaf gas exchange were reduced (11 - 45 %) under the elevated O3 regime. However, due to large variation between the plants, no statistically significant O3 effect was found. Even though the amount of primary metabolites, such as sugar and starch, was not influenced by elevated O3 concentrations, the reduced photosynthetic performance was reflected in leaf biochemistry in the form of a reduction in soluble phenolic metabolites. The rhizosphere microbial community also responded to the O3 treatment. Both community structure and function were affected, with a tendency towards a lower diversity and a significant reduction in the potential nutrient turnover. In contrast, litter degradation was unaffected by the fumigation, indicating that in situ microbial functionality of the bulk soil did not change.

The use of immunological methods to detect and identify bacteria in the environment.

Immunological detection methods have become increasingly important in microbial ecology for the tracking of specific microorganisms and for community analysis. For a reliable application of these techniques, the monoclonal antibodies or polyclonal antisera used have to fulfill several quality criteria. Cross reactivity, cellular localization of the antigenic determinant, affinity characteristics and the expression of the antigenic determinant at environmental conditions have to be determined. Immunological methods can be used for the identification, quantification and enrichment of specific bacteria in extracts as well as for the visualization of cells in situ. The sensitivity of advanced immunological methods can be compared to PCR techniques. Using image processing of epifluorescence micrographs or confocal laser scanning microscopy, the immunofluorescence approach can now be applied to study complex environmental samples.

RNA fingerprinting of microbial community in the rhizosphere soil of grain legumes.

Microbial structural and expression profiles of the rhizospheres of three legumes, faba beans, peas and white lupin, were compared by RNA-arbitrarily primed PCR technique. Two different primers, M13 reverse and 10-mer primers, were used in the amplification and products resolved on non-denaturing polyacrylamide gel. With both DNA and RNA profiles Lupinus and Pisum rhizospheres were more similar to each other than to Vicia rhizosphere. The RAP-PCR products were also dot blotted and probed for bacterial peptidase transcripts. Plant-dependent rhizosphere effect was evident by the marked absence of transcripts for bacterial neutral metallopeptidase in Lupinus rhizosphere. The results of dot blot were further confirmed by RT-PCR for the expression of bacterial neutral metallopeptidase in the three rhizospheres.

Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification.

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.

A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies.

Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run. The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies. Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles. Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR. Only Cm species were detected and there was clear differentiation among the subspecies C. michiganensis sepedonicus (Cms), C. michiganensis michiganensis (Cmm), C. michiganensis nebraskensis (Cmn), C. michiganensis insidiosus (Cmi) and C. michiganensis tessellarius (Cmt). The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies. Validity is shown by comparison with cell counts.

PCR primers and functional probes for amplification and detection of bacterial genes for extracellular peptidases in single strains and in soil.

A set of primers and functional probes was developed for the detection of peptidase gene fragments of proteolytic bacteria. Based on DNA sequence data, degenerate PCR primers and internal DIG-labeled probes specific for genes encoding alkaline metallopeptidases (apr) (E.3.4.24), neutral metallopeptidases (npr) (E.3.4.24) and serine peptidases (sub) (E.3.4.21) were derived by multiple sequence alignments. Type strains with known peptidase genes and proteolytic bacteria from a grassland rhizosphere soil, a garden soil and an arable field were investigated for their genotypic proteolytic potential. For 52 out of 53 proteolytic bacterial isolates, at least one of the three peptidase classes could be identified by this approach. The amplified gene fragments were of the expected sizes with each of the three primer sets. The functional probes APR, NPR and SUB have been shown to hybridize specifically to the corresponding gene fragments. sub and npr genes were mainly found in Bacillus species. apr genes were only found in the Pseudomonas fluorescens biotypes and in two morphologically identical Flavobacterium-Cytophaga strains from two different sites. In most of the Bacillus spp., both sub and the npr and in the Flavobacterium-Cytophaga strains even all the three genes could be detected. PCR with DNA isolated from soil led to one main product of the expected size with each primer pair whose identity was additionally confirmed by Southern blot hybridization with the corresponding probes.

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

Influence of nonylphenol on the microbial community of lake sediments in microcosms.

In this study the impact of nonylphenol, an estrogenic degradation product of alkylphenol polyethoxylates, on the microbial community structure in contaminated sediments of aquatic microcosms using in situ hybridization with fluorescently labeled oligonucleotides probes was investigated. A positive correlation between nonylphenol concentration and cell numbers of bacteria and microfungi as well as an increase in the numbers of active bacteria was found. However, the ratio between total microorganisms and active bacteria remained unchanged. A large fraction of the cells could be identified using group specific oligonucleotide probes. A slight change in the composition of the microbial community structure was observed, with Gram-positive bacteria with high DNA G + C-content becoming more abundant at higher concentrations of nonylphenol.