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PubMed2025

Establishing the ELIXIR Microbiome Community.

Microbiome research has grown substantially over the past decade in terms of the range of biomes sampled, identified taxa, and the volume of data derived from the samples. In particular, experimental approaches such as metagenomics, metabarcoding, metatranscriptomics and metaproteomics have provided profound insights into the vast, hitherto unknown, microbial biodiversity. The ELIXIR Marine Metagenomics Community, initiated amongst researchers focusing on marine microbiomes, has concentrated on promoting standards around microbiome-derived sequence analysis, as well as understanding the gaps in methods and reference databases, and identifying solutions to the computational overheads of performing such analyses. Nevertheless, the methods used and the challenges faced are not confined to marine microbiome studies, but are broadly applicable to other biomes. Thus, expanding this Marine Metagenomics Community to a more inclusive ELIXIR Microbiome Community will enable it to encompass a broader range of biomes and link expertise across 'omics technologies. Furthermore, engaging with a large number of researchers will improve the efficiency and sustainability of bioinformatics infrastructure and resources for microbiome research (standards, data, tools, workflows, training), which will enable a deeper understanding of the function and taxonomic composition of the different microbial communities.

Computational Biology↗

PubMed1994

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4 degrees C.

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4 degrees C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.

Journal Article↗

PubMed2001

Population dynamics of anaerobic microbial consortia in thermophilic granular sludge in response to feed composition change.

A thermophilic UASB reactor was operated at 55 degrees C for greater than 470 days in order to investigate the effects of feed composition on the changes in microbial community structure where thermophilic granular sludge was used as the inoculum source. The feed compositions were changed with cultivation days; phase 1 (1-70 days), alcohol distillery wastewater; phase 2 (71-281 days), artificial acetate wastewater; phase 3 (282-474 days), artificial sucrose wastewater. During the first one month of each phase, the methanogenic activity and cell density of methanogens quantified by fluorescence in situ hybridization (FISH) drastically changed as a result of shift in feed composition. When artificial acetate wastewater was used as feed, retained granular sludge was partially disintegrated due to a decrease in the number of symbiotic bacterial community members: acetogens (acidogens) and hydrogenotrophic methanogens. In contrast, when the feed was shifted to sucrose (phase 3), granulation of biomass was promoted by a remarkable proliferation of the symbiotic community. The presence of hydrogen-utilizing methanogens and acetogens (acidogens) are shown to be effective for the enhancement of thermophilic granulation. The cell density of methanogens determined by FISH was strongly correlated with the methane-producing potential of the retained thermophilic granular sludge.

Acetates↗

PubMed1996

Broad-Scale Approaches to the Determination of Soil Microbial Community Structure: Application of the Community DNA Hybridization Technique

Broad-scale approaches seek to integrate information on whole microbial communities. It is widely recognized that culture techniques are too selective and unrepresentative to allow a realistic assessment of the overall structure of microbial communities. Techniques based on fatty acid or metabolic profiles determine the phenotypic composition of the community. Complementary information about the genotypic structure of soil microbial communities necessitates analysis of community DNA. To determine broad-scale differences in soil microbial community structure (i.e., differences at the whole community level, rather than specific differences in species composition), we have applied a community hybridization technique to determine the similarity and relative diversity of two samples by cross hybridization. In previous studies this assay failed with whole-soil community DNA. Usable hybridization signals were obtained using whole-soil DNA, in this study, by digesting the DNA with restriction enzymes before the labeling with a random-primer reaction. The community hybridization technique was tested using a graded series of microbial fractions, increasing in complexity, all isolated from the same soil sample. This demonstrated that single bacterial species and a mixture of cultivable bacteria were less complex and only 5% similar to whole-community DNA or bacteria directly extracted from the soil. Extracted bacterial and whole-community DNA were 75% similar to each other and equally complex. When DNA was extracted from four different agricultural soils, their similarities ranged from 35 to 75%. The potential usefulness of community hybridization applied to soil microbial communities is discussed.

Journal Article↗

PubMed2005

Nitrogenase activity and nifH expression in a marine intertidal microbial mat.

N(2) fixation, diazotrophic community composition, and organisms actively expressing genes for N(2) fixation were examined over at 3-year period (1997-1999) for intertidal microbial mats on a sand flat located in the Rachel Carson National Estuarine Research Reserve (RCNERR) (Beaufort, NC, USA). Specifically, diel variations of N(2) fixation in the mats from the RCNERR were examined. Three distinct diel patterns of nitrogenase activity (NA) were observed. NA responses to short-term inhibitions of photosynthesis corresponded to one of the three patterns. High rates of NA were observed during peak O(2) production periods for diel experiments during summer months. Different types of NA diel variations correspond to different stages of mat development. Chloramphenicol treatments indicated that the mechanism of protein synthesis supporting NA changed throughout the day. Analysis of mat DNA and RNA gave further evidence suggesting that in addition to cyanobacteria, other functional groups were responsible for the NA observed in the RCNERR mats. The role of microbial diversity in the N(2) fixation dynamics of these mats is discussed.

Bacteria↗

PubMed2004

Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis.

BACKGROUND: The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora. RESULTS: A total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples. CONCLUSIONS: Although historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study--like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species--indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV.

Actinobacteria↗

PubMed2004

Wheat cultivar-specific selection of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas species from resident soil populations.

An emerging body of evidence indicates a role for plant genotype as a determinant of the species and genetic composition of the saprophytic microbial community resident to the rhizosphere. In this study, experiments were conducted to determine the capacity of five different wheat cultivars to enhance resident populations and support introduced strains of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent pseudomonads, a group of bacteria known to provide biological control of several soilborne diseases. When soils were cropped with three successive 28-day growth cycles of wheat, the 2,4-DAPG-producing strains were consistently recovered from the rhizosphere of the cultivar Lewjain, and commonly were present at populations higher than those recovered from other wheat cultivars. Based on restriction fragment length polymorphism and sequence analyses of phlD, a key gene involved in 2,4-DAPG production, two previously undefined phlD+ genotypes, referred to as genotypes PfZ and PfY, were discovered. Wheat cultivar Lewjain was the primary source of genotype PfY while cultivar Penawawa yielded the majority of genotype PfZ. Based on 16S rDNA sequence analysis, both new phlD genotypes were classified as P. fluorescens. Comparison of the rhizosphere competence of 2,4-DAPG-producing P. fluorescens Q2-87 (genotype B) and P. fluorescens LR3-A28 (genotype PfY) showed that both strains persisted at similar populations in the rhizosphere of all cultivars tested over a 30 day period when introduced as a seed inoculant. However, when strain LR3-A28 was applied as a soil inoculant, this strain was recovered at higher populations from the rhizosphere of wheat cultivar Lewjain than from the rhizospheres of two other cultivars. No cultivar effects were shown for strain Q2-87. Collectively, these results add further to evidence indicating a degree of specificity in interactions between plant cultivars and specific members of the saprophytic microbial community. Furthermore, as 2,4-DAPG-producing fluorescent Pseudomonas spp. have a central role in the spontaneous reduction in severity of take-all disease of wheat in response to continuous wheat monoculture, we postulate that the use of specific cultivars, such as Lewjain, which possess a superior capacity to enhance resident soil populations of these bacteria may have potential to reduce the length of the monoculture period required to induce natural suppressiveness of soils toward this disease.

Bacterial Proteins↗

PubMed2001

Comparative measurements of microbial activity in drinking water biofilters.

Tetrazolium reduction assays, phospholipid analysis, and 16S rRNA (rDNA) sequence analysis were applied to assess the distribution, composition and activity of microbial communities developing in biofilters treating non-ozonated and ozonated drinking water. The response of media-attached biomass to both operating temperature (3 degrees C vs. > 12 degrees C) and ozone application point was assessed. As judged by 2-(p-iodo-phenyl)-3-(p-nitrophenyl)-s-phenyl tetrazolium chloride (INT) reduction, the dehydrogenase activity in biofilter systems that were operated with non-ozonated water was 55% lower than in identical filters operating with ozonated water. There was no significant difference between the microbiological activity measured in a biofilter series treating ozonated water and an identical series where ozonated water was introduced at an intermediate point. The biomass levels in biofilter systems that were operated with ozonated water were 47% higher on average than identical systems operated with non-ozonated water. Operating temperature had no significant impact on total biomass levels; however, specific dehydrogenase activity was 70% higher in systems operated at ambient temperatures (> 12 degrees C) than in systems held at 3 degrees C. Phospholipid and rDNA analysis suggests that there was a community structure response to ozone application and operating temperature, but no response to different ozone application points.

Biomass↗

PubMed2005

In situ functional gene analysis: recognition of individual genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) using specific probes certainly is one of the most commonly applied molecular techniques in microbial ecology. Monitoring of community composition and dynamics can be combined with localization and identification of individual cells in situ. However, the resolution power of the method is limited by the need for high target numbers per cell. Apart from standard targets (ribosomal RNAs), mRNAs could be used successfully for in situ visualization in some cases. A new promising variant of in situ hybridization could be established that should provide access to any low copy number nucleic acid targets, such as chromosomal genes. The recognition of individual genes by FISH (RING-FISH) technology is based on target visualization mediated by polynucleotide probe network formation. The specificity of the approach is provided by intracellular probe-target hybridization. This initial hybridization apparently acts as a focal point for inter-probe hybridization within and mainly in the periphery of the cells. Probe-conferred fluorescence typically appears halo-like around the cells. RING-FISH can be used in combination with conventional oligonucleotide FISH. Thus, genetic potential can be assigned to in situ identified cells.

Escherichia coli↗

PubMed1998

Recovery of a Nitrosomonas-like 16S rDNA sequence group from freshwater habitats.

In order to study the diversity of ammonia-oxidising bacteria in freshwater habitats, including sediments, a molecular approach focused on the sequencing of 16S rDNA was adopted. 16S rDNA sequences showing affinity with the beta-subgroup of ammonia-oxidising bacteria were recovered by specific PCR of directly isolated DNA from freshwater samples, and samples from brackish water and Glyceria maxima rhizosphere were included in the analysis for comparison. The ammonia oxidiser-like sequences recovered from several locations, which exhibit differences in the composition of their total microbial communities as indicated by denaturing gradient gel electrophoresis, formed a strong monophyletic cluster including Nitrosomonas ureae. This is the first report presenting sequences from an apparently dominant group of Nitrosomonas-like organisms among the beta-subdivision of ammonia-oxidising bacteria in freshwater environments. This group of sequences extends the known diversity within the beta-subgroup of ammonia-oxidisers. The new sequences related to Nitrosomonas ureae do not match with some published primers and probes designed for the detection of Nitrosomonas species, which may explain why these sequences have not previously been detected in freshwater habitats. The sequence diversity detected within this group of sequences was minimal across the environments examined, and no patterns of distribution were indicated with respect to environmental factors such as sediment depth or location.

Base Sequence↗

PubMed2000

Microbial reductive dehalogenation of polychlorinated biphenyls.

Under anaerobic conditions, microbial reductive dechlorination of polychlorinated biphenyls (PCBs) occurs in soils and aquatic sediments. In contrast to dechlorination of supplemented single congeners for which frequently ortho dechlorination has been observed, reductive dechlorination mainly attacks meta and/or para chlorines of PCB mixtures in contaminated sediments, although in a few instances ortho dechlorination of PCBs has been observed. Different microorganisms appear to be responsible for different dechlorination activities and the occurrence of various dehalogenation routes. No axenic cultures of an anaerobic microorganism have been obtained so far. Most probable number determinations indicate that the addition of PCB congeners, as potential electron acceptors, stimulates the growth of PCB-dechlorinating microorganisms. A few PCB-dechlorinating enrichment cultures have been obtained and partially characterized. Temperature, pH, availability of naturally occurring or of supplemented carbon sources, and the presence or absence of H(2) or other electron donors and competing electron acceptors influence the dechlorination rate, extent and route of PCB dechlorination. We conclude from the sum of the experimental data that these factors influence apparently the composition of the active microbial community and thus the routes, the rates and the extent of the dehalogenation. The observed effects are due to the specificity of the dehalogenating bacteria which become active as well as changing interactions between the dehalogenating and non-dehalogenating bacteria. Important interactions include the induced changes in the formation and utilization of H(2) by non-dechlorinating and dechlorinating bacteria, competition for substrates and other electron donors and acceptors, and changes in the formation of acidic fermentation products by heterotrophic and autotrophic acidogenic bacteria leading to changes in the pH of the sediments.

Journal Article↗

PubMed2006

The oral microflora--friend or foe? Can we decide?

This paper discusses the factors that determine whether the oral microflora play a beneficial or detrimental role in the health of an individual during their life. The resident microflora of the mouth is diverse, and distinct from that found elsewhere in the body due to its unique biological and physical properties. This natural microflora is essential for the normal development of the physiology of the host, and contributes to the host defences by excluding exogenous micro-organisms. The oral microflora varies in composition on distinct surfaces (e.g. teeth, mucosa), and at sites on a specific surface (e.g. fissures, gingival crevice), demonstrating that subtle properties of a habitat influence the ability of individual species to colonise and dominate. The composition of these oral microbial communities remains relatively stable over time (microbial homeostasis). This stability does not indicate a passive relationship with the host, but reflects a dynamic balance among the component species. However, this stability can be perturbed by significant changes to the oral environment or in a person's life-style that occur during the life of an individual. Alterations in diet, medication, smoking, saliva flow, denture wearing, general health, etc, can lead to overgrowth by previously minor components of the oral microflora, which can predispose a site to disease. Likewise, the immune response can wane in old age, which may result in colonisation by exogenous and often pathogenic micro-organisms. Oral micro-organisms can also act as opportunistic pathogens, and cause serious disease elsewhere in the body. Therefore, active oral health care management is needed in order to maintain microbial homeostasis throughout life to ensure that we reap the benefits of our resident oral bacteria and not suffer from their mischief.

Age Factors↗

PubMed2006

Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea.

Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by gamma-proteobacteria (53 to 64%), followed by beta-proteobacteria (18 to 21%) and alpha-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by alpha-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of delta-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.

Animals↗

PubMed2006

mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray.

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.

Ecosystem↗

PubMed2002

Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR.

The use of real-time quantitative PCR (5' nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5' nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5' nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.

Bacterial Adhesion↗

PubMed2003

Complexity in natural microbial ecosystems: the Guerrero Negro experience.

The goal of this project is to describe and understand the organismal composition, structure, and physiology of microbial ecosystems from hypersaline environments. One collection of such ecosystems occurs at North America's largest saltworks, the Exportadora de Sal, in Guerrero Negro, Baja California Sur. There, seawater flows through a series of evaporative basins with an increase in salinity until saturation is reached and halite crystallization begins. Several of these ponds are lined with thick (10 cm) microbial mats that have received some biological study. To determine the nature and extent of diversity of the microbial organisms that constitute these ecosystems, we are conducting a phylogenetic analysis using molecular approaches, based on cloning and sequencing of small subunit (SSU) rRNA genes (16S for Bacteria and Archaea, 18S for Eukarya). In addition, we report preliminary results on the microbial composition of a laminated community that occurs in a crystallized gypsum-halite matrix in near-saturated salt water. Exposure of the interior of these large (kilogram) wet, endoevaporite crystals reveals a multitude of colors: layers of yellow, green, pink, and purple microbiota. To date, analyses of these two environments indicate the ubiquitous dominance of uncultured organisms of phylogenetic kinds not generally thought to be associated with hypersaline environments.

Bacteria↗

PubMedDate not supplied

[Dynamics of organic matter decomposition and microflora composition of forest litter in artificial biogeocenoses].

The dynamics of litter stock, microbial biomass, and composition and structure of microbial communities, were studied in the course of soil organic matter transformation during vegetation season. The dynamics of litter stock in coniferous and deciduous forests proved to correlate with the biomass and total abundance of microorganisms, particularly, with the proportion of microfungi in the microbial community.

Biodegradation, Environmental↗

PubMed2001

DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems.

Culture-independent molecular techniques are now available to study microbial ecosystems. They are opening interesting perspectives to problems related to composition and population dynamics of microbial communities in various environmental niches (e.g., soil, water) and foods. In fermented food products, estimates of true microbial diversity is often difficult chiefly on account of the inability to cultivate most of the viable bacteria. The increasing knowledge of gene sequences and the concomitant development of new culture-independent molecular techniques are providing new and effective tools to compare the diversity of microbial communities and to monitor population dynamics in minimally disturbed samples. In this review, recent advances in these techniques are reported. Possible applications to food-associated microbial ecosystems are emphasised.

Bacteria↗