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PubMed2006

Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota.

Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.

Adult↗
PubMed2003

In situ exposure to low herbicide concentrations affects microbial population composition and catabolic gene frequency in an aerobic shallow aquifer.

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 micro g l(-1)) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10(0) to 10(4) g(-1) sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (10(2) to 10(3) gene copies g(-1) sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4',6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2025

Reduction in ARGs and Mobile Genetic Elements Using 2-Bromoethane Sulfonate in an MFC-Powered Fenton System.

The integration of an MFC-powered Fenton (MFC-Fenton) system into the traditional anaerobic composting process can promote excess dewatered sludge (ES) decomposition. However, the antibiotic resistance gene (ARG) profiles in ES treated by MFC-Fenton systems remain poorly understood; in addition, the effect of adding 2-bromoethane sulfonate (BES, a methane inhibitor) during ES treatment using an MFC-Fenton system on ARG levels is largely unexplored. The present work focused on investigating the effects of BES and bioelectrochemical processes on ARG and MGE abundances and unraveling the ARG attenuation mechanism. According to our findings, adding BES promoted ARG reduction in ES in an MFC-Fenton system. The average ARG levels in the MFC-Fenton samples containing high BES contents (0.4 or 0.5 g BES/g VSS) markedly declined relative to those in samples containing lower BES levels. Moreover, macrolide transporter ATP-binding protein, macrolide-efflux protein, and macB levels markedly decreased as BES levels increased. BES supplementation and bioelectrochemical assistance were crucial for altering the ARG composition in the MFC-Fenton system. Changes in the microbial community composition had the greatest effect on the variation in ARG composition. Furthermore, the Actinobacteria and Firmicutes levels accounted for 52.8% of the overall ARG variation. Among MGEs, plasmids, insertion sequences, and integrons showed lower levels within the sludge metagenomes. Typically, sulI, sulII, tetG, and bla TEM levels were positively correlated with metal resistance genes (MRGs), and their levels markedly declined following the MFC-Fenton process. Thus, the collective evidence indicates that BES synergizes with bioelectrogenesis to reduce ARG abundance.

Sewage↗
PubMed2000

Influence of TNT transformation on microbial community structure in four different lake microcosms.

After World War II, large amounts of obsolete ammunition were dumped in various lakes in Sweden. Trinitrotoluene, TNT, was one of the main components of the dumped explosives. In this study, four different lake microcosms originating from lakes where relatively large amounts of ammunition were dumped were used to mimic the effect of TNT release on the natural microbial community. Increased microbial growth was found in lake microcosms amended with TNT. However, negligible mineralization of TNT was detected, suggesting that TNT was not utilized as a carbon source, but as a nitrogen source. Random amplified polymorphic DNA (RAPD) analysis indicated that the TNT induced no significant differences in microbial community composition and therefore, no major changes in natural selection, despite the increased microbial growth in the presence of the compound. More than 95% of the added TNT bound irreversibly to the sediments, possibly as a result of microbial transformation to reactive metabolites that subsequently bound covalently to components of the sediment. The results, taken together, suggest that no permanent change in the microbial ecology occurred as a result of the TNT amendment. This was probably due partly to the transient exposure of the microbial communities to the TNT before it became irreversibly bound to the sediment, and partly to the fact that TNT was not a primary growth substrate that strongly affects natural selection.

Bacteria↗
PubMed2002

Effect of starvation length upon microbial activity in a biomass recycle reactor.

The kinetics of substrate degradation and bacterial growth was determined in a microbial community from a biomass recycle reactor that had been deprived of substrate feed for 0-32 days. Starvation caused changes in bacterial numbers, community composition, and physiological state. Substrate starvation for less than 1 day resulted in modest (less than threefold) changes in endogenous respiration rate, ATP content, and biomass level. During a starvation period of 32 days, there were substantial changes in microbial community composition, as assessed by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR amplicons of a portion of the 16S rDNA or by phospholipid fatty acid (PLFA) analysis. When the starved communities were stimulated with organic nutrients, the growth kinetics was a function of the length of the starvation period. For starvation periods of 2-8 days prior to nutrient addition, there was a phase of suboptimal exponential growth (S-phase) in which the exponential growth rate was about 30% of the ultimate unrestricted growth rate. S-phase lasted for 2-8 h and then unrestricted growth occurred at rates of 0.3-0.4 h(-1). At starvation times of 12 and 20 days, a lag phase preceded S-phase and the unrestricted growth phase.

Bacteria↗
PubMed2005

Strain stability in biological systems treating recalcitrant organic compounds.

The availability of molecular probing technology in recent years has facilitated investigation of microbial community composition during bio-treatment of organic wastes. Particularly, it has allowed the study of microbial culture stability and correlation between stability and treatment performance. However, most studies to date have only addressed mixed cultures and there is limited information regarding single strain stability. Here we have investigated the microbial community dynamics in two bioreactors, each inoculated with a pure bacterial strain capable of degrading a recalcitrant substrate, namely Xanthobacter aut. GJ10 degrading 1,2-dichloroethane (DCE) and Burkholderia sp. JS150 degrading monochlorobenzene (MCB). Universal and strain specific 16S rRNA oligonucleotide probes were designed and used to follow strain stability. The bioreactor fed with DCE was functionally stable and the percentage of GJ10 cells in the community remained high (around 95% of total cells) throughout, even after introduction of foreign microorganisms. The bioreactor fed with MCB was also functionally stable, but in contrast to the DCE bioreactor, probing results revealed the disappearance of strain JS150 from the bioreactor within a week. The difference in behavior between the two systems is attributed to the specific pathway required to degrade DCE.

Bioreactors↗
PubMed2006

A dynamic programming algorithm for binning microbial community profiles.

MOTIVATION: A number of community profiling approaches have been widely used to study the microbial community composition and its variations in environmental ecology. Automated Ribosomal Intergenic Spacer Analysis (ARISA) is one such technique. ARISA has been used to study microbial communities using 16S-23S rRNA intergenic spacer length heterogeneity at different times and places. Owing to errors in sampling, random mutations in PCR amplification, and probably mostly variations in readings from the equipment used to analyze fragment sizes, the data read directly from the fragment analyzer should not be used for down stream statistical analysis. No optimal data preprocessing methods are available. A commonly used approach is to bin the reading lengths of the 16S-23S intergenic spacer. We have developed a dynamic programming algorithm based binning method for ARISA data analysis which minimizes the overall differences between replicates from the same sampling location and time. RESULTS: In a test example from an ocean time series sampling program, data preprocessing identified several outliers which upon re-examination were found to be because of systematic errors. Clustering analysis of the ARISA from different times based on the dynamic programming algorithm binned data revealed important features of the biodiversity of the microbial communities.

Algorithms↗
PubMed2024

Metagenomic analysis reveals gene taxonomic and functional diversity response to microplastics and cadmium in an agricultural soil.

Both microplastics (MPs) and heavy metals are common soil pollutants and can interact to generate combined toxicity to soil ecosystems, but their impact on soil microbial communities (e.g., archaea and viruses) remains poorly studied. Here, metagenomic analysis was used to explore the response of soil microbiome in an agricultural soil exposed to MPs [i.e., polyethylene (PE), polystyrene (PS), and polylactic acid (PLA)] and/or Cd. Results showed that MPs had more profound effects on microbial community composition, diversity, and gene abundances when compared to Cd or their combination. Metagenomic analysis indicated that the gene taxonomic diversity and functional diversity of microbial communities varied with MPs type and dose. MPs affected the relative abundance of major microbial phyla and genera, while their coexistence with Cd influenced dominant fungi and viruses. Nitrogen-transforming and pathogenic genera, which were more sensitive to MPs variations, could serve as the indicative taxa for MPs contamination. High-dose PLA treatments (10%, w/w) not only elevated nitrogen metabolism and pathogenic genes, but also enriched copiotrophic microbes from the Proteobacteria phylum. Overall, MPs and Cd showed minimal interactions on soil microbial communities. This study highlights the microbial shifts due to co-occurring MPs and Cd, providing evidence for understanding their environmental risks.

Soil Microbiology↗
PubMed2004

Does smearing inoculum reflect the bacterial composition of the smear at the end of the ripening of a French soft, red-smear cheese?

The microbial community composition and dynamics during the production of a French soft, red-smear cheese were investigated. The colonization efficiency of the smearing inoculum was followed, and the parts played by the inoculum used and the resident microflora were tentatively estimated. Single-strand conformation polymorphism analysis (SSCP) was applied to 2 productions of a soft, red-smear cheese produced by the same dairy plant at 4-mo intervals. Microbial composition of the different cheese samples analyzed was found to be reproducible from one production to another. However, the composition of the surface flora of both cheeses at the end of the ripening did not reflect the composition of the smearing inoculum used, qualitatively as well as quantitatively. These results were confirmed by those obtained when assessing the microbial composition of the culturable flora by the spread plate technique. The inoculum used by the industry had low resiliency potentialities against colonization of cheeses by resident organisms. Therefore, fitness and colonization potential of smearing inocula should be carefully assessed by the industry before use. The use of Arthrobacter strains as part of the smearing inoculum should be evaluated.

Arthrobacter↗
PubMed2024

Environmental stress mediates groundwater microbial community assembly.

Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.

Phylogeny↗
PubMed1980

Effects of grazing by estuarine gammaridean amphipods on the microbiota of allochthonous detritus.

Estuarine gammaridean amphipods grazing at natural population density on detrital microbiota affected the microbial community composition, biomass, and metabolic activity without affecting the physical structure of the leaves. Total microbial biomass estimated by adenosine triphosphate and lipid phosphate or observed by scanning electron microscopy was greater on grazed than on ungrazed detritus. The rates of oxygen consumption, poly-beta-hydroxybutyrate synthesis, total lipid biosynthesis, and release of CO(2) from radioactively prelabeled microbiota were higher on grazed than on ungrazed leaves, indicating stimulation of the metabolic activity of grazed detrital microbes. This was true with rates based either on the dry leaf weight or microbial biomass. Alkaline phosphatase activity was lower in the grazed system, consistent with enhanced inorganic phosphate cycling. The loss of C from both total lipid and poly-beta-hydroxybutyrate of microorganisms prelabeled with C was greater from grazed than ungrazed microbes. There was a faster decrease in the C-glycolipid than in the C-neutral lipid or C-phospholipid fractions. Analysis of specific phospholipids showed losses of the metabolically stable [C]glycerolphosphorylcholine derived from phosphatidylcholine and much more rapid metabolism of the bacterial lipid phosphatidylglycerol measured as [C]glycerolphosphorylglycerol with amphipod grazing. The biochemical data supported scanning electron microscopy observations of a shift as the grazing proceeded from a bacterial/fungal community to one dominated by bacteria.

Journal Article↗
PubMed2003

Microbial community responses to atrazine exposure and nutrient availability: linking degradation capacity to community structure.

Repeated pesticide exposure may enhance biodegradation through selective enrichment of pesticide-metabolizing microorganisms, particularly when the compound is used as a C and energy source. The relationship between pesticide application history and degradation rate is unclear when the chemical is utilized as a nutrient source other than C. Atrazine, a poor source of C and energy, was chosen as a model compound because it can serve as an N source for some microorganisms. Soils with (H-soil) and without (NH-soil) prior s-triazine treatment history were repeatedly exposed to atrazine and a variety of C and N source amendments. Exposure to atrazine and inorganic-N availability were the dominant factors leading to the development of microbial communities with an enhanced capacity to degrade atrazine. The density of the atrazine-degrading microorganisms increased immediately, up to 1000-fold, with atrazine exposure in the H-soil, but comparable increases were not observed in the NH-soil until 12 weeks following laboratory acclimation, despite high rates of atrazine mineralization in these soils immediately following the acclimation period. Whole-soil fatty acid methyl ester (FAME) analysis showed that the application of alternative C and N sources in addition to atrazine resulted in a microbial community composition that was distinctly different from that in either the atrazinealone treatment or water controls for both the H- and NH-soils. These data suggest that the microbial communities in both soils were altered differently in response to the treatments but developed a similar enhanced capacity to mineralize atrazine.

Atrazine↗
PubMed2004

Effects of elevated atmospheric CO2 concentrations on soil microorganisms.

Effects of elevated CO(2) on soil microorganisms are known to be mediated by various interactions with plants, for which such effects are relatively poorly documented. In this review, we summarize and synthesize results from studies assessing impacts of elevated CO(2) on soil ecosystems, focusing primarily on plants and a variety the of microbial processes. The processes considered include changes in microbial biomass of C and N, microbial number, respiration rates, organic matter decomposition, soil enzyme activities, microbial community composition, and functional groups of bacteria mediating trace gas emission such as methane and nitrous oxide. Elevated CO(2) in atmosphere may enhance certain microbial processes such as CH(4) emission from wetlands due to enhanced carbon supply from plants. However, responses of extracellular enzyme activities and microbial community structure are still controversy, because interferences with other factors such as the types of plants, nutrient availabilitial in soil, soil types, analysis methods, and types of CO(2) fumigation systems are not fully understood.

Atmosphere↗
PubMed2005

Microbial assemblages in soil microbial succession after glacial retreat in Svalbard (high arctic).

Microbial community composition (cyanobacteria and eukaryotic microalgae abundance and diversity, bacterial abundance, and soil respiration) was studied in subglacial and periglacial habitats on five glaciers near Ny-Alesund, Svalbard (79 degrees N). Soil microbial communities from nonvegetated sites (subglacial, recently deglaciated, and cryoconite sediments) and sites with plant cover (deglaciated some hundreds of years ago) were analyzed. Physicochemical analyses (pH, texture, water content, organic matter, total C and N content) were also performed on the samples. In total, 57 taxa of 23 genera of cyanobacteriaand algae were identified. Algae from the class Chlorophyceae (25 species) and cyanobacteria (23 species) were richest in biodiversity. The numbers of identified species in single habitat types were 23 in subglacial, 39 inbarren, 22 in cryoconite, and 24 in vegetated soils. The highest cyanobacterial and algal biovolume and cell numbers, respectively, were present in cryoconite (13x10(4) microm3 mg-1 soil and 508 cells per mg of soil), followed by barren (5.7x10(4) and 188), vegetated (2.6x10(4) and 120), and subglacial (0.1x10(4) and 5) soils. Cyanobacteria prevailed in all soil samples. Algae (mainly green algae) were present only as accessory organisms. The density of bacteria showed a slightly different trend to that of the cyanobacterial and algal assemblages. The highest number of bacteria was present in vegetated (mean: 13,722x10(8) cells per mg of soil dry wt.), followed by cryoconite (3802x10(8)), barren (654x10(8)), and subglacial (78x10(8)) soils. Response of cyanobacteria and algae to physical parameters showed that soil texture and water content are important for biomass development. In addition, it is shown that nitrogen and water content are the main factors affecting bacterial abundance and overall soil respiration. Redundancy analysis (RDA) with forward selection was used to create a model explaining variability in cyanobacterial, algal, and bacterial abundance. Cryoconites accounted for most of the variation in cyanobacteria and algae biovolume, followed by barren soils. Oscillatoriales, desmids, and green coccoid algae preferred cryoconites, whereas Nostocales and Chroococcales occurred mostly in barren soils. From the data obtained, it is evident that of the studied habitats cryoconite sediments are the most suitable ones for the development of microbial assemblages. Although subglacial sediments do not provide as good conditions as cryoconites, they support the survival of microbial communities. Both mentioned habitats are potential sources for the microbial recolonization of freshly deglaciated soil after the glacier retreat.

Arctic Regions↗
PubMed2025

Gut microbial and functional alterations lead to metagenomic signatures for midgut neuroendocrine tumor patients and for carcinoid syndrome.

Midgut neuroendocrine tumors (NET) derive from enterochromaffin cells, which have a close interrelationship with intestinal microbiota. Recently, we have utilized 16S rRNA sequencing to uncover that midgut NET patients have a depleted gut microbiome and a specific fecal microbial signature. This study aims to validate these findings and to further characterize the role of microbes and microbial metabolic pathways in midgut NET patients with and without carcinoid syndrome (CS). Fecal samples from 60 midgut NET patients and 20 household-matched controls were subjected to whole metagenome sequencing. The gut microbial community composition of midgut NET patients differed from that of controls, with 2 genera, 17 species and 9 microbial pathways showing differential abundance (P < 0.001). No differences in the microbial composition were observed between midgut NET patients with and without CS (P > 0.05). However, we did observe changes in inter-genus correlations of Bacteroides, Odoribacter, Parasutterella, Klebsiella, Ruminococcus and Proteobacteria when comparing these two patient groups. A signature of 16 microbial species (area under the receiver operating characteristics (AUROC) curve 0.892) or 18 microbial pathways (AUROC 0.909) accurately predicted the presence of a midgut NET. Furthermore, a microbial signature consisting of 14 functional microbial pathways distinguished CS patients from non-CS patients (AUROC 0.807). Thus, this study confirms that the gut microbiome of midgut NET patients is altered at the metagenomic level, which is not related to the presence of CS. A fecal microbial signature could constitute a novel biomarker for the diagnosis of midgut NET or CS.

Aged↗
PubMed2004

Enhancement of the biodegradability of aromatic groundwater contaminants.

Groundwater (GW) from the Bitterfeld industrial region, Central Germany, is contaminated mainly with monochlorobenzene (MCB). Accordingly, current research addresses the development of feasible in situ groundwater remediation technologies. Although easily degradable under aerobic conditions, MCB persists in the essentially anaerobic Bitterfeld aquifer. Therefore, we focused on primary oxidation of MCB and the subsequent anaerobic biodegradability of MCB oxidation products by the indigenous microbial community. In groundwater microcosms, most efficient MCB removal was observed upon treatment with Fenton's reagent (H2O2 + Fe2+), which produces the highly reactive hydroxyl radical and Fe3+ simultaneously. Phospholipid fatty acid analysis following different treatments suggested respective shifts of the microbial community compositions, and indicated that Fenton's reagent had a rather beneficial than an adverse effect on biomass development. Potential metabolites of hydroxyl radical attack on MCB such as chlorohydroquinone, hydroquinone, catechol, resorcinol, and phenol were anaerobically degraded by the groundwater microbial community under Fe3+ -reducing conditions.

Anaerobiosis↗
PubMed2006

Local similarity analysis reveals unique associations among marine bacterioplankton species and environmental factors.

MOTIVATION: Characterizing the diversity of microbial communities and understanding the environmental factors that influence community diversity are central tenets of microbial ecology. The development and application of cultivation independent molecular tools has allowed for rapid surveying of microbial community composition at unprecedented resolutions and frequencies. There is a growing need to discern robust patterns and relationships within these datasets which provide insight into microbial ecology. Pearson correlation coefficient (PCC) analysis is commonly used for identifying the linear relationship between two species, or species and environmental factors. However, this approach may not be able to capture more complex interactions which occur in situ; thus, alternative analyses were explored. RESULTS: In this paper we introduced local similarity analysis (LSA), which is a technique that can identify more complex dependence associations among species as well as associations between species and environmental factors without requiring significant data reduction. To illustrate its capability of identifying relationships that may not otherwise be identified by PCC, we first applied LSA to simulated data. We then applied LSA to a marine microbial observatory dataset and identified unique, significant associations that were not detected by PCC analysis. LSA results, combined with results from PCC analysis were used to construct a theoretical ecological network which allows for easy visualization of the most significant associations. Biological implications of the significant associations detected by LSA were discussed. We also identified additional applications where LSA would be beneficial. AVAILABILITY: The algorithms are implemented in Splus/R and they are available upon request from the corresponding author.

Bacteria↗
PubMed2002

Phylogenetic analysis of intestinal microflora indicates a novel Mycoplasma phylotype in farmed and wild salmon.

All studies of the microbial community of the gastrointestinal tract of salmon to date have employed culture-based approaches, typically on pond- or tank-raised, freshwater animals. We present a phylogenetic survey of the bacterial populations present in the distal intestine of salmon from three different marine locations in Europe. This was accomplished through PCR amplification, cloning, and sequencing of partial 16S rDNA genes from microbial community DNA isolated from the contents of the GI tract distal to the pyloric ceca. Using this approach, the intestinal microbial communities of wild salmon from Scotland and pen-raised salmon from Scotland and Norway were compared. The predominating bacterial populations detected were Acinetobacter junii and a novel Mycoplasma phylotype. This Mycoplasma phylotype apparently comprised approximately 96% of the total microbes in the distal intestine of wild salmon. Substantial differences in intestinal microbial community composition and diversity were observed between the two groups of pen-raised salmon, which, in addition to geographical separation, were raised on different feeds. The microbial profiles found in this study were substantially different from those indicated in earlier culture-based studies for several species of fish, presumably because of the culture-independent techniques employed. Further, analysis of short-chain fatty acids in the digestive tract indicated that the decreasing redox gradient from proximal to distal reaches common to homeothermic animals was absent in salmon, and that the bacterial fermentation levels were much lower than are reported in homeothermic animals.

Animals↗