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Spatial scaling of metagenomic diversity reveals ecological disruption in the gut microbiome of gout patients.

Gout, a painful inflammatory arthritis, is characterized by hyperuricemia and monosodium urate crystal deposition, with growing evidence linking its pathogenesis to gut microbiome dysbiosis. However, traditional diversity metrics fail to capture the complex spatial organization of microbial communities. This study addresses this gap by applying the novel metagenomic Diversity-Area Relationship (m-DAR) model to investigate scaling laws in the gout microbiome-quantifying how metagenomic diversity changes with the number of individuals sampled. Our analysis of gut microbiomes from gout patients and healthy controls revealed fundamental ecological disruptions. We found that gout microbiomes exhibited significantly altered scaling patterns: they showed greater inter-individual dissimilarity (higher z-values) at the level of rare genes (q = 0), but weaker scaling of dominant genes (q = 1-3) compared to healthy controls. Crucially, the maximal accrual diversity (MAD) was substantially lower in gout patients, indicating a severely constrained potential for total microbial gene diversity. Furthermore, profiling of metagenomic functional gene clusters (MFGCs) uncovered widespread functional perturbations, including increased diversity scaling for carbohydrate-active enzymes (CAZy) but decreased scaling in essential metabolic pathways (KEGG, KO). These results demonstrate that the gout gut microbiome is defined by a loss of ecological structure, featuring reduced homogeneity in dominant taxa, expanded rare biosphere variation, and an overall collapsed diversity capacity. This work introduces an ecological framework for characterizing dysbiosis in gout that complements traditional diversity metrics and may inform the development of microbiome-based therapeutic strategies. Further research is needed to translate these ecological patterns into clinical applications.

Humans↗

Bioaugmentation for bioremediation: the challenge of strain selection.

Despite its long-term use in bioremediation, bioaugmentation of contaminated sites with microbial cells continues to be a source of controversy within environmental microbiology. This largely results from its notoriously unreliable performance record. In this article, we argue that the unpredictable nature of the approach comes from the initial strain selection step. Up until now, this has been dictated by the search for catabolically competent microorganisms, with little or no consideration given to other essential features that are required to be functionally active and persistent in target habitats. We describe how technical advances in molecular biology and analytical chemistry, now enable assessments of the functional diversity and spatial distribution of microbial communities to be made in situ. These advances now enable microbial populations, targeted for exploitation, to be differentiated to the cell level, an advance that is bound to improve microbial selection and exploitation. We argue that this information-based approach is already proving to be more effective than the traditional 'black-box' approach of strain selection. The future perspectives and opportunities for improving selection of effective microbial strains for bioaugmentation are also discussed.

Bacteria↗

Using the CATH domain database to assign structures and functions to the genome sequences.

The CATH database of protein structures contains approximately 18000 domains organized according to their (C)lass, (A)rchitecture, (T)opology and (H)omologous superfamily. Relationships between evolutionary related structures (homologues) within the database have been used to test the sensitivity of various sequence search methods in order to identify relatives in Genbank and other sequence databases. Subsequent application of the most sensitive and efficient algorithms, gapped blast and the profile based method, Position Specific Iterated Basic Local Alignment Tool (PSI-BLAST), could be used to assign structural data to between 22 and 36 % of microbial genomes in order to improve functional annotation and enhance understanding of biological mechanism. However, on a cautionary note, an analysis of functional conservation within fold groups and homologous superfamilies in the CATH database, revealed that whilst function was conserved in nearly 55% of enzyme families, function had diverged considerably, in some highly populated families. In these families, functional properties should be inherited far more cautiously and the probable effects of substitutions in key functional residues carefully assessed.

Algorithms↗

Microbial transformation of esters of chlorinated carboxylic acids.

Two groups of compounds were selected for microbial transformation studies. In the first group were carboxylic acid esters having a fixed aromatic moiety and an increasing length of the alkyl component. Ethyl esters of chlorine-substituted carboxylic acids were in the second group. Microorganisms from environmental waters and a pure culture of Pseudomonas putida U were used. The bacterial populations were monitored by plate counts, and disappearance of the parent compound was followed by gas-liquid chromatography as a function of time. The products of microbial hydrolysis were the respective carboxylic acids. Octanol-water partition coefficients (K(ow)) for the compounds were measured. These values spanned three orders of magnitude, whereas microbial transformation rate constants (k(b)) varied only 50-fold. The microbial rate constants of the carboxylic acid esters with a fixed aromatic moiety increased with an increasing length of alkyl substituents. The regression coefficient for the linear relationships between log k(b) and log K(ow) was high for group 1 compounds, indicating that these parameters correlated well. The regression coefficient for the linear relationships for group 2 compounds, however, was low, indicating that these parameters correlated poorly.

Journal Article↗

Microbial community variation and its relationship with nitrogen mineralization in historically altered forests.

Past land use can impart soil legacies that have important implications for ecosystem function. Although these legacies have been linked with microbially mediated processes, little is known about the long-term influence of land use on soil microbial communities themselves. We examined whether historical land use affected soil microbial community composition (lipid profiles) and whether community composition was related to potential net nitrogen (N) mineralization rates in southern Appalachian (USA) forest stands abandoned from agriculture or logging and reforested >50 yr ago. Microbial community composition was determined by a hybrid procedure of phospholipid fatty acid (PLFA) and fatty acid methyl ester (FAME) analysis. We found that community composition varied significantly with past land use. Communities in formerly farmed stands had a higher relative abundance of markers for gram-negative bacteria and a lower abundance of markers for fungi compared with previously logged and reference (i.e., no disturbance history) stands. Potential net N mineralization rates were negatively correlated with fungal and gram-negative bacterial markers in both farmed and reference stands, and fungal abundance and soil bulk density effectively predicted mineralization rates in all stands. Our results indicate that the alteration of microbial communities by historical land use may influence the ecosystem processes they mediate. This is in contrast to typical expectations about microbial community resilience to change. Here, the decrease in fungal abundance observed from disturbance appeared to result in decreased nitrogen mineralization over the long term.

Agriculture↗

Effects on the immune system.

Marijuana and other exogenous cannabinoids alter immune function and decrease host resistance to microbial infections in experimental animal models and in vitro. Two modes of action by which delta9-tetrahydrocannabinol (THC) and other cannabinoids affect immune responses have been proposed. First, cannabinoids may signal through the cannabinoid receptors CB1 and CB2. Second, at sites of direct exposure to high concentrations of cannabinoids, such as the lung, membrane perturbation may be involved. In addition, endogenous cannabinoids or endocannabinoids have been identified and have been proposed as native modulators of immune functions through cannabinoid receptors. Exogenously introduced cannabinoids may disturb this homoeostatic immune balance. A mode by which cannabinoids may affect immune responses and host resistance maybe by perturbing the balance of T helper (Th)1 pro-inflammatory versus Th2 anti-inflammatory cytokines. While marijuana and various cannabinoids have been documented to alter immune functions in vitro and in experimental animals, no controlled longitudinal epidemiological studies have yet definitively correlated immunosuppressive effects with increased incidence of infections or immune disorders in humans. However, cannabinoids by virtue of their immunomodulatory properties have the potential to serve as therapeutic agents for ablation of untoward immune responses.

Acquired Immunodeficiency Syndrome↗

Exacerbation of facial motoneuron loss after facial nerve transection in severe combined immunodeficient (scid) mice.

The immune system functions to protect an organism against microbial infections and may be involved in the reparative response to nerve injury. The goal of this study was to determine whether the immune system plays a role in regulating motoneuron survival after a peripheral nerve injury. After a right facial nerve axotomy, facial motoneuron (FMN) survival in C.B-17 (+/+) wild-type mice was found to be 87 +/- 3.0% of the unaxotomized left side control. In contrast, facial nerve axotomy in C.B-17 (-/-) severe combined immunodeficient (scid) mice, lacking functional T and B lymphocytes, resulted in an average FMN survival of 55 +/- 3.5% relative to the unaxotomized left side control. This represented an approximately 40% decrease in FMN survival compared with wild-type controls. The reconstitution of scid mice with wild-type splenocytes containing T and B lymphocytes restored FMN survival in these mice to the level of the wild-type controls. These results suggest that immune cells associated with acquired immunity play a role in regulating motoneuron survival after a peripheral nerve injury.

Animals↗

Dental plaque as a biofilm and a microbial community - implications for health and disease.

Dental plaque is a structurally- and functionally-organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that, in health, remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift toward community dominance by acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other species with relevant traits may be involved. Strategies to control caries could include inhibition of biofilm development (e.g. prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.), or enhancement of the host defenses. Additionally, these more conventional approaches could be augmented by interference with the factors that enable the cariogenic bacteria to escape from the normal homeostatic mechanisms that restrict their growth in plaque and out compete the organisms associated with health. Evidence suggests that regular conditions of low pH in plaque select for mutans streptococci and lactobacilli. Therefore, the suppression of sugar catabolism and acid production by the use of metabolic inhibitors and non-fermentable artificial sweeteners in snacks, or the stimulation of saliva flow, could assist in the maintenance of homeostasis in plaque. Arguments will be presented that an appreciation of ecological principles will enable a more holistic approach to be taken in caries control.

Journal Article↗

Improving phosphorus availability in soybean meal for broilers by supplemental phytase.

A 21-d experiment was conducted with day-old male broilers (n=840) to evaluate the effectiveness of supplemental phytase for improving the availability of phytate P in soybean meal when varying levels of P were fed. The semi-purified basal diet (.18% phytate P) contained soybean meal as the only protein source. Seven levels of phytase (0, 200, 400, 600, 800, 1,000, and 1,200 U/kg diet) were added to diets formulated to contain .20, .27, or .34% nonphytate P (nP; or .38, .45, and .52% total P, respectively). The desired levels of nP in the three basal P diets were achieved by adding varying amounts of defluorinated phosphate. A 2:1 Ca:total P ratio was maintained in all diets. Body weight gains and feed intake were improved (P < .001) by phytase at all nP levels, but the magnitude of response was greatest at low nP levels, resulting in an nP by phytase interaction (P < .01). Gain:feed was unaffected by phytase addition. A high mortality (35 to 45%) was observed for the .20 and .27% nP diets without added phytase, but this declined to normal levels with the addition of 200 to 400 U phytase/kg diet. Ash percentage of toes and tibia and shear force and stress of tibia increased with added phytase. These responses clearly show that the phytate-bound P in soybean meal was made more available to broilers by microbial phytase, and the total response was related to the phytase and nP/total P levels. Based on the high R2 values for the second order translog equations, BW gain, feed intake, and toe ash percentage were the most sensitive indicators to assess P availability, followed by tibia force and ash percentage. Derived nonlinear and linear equations for BW gain and toe ash percentage at the two lower nP levels were used to calculate P equivalency values of phytase for inorganic P. Using the average function of P released ( gamma ) by microbial phytase ( chi ) derived with nP levels of .20 and .27% for BW gain and toe ash percentage, gamma = 1.120 - 1.102e-.0027chi, 1 g of P could be released with 821 U of phytase. The amount of P released increased with increasing levels of phytase, but the amount of P released per 100 U of phytase decreased. Released P ranged from 31 to 58% of phytate P for 250 to 1,000 U of phytase/kg of diet.

6-Phytase↗

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo.

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

Biopsy↗

Sedimentary microbial oxygen demand for laminar flow over a sediment bed of finite length.

Dead organic material accumulated on the bed of a lake, reservoir or wetland often provides the substrate for substantial microbial activity as well as chemical processes that withdraw dissolved oxygen (DO) from the water column. A model to estimate the actual DO profile and the "sedimentary oxygen demand (SOD)" must specify the rate of microbial or chemical activity in the sediment as well as the diffusive supply of DO from the water column through the diffusive boundary layer into the sediment. Most previous experimental and field studies have considered this problem with the assumptions that the diffusive boundary layer is (a) turbulent and (b) fully developed. These assumptions require that (a) the flow velocity above the sediment bed is fast enough to produce turbulent mixing in the boundary layer, and (b) the sediment bed is long. In this paper a model for laminar flow and SOD over a sediment bed of finite length is presented and the results are compared with those for turbulent flow. Laminar flow near a sediment bed is encountered in quiescent water bodies such as lakes, reservoirs, river backwaters, wetlands and ponds under calm wind conditions. The diffusive oxygen transfer through the laminar diffusive boundary layer above the sediment surface can restrict the microbial or chemical oxygen uptake inside the sediment significantly. The developing laminar diffusive boundary layer above the sediment/water interface is modeled based on the analogy with heat transfer, and DO uptake inside the sediment is modeled by Michaelis-Menten microbial growth kinetics. The model predicts that the rate of SOD at the beginning of the reactive sediment bed is solely dependent on microbial density in the sediment regardless of flow velocity and type. The rate of SOD, and the DO penetration depth into the sediment decrease in stream-wise direction over the length of the sediment bed, as the diffusive boundary layer above the sediment/water interface thickens. With increasing length of the sediment bed both SOD rate and DO penetration depth into the sediment tend towards zero if the flow is laminar, but tend towards a finite value if the flow is turbulent. That value can be determined as a function of both flow velocity and microbial density. The effect of the developing laminar boundary layer on SOD is strongest at the very lowest flow velocity and/or highest microbial density inside the sediment. Under quiescent conditions, the effective SOD exerted by a reactive sediment bed of a lake or wetland approaches zero, i.e. no or very little oxygen demand is exerted on the overlying water column, except at the leading edge.

Environmental Monitoring↗

Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities.

Biological soil characteristics such as microbial biomass, community structures, activities, and functions may provide important information on environmental and anthropogenic influences on agricultural soils. Diagnostic tools and detailed statistical approaches need to be developed for a reliable evaluation of these parameters, in order to allow classification and quantification of the magnitude of such effects. The DOK long-term agricultural field experiment was initiated in 1978 in Switzerland for the evaluation of organic and conventional farming practices. It includes three representative Swiss farming systems with biodynamic, bio-organic and conventional fertilization and plant protection schemes along with minerally fertilized and unfertilized controls. Effects on microbial soil characteristics induced by the long-term management at two different stages in the crop rotation, i.e. winter wheat after potato or corn, were investigated by analyzing soil bacterial community structures using analysis of PCR-amplified rRNA genes by terminal restriction fragment length polymorphism and ribosomal intergenic spacer analysis. Application of farmyard manure consistently revealed the strongest influence on bacterial community structures and biomass contents. Effects of management and plant protection regimes occurred on an intermediate level, while the two stages in the crop rotation had a marginal influence that was not significant.

Agriculture↗

A quasi-chemical model for the growth and death of microorganisms in foods by non-thermal and high-pressure processing.

Predictive microbial models generally rely on the growth of bacteria in laboratory broth to approximate the microbial growth kinetics expected to take place in actual foods under identical environmental conditions. Sigmoidal functions such as the Gompertz or logistics equation accurately model the typical microbial growth curve from the lag to the stationary phase and provide the mathematical basis for estimating parameters such as the maximum growth rate (MGR). Stationary phase data can begin to show a decline and make it difficult to discern which data to include in the analysis of the growth curve, a factor that influences the calculated values of the growth parameters. In contradistinction, the quasi-chemical kinetics model provides additional capabilities in microbial modelling and fits growth-death kinetics (all four phases of the microbial lifecycle continuously) for a general set of microorganisms in a variety of actual food substrates. The quasi-chemical model is differential equations (ODEs) that derives from a hypothetical four-step chemical mechanism involving an antagonistic metabolite (quorum sensing) and successfully fits the kinetics of pathogens (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes) in various foods (bread, turkey meat, ham and cheese) as functions of different hurdles (a(w), pH, temperature and anti-microbial lactate). The calculated value of the MGR depends on whether growth-death data or only growth data are used in the fitting procedure. The quasi-chemical kinetics model is also exploited for use with the novel food processing technology of high-pressure processing. The high-pressure inactivation kinetics of E. coli are explored in a model food system over the pressure (P) range of 207-345 MPa (30,000-50,000 psi) and the temperature (T) range of 30-50 degrees C. In relatively low combinations of P and T, the inactivation curves are non-linear and exhibit a shoulder prior to a more rapid rate of microbial destruction. In the higher P, T regime, the inactivation plots tend to be linear. In all cases, the quasi-chemical model successfully fit the linear and curvi-linear inactivation plots for E. coli in model food systems. The experimental data and the quasi-chemical mathematical model described herein are candidates for inclusion in ComBase, the developing database that combines data and models from the USDA Pathogen Modeling Program and the UK Food MicroModel.

Bacteria↗

Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyoctanoate.

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudomonas oleovorans when grown on n-octanoic acid, are complex macromolecular structures consisting of polyester, organized paracrystalline lattice arrays and lipids. While it is known that the polymer in the granules maintains its native, amorphous state while it is surrounded by the components of this complex, the precise functions of the various components during polymer production and utilization have yet to be established. By utilizing electron microscopy, SDS-PAGE, and gel filtration chromatography along with in vitro assays for depolymerase activity, the present study demonstrates that a protein species with molecular weight of approximately 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability required utilization of the stored polyester. Under these conditions, the concentration of the depolymerase increased while the concentrations of the polymerase decreased. Thus, the association of the depolymerase with the granules was shown to be under metabolic regulation relative to the polymerase. The results from the present studies show that careful manipulation of the substrate concentration can selectively, and differentially, alter the level of inclusion associated proteins as well as the quantity and quality of the polyester which is accumulated.

Bacterial Proteins↗

[Bacterial infections and toll-like receptors].

Toll-like receptors are type-1 transmembrane receptors involved in microbial recognition. TLR4 has been shown to function as the lipopolysaccharide signaling receptor, while TLR2 recognizes peptidoglycans from Gram-positive bacteria, and lipoproteins. TLR9 is involved in the recognition of bacterial DNA (CpG DNA). Although various microbial cell wall components are recognized by different receptors, all of these responses are abrogated in MyD88-deficient cells. These results show that different TLRs recognize different microbial cell wall components, and that MyD88 is an essential signaling molecule shared among interleukin-1 receptor/Toll family members. However, in LPS signaling MyD88-independent pathway is present in addition to MyD88-dependent pathway.

Adaptor Proteins, Signal Transducing↗

Stable carbon isotope ratio and composition of microbial fatty acids in tropical soils.

The soil microbial community plays a critical part in tropical ecosystem functioning through its role in the soil organic matter (SOM) cycle. This study evaluates the relative effects of soil type and land use on (i) soil microbial community structure and (ii) the contribution of SOM derived from the original forest vegetation to the functioning of pasture and sugarcane (Saccharum spp.) ecosystems. We used principal components analysis (PCA) of soil phospholipid fatty acid (PLFA) profiles to evaluate microbial community structure and PLFA stable carbon isotope ratios (delta13C) as indicators of the delta13C of microbial substrates. Soil type mainly determined the relative proportions of gram positive versus gram negative bacteria whereas land use primarily determined the relative proportion of fungi, protozoa, and actinomycetes versus other types of microorganisms. Comparison of a simple model to our PLFA delta13C data from land use chronosequences indicates that forest-derived SOM is actively cycled for appreciably longer times in sugarcane ecosystems developed on Andisols (mean turnover time = 50 yr) than in sugarcane ecosystems developed on an Oxisol (mean turnover time = 13 yr). Our analyses indicate that soil chronosequence PLFA delta13C measurements can be useful indicators of the contribution that SOM derived from the original vegetation makes to continued ecosystem function under the new land use.

Agriculture↗

Effect of a local immune reaction on peripheral blood polymorphonuclear neutrophil microbicidal function: studies with fungal targets.

Peripheral blood polymorphonuclear neutrophils (PMN) from mice immunized with Blastomyces dermatitidis and then stimulated locally (intraperitoneally, ip) with B. dermatitidis antigen had enhanced killing of B. dermatitidis in vitro (54.4 +/- 19.49 of inoculum) compared to nonimmune mice (32.7 +/- 8.7%; P less than 0.02), nonimmune mice given antigen ip (30.6 +/- 14.0%; P less than 0.05), or immune mice not given antigen ip (15.4 +/- 9.9%; P less than 0.01). Peripheral blood PMN from all four groups had marked killing ability against Candida albicans (91.8-99.3% of inoculum). That the killing of B. dermatitidis was due to PMNs was demonstrated by lack of killing by isolated peripheral blood mononuclear cells from all four groups. A local immune reaction can result in enhancement of PMN fungicidal activity, and this is reflected even in peripheral blood PMN. We hypothesize this is an important component of normal host defenses against fungal infection, and likely other microbial infections. Enhancement of PMN microbicidal function by the soluble mediators presumed to be responsible for the effects observed may be an approach to immunomodulating therapy or prophylaxis of infection.

Animals↗

The superantigen Pseudomonas exotoxin A requires additional functions from accessory cells for T lymphocyte proliferation.

We have examined the functions required of accessory cells (AC) for murine thymocyte proliferation induced by Pseudomonas exotoxin A (PE) and have compared these functions to those required of a known superantigen, staphylococcal enterotoxin B (SEB). We demonstrate that PE, like SEB, preferentially stimulates PNA+ thymocytes expressing a specific V beta element within the T cell receptor. However, PE requires functions from AC that are distinct from those required by SEB. AC treated with paraformaldehyde (PCHO) prior to stimulation supported thymocyte proliferation induced by SEB but not PE. However, when AC were treated with PCHO subsequent to stimulation with PE, thymocyte proliferation was observed, which suggests that PE requires antigen processing in addition to presentation. Furthermore, treatment of AC with lysosomotropic agents abrogated thymocyte proliferation induced by PE but not SEB. Antibodies to MHC class II molecules inhibited thymocyte proliferation induced by both PE and SEB. In addition, we observed that interleukin 1 alpha (IL-1 alpha) participated in the proliferation of thymocytes induced by PE but not SEB. Thus, our data indicate that PE is a unique microbial superantigen that requires additional AC functions for T lymphocyte proliferation.

ADP Ribose Transferases↗