Long-term cultivation-independent microbial diversity analysis demonstrates that bacterial communities infecting the adult cystic fibrosis lung show stability and resilience.

Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

Characterization of para-Nitrophenol-degrading bacterial communities in river water by using functional markers and stable isotope probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [13C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

Arbuscular mycorrhizal modulation of diazotrophic and denitrifying microbial communities in the (mycor)rhizosphere of Plantago lanceolata

Impacts of divergent arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Gigaspora margarita, on denitrifying and diazotrophic bacterial communities of Plantago lanceolata in nutrient-limited dune soil were assessed. We hypothesized AM species-related modifications that were confirmed in respective bacterial nirK and nifH sequence polymorphism -based community clustering and community variance allocation. The denitrifying community appeared more responsive to AM fungi than the nitrogen-fixing community. Nevertheless, the main explanatory variable, in both cases, was plant age. We conclude that AM fungi can modify N-cycling microbial rhizosphere communities and future work should aim to verify the functional significance and mechanistic basis.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

In vitro examination of the effect of Orlistat on the ability of the faecal microbiota to utilize dietary lipids

Orlistat is an anti-obesity treatment with which several gastrointestinal (GI) side-effects are commonly associated in the initial stages of therapy. There is no physiological explanation as to why two-thirds of those who take the drug experience one or more side-effects. It has been hypothesized that the GI microbiota may protect from or contribute to these GI disturbances. Using in vitro batch culture and human gut model systems, studies were conducted to determine whether increased availability of dietary lipids and/or orlistat affect the composition and/or activity of the faecal microbiota. Results from 24-h batch culture fermentation experiments demonstrated no effect of orlistat in the presence or absence of a dietary lipid (olive oil) on the composition of bacterial communities [as determined by fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses], but did show there was great variability in the lipolytic activities of the microbiotas of individuals, as determined by gas chromatography analysis of long-chain fatty acids in samples. Subsequent studies focused on the effect of orlistat in the presence and absence of lipid in in vitro human gut model systems. Systems were run for 14 days with gut model medium (GMM) only (to steady state, SS), then fed at 12-h intervals with 50 mg orlistat, 2 g olive oil or a mixture of both for 14 days. FISH and DGGE were used to monitor changes in bacterial populations. Bacteria were cultivated from the GMM only (control) systems at SS. All strains isolated were screened for lipolytic activity using tributyrin agar. FISH and DGGE demonstrated that none of the compounds (singly or in combination) added to the systems had any notable effect on microbial population dynamics for any of the donors, although Subdoligranulum populations appeared to be inhibited by orlistat in the presence or absence of lipid. Orlistat had little or no effect on the metabolism of indigenous and added lipids in the fermentation systems, but there was great variability in the way the faecal microbiotas of the donors were able to degrade added lipids. Variability in lipid degradation could be correlated with the number and activity of isolated lipolytic bacteria. The mechanism by which orlistat and the GI microbiota cause side-effects in individuals is unknown, but several hypotheses have been proposed to account for their manifestation. The demonstration of great variability in the lipolytic activity of microbiotas to degrade lipids led to a large-scale cultivation-based study of lipolytic/lipase-positive bacteria present in the human faecal microbiota. Of 4,000 colonies isolated from 15 donors using five different agars, 378 strains were identified that had lipase activity. Molecular identification of strains isolated from five donors demonstrated that lipase activity is more prevalent in the human GI microbiota than previously thought, with members of the phyla Firmicutes, Bacteroidetes and Actinobacteria identified. Molecular identification and characterization of the substrate specificities of the strains will be carried out as part of ongoing work.

Community patterns of soil bacteria and nematodes in relation to geographic distance

Ecosystems consist of aboveground and belowground subsystems and the structure of their communities is known to change with distance. However, most of this knowledge originates from visible, aboveground components, whereas relatively little is known about how soil community structure varies with distance and if this variability depends on the group of organisms considered. In the present study, we analyzed 30 grasslands from three neighboring chalk hill ridges in southern UK to determine the effect of geographic distance (1e198 km) on the similarity of bacterial communities and of nematode communities in the soil. We found that for both groups, community similarity decayed with distance and that this spatial pattern was not related to changes either in plant community composition or soil chemistry. Site history may have contributed to the observed pattern in the case of nematodes, since the distance effect depended on the presence of different nematode taxa at one of the hill ridges. On the other hand, site-related differences in bacterial community composition alone could not explain the spatial turnover, suggesting that other factors, such as biotic gradients and local dispersal processes that we did not include in our analysis, may be involved in the observed pattern. We conclude that, independently of the variety of causal factors that may be involved, the decay in similarity with geographic distance is a characteristic feature of both communities of soil bacteria and nematodes.

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 102 to 1.4 × 103 per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of “Candidatus Cardinium hertigii” and as a separate novel cluster.

Viruses in soils: morphological diversity and abundance in the rhizosphere

Soil viruses are potentially of great importance as they may influence the ecology and evolution of soil biological communities through both an ability to transfer genes from host to host and as a potential cause of microbial mortality. Despite this importance, the area of soil virology is understudied. Here, we report the isolation and preliminary characterisation of viruses from soils in the Dundee area of Scotland. Different virus morphotypes including tailed, polyhedral (spherical), rod shaped, filamentous and bacilliform particles were detected in the soil samples. An apparent predominance of small spherical and filamentous bacteriophages was observed, whereas tailed bacteriophages were significantly less abundant. In this report, we also present observations and characterisation of viruses from different soil functional domains surrounding wheat roots: rhizosheath, rhizosphere and bulk soil. In spite of the differences in abundance of bacterial communities in these domains, no significant variations in viral population structure in terms of morphology and abundance were found. Typically, there were approximately 1.1–1.2 × 109 virions g−1 dry weight, implicating remarkable differences in virus-to-bacteria ratios in domains close to roots, rhizosphere and rhizosheath (approximately 0.27) and in bulk soil (approximately 4.68).

Impacts of plant-based foods in ancestral hominin diets on the metabolism and function of gut microbiota in vitro

Ancestral human populations had diets containing more indigestible plant material than present-day diets in industrialized countries. One hypothesis for the rise in prevalence of obesity is that physiological mechanisms for controlling appetite evolved to match a diet with plant fiber content higher than that of present-day diets. We investigated how diet affects gut microbiota and colon cells by comparing human microbial communities with those from a primate that has an extreme plant-based diet, namely, the gelada baboon, which is a grazer. The effects of potato (high starch) versus grass (high lignin and cellulose) diets on human-derived versus gelada-derived fecal communities were compared in vitro. We especially focused on the production of short-chain fatty acids, which are hypothesized to be key metabolites influencing appetite regulation pathways. The results confirmed that diet has a major effect on bacterial numbers, short-chain fatty acid production, and the release of hormones involved in appetite suppression. The potato diet yielded greater production of short-chain fatty acids and hormone release than the grass diet, even in the gelada cultures, which we had expected should be better adapted to the grass diet. The strong effects of diet on hormone release could not be explained, however, solely by short-chain fatty acid concentrations. Nuclear magnetic resonance spectroscopy found changes in additional metabolites, including betaine and isoleucine, that might play key roles in inhibiting and stimulating appetite suppression pathways. Our study results indicate that a broader array of metabolites might be involved in triggering gut hormone release in humans than previously thought. IMPORTANCE: One theory for rising levels of obesity in western populations is that the body's mechanisms for controlling appetite evolved to match ancestral diets with more low-energy plant foods. We investigated this idea by comparing the effects of diet on appetite suppression pathways via the use of gut bacterial communities from humans and gelada baboons, which are modern-day primates with an extreme diet of low-energy plant food, namely, grass. We found that diet does play a major role in affecting gut bacteria and the production of a hormone that suppresses appetite but not in the direction predicted by the ancestral diet hypothesis. Also, bacterial products were correlated with hormone release that were different from those normally thought to play this role. By comparing microbiota and diets outside the natural range for modern humans, we found a relationship between diet and appetite pathways that was more complex than previously hypothesized on the basis of more-controlled studies of the effects of single compounds.

Prebiotics modulate the effects of antibiotics on gut microbial diversity and functioning in vitro

Intestinal bacteria carry out many fundamental roles, such as the fermentation of non-digestible dietary carbohydrates to produce short chain fatty acids (SCFAs), which can affect host energy levels and gut hormone regulation. Understanding how to manage this ecosystem to improve human health is an important but challenging goal. Antibiotics are the front line of defence against pathogens, but in turn they have adverse effects on indigenous microbial diversity and function. Here, we have investigated whether dietary supplementation—another method used to modulate gut composition and function—could be used to ameliorate the side effects of antibiotics. We perturbed gut bacterial communities with gentamicin and ampicillin in anaerobic batch cultures in vitro. Cultures were supplemented with either pectin (a non-fermentable fibre), inulin (a commonly used prebiotic that promotes the growth of beneficial bacteria) or neither. Although antibiotics often negated the beneficial effects of dietary supplementation, in some treatment combinations, notably ampicillin and inulin, dietary supplementation ameliorated the effects of antibiotics. There is therefore potential for using supplements to lessen the adverse effects of antibiotics. Further knowledge of such mechanisms could lead to better therapeutic manipulation of the human gut microbiota.

A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects

This placebo-controlled, randomised, double-blind, cross-over human feeding study aimed to determine the prebiotic effect of agave fructans. A total of thirty-eight volunteers completed this trial. The treatment consisted of 3 weeks' supplementation with 5 g/d of prebiotic agave fructan (Predilife) or equivalent placebo (maltodextrin), followed by a 2-week washout period following which subjects were crossed over to alternate the treatment arm for 3 weeks followed by a 2-week washout. Faecal samples were collected at baseline, on the last day of treatment (days 22 and 58) and washout (days 36 and 72), respectively. Changes in faecal bacterial populations, SCFA and secretory IgA were assessed using fluorescent in situ hybridisation, GC and ELISA, respectively. Bowel movements, stool consistencies, abdominal comfort and mood changes were evaluated by a recorded daily questionnaire. In parallel, the effect of agave fructans on different regions of the colon using a three-stage continuous culture simulator was studied. Predilife significantly increased faecal bifidobacteria (log10 9·6 (sd 0·4)) and lactobacilli (log10 7·7 (sd 0·8)) compared with placebo (log10 9·2 (sd 0·4); P = 0·00) (log10 7·4 (sd 0·7); P = 0·000), respectively. No change was observed for other bacterial groups tested, SCFA, secretory IgA, and PGE2 concentrations between the treatment and placebo. Denaturing gradient gel electrophoresis analysis indicated that bacterial communities were randomly dispersed and no significant differences were observed between Predilife and placebo treatments. The in vitro models showed similar increases in bifidobacterial and lactobacilli populations to that observed with the in vivo trial. To conclude, agave fructans are well tolerated in healthy human subjects and increased bifidobacteria and lactobacilli numbers in vitro and in vivo but did not influence other products of fermentation

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.