MICROBIAL COMMUNITY COMPOSITION IN THE LUNGS OF PATIENTS WITH CYSTIC FIBROSIS AND NON-CF BRONCHIECTASIS

Introduction and Aims: Persistent bacterial infection is a major cause of morbidity and mortality in patients with both Cystic Fibrosis (CF) and non-CF Bronchiectasis (non-CFBX). Numerous studies have shown that CF and non-CFBX airways are colonised by a complex microbiota. However, many bacteria are difficult, if not impossible, to culture by conventional laboratory techniques. Therefore, molecular detection techniques offer a more comprehensive view of bacterial diversity within clinical specimens. The objective of this study was to characterise and compare bacterial diversity and relative abundance in patients with CF and non-CFBX during exacerbation and when clinically stable.

Methods: Sputum samples were collected from CF (n=50 samples) and non-CFBX (n=52 samples) patients at the start and end of treatment for an infective exacerbation and when clinically stable. Pyrosequencing was used to assess the microbial diversity and relative genera (or the closest possibly taxonomic order) abundance within the samples. Each sequence read was defined based on 3% difference.

Results: High-throughput pyrosequencing allowed a sensitive and detailed examination of microbial community composition. Rich microbial communities were apparent within both CF (171 species-level phylotypes per genus) and non-CFBX airways (144 species-level phylotypes per genus). Relative species distribution within those two environments was considerably different; however, relatively few genera formed a core of microorganisms, representing approximately 90% of all sequences, which dominated both environments. Relative abundance based on observed operational taxonomic units demonstrated that the most abundant bacteria in CF were Pseudomonas (28%), Burkholderia (22%), Streptococcus (13%), family Pseudomonadaceae (8%) and Prevotella (6%). In contrast, the most commonly detected operational taxonomic units in non-CFBX were Haemophilus (22%), Streptococcus (14%), other (unassigned taxa) (11%), Pseudomonas (10%), Veillonella (7%) and Prevotella (6%).

Conclusions: These results suggest that distinctive microbial communities are associated with infection and/or colonisation in patients with both CF and non-CFBX. Although relatively high species richness was observed within the two environments, each was dominated by different core taxa. This suggests that differences in the lung environment of these two diseases may affect adaptability of the relevant bacterial taxa.

Interchange of entire communities: microbial community coalescence

Microbial communities are enigmatically diverse. We propose a novel view of processes likely affecting microbial assemblages, which could be viewed as the Great American Interchange en miniature: the wholesale exchange among microbial communities resulting from moving pieces of the environment containing entire assemblages. Incidental evidence for such ‘community coalescence’ is accumulating, but such processes are rarely studied, likely because of the absence of suitable terminology or a conceptual framework. We provide the nucleus for such a conceptual foundation for the study of community coalescence, examining factors shaping these events, links to bodies of ecological theory, and we suggest modeling approaches for understanding coalescent communities. We argue for the systematic study of community coalescence because of important functional and applied consequences.

Microbial community composition and mercury cycling in sediments of tagus estuary

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2016

Spatial and Temporal Variation of Microbial Community Structure In Surficial Sediments of Cochin Estuary

This thesis entitled spatial and temporal variarion of microbial community structure in surficial sediments of cochin estuary.In the estuarine and coastal systems, organic matter (OM) is derived not only from autochthonous primary production, but also from allochthonous (terrestrial) organic matter (OM) delivered by river discharge and runoff. A significant portion of the OM sinks through the water column and is ultimately stored in carbon pool in the sediments.Analysis of spatial and temporal variation in benthic microbial community of a tropical estuary was conducted for the first time using non selective measures that affirms that PLFA approach is a sensitive and reliable method in determining microbial community structures of surficial sediments of estuary.The close relationship between the concentrations of the microbial fatty acids and total biomass indicates that bacteria could account for the largest proportion of the biomass in the sediments.This is first study that has documented the changes in microbial community composition linkage to biotic and abiotic variables in benthic estuarine ecosystem. This contemporaneous community will be the backdrop for understanding the response of autochthonous community to increasing anthropogenic stress.

Influences of space, soil, nematodes and plants on microbial community composition of chalk grassland soils

Microbial communities respond to a variety of environmental factors related to resources (e.g. plant and soil organic matter), habitat (e.g. soil characteristics) and predation (e.g. nematodes, protozoa and viruses). However, the relative contribution of these factors on microbial community composition is poorly understood. Here, we sampled soils from 30 chalk grassland fields located in three different chalk hill ridges of Southern England, using a spatially explicit sampling scheme. We assessed microbial communities via phospholipid fatty acid (PLFA) analyses and PCR-denaturing gradient gel electrophoresis (DGGE) and measured soil characteristics, as well as nematode and plant community composition. The relative influences of space, soil, vegetation and nematodes on soil microorganisms were contrasted using variation partitioning and path analysis. Results indicate that soil characteristics and plant community composition, representing habitat and resources, shape soil microbial community composition, whereas the influence of nematodes, a potential predation factor, appears to be relatively small. Spatial variation in microbial community structure was detected at broad (between fields) and fine (within fields) scales, suggesting that microbial communities exhibit biogeographic patterns at different scales. Although our analysis included several relevant explanatory data sets, a large part of the variation in microbial communities remained unexplained (up to 92% in some analyses). However, in several analyses, significant parts of the variation in microbial community structure could be explained. The results of this study contribute to our understanding of the relative importance of different environmental and spatial factors in driving the composition of soil-borne microbial communities.

Comparison of the euryarchaeal microbial community in guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant affect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physico-chemical conditions and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and Terminal-restriction Fragment Length Polymorphism (T-RFLP) analysis to examine methanogenic Archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If they are strictly vertically inherited, then MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus or Rice Cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities but these may represent transient populations in either guts or soil. Our data does not support the hypothesis that termite gut MA are derived from their food soil but also does not support a purely vertical transmission of gut microflora.

Extracellular release of a heterologous phytase from roots of transgenic plants: does manipulation of rhizosphere biochemistry impact microbial community structure?

To maintain the sustainability of agriculture, it is imperative that the reliance of crops on inorganic phosphorus (P) fertilizers is reduced. One approach is to improve the ability of crop plants to acquire P from organic sources. Transgenic plants that produce microbial phytases have been suggested as a possible means to achieve this goal. However, neither the impact of heterologous expression of phytase on the ecology of microorganisms in the rhizosphere nor the impact of rhizosphere microorganisms on the efficacy of phytases in the rhizosphere of transgenic plants has been tested. In this paper, we demonstrate that the presence of rhizosphere microorganisms reduced the dependence of plants oil extracellular secretion of phytase from roots when grown in a P-deficient soil. Despite this, the expression of phytase in transgenic plants had little or no impact on the microbial community structure as compared with control plant lines, whereas soil treatments, such as the addition of inorganic P, had large effects. The results demonstrate that soil microorganisms are explicitly involved in the availability of P to plants and that the microbial community in the rhizosphere appears to be resistant to the impacts of single-gene changes in plants designed to alter rhizosphere biochemistry and nutrient cycling.

Microbial community dynamics in the forefield of glaciers

Retreating ice fronts (as a result of a warming climate) expose large expanses of deglaciated forefield, which become colonized by microbes and plants. There has been increasing interest in characterizing the biogeochemical development of these ecosystems using a chronosequence approach. Prior to the establishment of plants, microbes use autochthonously produced and allochthonously delivered nutrients for growth. The microbial community composition is largely made up of heterotrophic microbes (both bacteria and fungi), autotrophic microbes and nitrogen-fixing diazotrophs. Microbial activity is thought to be responsible for the initial build-up of labile nutrient pools, facilitating the growth of higher order plant life in developed soils. However, it is unclear to what extent these ecosystems rely on external sources of nutrients such as ancient carbon pools and periodic nitrogen deposition. Furthermore, the seasonal variation of chronosequence dynamics and the effect of winter are largely unexplored. Modelling this ecosystem will provide a quantitative evaluation of the key processes and could guide the focus of future research. Year-round datasets combined with novel metagenomic techniques will help answer some of the pressing questions in this relatively new but rapidly expanding field, which is of growing interest in the context of future large-scale ice retreat.

Hydrocarbon biodegradation and soil microbial community response to repeated oil exposure

Bioremediation strategies continue to be developed to mitigate the environmental impact of petroleum hydrocarbon contamination. This study investigated the ability of soil microbiota, adapted by prior exposure, to biodegrade petroleum. Soils from Barrow Is. (W. Australia), a class A nature reserve and home to Australia’s largest onshore oil field, were exposed to Barrow production oil (50 ml/kg soil) and incubated (25 °C) for successive phases of 61 and 100 days. Controls in which oil was not added at Phase I or II were concurrently studied and all treatments were amended with the same levels of additional nutrient and water to promote microbial activity. Prior exposure resulted in accelerated biodegradation of most, but not all, hydrocarbon constituents in the production oil. Molecular biodegradation parameters measured using gas chromatography–mass spectrometry (GC–MS) showed that several aromatic constituents were degraded more slowly with increased oil history. The unique structural response of the soil microbial community was reflected by the response of different phospholipid fatty acid (PLFA) sub-classes (e.g. branched saturated fatty acids of odd or even carbon number) measured using a ratio termed Barrow PLFA ratio (B-PLFAr). The corresponding values of a previously proposed hydrocarbon degrading alteration index showed a negative correlation with hydrocarbon exposure, highlighting the site specificity of PLFA-based ratios and microbial community dynamics. B-PLFAr values increased with each Phase I and II addition of production oil. The different hydrocarbon biodegradation rates and responses of PLFA subclasses to the Barrow production oil probably relate to the relative bioavailability of production oil hydrocarbons. These different effects suggest preferred structural and functional microbial responses to anticipated contaminants may potentially be engineered by controlled pre-exposure to the same or closely related substrates. The bioremediation of soils freshly contaminated with petroleum could benefit from the addition of exhaustively bioremediated soils rich in biota primed for the impacting hydrocarbons.

Characterization of the extracellular polymeric substances and microbial community of aerobic granulation sludge exposed to cefalexin

This study characterizes the extracellular polymeric substances and bacterial community composition of aerobic granules exposed to cefalexin (CLX). The presence of CLX potentially decreases granular stabilities, resulting in a lowered granule diameter. Chemical oxygen demand and NH4+-N removal efficiencies were slightly decreased and the denitrification process was inhibited with CLX addition. Extracellular polymeric substance contents were significantly increased in aerobic granules exposed to CLX. The shifts of fluorescence intensities and peak locations in 3D-EEM fluorescence spectra indicated changes of EPS components. High-throughput sequencing analysis showed aerobic granules with CLX addition in synthetic wastewater had superior diversity of microbial species, and this was the reason that the level and components of EPS changed. The species richness for bacteria was increased from 341 to 352, which was revealed by Chao1. The Shannon index of diversity rose slightly from 3.59 to 3.73 with CLX addition. The abundance of Proteobacteria significantly decreased, while the abundance of Bacteroidetes and Chloroflexi underwent a highly significant increase in aerobic granules exposed to CLX.

Methanogenic potential and microbial community of anaerobic batch reactors at different ethylamine/sulfate ratios

Methylamine and sulfate are compounds commonly found in wastewaters. This study aimed to determine the methanogenic potential of anaerobic reactors containing these compounds and to correlate it with their microbial communities. Batch experiments were performed at different methylamine/sulfate ratios of 0.71, 1.26 and 2.18 (with respect to mass concentration). Control and experimental runs were inoculated with fragmented granular sludge. The maximum specific methane formation rates were approximately 2.3 mmol CH4 L-1 g TVS-1 day-1 for all conditions containing methylamine, regardless of sulfate addition. At the end of the experiment, total ammonium-N and methane formation were proportional to the initial concentrations of methylamine. In the presence of methylamine and sulfate, Firmicutes (46%), Deferribacteres (13%) and Proteobacteria (12%) were the predominant phyla of the Bacteria domain, while Spirochaetes (40%), Deferribacteres (17%) and Bacteroidetes (16%) predominated in the presence of methylamine only. There was no competition for methylamine between sulfate-reducing bacteria and methanogenic archaea.

Analysis of a microbial community associated with polychlorinated biphenyl degradation in anaerobic batch reactors

The degradation of polychlorinated biphenyls (PCBs) was investigated under fermentativemethanogenic conditions for up to 60 days in the presence of anaerobic biomass from a full-scale UASB reactor. The low methane yields in the PCBs-spiked batch reactors suggested that the biomass had an inhibitory effect on the methanogenic community. Reactors containing PCBs and co-substrates (ethanol/ sodium formate) exhibited substantial PCB reductions from 0.7 to 0.2 mg mL-1 . For the Bacteria domain, the PCBs-spiked reactors were grouped with the PCB-free reactors with a similarity of 55 %, which suggested the selection of a specific population in the presence of PCBs. Three genera of bacteria were found exclusively in the PCB-spiked reactors and were identified using pyrosequencing analysis, Sedimentibacter, Tissierela and Fusibacter. Interestingly, the Sedimentibacter, which was previously correlated with the reductive dechlorination of PCBs, had the highest relative abundance in the RCS-PCB (7.4 %) and RCS-PCB-PF (12.4 %) reactors. Thus, the anaerobic sludge from the UASB reactor contains bacteria from the Firmicutes phylum that are capable of degrading PCBs.

Correlation of soil microbial community responses to contamination with crude oil with and without chromium and copper

Soil microcosms contaminated with crude oil with or without chromium and copper were monitored over a period of 90 days for microbial respiration, biomass, and for dehydrogenase, lipase, acid phosphatase, and arylsulfatase activities. In addition, the community structure was followed by enumerating the total heterotrophic and oil-degrading viable bacteria and by performing a denaturing gradient gel electrophoresis (DGGE) of the PCR amplified 16S rDNA. A significant difference was observed for biochemical activities and microbial community structures between the microcosms comprised of uncontaminated soil, soil contaminated with crude oil and soil contaminated with crude oil and heavy metals. The easily measured soil enzyme activities correlated well with microbial population levels, community structures and rates of respiration (CO2 production). The estimation of microbial responses to soil contamination provides a more thorough understanding of the microbial community function in contaminated soil, in situations where technical and financial resources are limited and may be useful in addressing bioremediation treatability and effectiveness. (C) 2012 Published by Elsevier Ltd.

Effects of tannery sludge application on physiological and fatty acid profiles of the soil microbial community

The impact of tannery sludge application on soil microbial community and diversity is poorly understood. We studied the microbial community in an agricultural soil following two applications (2006 and 2007) of tannery sludge with annual application rates of 0.0,2.3 and 22.6 Mg ha(-1). The soil was sampled 12 and 271 days after the second (2007) application. Community structure was assessed via a phospholipid fatty acid analysis, and the physiological profile of the soil microbial community via the Biolog method. Tannery sludge application changed soil chemical properties, increasing the soil pH and electrical conductivity as well as available P and mineral N concentrations. The higher sludge application rate changed the community structure and the physiological profile of the microbial community at both sampling dates. However, there is no clear link between community structure and carbon substrate utilization. According to the Distance Based Linear Models Analysis, the fatty acids 16:0 and 117:0 together contributed 84% to the observed PLFA patterns, whereas the chemical properties available P, mineral N, and Ca, and pH together contributed 54%. At 12 days, tannery sludge application increased the average well color development from 0.46 to 0.87 after 48 h, and reduced the time elapsed before reaching the midpoint carbon substrate utilization (s) from 71 to 44 h, an effect still apparent nine months after application of the higher sludge application rate. The dominant signature fatty acids and kinetic parameters (r and s) were correlated to the concentrations of available P. Ca, mineral N, pH and EC. (c) 2012 Elsevier B.V. All rights reserved.