Showing papers by "Eoin L. Brodie published in 2011"

Airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma.

Abstract: Background Improvement in lung function after macrolide antibiotic therapy has been attributed to reduction in bronchial infection by specific bacteria. However, the airway might be populated by a more diverse microbiota, and clinical features of asthma might be associated with characteristics of the airway microbiota present. Objective We sought to determine whether relationships exist between the composition of the airway bacterial microbiota and clinical features of asthma using culture-independent tools capable of detecting the presence and relative abundance of most known bacteria. Methods In this pilot study bronchial epithelial brushings were collected from 65 adults with suboptimally controlled asthma participating in a multicenter study of the effects of clarithromycin on asthma control and 10 healthy control subjects. A combination of high-density 16S ribosomal RNA microarray and parallel clone library-sequencing analysis was used to profile the microbiota and examine relationships with clinical measurements. Results Compared with control subjects, 16S ribosomal RNA amplicon concentrations (a proxy for bacterial burden) and bacterial diversity were significantly higher among asthmatic patients. In multivariate analyses airway microbiota composition and diversity were significantly correlated with bronchial hyperresponsiveness. Specifically, the relative abundance of particular phylotypes, including members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae, and other bacterial families were highly correlated with the degree of bronchial hyperresponsiveness. Conclusion: The composition of bronchial airway microbiota is associated with the degree of bronchial hyperresponsiveness among patients with suboptimally controlled asthma. These findings support the need for further functional studies to examine the potential contribution of members of the airway microbiota in asthma pathogenesis.

Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance

Abstract: Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeled DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin-protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial composition.

Structure of the human gastric bacterial community in relation to Helicobacter pylori status.

Abstract: The human stomach is naturally colonized by Helicobacter pylori, which, when present, dominates the gastric bacterial community. In this study, we aimed to characterize the structure of the bacterial community in the stomach of patients of differing H. pylori status. We used a high-density 16S rRNA gene microarray (PhyloChip, Affymetrix, Inc.) to hybridize 16S rRNA gene amplicons from gastric biopsy DNA of 10 rural Amerindian patients from Amazonas, Venezuela, and of two immigrants to the United States (from South Asia and Africa, respectively). H. pylori status was determined by PCR amplification of H. pylori glmM from gastric biopsy samples. Of the 12 patients, 8 (6 of the 10 Amerindians and the 2 non-Amerindians) were H. pylori glmM positive. Regardless of H. pylori status, the PhyloChip detected Helicobacteriaceae DNA in all patients, although with lower relative abundance in patients who were glmM negative. The G2-chip taxonomy analysis of PhyloChip data indicated the presence of 44 bacterial phyla (of which 16 are unclassified by the Taxonomic Outline of the Bacteria and Archaea taxonomy) in a highly uneven community dominated by only four phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Positive H. pylori status was associated with increased relative abundance of non-Helicobacter bacteria from the Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes. The PhyloChip detected richness of low abundance phyla, and showed marked differences in the structure of the gastric bacterial community according to H. pylori status.

Soil microbial community successional patterns during forest ecosystem restoration.

Abstract: Soil microbial community characterization is increasingly being used to determine the responses of soils to stress and disturbances and to assess ecosystem sustainability. However, there is little experimental evidence to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration. This study utilized a chronosequence of developing jarrah (Eucalyptus marginata) forest ecosystems, rehabilitated after bauxite mining (up to 18 years old), to examine changes in soil bacterial and fungal community structures (by automated ribosomal intergenic spacer analysis [ARISA]) and changes in specific soil bacterial phyla by 16S rRNA gene microarray analysis. This study demonstrated that mining in these ecosystems significantly altered soil bacterial and fungal community structures. The hypothesis that the soil microbial community structures would become more similar to those of the surrounding nonmined forest with rehabilitation age was broadly supported by shifts in the bacterial but not the fungal community. Microarray analysis enabled the identification of clear successional trends in the bacterial community at the phylum level and supported the finding of an increase in similarity to nonmined forest soil with rehabilitation age. Changes in soil microbial community structure were significantly related to the size of the microbial biomass as well as numerous edaphic variables (including pH and C, N, and P nutrient concentrations). These findings suggest that soil bacterial community dynamics follow a pattern in developing ecosystems that may be predictable and can be conceptualized as providing an integrated assessment of numerous edaphic variables.

PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip

Abstract: Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.

Comparative genomics of two newly isolated Dehalococcoides strains and an enrichment using a genus microarray

Abstract: Comparative genomics of Dehalococcoides strains and an enrichment were performed using a microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus. The microarray was designed with 4305 probe sets to target 98.6% of the open-reading frames from strains 195, CBDB1, BAV1 and VS. The microarrays were validated and applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2, and their enrichment source (ANAS) to understand the genome–physiology relationships. Strains ANAS1 and ANAS2 can both couple the reduction of trichloroethene, cis-dichloroethene (DCE) and 1,1-DCE, but not tetrachloroethene and trans-DCE with growth, whereas only strain ANAS2 couples vinyl chloride reduction to growth. Comparative genomic analysis showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements or high-plasticity regions. Combined results of the two isolates closely matched the results obtained using genomic DNA of the ANAS enrichment. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny seems to be related to the presence of distinct reductive dehalogenase-encoding genes with assigned chlorinated ethene functions (pceA, tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Overall, the microarrays are a valuable high-throughput tool for comparative genomics of unsequenced Dehalococcoides-containing samples to provide insights into their gene content and dechlorination functions.

Bacterial diversity of terrestrial crystalline volcanic rocks, Iceland

Abstract: Bacteria inhabiting crystalline rocks from two terrestrial Icelandic volcanic lava flows of similar age and from the same geographical region, but differing in porosity and mineralogy, were characterised. Microarray (PhyloChip) and clone library analysis of 16S rRNA genes revealed the presence of a diverse assemblage of bacteria in each lava flow. Both methods suggested a more diverse community at the Domadalshraun site (rhyolitic/andesitic lava flow) than that present at the Hnausahraun site (basaltic lava flow). Proteobacteria dominated the clone library at the Domadalshraun site, while Acidobacteria was the most abundant phylum in the Hnausahraun site. Although analysis of similarities of denaturing gradient gel electrophoresis profiles suggested a strong correlation of community structure with mineralogy, rock porosity may also play an important role in shaping the bacterial community in crystalline volcanic rocks. Clone sequences were most similar to uncultured microorganisms, mainly from soil environments. Of these, Antarctic soils and temperate rhizosphere soils were prominent, as were clones retrieved from Hawaiian and Andean volcanic soils. The novel diversity of these Icelandic microbial communities was supported by the finding that up to 46% of clones displayed <85% sequence identities to sequences currently deposited in the RDP database.

Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane.

Abstract: Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 107 to 108 cells cm−3. This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (−35 to −43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.

Systems and methods for detecting antibiotic resistance

Abstract: A robust, automated computational pipeline was used to design a system comprising a microarray for the identification of microorganisms and theira antibiotic resistance profiles.. This system and methods will facilitate the study of the epidemiology and microbial ecology of antibiotic resistance and be an invaluable tool to rapidly and simultaneously identify organisms and their antimicrobial resistance elements in environmental, food and clinical samples.

Simrank: Rapid and sensitive general-purpose k-mer search tool

Abstract: Terabyte-scale collections of string-encoded data are expected from consortia efforts such as the Human Microbiome Project http://nihroadmap.nih.gov/hmp . Intra- and inter-project data similarity searches are enabled by rapid k-mer matching strategies. Software applications for sequence database partitioning, guide tree estimation, molecular classification and alignment acceleration have benefited from embedded k-mer searches as sub-routines. However, a rapid, general-purpose, open-source, flexible, stand-alone k-mer tool has not been available. Here we present a stand-alone utility, Simrank, which allows users to rapidly identify database strings the most similar to query strings. Performance testing of Simrank and related tools against DNA, RNA, protein and human-languages found Simrank 10X to 928X faster depending on the dataset. Simrank provides molecular ecologists with a high-throughput, open source choice for comparing large sequence sets to find similarity.

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

Abstract: The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

Microbial community structure of a slow sand filter schmutzdecke: a phylogenetic snapshot based on rRNA sequence analysis

Abstract: Slow sand filters (SSF) are widely used to treat water for potable use. The process is dependent on the activities of complex microbial communities in the biofilm (schmutzdecke) layer. In this study, we generated a comprehensive view of three-domain microbiological complexity within a model SSF. DNA was analysed using a high-density microarray (PhyloChip) and rRNA library analysis. The Eukaryotic community was dominated by Cercozoa (Ebridd-type protists); these are likely to be involved in predation of other organisms in the schmutzdecke layer, thus proving opportunity for successional development. Ciliate protozoa, green microalgae, stramenopiles, amoeboid protozoa and fungi in the Phylum Ascomycota and the deep-branching Chytridiomycota were also detected. The Archaeal community was dominated by Euryarchaeota, and most were Halobacteriales. These organisms may contribute to filter function through removal of dissolved organic carbon, a primary treatment goal of these filters. Given that the Eukaryotic and Archaeal communities were examined using clone libraries, the expected richness of taxa present is expected to be greater than detected here and dependent on sampling effort (library size). The bacterial community was rich in taxa (21 Phyla) but was not dominated by any phylogenetic group. The successful function of SSF’s relies on interactions between taxa (e.g. grazing of Cercozoa protists on bacteria), and removal of dissolved organic matter from the influent. Understanding the taxa and functions in these systems will aid in monitoring and managing SSF for optimal water quality.

16S rRNA gene microarray analysis of microbial communities in ethanol-stimulated subsurface sediment.

Abstract: A high-density 16S rRNA gene microarray was used to analyze microbial communities in a slurry of ethanol-amended, uranium-contaminated subsurface sediment. Of specific interest was the extent to which the microarray could detect temporal patterns in the relative abundance of major metabolic groups (nitrate-reducing, metal-reducing, sulfate-reducing, and methanogenic taxa) that were stimulated by ethanol addition. The results show that the microarray, when used in conjunction with geochemical data and knowledge of the physiological properties of relevant taxa, provided accurate assessment of the response of key functional groups to biostimulation.

Devices, methods and systems for target detection

Abstract: Polymer arrays suitable to perform quantitative and qualitative detection as well as sorting of a target molecules and related devices methods and systems.

Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

Abstract: Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

Short Communication 16S rRNA Gene Microarray Analysis of Microbial Communities in Ethanol-Stimulated Subsurface Sediment

Abstract: A high-density 16S rRNA gene microarray was used to analyze microbial communities in a slurry of ethanol-amended, uranium-contaminated subsurface sediment. Of specific interest was the extent to which the microarray could detect temporal patterns in the relative abundance of major metabolic groups (nitrate-reducing, metal-reducing, sulfate-reducing, and methanogenic taxa) that were stimulated by ethanol addition. The results show that the microarray, when used in conjunction with geochemical data and knowledge of the physiological properties of relevant taxa, provided accurate assessment of the response of key functional groups to biostimulation.