A pilot study characterizing longitudinal changes in fecal microbiota of patients with Hirschsprung-associated enterocolitis.

PURPOSE: Hirschsprung disease is a neurointestinal disease that occurs due to failure of enteric neural crest-derived cells to complete their rostrocaudal migration along the gut mesenchyme, resulting in aganglionosis along variable lengths of the distal bowel. Despite the effective surgery that removes the aganglionic segment, children with Hirschsprung disease remain at high risk for developing a potentially life-threatening enterocolitis (Hirschsprung-associated enterocolitis). Although the etiology of this enterocolitis remains poorly understood, several recent studies in both mouse models and in human subjects suggest potential involvement of gastrointestinal microbiota in the underlying pathogenesis of Hirschsprung-associated enterocolitis. METHODS: We present the first study to exploit the Illumina MiSeq next-generation sequencing platform within a longitudinal framework focused on microbiomes of Hirschsprung-associated enterocolitis in five patients. We analyzed bacterial communities from fecal samples collected at different timepoints starting from active enterocolitis and progressing into remission. RESULTS: We observed compositional differences between patients largely attributable to variability in age at the time of sample collection. Remission samples across patients exhibited compositional similarity, including enrichment of Blautia, while active enterocolitis samples showed substantial variability in composition. CONCLUSIONS: Overall, our findings provide continued support for the role of GI microbiota in the pathogenesis of Hirschsprung-associated enterocolitis.

Maternal psycho-social risk factors associated with maternal alcohol consumption and Fetal Alcohol Spectrum Disorder: a systematic review.

PURPOSE: Fetal Alcohol Spectrum Disorder (FASD) is a preventable range of neurocognitive disorders associated with the biological mother's consumption of alcohol during pregnancy. However, on average, 45% of Australian women continue to consume alcohol during pregnancy resulting in a high rate of alcohol-exposed pregnancies and risk of FASD. This level of exposure is higher than the estimated global average of alcohol-exposed pregnancies (9.8%). This systematic literature review aims to identify demographic, health and psycho-social variables associated with alcohol consumption during pregnancy which may lead to FASD. METHODS: Using PRISMA principles, this systematic literature review reports on psycho-social factors which increase the risk of alcohol consumption during pregnancy thereby increasing the risk of FASD. RESULTS: Fourteen studies were accepted into this review. Studies were conducted across several countries and included a total of 386,067 cases. Seven studies were case-controlled and seven were cross-sectional design. Multiple studies identified the significance of prior mental illness, anxiety, depression, exposure to abuse and/or domestic violence and alcohol consumption behaviours of partners and family members as strong predictors of risky alcohol consumption during pregnancy and therefore associated risk of FASD. CONCLUSION: Clinical services may be able to use the evidence-based findings from this review to improve assessment and treatment services for vulnerable women to reduce alcohol-exposed pregnancies.

Analysis of the Duodenal Microbiome in Autistic Individuals: Association With Carbohydrate Digestion.

OBJECTIVES: There is evidence that symptoms of maldigestion or malabsorption in autistic individuals are related to changes in the indigenous microbiota. Analysis of colonic bacteria has revealed microbial dysbiosis in children with autism; however, characteristics of the duodenal microbiome are not well described. In the present study the microbiome of the duodenal mucosa of subjects with autism was evaluated for dysbiosis, bacteria overgrowth, and microbiota associated with carbohydrate digestion. The relationship between the duodenal microbiome and disaccharidase activity was analyzed in biopsies from 21 autistic subjects and 19 children without autism. METHODS: Microbiota composition was determined by 16S ribosomal RNA gene sequencing, and disaccharidase activity via biochemical assays. RESULTS: Although subjects with autism had a higher frequency of constipation (P < 0.005), there was no difference in disaccharidase activity between groups. In addition, no differences in microbiome diversity (species richness and evenness) were observed. Bacteria belonging to the genus Burkholderia were more abundant in subjects with autism, whereas members of the genus Neisseria were less abundant. At the species level, a relative decrease in abundance of 2 Bacteroides species and Escherichia coli was found in autistic individuals. There was a positive correlation between the abundance of Clostridium species, and disaccharidase activity, in autistic individuals. CONCLUSIONS: There are a variety of changes at the genus and species level in duodenal microbiota in children with autism that could be influenced by carbohydrate malabsorption. These observations could be affected by variations in individual diets, but also may represent a more pervasive dysbiosis that results in metabolites that affect the behavior of autistic children.

Spatially segregated transcription and translation in cells of the endomembrane-containing bacterium Gemmata obscuriglobus.

The dogma of coupled transcription and translation in bacteria has been challenged by recent reports of spatial segregation of these processes within the relatively simple cellular organization of the model organisms Escherichia coli and Bacillus subtilis. The bacterial species Gemmata obscuriglobus possesses an extensive endomembrane system. The membranes generate a very convoluted intracellular architecture in which some of the cell's ribosomes appear to have less direct access to the cell's nucleoid(s) than others. This observation prompted us to test the hypothesis that a substantial proportion of G. obscuriglobus translation may be spatially segregated from transcription. Using immunofluorescence and immunoelectron microscopy, we showed that translating ribosomes are localized throughout the cell, with a quantitatively greater proportion found in regions distal to nucleoid(s). Our results extend information about the phylogenetic and morphological diversity of bacteria in which the spatial organization of transcription and translation has been studied. These findings also suggest that endomembranes may provide an obstacle to colocated transcription and translation, a role for endomembranes that has not been reported previously for a prokaryotic organism. Our studies of G. obscuriglobus may provide a useful background for consideration of the evolutionary development of eukaryotic cellular complexity and how it led to decoupled processes of gene expression in eukaryotes.

Implementation of an Evidence-Based and Content Validated Standardized Ostomy Algorithm Tool in Home Care: A Quality Improvement Project.

Many nurses have limited experience with ostomy management. We sought to provide a standardized approach to ostomy education and management to support nurses in early identification of stomal and peristomal complications, pouching problems, and provide standardized solutions for managing ostomy care in general while improving utilization of formulary products. This article describes development and testing of an ostomy algorithm tool.

Global and Targeted Lipid Analysis of Gemmata obscuriglobus Reveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane.

UNLABELLED: Planctomycete bacteria possess many unusual cellular properties, contributing to a cell plan long considered to be unique among the bacteria. However, data from recent studies are more consistent with a modified Gram-negative cell plan. A key feature of the Gram-negative plan is the presence of an outer membrane (OM), for which lipopolysaccharide (LPS) is a signature molecule. Despite genomic evidence for an OM in planctomycetes, no biochemical verification has been reported. We attempted to detect and characterize LPS in the planctomycete Gemmata obscuriglobus. We obtained direct evidence for LPS and lipid A using electrophoresis and differential staining. Gas chromatography-mass spectrometry (GC-MS) compositional analysis of LPS extracts identified eight different 3-hydroxy fatty acids (3-HOFAs), 2-keto 3-deoxy-d-manno-octulosonic acid (Kdo), glucosamine, and hexose and heptose sugars, a chemical profile unique to Gram-negative LPS. Combined with molecular/structural information collected from matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS analysis of putative intact lipid A, these data led us to propose a heterogeneous hexa-acylated lipid A structure (multiple-lipid A species). We also confirmed previous reports of G. obscuriglobus whole-cell fatty acid (FA) and sterol compositions and detected a novel polyunsaturated FA (PUFA). Our confirmation of LPS, and by implication an OM, in G. obscuriglobus raises the possibility that other planctomycetes possess an OM. The pursuit of this question, together with studies of the structural connections between planctomycete LPS and peptidoglycans, will shed more light on what appears to be a planctomycete variation on the Gram-negative cell plan. IMPORTANCE: Bacterial species are classified as Gram positive or negative based on their cell envelope structure. For 25 years, the envelope of planctomycete bacteria has been considered a unique exception, as it lacks peptidoglycan and an outer membrane (OM). However, the very recent detection of peptidoglycan in planctomycete species has provided evidence for a more conventional cell wall and raised questions about other elements of the cell envelope. Here, we report direct evidence of lipopolysaccharide in the planctomycete G. obscuriglobus, suggesting the presence of an OM and supporting the proposal that the planctomycete cell envelope is an extension of the canonical Gram-negative plan. This interpretation features a convoluted cytoplasmic membrane and expanded periplasmic space, the functions of which provide an intriguing avenue for future investigation.

Random transposon mutagenesis of Verrucomicrobium spinosum DSM 4136(T).

The Verrucomicrobia are a bacterial group of growing interest due to their environmental ubiquity as free-living and host-associated microbes. They also exhibit an unusual compartmentalized cell plan, shared with members of neighboring phyla that include the Planctomycete bacteria. However, Verrucomicrobia are currently difficult to study, due to a lack of available genetic tools that would permit robust testing of hypotheses formulated from ecological and genomic data. To our knowledge, there are no published studies describing the transformation of exogenous DNA into any members of the Verrucomicrobia (or the neighboring phylum containing Planctomycetes). Here, we present a procedure for the transformation of DNA into Verrucomicrobium spinosum DSM 4136(T) via electroporation and the first description of a random transposon mutant library in this organism. We anticipate that this approach could be applied successfully to other Verrucomicrobia, providing opportunities to test the role of predicted gene function in ecological interactions and identify genes associated with the distinctive Planctomycete-Verrucomicrobial cell plan.

Genome sequence of Silicibacter pomeroyi reveals adaptations to the marine environment.

Since the recognition of prokaryotes as essential components of the oceanic food web, bacterioplankton have been acknowledged as catalysts of most major biogeochemical processes in the sea. Studying heterotrophic bacterioplankton has been challenging, however, as most major clades have never been cultured or have only been grown to low densities in sea water. Here we describe the genome sequence of Silicibacter pomeroyi, a member of the marine Roseobacter clade (Fig. 1), the relatives of which comprise approximately 10-20% of coastal and oceanic mixed-layer bacterioplankton. This first genome sequence from any major heterotrophic clade consists of a chromosome (4,109,442 base pairs) and megaplasmid (491,611 base pairs). Genome analysis indicates that this organism relies upon a lithoheterotrophic strategy that uses inorganic compounds (carbon monoxide and sulphide) to supplement heterotrophy. Silicibacter pomeroyi also has genes advantageous for associations with plankton and suspended particles, including genes for uptake of algal-derived compounds, use of metabolites from reducing microzones, rapid growth and cell-density-dependent regulation. This bacterium has a physiology distinct from that of marine oligotrophs, adding a new strategy to the recognized repertoire for coping with a nutrient-poor ocean.

Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils.

The complete genomes of three strains from the phylum Acidobacteria were compared. Phylogenetic analysis placed them as a unique phylum. They share genomic traits with members of the Proteobacteria, the Cyanobacteria, and the Fungi. The three strains appear to be versatile heterotrophs. Genomic and culture traits indicate the use of carbon sources that span simple sugars to more complex substrates such as hemicellulose, cellulose, and chitin. The genomes encode low-specificity major facilitator superfamily transporters and high-affinity ABC transporters for sugars, suggesting that they are best suited to low-nutrient conditions. They appear capable of nitrate and nitrite reduction but not N(2) fixation or denitrification. The genomes contained numerous genes that encode siderophore receptors, but no evidence of siderophore production was found, suggesting that they may obtain iron via interaction with other microorganisms. The presence of cellulose synthesis genes and a large class of novel high-molecular-weight excreted proteins suggests potential traits for desiccation resistance, biofilm formation, and/or contribution to soil structure. Polyketide synthase and macrolide glycosylation genes suggest the production of novel antimicrobial compounds. Genes that encode a variety of novel proteins were also identified. The abundance of acidobacteria in soils worldwide and the breadth of potential carbon use by the sequenced strains suggest significant and previously unrecognized contributions to the terrestrial carbon cycle. Combining our genomic evidence with available culture traits, we postulate that cells of these isolates are long-lived, divide slowly, exhibit slow metabolic rates under low-nutrient conditions, and are well equipped to tolerate fluctuations in soil hydration.

Characterization of the intestinal microbiota of two Antarctic notothenioid fish species.

The fish fauna of the Southern Ocean is dominated by species of the perciform suborder Notothenioidei, which constitute 46% of fish species and 90% of biomass. Notothenioids have undergone rapid morphological and ecological diversification and developed physiological adaptations to a cold, highly oxygenated environment. Microbes inhabiting animal intestines include those that perform essential nutritional functions, but notothenioid gut microbial communities have not been investigated using cultivation-independent approaches. We analyzed bacterial 16S rRNA gene sequences obtained from the intestinal tract of Notothenia coriiceps and Chaenocephalus aceratus, which differ in their pelagic distribution and feeding strategies. Both samples showed dominance of Gammaproteobacteria (mostly Vibrionaceae), as has been reported for temperate teleost species. Both samples showed low diversity relative to that reported for other fish microbiota studies, with C. aceratus containing fewer OTUs than N. coriiceps. Despite the small sample size of this preliminary study, our findings suggest that Antarctic notothenioids carry a gut microbiota similar in composition to that of temperate fish, but exhibiting lower species-level diversity. The omnivorous N. coriiceps individual exhibited greater diversity than the exclusively carnivorous C. aceratus individual, which may indicate that increasing herbivory in fish leads to gut microbe diversification, as found in mammals. Lastly, we detected members of taxa containing known microbial pathogens, which have not been previously reported in Antarctic notothenioid fish.

Essentiality of sterol synthesis genes in the planctomycete bacterium Gemmata obscuriglobus.

Sterols and hopanoids are chemically and structurally related lipids mostly found in eukaryotic and bacterial cell membranes. Few bacterial species have been reported to produce sterols and this anomaly had originally been ascribed to lateral gene transfer (LGT) from eukaryotes. In addition, the functions of sterols in these bacteria are unknown and the functional overlap between sterols and hopanoids is still unclear. Gemmata obscuriglobus is a bacterium from the Planctomycetes phylum that synthesizes sterols, in contrast to its hopanoid-producing relatives. Here we show that sterols are essential for growth of G. obscuriglobus, and that sterol depletion leads to aberrant membrane structures and defects in budding cell division. This report of sterol essentiality in a prokaryotic species advances our understanding of sterol distribution and function, and provides a foundation to pursue fundamental questions in evolutionary cell biology.

Microbiome Composition in Both Wild-Type and Disease Model Mice Is Heavily Influenced by Mouse Facility.

Murine models have become essential tools for understanding the complex interactions between gut microbes, their hosts, and disease. While many intra-facility factors are known to influence the structure of mouse microbiomes, the contribution of inter-facility variation to mouse microbiome composition, especially in the context of disease, remains under-investigated. We replicated microbiome experiments using identical mouse lines housed in two separate animal facilities and report drastic differences in composition of microbiomes based upon animal facility of origin. We observed facility-specific microbiome signatures in the context of a disease model [the Ednrb (endothelin receptor type B) Hirschsprung disease mouse] and in normal C57BL/6J mice. Importantly, these facility differences were independent of cage, sex, or sequencing-related influence. In addition, we investigated the reproducibility of microbiome dysbiosis previously associated with Ednrb-/- (knock-out; KO) mice. While we observed genotype-based differences in composition between wild-type (WT) and KO mice, these differences were inconsistent with the previously reported conclusions. Furthermore, the genotype-based differences were not identical across animal facilities. Despite this, through differential abundance testing, we identified several conserved candidate taxa and candidate operational taxonomic units that may play a role in disease promotion or protection. Overall, our findings raise the possibility that previously reported microbiome-disease associations from murine studies conducted in a single facility may be heavily influenced by facility-specific effects. More generally, these results provide a strong rationale for replication of mouse microbiome studies at multiple facilities, and for the meticulous collection of metadata that will allow the confounding effects of facility to be more specifically identified.

Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath).

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encoding enzymes for energy transduction, together with inferred cellular location of the enzymes, provides a basis for proposing an expansion to the 'hydrogen-cycling' model for increasing energy efficiency in this bacterium. Plasmid-encoded functions include modification of cell surface components, nitrogen fixation and a type-III protein secretion system. This genome sequence represents a substantial step toward the elucidation of pathways for reduction (and bioremediation) of pollutants such as uranium and chromium and offers a new starting point for defining this organism's complex anaerobic respiration.

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis.

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803-base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613-base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organism's complex electron transport systems and metal ion-reducing capabilities.

How genomics has affected the concept of microbiology.

Genomics influences multiple areas of microbiology, and thus affects key microbiological concepts. Recent reports that describe the large genome and unusual coding capacity of mimivirus, the minimized fungal genomes that contain elements of bacterial metabolism, and the 'signature' eukaryotic proteins in bacteria are introducing grey shades into the black-and-white distinctions between microbial domains. The concept of the 'universal' minimal genome is being challenged, and the ability of minimal genomes to support cellular complexity is under investigation. There have been intriguing insights into microbe-microbe relationships, for example conflict mediation in competing bacteriophages that rapidly evolve survival mechanisms when cooperation is experimentally enforced. Genomics has given birth to metagenomics, but has also stimulated the development of improved cultivation techniques. Lastly, the taxonomic potential of genomics is emerging, as studies of multiple strains allow us to revise and refine the bacterial species concept as well as the idea of a static genome.

Exopolysaccharide-associated protein sorting in environmental organisms: the PEP-CTERM/EpsH system. Application of a novel phylogenetic profiling heuristic.

BACKGROUND: Protein translocation to the proper cellular destination may be guided by various classes of sorting signals recognizable in the primary sequence. Detection in some genomes, but not others, may reveal sorting system components by comparison of the phylogenetic profile of the class of sorting signal to that of various protein families. RESULTS: We describe a short C-terminal homology domain, sporadically distributed in bacteria, with several key characteristics of protein sorting signals. The domain includes a near-invariant motif Pro-Glu-Pro (PEP). This possible recognition or processing site is followed by a predicted transmembrane helix and a cluster rich in basic amino acids. We designate this domain PEP-CTERM. It tends to occur multiple times in a genome if it occurs at all, with a median count of eight instances; Verrucomicrobium spinosum has sixty-five. PEP-CTERM-containing proteins generally contain an N-terminal signal peptide and exhibit high diversity and little homology to known proteins. All bacteria with PEP-CTERM have both an outer membrane and exopolysaccharide (EPS) production genes. By a simple heuristic for screening phylogenetic profiles in the absence of pre-formed protein families, we discovered that a homolog of the membrane protein EpsH (exopolysaccharide locus protein H) occurs in a species when PEP-CTERM domains are found. The EpsH family contains invariant residues consistent with a transpeptidase function. Most PEP-CTERM proteins are encoded by single-gene operons preceded by large intergenic regions. In the Proteobacteria, most of these upstream regions share a DNA sequence, a probable cis-regulatory site that contains a sigma-54 binding motif. The phylogenetic profile for this DNA sequence exactly matches that of three proteins: a sigma-54-interacting response regulator (PrsR), a transmembrane histidine kinase (PrsK), and a TPR protein (PrsT). CONCLUSION: These findings are consistent with the hypothesis that PEP-CTERM and EpsH form a protein export sorting system, analogous to the LPXTG/sortase system of Gram-positive bacteria, and correlated to EPS expression. It occurs preferentially in bacteria from sediments, soils, and biofilms. The novel method that led to these findings, partial phylogenetic profiling, requires neither global sequence clustering nor arbitrary similarity cutoffs and appears to be a rapid, effective alternative to other profiling methods.

New directions and interactions in metagenomics research.

Metagenomics, which aims to access the genomic potential of an environmental sample directly, is a burgeoning area that is generating enormous amounts of biological information. An examination of recent metagenomics literature reveals the discipline to be heading in new and interesting directions, including the investigation of the normal flora of mammals, analysis of ancient genomes, and exploration of the distribution of novel pathways. In addition, the development of new bioinformatics approaches and tools is allowing innovative mining of both existing and new data. Finally, there are indications that the integration of metagenomics with complementary approaches in microbial ecology is beginning.

Antibiotic Treatment Induces Long-lasting Changes in the Fecal Microbiota that Protect Against Colitis.

BACKGROUND: The interplay between host genetics, immunity, and microbiota is central to the pathogenesis of inflammatory bowel disease. Previous population-based studies suggested a link between antibiotic use and increased inflammatory bowel disease risk, but the mechanisms are unknown. The purpose of this study was to determine the long-term effects of antibiotic administration on microbiota composition, innate immunity, and susceptibility to colitis, as well as the mechanism by which antibiotics alter host colitogenicity. METHODS: Wild-type mice were given broad-spectrum antibiotics or no antibiotics for 2 weeks, and subsequent immunophenotyping and 16S rRNA gene sequencing-based analysis of the fecal microbiome were performed 6 weeks later. In a separate experiment, control and antibiotic-treated mice were given 7 days of dextran sulfate sodium, 6 weeks after completing antibiotic treatment, and the severity of colitis scored histologically. Fecal transfer was performed from control or antibiotic-treated mice to recipient mice whose endogenous microbiota had been cleared with antibiotics, and the susceptibility of the recipients to dextran sulfate sodium-induced colitis was analyzed. Naive CD4 T cells were transferred from control and antibiotic-treated mice to immunodeficient Rag-1 recipients and the severity of colitis compared. RESULTS: Antibiotics led to sustained dysbiosis and changes in T-cell subpopulations, including reductions in colonic lamina propria total T cells and CD4 T cells. Antibiotics conferred protection against dextran sulfate sodium colitis, and this effect was transferable by fecal transplant but not by naive T cells. CONCLUSIONS: Antibiotic exposure protects against colitis, and this effect is transferable with fecal microbiota from antibiotic-treated mice, supporting a protective effect of the microbial community.