Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria with Immunoglobulin A.

Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

Early-Life Microbial Restitution Reduces Colitis Risk Promoted by Antibiotic-Induced Gut Dysbiosis in Interleukin 10-/- Mice.

BACKGROUND & AIMS: Perturbations in the early-life gut microbiome are associated with increased risk for complex immune disorders like inflammatory bowel diseases. We previously showed that maternal antibiotic-induced gut dysbiosis vertically transmitted to offspring increases experimental colitis risk in interleukin (IL) 10 gene deficient (IL10-/-) mice, a finding that may result from the loss/lack of essential microbes needed for appropriate immunologic education early in life. Here, we aimed to identify key microbes required for proper development of the early-life gut microbiome that decrease colitis risk in genetically susceptible animals. METHODS: Metagenomic sequencing followed by reconstruction of metagenome-assembled genomes was performed on fecal samples of IL10-/- mice with and without antibiotic-induced dysbiosis to identify potential missing microbial members needed for immunologic education. One high-value target strain was then engrafted early and/or late into the gut microbiomes of IL10-/- mice with antibiotic-induced dysbiosis. RESULTS: Early-, but not late-, life engraftment of a single dominant Bacteroides strain of non-antibiotic-treated IL10-/- mice was sufficient to restore the development of the gut microbiome, promote immune tolerance, and prevent colitis in IL10-/- mice that had antibiotic-induced dysbiosis. CONCLUSIONS: Restitution of a keystone microbial strain missing in the early-life antibiotic-induced gut dysbiosis results in recovery of the microbiome, proper development of immune tolerance, and reduced risk for colitis in genetically prone hosts.

PATRIC as a unique resource for studying antimicrobial resistance.

The Pathosystems Resource Integration Center (PATRIC, www.patricbrc.org) is designed to provide researchers with the tools and services that they need to perform genomic and other 'omic' data analyses. In response to mounting concern over antimicrobial resistance (AMR), the PATRIC team has been developing new tools that help researchers understand AMR and its genetic determinants. To support comparative analyses, we have added AMR phenotype data to over 15 000 genomes in the PATRIC database, often assembling genomes from reads in public archives and collecting their associated AMR panel data from the literature to augment the collection. We have also been using this collection of AMR metadata to build machine learning-based classifiers that can predict the AMR phenotypes and the genomic regions associated with resistance for genomes being submitted to the annotation service. Likewise, we have undertaken a large AMR protein annotation effort by manually curating data from the literature and public repositories. This collection of 7370 AMR reference proteins, which contains many protein annotations (functional roles) that are unique to PATRIC and RAST, has been manually curated so that it projects stably across genomes. The collection currently projects to 1 610 744 proteins in the PATRIC database. Finally, the PATRIC Web site has been expanded to enable AMR-based custom page views so that researchers can easily explore AMR data and design experiments based on whole genomes or individual genes.

Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice.

The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia. This was associated with a pro-inflammatory T helper type 1 (T(H)1) immune response and increased incidence of colitis in genetically susceptible Il10(−/−), but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10(−/−) mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts.

Commensal bacteria protect against food allergen sensitization.

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

Insights into the pathogenesis of ulcerative colitis from a murine model of stasis-induced dysbiosis, colonic metaplasia, and genetic susceptibility.

Gut dysbiosis, host genetics, and environmental triggers are implicated as causative factors in inflammatory bowel disease (IBD), yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis (UC) patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically created blind self-filling (SFL) and self-emptying (SEL) ileal loops using wild-type (WT), IL-10 knockout (KO) (IL-10), TLR4 KO (T4), and IL-10/T4 double KO mice. After 5 wk, loop histology, host gene/protein expression, and bacterial 16s rRNA profiles were examined. SFL exhibit fecal stasis due to directional motility oriented toward the loop end, whereas SEL remain empty. In WT mice, SFL, but not SEL, develop pouchlike microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. The inflammation associated with IL-10 required TLR4, as animals lacking both pathways displayed little disease. Furthermore, germ-free IL-10 mice conventionalized with SFL, but not SEL, microbiota populations develop severe colitis. These data support essential roles of stasis-induced, colon-like microbiota, TLR4-mediated colonic metaplasia, and genetic susceptibility in the development of pouchitis and possibly UC. However, these factors by themselves are not sufficient. Similarities between this model and human UC/pouchitis provide opportunities for gaining insights into the mechanistic basis of IBD and for identification of targets for novel preventative and therapeutic interventions.

Spatial scale drives patterns in soil bacterial diversity.

Soil microbial communities are essential for ecosystem function, but linking community composition to biogeochemical processes is challenging because of high microbial diversity and large spatial variability of most soil characteristics. We investigated soil bacterial community structure in a switchgrass stand planted on soil with a history of grassland vegetation at high spatial resolution to determine whether biogeographic trends occurred at the centimeter scale. Moreover, we tested whether such heterogeneity, if present, influenced community structure within or among ecosystems. Pronounced heterogeneity was observed at centimeter scales, with abrupt changes in relative abundance of phyla from sample to sample. At the ecosystem scale (> 10 m), however, bacterial community composition and structure were subtly, but significantly, altered by fertilization, with higher alpha diversity in fertilized plots. Moreover, by comparing these data with data from 1772 soils from the Earth Microbiome Project, it was found that 20% of bacterial taxa were shared between their site and diverse globally sourced soil samples, while grassland soils shared approximately 40% of their operational taxonomic units with the current study. By spanning several orders of magnitude, the analysis suggested that extreme patchiness characterized community structure at smaller scales but that coherent patterns emerged at larger length scales.

A novel conjugative transposon carrying an autonomously amplified plasmid.

Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.IMPORTANCEThe exchange of antibiotic production and resistance genes between microorganisms can lead to the emergence of new pathogens. In this study, short-read mapping of metagenomic samples taken over time from the illeal pouch of a patient with ulcerative colitis to a Bacteroides fragilis metagenome-assembled genome revealed two distinct genomic arrangements of a novel conjugative transposon, CTn214, that encodes tetracycline resistance. The autonomous amplification of a plasmid-like circular form from CTn214 that includes tetQ potentially provides consistent ribosome protection against tetracycline. This mode of antibiotic resistance offers a novel mechanism for understanding the emergence of pathobionts like B. fragilis and their persistence for extended periods of time in patients with inflammatory bowel disease.

Maternal activation of gap genes in the hover fly Episyrphus.

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, we used loss- and gain-of-function experiments in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. We find that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, we find that torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. We conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha).

Microbiota-dependent proteolysis of gluten subverts diet-mediated protection against type 1 diabetes.

Diet and commensals can affect the development of autoimmune diseases like type 1 diabetes (T1D). However, whether dietary interventions are microbe-mediated was unclear. We found that a diet based on hydrolyzed casein (HC) as a protein source protects non-obese diabetic (NOD) mice in conventional and germ-free (GF) conditions via improvement in the physiology of insulin-producing cells to reduce autoimmune activation. The addition of gluten (a cereal protein complex associated with celiac disease) facilitates autoimmunity dependent on microbial proteolysis of gluten: T1D develops in GF animals monocolonized with Enterococcus faecalis harboring secreted gluten-digesting proteases but not in mice colonized with protease deficient bacteria. Gluten digestion by E. faecalis generates T cell-activating peptides and promotes innate immunity by enhancing macrophage reactivity to lipopolysaccharide (LPS). Gnotobiotic NOD Toll4-negative mice monocolonized with E. faecalis on an HC + gluten diet are resistant to T1D. These findings provide insights into strategies to develop dietary interventions to help protect humans against autoimmunity.

Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae).

BACKGROUND: In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in Drosophila melanogaster, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of D. melanogaster with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid Episyrphus balteatus is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of E. balteatus gives rise to two extraembryonic tissues (serosa and amnion), whereas in D. melanogaster, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of D. melanogaster, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa. RESULTS: To search for BMP signaling components in E. balteatus, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative E. balteatus orthologues of 43% of all annotated D. melanogaster genes, including the genes of all BMP ligands and other BMP signaling components. CONCLUSION: The diversification of several BMP signaling components in the dipteran linage of D. melanogaster preceded the origin of the amnioserosa.[Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (JN006969-JN006986).].

Reduction of Sb(V) by coupled biotic-abiotic processes under sulfidogenic conditions.

Increasing use and mining of antimony (Sb) has resulted in greater concern involving its fate and transport in the environment. Antimony(V) and (III) are the two most environmentally relevant oxidation states, but little is known about the redox transitions between the two in natural systems. To better understand the behavior of antimony in anoxic environments, the redox transformations of Sb(V) were studied in biotic and abiotic reactors. The biotic reactors contained Sb(V) (2 mM as KSb(OH)6), ferrihydrite (50 mM Fe(III)), sulfate (10 mM), and lactate (10 mM), that were inoculated with sediment from a wetland. In the abiotic reactors, The interaction of Sb(V) with green rust, magnetite, siderite, vivianite or mackinawite was examined under abiotic conditions. Changes in the concentrations of Sb, Fe(II), sulfate, and lactate, as well as the microbial community composition were monitored over time. Lactate was rapidly fermented to acetate and propionate in the bioreactors, with the latter serving as the primary electron donor for dissimilatory sulfate reduction (DSR). The reduction of ferrihydrite was primarily abiotic, being driven by biogenic sulfide. Sb and Fe K-edge X-ray absorption near edge structure (XANES) analysis showed reduction of Sb(V) to Sb(III) within 4 weeks, concurrent with DSR and the formation of FeS. Sb K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy analysis indicated that the reduced phase was a mixture of S- and O-coordinated Sb(III). Reduction of Sb(V) was not observed in the presence of magnetite, siderite, or green rust, and limited reduction occurred with vivianite. However, reduction of Sb(V) to amorphous Sb(III) sulfide occurred with mackinawite. These results are consistent with abiotic reduction of Sb(V) by biogenic sulfide and reveal a substantial influence of Fe oxides on the speciation of Sb(III), which illustrates the tight coupling of Sb speciation with the biogeochemical cycling of S and Fe.

Metagenomic Alterations in Gut Microbiota Precede and Predict Onset of Colitis in the IL10 Gene-Deficient Murine Model.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBD) are chronic inflammatory disorders where predictive biomarkers for the disease development and clinical course are sorely needed for development of prevention and early intervention strategies that can be implemented to improve clinical outcomes. Since gut microbiome alterations can reflect and/or contribute to impending host health changes, we examined whether gut microbiota metagenomic profiles would provide more robust measures for predicting disease outcomes in colitis-prone hosts. METHODS: Using the interleukin (IL) 10 gene-deficient (IL10 KO) murine model where early life dysbiosis from antibiotic (cefoperozone [CPZ]) treated dams vertically transferred to pups increases risk for colitis later in life, we investigated temporal metagenomic profiles in the gut microbiota of post-weaning offspring and determined their relationship to eventual clinical outcomes. RESULTS: Compared to controls, offspring acquiring maternal CPZ-induced dysbiosis exhibited a restructuring of intestinal microbial membership in both bacteriome and mycobiome that was associated with alterations in specific functional subsystems. Furthermore, among IL10 KO offspring from CPZ-treated dams, several functional subsystems, particularly nitrogen metabolism, diverged between mice that developed spontaneous colitis (CPZ-colitis) versus those that did not (CPZ-no-colitis) at a time point prior to eventual clinical outcome. CONCLUSIONS: Our findings provide support that functional metagenomic profiling of gut microbes has potential and promise meriting further study for development of tools to assess risk and manage human IBD.

Biogeochemical dynamics and microbial community development under sulfate- and iron-reducing conditions based on electron shuttle amendment.

Iron reduction and sulfate reduction are two of the major biogeochemical processes that occur in anoxic sediments. Microbes that catalyze these reactions are therefore some of the most abundant organisms in the subsurface, and some of the most important. Due to the variety of mechanisms that microbes employ to derive energy from these reactions, including the use of soluble electron shuttles, the dynamics between iron- and sulfate-reducing populations under changing biogeochemical conditions still elude complete characterization. Here, we amended experimental bioreactors comprised of freshwater aquifer sediment with ferric iron, sulfate, acetate, and the model electron shuttle AQDS (9,10-anthraquinone-2,6-disulfonate) and monitored both the changing redox conditions as well as changes in the microbial community over time. The addition of the electron shuttle AQDS did increase the initial rate of FeIII reduction; however, it had little effect on the composition of the microbial community. Our results show that in both AQDS- and AQDS+ systems there was an initial dominance of organisms classified as Geobacter (a genus of dissimilatory FeIII-reducing bacteria), after which sequences classified as Desulfosporosinus (a genus of dissimilatory sulfate-reducing bacteria) came to dominate both experimental systems. Furthermore, most of the ferric iron reduction occurred under this later, ostensibly "sulfate-reducing" phase of the experiment. This calls into question the usefulness of classifying subsurface sediments by the dominant microbial process alone because of their interrelated biogeochemical consequences. To better inform models of microbially-catalyzed subsurface processes, such interactions must be more thoroughly understood under a broad range of conditions.

The GAAS metagenomic tool and its estimations of viral and microbial average genome size in four major biomes.

Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon.

Metagenomic analysis of colonic mucosa-associated microbes has been complicated by technical challenges that disrupt or alter community structure and function. In the present study, we determined the feasibility of laser capture microdissection (LCM) of intact regional human colonic mucosa-associated microbes followed by phi29 multiple displacement amplification (MDA) and massively parallel sequencing for metagenomic analysis. Samples were obtained from the healthy human subject without bowel preparation and frozen sections immediately prepared. Regional mucosa-associated microbes were successfully dissected using LCM with minimal contamination by host cells, their DNA extracted and subjected to phi29 MDA with a high fidelity, prior to shotgun sequencing using the GS-FLX DNA sequencer. Metagenomic analysis of approximately 67 million base pairs of DNA sequences from two samples revealed that the metabolic functional profiles in mucosa-associated microbes were as diverse as those reported in feces, specifically the representation of functional genes associated with carbohydrate, protein, and nucleic acid utilization. In summary, these studies demonstrate the feasibility of the approach to study the structure and metagenomic profiles of human intestinal mucosa-associated microbial communities at small spatial scales.

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation.

Shifts in microbial communities are implicated in the pathogenesis of a number of gastrointestinal diseases, but we have limited understanding of the mechanisms that lead to altered community structures. One difficulty with studying these mechanisms in human subjects is the inherent baseline variability of the microbiota in different individuals. In an effort to overcome this baseline variability, we employed a mouse model to control the host genotype, diet, and other possible influences on the microbiota. This allowed us to determine whether the indigenous microbiota in such mice had a stable baseline community structure and whether this community exhibited a consistent response following antibiotic administration. We employed a tag-sequencing strategy targeting the V6 hypervariable region of the bacterial small-subunit (16S) rRNA combined with massively parallel sequencing to determine the community structure of the gut microbiota. Inbred mice in a controlled environment harbored a reproducible baseline community that was significantly impacted by antibiotic administration. The ability of the gut microbial community to recover to baseline following the cessation of antibiotic administration differed according to the antibiotic regimen administered. Severe antibiotic pressure resulted in reproducible, long-lasting alterations in the gut microbial community, including a decrease in overall diversity. The finding of stereotypic responses of the indigenous microbiota to ecologic stress suggests that a better understanding of the factors that govern community structure could lead to strategies for the intentional manipulation of this ecosystem so as to preserve or restore a healthy microbiota.

Perturbation of the small intestine microbial ecology by streptomycin alters pathology in a Salmonella enterica serovar typhimurium murine model of infection.

The small intestine is an important site of infection for many enteric bacterial pathogens, and murine models, including the streptomycin-treated mouse model of infection, are frequently used to study these infections. The environment of the mouse small intestine and the microbiota with which enteric pathogens are likely to interact, however, have not been well described. Therefore, we compared the microbiota and the concentrations of short-chain fatty acids (SCFAs) present in the ileum and cecum of streptomycin-treated mice and untreated controls. We found that the microbiota in the ileum of untreated mice differed greatly from that of the cecum of the same mice, primarily among families of the phylum Firmicutes. Upon treatment with streptomycin, substantial changes in the microbial composition occurred, with a marked loss of population complexity. Characterization of the metabolic products of the microbiota, the SCFAs, showed that formate was present in the ileum but low or not detectable in the cecum while butyrate was present in the cecum but not the ileum. Treatment with streptomycin altered the SCFAs in the cecum, significantly decreasing the concentration of acetate, propionate, and butyrate. In this work, we also characterized the pathology of Salmonella infection in the ileum. Infection of streptomycin-treated mice with Salmonella was characterized by a significant increase in the relative and absolute levels of the pathogen and was associated with more severe ileal inflammation and pathology. Together these results provide a better understanding of the ileal environment in the mouse and the changes that occur upon streptomycin treatment.