Deciphering interactions between the gut microbiota and the immune system via microbial cultivation and minimal microbiomes.

The community of microorganisms in the mammalian gastrointestinal tract, referred to as the gut microbiota, influences host physiology and immunity. The last decade of microbiome research has provided significant advancements for the field and highlighted the importance of gut microbes to states of both health and disease. Novel molecular techniques have unraveled the tremendous diversity of intestinal symbionts that potentially influence the host, many proof-of-concept studies have demonstrated causative roles of gut microbial communities in various pathologies, and microbiome-based approaches are beginning to be implemented in the clinic for diagnostic purposes or for personalized treatments. However, several challenges for the field remain: purely descriptive reports outnumbering mechanistic studies and slow translation of experimental results obtained in animal models into the clinics. Moreover, there is a dearth of knowledge regarding how gut microbes, including novel species that have yet to be identified, impact host immune responses. The sheer complexity of the gut microbial ecosystem makes it difficult, in part, to fully understand the microbiota-host networks that regulate immunity. In the present manuscript, we review key findings on the interactions between gut microbiota members and the immune system. Because culturing microbes allows performing functional studies, we have emphasized the impact of specific taxa or communities thereof. We also highlight underlying molecular mechanisms and discuss opportunities to implement minimal microbiome-based strategies.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Epidemiological investigation of Candida species causing bloodstream infection in paediatric small bowel transplant recipients.

Small bowel transplantation (SBT) can be a life-saving medical procedure. However, these recipients experience high risk of bloodstream infections caused by Candida. This research aims to characterise the SBT recipient gut microbiota over time following transplantation and investigate the epidemiology of candidaemia in seven paediatric patients. Candida species from the recipients' ileum and bloodstream were identified by internal transcribed spacer sequence and distinguished to strain by multilocus sequence typing and randomly amplified polymorphic DNA. Antifungal susceptibility of bloodstream isolates was determined against nine antifungals. Twenty-two ileostomy samples harboured at least one Candida species. Fungaemia were caused by Candida parapsilosis, Candida albicans, Candida glabrata, Candida orthopsilosis and Candida pelliculosa. All but three bloodstream isolates showed susceptibility to all the antifungals tested. One C. glabrata isolate showed multidrug resistance to itraconazole, amphotericin B and posaconazole and intermediate resistance to caspofungin. Results are congruent with both endogenous (C. albicans, C. glabrata) and exogenous (C. parapsilosis) infections; results also suggest two patients were infected by the same strain of C. parapsilosis. Continuing to work towards a better understanding of sources of infection-particularly the exogenous sources-would lead to targeted prevention strategies.

Reconsolidation and update of morphine-associated contextual memory in mice.

Drug addiction can be viewed as a pathological memory that is constantly retrieved and reconsolidated. Since drug abuse takes place in different contexts, it could be considered that reconsolidation plays a role in memory updating. There is consistent evidence supporting the role of reconsolidation in the strength and maintenance of contextual memories induced by drugs of abuse. However, this role is not well established in memory update. The purpose of the current study was to assess the reconsolidation process over memory update. C57BL6 mice were subjected to a morphine-induced, conditioned place preference (CPP) paradigm. Based on CPP results, animals were divided into distinct experimental groups, according to the contextual characteristics of the re-exposure and a second CPP Test. Re-exposure in the original context was important for memory maintenance and re-exposure under discrete contextual changes resulted in memory updating, although original memory was maintained. Interestingly, cycloheximide, an inhibitor of protein synthesis, had different outcomes in our protocol. When the re-exposure was done under discrete contextual changes, cycloheximide treatment just after re-exposure blocked memory updating, without changes in memory maintenance. When re-exposure was done under the original context, only two subsequent cycloheximide injections (3 and 6h) disrupted later CPP expression. Considering the temporal window of protein synthesis in consolidation and reconsolidation, these findings suggest that re-exposure, according to the contextual characteristics in our protocol, could trigger both phenomena. Furthermore, when new information is present on retrieval, reconsolidation plays a pivotal role in memory updating.

Chronic light deprivation inhibits appetitive associative learning induced by ethanol and its respective c-Fos and pCREB expression.

To address the role of mixed anxiety/mood disorder on appetitive associative learning, we verify whether previous chronic light deprivation changes ethanol-induced conditioned place preference and its respective expression of c-Fos and pCREB, markers of neuronal activity and plasticity. The experimental group was maintained in light deprivation for 24 h for a period of 4 wk. Subsequently, it was adapted to a standard light-dark cycle for 1 wk. As a control, some mice were maintained in standard cycle for a period of 4 wk (Naïve group). Then, all animals were submitted to behavioral tests to assess emotionality: elevated plus maze; open field; and forced swim. After that, they were submitted to ethanol-induced conditioned place preference. Ninety minutes after the place preference test, they were perfused, and their brains processed for c-Fos and pCREB immunohistochemistry. Light deprivation induced anxiety-like trait (elevated plus maze), despair (forced swim), and hyperlocomotion (open field), common features seen in other animal models of depression. Ethanol-induced conditioned place preference was accompanied by increases on c-Fos and pCREB in the hippocampus, prefrontal cortex and striatum. Interestingly, mice previously submitted to light deprivation did not develop either acquisition and/or expression of ethanol-induced conditioned place preference or increases in c-Fos and pCREB. Therefore, chronic light deprivation mimics several behavioral aspects of other animal models of depression. Furthermore, it could be useful to study the neurochemical mechanisms involved in the dual diagnosis. However, given its likely deleterious effects on appetitive associative memory, it should be used with caution to investigate the cognitive aspects related to the dual diagnosis.

Increased Dietary Trp, Thr, and Met Supplementation Improves Performance, Health, and Protein Metabolism of Weaned Piglets under Mixed Management and Poor Housing Conditions.

A sanitary challenge was carried out to induce suboptimal herd health while investigating the effect of amino acids supplementation on piglet responses. Weaned piglets of high sanitary status (6.33 ± 0.91 kg of BW) were distributed in a 2 × 2 factorial arrangement into two similar facilities with contrasting sanitary conditions and two different diets. Our results suggest that increased Trp, Thr, and Met dietary supplementation could support the immune systems of piglets under a sanitary challenge. In this manner, AA+ supplementation improved the performance and metabolism of piglets under mixed management and poor sanitary conditions. No major temporal microbiome changes were associated with differences in performance regardless of sanitary conditions or diets. Since piglets often become mixed in multiple-site production systems and facility hygiene is also often neglected, this study suggests that increased Trp, Thr, and Met (AA+) dietary supplementation could contribute to mitigating the side effects of these harmful risk factors in modern pig farms.

AI-based approach for transcribing and classifying unstructured emergency call data: A methodological proposal.

Emergency care-sensitive conditions (ECSCs) require rapid identification and treatment and are responsible for over half of all deaths worldwide. Prehospital emergency care (PEC) can provide rapid treatment and access to definitive care for many ECSCs and can reduce mortality in several different settings. The objective of this study is to propose a method for using artificial intelligence (AI) and machine learning (ML) to transcribe audio, extract, and classify unstructured emergency call data in the Serviço de Atendimento Móvel de Urgência (SAMU) system in southern Brazil. The study used all "1-9-2" calls received in 2019 by the SAMU Novo Norte Emergency Regulation Center (ERC) call center in Maringá, in the Brazilian state of Paraná. The calls were processed through a pipeline using machine learning algorithms, including Automatic Speech Recognition (ASR) models for transcription of audio calls in Portuguese, and a Natural Language Understanding (NLU) classification model. The pipeline was trained and validated using a dataset of labeled calls, which were manually classified by medical students using LabelStudio. The results showed that the AI model was able to accurately transcribe the audio with a Word Error Rate of 42.12% using Wav2Vec 2.0 for ASR transcription of audio calls in Portuguese. Additionally, the NLU classification model had an accuracy of 73.9% in classifying the calls into different categories in a validation subset. The study found that using AI to categorize emergency calls in low- and middle-income countries is largely unexplored, and the applicability of conventional open-source ML models trained on English language datasets is unclear for non-English speaking countries. The study concludes that AI can be used to transcribe audio and extract and classify unstructured emergency call data in an emergency system in southern Brazil as an initial step towards developing a decision-making support tool.

Salmonella enterica induces biogeography-specific changes in the gut microbiome of pigs.

Swine are a major reservoir of an array of zoonotic Salmonella enterica subsp. enterica lineage I serovars including Derby, Typhimurium, and 4,[5],12:i:- (a.k.a. Monophasic Typhimurium). In this study, we assessed the gastrointestinal (GI) microbiome composition of pigs in different intestinal compartments and the feces following infection with specific zoonotic serovars of S. enterica (S. Derby, S. Monophasic, and S. Typhimurium). 16S rRNA based microbiome analysis was performed to assess for GI microbiome changes in terms of diversity (alpha and beta), community structure and volatility, and specific taxa alterations across GI biogeography (small and large intestine, feces) and days post-infection (DPI) 2, 4, and 28; these results were compared to disease phenotypes measured as histopathological changes. As previously reported, only S. Monophasic and S. Typhimurium induced morphological alterations that marked an inflammatory milieu restricted to the large intestine in this experimental model. S. Typhimurium alone induced significant changes at the alpha- (Simpson's and Shannon's indexes) and beta-diversity levels, specifically at the peak of inflammation in the large intestine and feces. Increased community dispersion and volatility in colonic apex and fecal microbiomes were also noted for S. Typhimurium. All three Salmonella serovars altered community structure as measured by co-occurrence networks; this was most prominent at DPI 2 and 4 in colonic apex samples. At the genus taxonomic level, a diverse array of putative short-chain fatty acid (SCFA) producing bacteria were altered and often decreased during the peak of inflammation at DPI 2 and 4 within colonic apex and fecal samples. Among all putative SCFA producing bacteria, Prevotella showed a broad pattern of negative correlation with disease scores at the peak of inflammation. In addition, Prevotella 9 was found to be significantly reduced in all Salmonella infected groups compared to the control at DPI 4 in the colonic apex. In conclusion, this work further elucidates that distinct swine-related zoonotic serovars of S. enterica can induce both shared (high resilience) and unique (altered resistance) alterations in gut microbiome biogeography, which helps inform future investigations of dietary modifications aimed at increasing colonization resistance against Salmonella through GI microbiome alterations.

Electroacupuncture reverses ethanol-induced locomotor sensitization and subsequent pERK expression in mice.

Extracellular signal-regulated kinase (ERK) plays a role in neuronal changes induced by repeated drug exposure. Given that electroacupuncture reverses locomotor sensitization induced by ethanol, we investigated whether this effect is parallel to ERK signalling. Mice received daily ethanol (2 g/kg i.p), for 21 d. Electroacupuncture was performed daily, during four (subsequent) days of ethanol withdrawal. The stimulus of 2 Hz or 100 Hz was provided in combinations of two acupoints: Ea1 (ST-36/Zusanli and PC-6/Neiguan) or Ea2 (Du-14/Dazhui and Du-20/Baihui). The specificity of acupoint effects were assessed by the inclusion of additional groups: Ea3 (ST-25/Tianshu--acupoint used for other non-related disorders), Sham1 or Sham2 (transdermic stimulation near the respective acupoints). The control group was only handled during withdrawal and the saline group was chronically treated with saline and handled similarly to controls. At day 5 of withdrawal, each group was divided in two subgroups, according to the presence or absence of ethanol challenge. The animals were perfused and their brains processed for pERK immunohistochemistry. Only Ea1 at 100 Hz (Ea1_100) and Ea2 at 2 Hz (Ea2_2) reversed locomotor sensitization induced by ethanol. Ethanol withdrawal decreases pERK in the dorsomedial striatum. This decrease is not abolished by electroacupuncture. Conversely, ethanol challenge increases pERK in the dorsomedial striatum, infralimbic cortex and central nucleus of amygdala. The specificity of acupoint stimulation to reverse these increases was seen only for Ea2_2, in the infralimbic cortex and dorsomedial striatum. Therefore, behavioural effects of Ea2_2 (but not Ea1_100) depend, at least in part, on ERK signalling.

Systems-Based Approach for Optimization of Assembly-Free Bacterial MLST Mapping.

Epidemiological surveillance of bacterial pathogens requires real-time data analysis with a fast turnaround, while aiming at generating two main outcomes: (1) species-level identification and (2) variant mapping at different levels of genotypic resolution for population-based tracking and surveillance, in addition to predicting traits such as antimicrobial resistance (AMR). Multi-locus sequence typing (MLST) aids this process by identifying sequence types (ST) based on seven ubiquitous genome-scattered loci. In this paper, we selected one assembly-dependent and one assembly-free method for ST mapping and applied them with the default settings and ST schemes they are distributed with, and systematically assessed their accuracy and scalability across a wide array of phylogenetically divergent Public Health-relevant bacterial pathogens with available MLST databases. Our data show that the optimal k-mer length for stringMLST is species-specific and that genome-intrinsic and -extrinsic features can affect the performance and accuracy of the program. Although suitable parameters could be identified for most organisms, there were instances where this program may not be directly deployable in its current format. Next, we integrated stringMLST into our freely available and scalable hierarchical-based population genomics platform, ProkEvo, and further demonstrated how the implementation facilitates automated, reproducible bacterial population analysis.

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations.

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried out by Public Health laboratories and regulatory agencies. Large volumes of publicly available WGS data can be used to study pathogenic populations at a large scale. Recently, a freely available computational platform called ProkEvo was published to enable reproducible, automated, and scalable hierarchical-based population genomic analyses using bacterial WGS data. This implementation of ProkEvo demonstrated the importance of combining standard genotypic mapping of populations with mining of accessory genomic content for ecological inference. In particular, the work highlighted here used ProkEvo-derived outputs for population-scaled hierarchical analyses using the R programming language. The main objective was to provide a practical guide for microbiologists, ecologists, and epidemiologists by showing how to: i) use a phylogeny-guided mapping of hierarchical genotypes; ii) assess frequency distributions of genotypes as a proxy for ecological fitness; iii) determine kinship relationships and genetic diversity using specific genotypic classifications; and iv) map lineage differentiating accessory loci. To enhance reproducibility and portability, R markdown files were used to demonstrate the entire analytical approach. The example dataset contained genomic data from 2,365 isolates of the zoonotic foodborne pathogen Salmonella Newport. Phylogeny-anchored mapping of hierarchical genotypes (Serovar -> BAPS1 -> ST -> cgMLST) revealed the population genetic structure, highlighting sequence types (STs) as the keystone differentiating genotype. Across the three most dominant lineages, ST5 and ST118 shared a common ancestor more recently than with the highly clonal ST45 phylotype. ST-based differences were further highlighted by the distribution of accessory antimicrobial resistance (AMR) loci. Lastly, a phylogeny-anchored visualization was used to combine hierarchical genotypes and AMR content to reveal the kinship structure and lineage-specific genomic signatures. Combined, this analytical approach provides some guidelines for conducting heuristic bacterial population genomic analyses using pan-genomic information.

ProkEvo: an automated, reproducible, and scalable framework for high-throughput bacterial population genomics analyses.

Whole Genome Sequence (WGS) data from bacterial species is used for a variety of applications ranging from basic microbiological research, diagnostics, and epidemiological surveillance. The availability of WGS data from hundreds of thousands of individual isolates of individual microbial species poses a tremendous opportunity for discovery and hypothesis-generating research into ecology and evolution of these microorganisms. Flexibility, scalability, and user-friendliness of existing pipelines for population-scale inquiry, however, limit applications of systematic, population-scale approaches. Here, we present ProkEvo, an automated, scalable, reproducible, and open-source framework for bacterial population genomics analyses using WGS data. ProkEvo was specifically developed to achieve the following goals: (1) Automation and scaling of complex combinations of computational analyses for many thousands of bacterial genomes from inputs of raw Illumina paired-end sequence reads; (2) Use of workflow management systems (WMS) such as Pegasus WMS to ensure reproducibility, scalability, modularity, fault-tolerance, and robust file management throughout the process; (3) Use of high-performance and high-throughput computational platforms; (4) Generation of hierarchical-based population structure analysis based on combinations of multi-locus and Bayesian statistical approaches for classification for ecological and epidemiological inquiries; (5) Association of antimicrobial resistance (AMR) genes, putative virulence factors, and plasmids from curated databases with the hierarchically-related genotypic classifications; and (6) Production of pan-genome annotations and data compilation that can be utilized for downstream analysis such as identification of population-specific genomic signatures. The scalability of ProkEvo was measured with two datasets comprising significantly different numbers of input genomes (one with ~2,400 genomes, and the second with ~23,000 genomes). Depending on the dataset and the computational platform used, the running time of ProkEvo varied from ~3-26 days. ProkEvo can be used with virtually any bacterial species, and the Pegasus WMS uniquely facilitates addition or removal of programs from the workflow or modification of options within them. To demonstrate versatility of the ProkEvo platform, we performed a hierarchical-based population structure analyses from available genomes of three distinct pathogenic bacterial species as individual case studies. The specific case studies illustrate how hierarchical analyses of population structures, genotype frequencies, and distribution of specific gene functions can be integrated into an analysis. Collectively, our study shows that ProkEvo presents a practical viable option for scalable, automated analyses of bacterial populations with direct applications for basic microbiology research, clinical microbiological diagnostics, and epidemiological surveillance.

Captive Common Marmosets (Callithrix jacchus) Are Colonized throughout Their Lives by a Community of Bifidobacterium Species with Species-Specific Genomic Content That Can Support Adaptation to Distinct Metabolic Niches.

The common marmoset (Callithrix jacchus) is an omnivorous New World primate whose diet in the wild includes large amounts of fruit, seeds, flowers, and a variety of lizards and invertebrates. Marmosets also feed heavily on tree gums and exudates, and they have evolved unique morphological and anatomical characteristics to facilitate gum feeding (gummivory). In this study, we characterized the fecal microbiomes of adult and infant animals from a captive population of common marmosets at the Callitrichid Research Center at the University of Nebraska at Omaha under their normal dietary and environmental conditions. The microbiomes of adult animals were dominated by species of Bifidobacterium, Bacteroides, Prevotella, Phascolarctobacterium, Megamonas, and Megasphaera. Culturing and genomic analysis of the Bifidobacterium populations from adult animals identified four known marmoset-associated species (B. reuteri, B. aesculapii, B. myosotis, and B. hapali) and three unclassified taxa of Bifidobacterium that are phylogenetically distinct. Species-specific quantitative PCR (qPCR) confirmed that these same species of Bifidobacterium are abundant members of the microbiome throughout the lives of the animals. Genomic loci in each Bifidobacterium species encode enzymes to support growth and major marmoset milk oligosaccharides during breastfeeding; however, metabolic islands that can support growth on complex polysaccharide substrates in the diets of captive adults (pectin, xyloglucan, and xylan), including loci in B. aesculapii that can support its unique ability to grow on arabinogalactan-rich tree gums, were species-specific. IMPORTANCEBifidobacterium species are recognized as important, beneficial microbes in the human gut microbiome, and their ability colonize individuals at different stages of life is influenced by host, dietary, environmental, and ecological factors, which is poorly understood. The common marmoset is an emerging nonhuman primate model with a short maturation period, making this model amenable to study the microbiome throughout a life history. Features of the microbiome in captive marmosets are also shared with human gut microbiomes, including abundant populations of Bifidobacterium species. Our studies show that several species of Bifidobacterium are dominant members of the captive marmoset microbiome throughout their life history. Metabolic capacities in genomes of the marmoset Bifidobacterium species suggest species-specific adaptations to different components of the captive marmoset diet, including the unique capacity in B. aesculapii for degradation of gum arabic, suggesting that regular dietary exposure in captivity may be important for preserving gum-degrading species in the microbiome.

Genome Sequences of Uncommon Shiga Toxin-Producing Escherichia coli Serotypes.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne disease with worldwide outbreaks. STEC serotypes O157, O26, O45, O103, O111, O121, and O145 cause the most outbreaks. There is little published information regarding the other serotypes. We report the draft genome sequences for 11 uncommon STEC serotypes from Nebraska.

Mucosal immunity and gut microbiota in dogs with chronic enteropathy.

Chronic enteropathy (CE) in dogs is a chronic and relapsing immunopathology, of unknown etiology, that usually manifests with a plethora of clinical signs reflecting the underlying heterogeneity in its pathogenesis. Alterations of the mucosal immune responses and the gut microbiota composition are thought to play an essential role in CE. Similar to humans, it is hypothesized that the breakdown in mucosal tolerance leads to aberrant and pathological immune responses toward the gut microbiota, that in turn, may contribute to the severity of disease, at least for certain CE subsets. Therefore, in this review, we discuss some of the most relevant and recent insights microbiological and immunological aspects characterizing CE in dogs.

The microbiota protects from viral-induced neurologic damage through microglia-intrinsic TLR signaling.

Symbiotic microbes impact the function and development of the central nervous system (CNS); however, little is known about the contribution of the microbiota during viral-induced neurologic damage. We identify that commensals aid in host defense following infection with a neurotropic virus through enhancing microglia function. Germfree mice or animals that receive antibiotics are unable to control viral replication within the brain leading to increased paralysis. Microglia derived from germfree or antibiotic-treated animals cannot stimulate viral-specific immunity and microglia depletion leads to worsened demyelination. Oral administration of toll-like receptor (TLR) ligands to virally infected germfree mice limits neurologic damage. Homeostatic activation of microglia is dependent on intrinsic signaling through TLR4, as disruption of TLR4 within microglia, but not the entire CNS (excluding microglia), leads to increased viral-induced clinical disease. This work demonstrates that gut immune-stimulatory products can influence microglia function to prevent CNS damage following viral infection.

Experimental evaluation of the importance of colonization history in early-life gut microbiota assembly.

The factors that govern assembly of the gut microbiota are insufficiently understood. Here, we test the hypothesis that inter-individual microbiota variation can arise solely from differences in the order and timing by which the gut is colonized early in life. Experiments in which mice were inoculated in sequence either with two complex seed communities or a cocktail of four bacterial strains and a seed community revealed that colonization order influenced both the outcome of community assembly and the ecological success of individual colonizers. Historical contingency and priority effects also occurred in Rag1-/- mice, suggesting that the adaptive immune system is not a major contributor to these processes. In conclusion, this study established a measurable effect of colonization history on gut microbiota assembly in a model in which host and environmental factors were strictly controlled, illuminating a potential cause for the high levels of unexplained individuality in host-associated microbial communities.

Commensal Escherichia coli Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production.

Escherichia coli is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal E. coli abundance in the intestine during inflammation; however, host immunological responses toward commensal E. coli during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal E. coli isolated from healthy conventional microbiota mice and representing distinct populations of E. coli elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-γ was a hallmark of the severe inflammation induced by E. coli strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with E. coli strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of E. coli-free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and in vivo neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage.

Lifestyle and Horizontal Gene Transfer-Mediated Evolution of Mucispirillum schaedleri, a Core Member of the Murine Gut Microbiota.

Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights into its lifestyle, we analyzed the genome and transcriptome of M. schaedleri ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant, M. schaedleri has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally, M. schaedleri reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species in vivo, which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note, M. schaedleri harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The M. schaedleri genome has been shaped by extensive horizontal gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria, indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem. IMPORTANCE Shifts in gut microbiota composition have been associated with intestinal inflammation, but it remains unclear whether inflammation-associated bacteria are commensal or detrimental to their host. Here, we studied the lifestyle of the gut bacterium Mucispirillum schaedleri, which is associated with inflammation in widely used mouse models. We found that M. schaedleri has specialized systems to handle oxidative stress during inflammation. Additionally, it expresses secretion systems and effector proteins and can modify the mucosal gene expression of its host. This suggests that M. schaedleri undergoes intimate interactions with its host and may play a role in inflammation. The insights presented here aid our understanding of how commensal gut bacteria may be involved in altering susceptibility to disease.

A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice.

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions.