The Microbial Metabolite Butyrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT10B Expression.

Nutritional supplementation with probiotics can prevent pathologic bone loss. Here we examined the impact of supplementation with Lactobacillus rhamnosus GG (LGG) on bone homeostasis in eugonadic young mice. Micro-computed tomography revealed that LGG increased trabecular bone volume in mice, which was due to increased bone formation. Butyrate produced in the gut following LGG ingestion, or butyrate fed directly to germ-free mice, induced the expansion of intestinal and bone marrow (BM) regulatory T (Treg) cells. Interaction of BM CD8+ T cells with Treg cells resulted in increased secretion of Wnt10b, a bone anabolic Wnt ligand. Mechanistically, Treg cells promoted the assembly of a NFAT1-SMAD3 transcription complex in CD8+ cells, which drove expression of Wnt10b. Reducing Treg cell numbers, or reconstitution of TCRß-/- mice with CD8+ T cells from Wnt10b-/- mice, prevented butyrate-induced bone formation and bone mass acquisition. Thus, butyrate concentrations regulate bone anabolism via Treg cell-mediated regulation of CD8+ T cell Wnt10b production.

Indoles from the commensal microbiota act via the AHR and IL-10 to tune the cellular composition of the colonic epithelium during aging.

The intestinal epithelium is a highly dynamic structure that rejuvenates in response to acute stressors and can undergo alterations in cellular composition as animals age. The microbiota, acting via secreted factors related to indole, appear to regulate the sensitivity of the epithelium to stressors and promote epithelial repair via IL-22 and type I IFN signaling. As animals age, the cellular composition of the intestinal epithelium changes, resulting in a decreased proportion of goblet cells in the colon. We show that colonization of young or geriatric mice with bacteria that secrete indoles and various derivatives or administration of the indole derivative indole-3 aldehyde increases proliferation of epithelial cells and promotes goblet cell differentiation, reversing an effect of aging. To induce goblet cell differentiation, indole acts via the xenobiotic aryl hydrocarbon receptor to increase expression of the cytokine IL-10. However, the effects of indoles on goblet cells do not depend on type I IFN or on IL-22 signaling, pathways responsible for protection against acute stressors. Thus, indoles derived from the commensal microbiota regulate intestinal homeostasis, especially during aging, via mechanisms distinct from those used during responses to acute stressors. Indoles may have utility as an intervention to limit the decline of barrier integrity and the resulting systemic inflammation that occurs with aging.

Commensal microbiota-induced redox signaling activates proliferative signals in the intestinal stem cell microenvironment.

A distinct taxon of the Drosophila microbiota, Lactobacillus plantarum, is capable of stimulating the generation of reactive oxygen species (ROS) within cells, and inducing epithelial cell proliferation. Here, we show that microbial-induced ROS generation within Drosophila larval stem cell compartments exhibits a distinct spatial distribution. Lactobacilli-induced ROS is strictly excluded from defined midgut compartments that harbor adult midgut progenitor (AMP) cells, forming a functional 'ROS sheltered zone' (RSZ). The RSZ is undiscernible in germ-free larvae, but forms following monocolonization with L. plantarumL. plantarum is a strong activator of the ROS-sensitive CncC/Nrf2 signaling pathway within enterocytes. Enterocyte-specific activation of CncC stimulated the proliferation of AMPs, which demonstrates that pro-proliferative signals are transduced from enterocytes to AMPs. Mechanistically, we show that the cytokine Upd2 is expressed in the gut following L. plantarum colonization in a CncC-dependent fashion, and may function in lactobacilli-induced AMP proliferation and intestinal tissue growth and development.

Butyrate supplementation to pregnant mice elicits cytoprotection against colonic injury in the offspring.

BACKGROUND: Maternal diet during pregnancy can impact progeny health and disease by influencing the offspring's gut microbiome and immune development. Gut microbial metabolism generates butyrate, a short-chain fatty acid that benefits intestinal health. Here we assess the effects of antenatal butyrate on the offspring's gastrointestinal health. We hypothesized that antenatal butyrate supplementation will induce protection against colitis in the offspring. METHODS: C57BL/6 mice received butyrate during pregnancy and a series of experiments were performed on their offspring. RNA sequencing was performed on colonic tissue of 3-week-old offspring. Six-8-week-old offspring were subjected to dextran sulfate sodium-induced colitis. Fecal microbiome analysis was performed on the 6-8-week-old offspring. RESULTS: Antenatal butyrate supplementation dampened transcript enrichment of inflammation-associated colonic genes and prevented colonic injury in the offspring. Antenatal butyrate increased the offspring's stool microbiome diversity and expanded the prevalence of specific gut microbes. CONCLUSIONS: Antenatal butyrate supplementation resulted in downregulation of genes in the offspring's colon that function in inflammatory signaling. In addition, antenatal butyrate supplementation was associated with protection against colitis and an expanded fecal microbiome taxonomic diversity in the offspring. IMPACT: Dietary butyrate supplementation to pregnant mice led to downregulation of colonic genes involved in inflammatory signaling and cholesterol synthesis, changes in the fecal microbiome composition of the offspring, and protection against experimentally induced colitis in the offspring. These data support the mounting evidence that the maternal diet during pregnancy has enduring effects on the offspring's long-term health and disease risk. Although further investigations are needed to identify the mechanism of butyrate's effects on fetal gut development, the current study substantiates the approach of dietary intervention during pregnancy to optimize the long-term gastrointestinal health of the offspring.

Lactococcus lactis Subspecies cremoris Elicits Protection Against Metabolic Changes Induced by a Western-Style Diet.

BACKGROUND & AIMS: A Western-style diet, which is high in fat and sugar, can cause significant dyslipidemia and nonalcoholic fatty liver disease; the diet has an especially strong effect in women, regardless of total calorie intake. Dietary supplementation with beneficial microbes might reduce the detrimental effects of a Western-style diet. We assessed the effects of Lactococcus lactis subspecies (subsp) cremoris on weight gain, liver fat, serum cholesterol, and insulin resistance in female mice on a high-fat, high-carbohydrate diet. METHODS: Female C57BL/6 mice were fed either a high-fat, high-carbohydrate (Western-style) diet that contained 40% fat (mostly milk fat) and 43% carbohydrate (mostly sucrose) or a calorie-matched-per-gram control diet. The diets of mice were supplemented with 1 × 109 colony-forming units of L lactis subsp cremoris ATCC 19257 or Lactobacillus rhamnosus GG ATCC 53103 (control bacteria) 3 times per week for 16 weeks. Body weights were measured, and fecal, blood, and liver tissues were collected and analyzed. Livers were analyzed for fat accumulation and inflammation, and blood samples were analyzed for cholesterol and glucose levels. Mice were housed within Comprehensive Lab Animal Monitoring System cages, and respiratory exchange ratio and activity were measured. Hepatic lipid profiles of L lactis subsp cremoris-supplemented mice were characterized by lipidomics mass spectrometry analysis. RESULTS: Mice fed L lactis subsp cremoris while on the Western-style diet gained less weight, developed less hepatic steatosis and inflammation, and had a lower mean serum level of cholesterol and body mass index than mice fed the control bacteria. Mice fed the L lactis subsp cremoris had increased glucose tolerance while on the Western-style diet compared to mice fed control bacteria and had alterations in hepatic lipids, including oxylipins. CONCLUSIONS: Dietary supplementation with L lactis subsp cremoris in female mice on a high-fat, high-carbohydrate (Western-style) diet caused them to gain less weight, develop less liver fat and inflammation, reduce serum cholesterol levels, and increase glucose tolerance compared with mice on the same diet fed control bacteria. L lactis subsp cremoris is safe for oral ingestion and might be developed for persons with metabolic and liver disorders caused by a Western-style diet.

Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair.

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Vibrio parahaemolyticus VopA Is a Potent Inhibitor of Cell Migration and Apoptosis in the Intestinal Epithelium of Drosophila melanogaster.

Animal models have played a key role in providing an understanding of the mechanisms that govern the pathophysiology of intestinal diseases. To expand on the repertoire of organisms available to study enteric diseases, we report on the use of the Drosophila melanogaster model to identify a novel function of an effector protein secreted by Vibrio parahaemolyticus, which is an enteric pathogen found in contaminated seafood. During pathogenesis, V. parahaemolyticus secretes effector proteins that usurp the host's innate immune signaling pathways, thus allowing the bacterium to evade detection by the innate immune system. One secreted effector protein, VopA, has potent inhibitory effects on mitogen-activated protein kinase (MAPK) signaling pathways via the acetylation of critical residues within the catalytic loops of mitogen-activated protein kinase kinases (MAPKKs). Using the Drosophila model and cultured mammalian cells, we show that VopA also has potent modulating activity on focal adhesion complex (FAC) proteins, where VopA markedly reduced the levels of focal adhesion kinase (FAK) phosphorylation at Ser910, whereas the phosphorylation levels of FAK at Tyr397 and Tyr861 were markedly increased. Cultured cells expressing VopA were also impaired in their ability to migrate and repopulate areas subjected to a scratch wound. Consistently, expression of VopA in Drosophila midgut enterocytes disrupted the normal enterocyte arrangement. Finally, VopA inhibited apoptosis in both Drosophila tissues and mammalian cultured cells. Together, our data show that VopA can alter normal intestinal homeostatic processes to facilitate opportunities for V. parahaemolyticus to prolong infection within the host.

Anemia induces gut inflammation and injury in an animal model of preterm infants.

BACKGROUND: While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. STUDY DESIGN AND METHODS: The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). RESULTS: Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. CONCLUSIONS: Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Cosmc is an X-linked inflammatory bowel disease risk gene that spatially regulates gut microbiota and contributes to sex-specific risk.

Inflammatory bowel disease (IBD) results from aberrant immune stimulation against a dysbiotic mucosal but relatively preserved luminal microbiota and preferentially affects males in early onset disease. However, factors contributing to sex-specific risk and the pattern of dysbiosis are largely unexplored. Core 1 ß3GalT-specific molecular chaperone (Cosmc), which encodes an X-linked chaperone important for glycocalyx formation, was recently identified as an IBD risk factor by genome-wide association study. We deleted Cosmc in mouse intestinal epithelial cells (IECs) and found marked reduction of microbiota diversity in progression from the proximal to the distal gut mucosa, but not in the overlying lumen, as seen in IBD. This loss of diversity coincided with local emergence of a proinflammatory pathobiont and distal gut restricted pathology. Mechanistically, we found that Cosmc regulates host genes, bacterial ligands, and nutrient availability to control microbiota biogeography. Loss of one Cosmc allele in males (IEC-Cosmc-/y) resulted in a compromised mucus layer, spontaneous microbe-dependent inflammation, and enhanced experimental colitis; however, females with loss of one allele and mosaic deletion of Cosmc in 50% of crypts (IEC-Cosmc+/-) were protected from spontaneous inflammation and partially protected from experimental colitis, likely due to lateral migration of normal mucin glycocalyx from WT cells over KO crypts. These studies functionally validate Cosmc as an IBD risk factor and implicate it in regulating the spatial pattern of dysbiosis and sex bias in IBD.

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species.

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

The Influence of the Gut Microbiota on Host Physiology: In Pursuit of Mechanisms.

The results generated from the NIH funded Human Microbiome Project (HMP) are necessarily tied to the overall mission of the agency, which is to foster scientific discoveries as a basis for protecting and improving health. The investment in the HMP phase 1 accomplished many of its goals including the preliminary characterization of the human microbiome and the identification of links between microbiome diversity and disease states. Going forward, the next step in these studies must involve the identification of the functional molecular elements that mediate the positive influence of a eubiotic microbiome on health and disease. This review will focus on recent advances describing mechanistic events in the intestine elicited by the microbiome. These include symbiotic bacteria-induced activation of redox-dependent cell signaling, the bacterial production of short chain fatty acids and ensuing cellular responses, and the secretion of bacteriocins by bacteria that have anti-microbial activities against potential pathogens.

Epithelial adhesion mediated by pilin SpaC is required for Lactobacillus rhamnosus GG-induced cellular responses.

Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.

A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

Gender-affirming hormone therapy preserves skeletal maturation in young mice via the gut microbiome.

Gender-affirming hormone therapy (GAHT) is often prescribed to transgender (TG) adolescents to alleviate gender dysphoria, but the effect of GAHT on the growing skeleton is unclear. We found GAHT to improve trabecular bone structure via increased bone formation in young male mice and not to affect trabecular structure in female mice. GAHT modified gut microbiome composition in both male and female mice. However, fecal microbiota transfers (FMTs) revealed that GAHT-shaped gut microbiome was a communicable regulator of bone structure and turnover in male, but not in female mice. Mediation analysis identified 2 species of Bacteroides as significant contributors to the skeletal effects of GAHT in male mice, with Bacteroides supplementation phenocopying the effects of GAHT on bone. Bacteroides have the capacity to expand Treg populations in the gut. Accordingly, GAHT expanded intestinal Tregs and stimulated their migration to the bone marrow (BM) in male but not in female mice. Attesting to the functional relevance of Tregs, pharmacological blockade of Treg expansion prevented GAHT-induced bone anabolism. In summary, in male mice GAHT stimulated bone formation and improved trabecular structure by promoting Treg expansion via a microbiome-mediated effect, while in female mice, GAHT neither improved nor impaired trabecular structure.

The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo.

The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild-type S. typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null-infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signalling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens.

Aeromonas salmonicida-secreted protein AopP is a potent inducer of apoptosis in a mammalian and a Drosophila model.

Some pathogens are able to establish themselves within the host because they have evolved mechanisms to disrupt host innate immunity. For example, a number of pathogens secrete preformed effector proteins via type III secretion apparatuses that influence innate immune or apoptotic signalling pathways. One group of effector proteins that usurp innate immune signalling is the YopJ-like family of bacterial effector proteins, which includes AopP from Aeromonas salmonicida. Aeromonas species are known to cause gastrointestinal disease in humans, and are associated mainly with subcutaneous wound infections and septicaemia in other metazoans, particularly fish. AopP has been reported to have inhibitory activity against the NF-κB pathway in cultured cells, although the pathological outcomes of AopP activity have not been examined. Here, we show that AopP has potent pro-apoptotic activity when expressed in cultured mammalian macrophage or epithelial cells, or when ectopically expressed in Drosophila melanogaster haemocytes or imaginal disk epithelial cells. Furthermore, apoptosis was significantly elevated upon concurrent AopP expression and TNF-α cellular stimulation. Together, our results demonstrate how the specificity of a YopJ-like protein towards signalling pathways directly governs cellular pathological outcome in disease.

Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1-mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

Intestinal barrier dysfunction in murine sickle cell disease is associated with small intestine neutrophilic inflammation, oxidative stress, and dysbiosis.

The intestinal microbiome has emerged as a potential contributor to the severity of sickle cell disease (SCD). We sought to determine whether SCD mice exhibit intestinal barrier dysfunction, inflammation, and dysbiosis. Using the Townes humanized sickle cell mouse model, we found a 3-fold increase in intestinal permeability as assessed via FITC-dextran (4 kDa) assay in SS (SCD) mice compared to AA (wild type) mice (n = 4, p < 0.05). This was associated with 25 to 50% decreases in claudin-1, 3, and 15 and zonula occludens-1 gene expression (n = 8-10, p < 0.05) in the small intestine. Increased Ly6G staining demonstrated more neutrophils in the SS small intestine (3-fold, n = 5, p < 0.05) associated with increased expression of TNFα, IL-17A, CXCL1, and CD68 (2.5 to 5-fold, n = 7-10, p < 0.05). In addition, we observed 30 to 55% decreases in superoxide dismutase-1, glutathione peroxidase-1, and catalase antioxidant enzyme expression (n = 7-8, p < 0.05) concomitant to an increase in superoxide (2-fold, n = 4, p < 0.05). Importantly, all significant observations of a leaky gut phenotype and inflammation were limited to the small intestine and not observed in the colon. Finally, characterization of the composition of the microbiome within the small intestine revealed dysbiosis in SS mice compared to their AA littermates with 47 phyla to species-level significant alterations in amplicon sequence variants. We conclude that the intestinal barrier is compromised in SCD, associated with decreased gene expression of tight junction proteins, enhanced inflammation, oxidative stress, and gut microbiome dysbiosis, all specific to the small intestine.

Microbial Changes occurring during oronasal fistula wound healing.

The oral microbiome is a complex community that matures with dental development while oral health is also a recognized risk factor for systemic disease. Despite the oral cavity having a substantial microbial burden, healing of superficial oral wounds occurs quickly and with little scarring. By contrast, creation of an oro-nasal fistula (ONF), often occurring after surgery to correct a cleft palate, is a significant wound healing challenge that is further complicated by a connection of the oral and nasal microbiome. In this study, we characterized the changes in the oral microbiome of mice following a freshly inflicted wound in the oral palate that results in an open and unhealed ONF. Creation of an ONF in mice significantly lowered oral microbiome alpha diversity, with concurrent blooms of Enterococcus faecalis, Staphylococcus lentus, and Staphylococcus xylosus in the oral cavity. Treatment of mice with oral antibiotics one week prior to ONF infliction resulted in a reduction in the alpha diversity, prevented E. faecalis and S. lentus, and S. xylosus blooms, but did not impact ONF healing. Strikingly, delivery of the beneficial microbe Lactococcus lactis subsp. cremoris (LLC) to the wound bed of the freshly inflicted ONF via a PEG-MAL hydrogel vehicle resulted in rapid healing of the ONF. Healing of the ONF was associated with the maintenance of relatively high microbiome alpha diversity, and limited the abundance of E. faecalis and S. lentus, and S. xylosus in the oral cavity. These data demonstrate that a freshly inflicted ONF in the murine palate is associated with a dysbiotic oral microbiome state that may prevent ONF healing, and a bloom of opportunistic pathogens. The data also demonstrate that delivery of a specific beneficial microbe, LLC, to the ONF can boost wound healing, can restore and/or preserve oral microbiome diversity, and inhibit blooms of opportunistic pathogens.

Fecal microbiota transplantation promotes reduction of antimicrobial resistance by strain replacement.

Multidrug-resistant organism (MDRO) colonization is a fundamental challenge in antimicrobial resistance. Limited studies have shown that fecal microbiota transplantation (FMT) can reduce MDRO colonization, but its mechanisms are poorly understood. We conducted a randomized, controlled trial of FMT for MDRO decolonization in renal transplant recipients called PREMIX (NCT02922816). Eleven participants were enrolled and randomized 1:1 to FMT or an observation period followed by delayed FMT if stool cultures were MDRO positive at day 36. Participants who were MDRO positive after one FMT were treated with a second FMT. At last visit, eight of nine patients who completed all treatments were MDRO culture negative. FMT-treated participants had longer time to recurrent MDRO infection versus PREMIX-eligible controls who were not treated with FMT. Key taxa (Akkermansia muciniphila, Alistipes putredinis, Phocaeicola dorei, Phascolarctobacterium faecium, Alistipes species, Mesosutterella massiliensis, Barnesiella intestinihominis, and Faecalibacterium prausnitzii) from the single feces donor used in the study that engrafted in recipients and metabolites such as short-chain fatty acids and bile acids in FMT-responding participants uncovered leads for rational microbiome therapeutic and diagnostic development. Metagenomic analyses revealed a previously unobserved mechanism of MDRO eradication by conspecific strain competition in an FMT-treated subset. Susceptible Enterobacterales strains that replaced baseline extended-spectrum ß-lactamase-producing strains were not detectable in donor microbiota manufactured as FMT doses but in one case were detectable in the recipient before FMT. These data suggest that FMT may provide a path to exploit strain competition to reduce MDRO colonization.