The Murine Lung Microbiome Changes During Lung Inflammation and Intranasal Vancomycin Treatment.
ABSTRACT: Most microbiome research related to airway diseases has focused on the gut microbiome. This is despite advances in culture independent microbial identification techniques revealing that even healthy lungs possess a unique dynamic microbiome. This conceptual change raises the question; if lung diseases could be causally linked to local dysbiosis of the local lung microbiota. Here, we manipulate the murine lung and gut microbiome, in order to show that the lung microbiota can be changed experimentally. We have used four different approaches: lung inflammation by exposure to carbon nano-tube particles, oral probiotics and oral or intranasal exposure to the antibiotic vancomycin. Bacterial DNA was extracted from broncho-alveolar and nasal lavage fluids, caecum samples and compared by DGGE. Our results show that: the lung microbiota is sex dependent and not just a reflection of the gut microbiota, and that induced inflammation can change lung microbiota. This change is not transferred to offspring. Oral probiotics in adult mice do not change lung microbiome detectible by DGGE. Nasal vancomycin can change the lung microbiome preferentially, while oral exposure does not. These observations should be considered in future studies of the causal relationship between lung microbiota and lung diseases.
Project description:BACKGROUND:Gut dysbiosis due to the adverse effects of antibiotics affects outcomes of lung infection. Previous murine models relied on significant depletion of both gut and lung microbiota, rendering the analysis of immune gut-lung cross-talk difficult. Here, we study the effects of antibiotic-induced gut dysbiosis without lung dysbiosis on lung immunity and the consequences on acute P. aeruginosa lung infection. METHODS:C57BL6 mice received 7?days oral vancomycin-colistin, followed by normal regimen or fecal microbial transplant or Fms-related tyrosine kinase 3 ligand (Flt3-Ligand) over 2?days, and then intra-nasal P. aeruginosa strain PAO1. Gut and lung microbiota were studied by next-generation sequencing, and lung infection outcomes were studied at 24?h. Effects of vancomycin-colistin on underlying immunity and bone marrow progenitors were studied in uninfected mice by flow cytometry in the lung, spleen, and bone marrow. RESULTS:Vancomycin-colistin administration induces widespread cellular immunosuppression in both the lung and spleen, decreases circulating hematopoietic cytokine Flt3-Ligand, and depresses dendritic cell bone marrow progenitors leading to worsening of P. aeruginosa lung infection outcomes (bacterial loads, lung injury, and survival). Reversal of these effects by fecal microbial transplant shows that these alterations are related to gut dysbiosis. Recombinant Flt3-Ligand reverses the effects of antibiotics on subsequent lung infection. CONCLUSIONS:These results show that gut dysbiosis strongly impairs monocyte/dendritic progenitors and lung immunity, worsening outcomes of P. aeruginosa lung infection. Treatment with a fecal microbial transplant or immune stimulation by Flt3-Ligand both restore lung cellular responses to and outcomes of P. aeruginosa following antibiotic-induced gut dysbiosis.
Project description:Transplanting beneficial bacteria to the gut microbiome can positively modulate the bacterial composition and remains of great interest in prevention and treatment. However, environmental assaults and rapid transit times in the gastrointestinal (GI) tract result in low oral bioavailability and limited intestinal colonization. Here, we describe a bioinspired strategy of self-coating with biofilms that endows the transplanted gut microbiota with superior resistance and adhesion capacity. Using clinical Bacillus subtilis as a model probiotic bacterium, biofilm-coated probiotics demonstrate substantially improved GI tract tolerance and mucoadhesion in mice and swine. In particular, coated probiotics exhibit a 125-fold higher oral bioavailability and a 17 times greater intestinal colonization than uncoated bacteria in the porcine model. With notable ability to survive and reside in the GI tract, coated bacteria further show a significantly enhanced decolonization effect in mice colonized with Staphylococcus aureus. Self-coating with biofilms suggests a robust platform for oral doses of gut microbiota.
Project description:The gut microbial community (Gut microbiota) is known to impact metabolic functions as well as immune responses in our body. Diet plays an important role in determining the composition of the gut microbiota. Gut microbes help in assimilating dietary nutrients which are indigestible by humans. The metabolites produced by them not only modulate gastro-intestinal immunity, but also impact distal organs like lung and brain. Micro-aspiration of gut bacteria or movement of sensitized immune cells through lymph or bloodstream can also influence immune response of other organs. Dysbiosis in gut microbiota has been implicated in several lung diseases, including allergy, asthma and cystic fibrosis. The bi-directional cross-talk between gut and lung (termed as Gut-Lung axis) is best exemplified by intestinal disturbances observed in lung diseases. Some of the existing probiotics show beneficial effects on lung health. A deeper understanding of the gut microbiome which comprises of all the genetic material within the gut microbiota and its role in respiratory disorders is likely to help in designing appropriate probiotic cocktails for therapeutic applications.
Project description:The gut bacteria producing metabolites like short-chain fatty acids (SCFAs; e.g., acetate, propionate and butyrate), are frequently reduced in Patients with diabetes, obesity, autoimmune disorders, and cancers. Hence, microbiome modulators such as probiotics may be helpful in maintaining or even restoring normal gut microbiome composition to benefit host health. Herein, we developed a human-origin probiotic cocktail with the ability to modulate gut microbiota to increase native SCFA production. Following a robust protocol of isolation, characterization and safety validation of infant gut-origin Lactobacillus and Enterococcus strains with probiotic attributes (tolerance to simulated gastric and intestinal conditions, adherence to intestinal epithelial cells, absence of potential virulence genes, cell-surface hydrophobicity, and susceptibility to common antibiotics), we select 10 strains (5 from each genera) out of total 321 isolates. A single dose (oral gavage) as well as 5 consecutive doses of this 10-strain probiotic cocktail in mice modulates gut microbiome and increases SCFA production (particularly propionate and butyrate). Inoculation of these probiotics in human feces also increases SCFA production along with microbiome modulation. Results indicate that human-origin probiotic lactobacilli and enterococci could ameliorate gut microbiome dysbiosis and hence may prove to be a potential therapy for diseases involving reduced SCFAs production in the gut.
Project description:Adoptive T cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome or treatment with antibiotics, or by heterologous fecal transfer. Depletion of bacteria with vancomycin decreased the rate of tumor growth in mice from The Jackson Laboratory receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8?+ DCs, which sustained systemic adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest potentially new avenues to improve response to ACT by altering the gut microbiota.
Project description:To investigate the contribution of the gut microbiota to the pathogenesis of uveitis.Experimental autoimmune uveitis (EAU) in B10.RIII mice was induced using interphotoreceptor binding protein peptide. Mice were treated with oral or intraperitoneal (IP) antibiotics. Effector (Teff) and regulatory (Treg) T lymphocytes were identified using flow cytometry; 16S rRNA gene sequencing and qPCR were performed on gastrointestinal (GI) contents.Broad-spectrum (four antibiotics given simultaneously) oral, but not IP, antibiotics reduced mean uveitis clinical scores significantly compared with water-treated animals (0.5 vs. 3.0, P < 0.0001 for oral; 3.4 vs. 3.4, P > 0.99 for IP). Both oral metronidazole (P = 0.02) and vancomycin (P < 0.0001) alone decreased inflammation, whereas neomycin (P = 0.7) and ampicillin (P = 0.4) did not change mean uveitis scores. Oral broad-spectrum antibiotics increased Tregs in the GI lamina propria of EAU animals at 1 week, and in extraintestinal lymphoid tissues later, whereas Teff and inflammatory cytokines were reduced. 16S sequencing of GI contents revealed altered microbiota in immunized mice compared with nonimmunized mice, and microbial diversity clustering in EAU mice treated with uveitis-protective antibiotics. Experimental autoimmune uveitis mice also demonstrated gut microbial diversity clustering associated with clinical score severity.Oral antibiotics modulate the severity of inducible EAU by increasing Tregs in the gut and extraintestinal tissues, as well as decreasing effector T cells and cytokines. 16S sequencing suggests that there may be protective and, conversely, potentially uveitogenic, gut microbiota. These findings may lead to a better understanding of how uveitis can be treated or prevented by modulating the gut microbiome.
Project description:Many patients with alcohol use disorder (AUD) consume alcohol chronically and in large amounts that alter intestinal microbiota, damage the gastrointestinal tract, and thereby injure other organs via malabsorption and intestinal inflammation. We hypothesized that alcohol consumption and subsequent abstinence would change the gut microbiome in adults admitted to a treatment program. Stool and oral specimens, diet data, gastrointestinal assessment scores, anxiety, depression measures and drinking amounts were collected longitudinally for up to 4 weeks in 22 newly abstinent inpatients with AUD who were dichotomized as less heavy drinkers (LHD, <10 drinks/d) and very heavy drinkers (VHD, 10 or more drinks/d). Next-generation 16 S rRNA gene sequencing was performed to measure the gut and oral microbiome at up to ten time points/subject and LHD and VHD were compared for change in principal components, Shannon diversity index and specific genera. The first three principal components explained 46.7% of the variance in gut microbiome diversity across time and all study subjects, indicating the change in gut microbiome following abstinence. The first time point was an outlier in three-dimensional principal component space versus all other time points. The gut microbiota in LHD and VHD were significantly dissimilar in change from day 1 to day 5 (p = .03) and from day 1 to week 3 (p = .02). The VHD drinking group displayed greater change from baseline. The Shannon diversity index of the gut microbiome changed significantly during abstinence in five participants. In both groups, the Shannon diversity was lower in the oral microbiome than gut. Ten total genera were shared between oral and stool in the AUD participants. These data were compared with healthy controls from the Human Microbiome Project to investigate the concept of a core microbiome. Rapid changes in gut microbiome following abstinence from alcohol suggest resilience of the gut microbiome in AUD and reflects the benefits of refraining from the highest levels of alcohol and potential benefits of abstinence.
Project description:<i>Clostridium difficile</i> infection (CDI), a common cause of hospital-acquired infections, typically occurs after disruption of the normal gut microbiome by broad-spectrum antibiotics. Fidaxomicin is a narrow-spectrum antibiotic that demonstrates a reduced impact on the normal gut microbiota and is approved for the treatment of CDI. To further explore the benefits of this property, we used a murine model to examine the effects of fidaxomicin versus vancomycin on gut microbiota and susceptibility to <i>C. difficile</i> colonization while tracking microbiota recovery over time. Mice were exposed to fidaxomicin or vancomycin by oral gavage for 3 days and subsequently challenged with <i>C. difficile</i> spores at predetermined time points up to 21 days postexposure to antibiotics. Fecal samples were subsequently collected for analysis. Twenty-four hours postchallenge, mice were euthanized and the colon contents harvested. The microbiota was characterized using 16S rRNA gene sequencing. All fidaxomicin-exposed mice (except for one at day 8) were resistant to <i>C. difficile</i> colonization. However, 9 of 15 vancomycin-exposed mice were susceptible to <i>C. difficile</i> colonization until day 12. All vancomycin-exposed mice recovered colonization resistance by day 16. Bacterial diversity was similar prior to antibiotic exposure in both arms and decreased substantially after exposure. A shift in taxonomic structure and composition occurred after both exposures; however, the shift was greater in vancomycin-exposed than in fidaxomicin-exposed mice. In summary, compared with vancomycin, fidaxomicin exposure had less impact on microbiota composition, promoted faster microbial recovery, and had less impact on the loss of <i>C. difficile</i> colonization resistance.
Project description:Accumulating evidence supports that the intestinal microbiome is involved in Type 1 diabetes (T1D) pathogenesis through the gut-pancreas nexus. Our aim was to determine whether the intestinal microbiota in the non-obese diabetic (NOD) mouse model played a role in T1D through the gut. To examine the effect of the intestinal microbiota on T1D onset, we manipulated gut microbes by: (1) the fecal transplantation between non-obese diabetic (NOD) and resistant (NOR) mice and (2) the oral antibiotic and probiotic treatment of NOD mice. We monitored diabetes onset, quantified CD4+T cells in the Peyer's patches, profiled the microbiome and measured fecal short-chain fatty acids (SCFA). The gut microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased T-helper type 1 (Th1) and reduced Th17 cells in the intestinal lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal SCFAs. In an attempt to correct the diabetogenic microbiome, we administered VLS#3 probiotics to NOD mice but found that VSL#3 colonized the intestine poorly and did not delay diabetes. We conclude that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3 are insufficient to overcome the effects of a diabetogenic microbiome.
Project description:Epithelial cells in the intestinal mucosa maintain gut homeostasis by interacting with different types of microbiota. Proper appropriate immune responses in the intestinal epithelium are essential for the preservation of the intestinal homeostasis. In the present study, we aimed to identify genotypic and phenotypic changes in mice following oral feeding of various substances which has been shown to differentially affect intestinal homeostasis. We orally fed C57BL/6 mice for either one or seven days with one of the four substances: dextran sulfate sodium (DSS); Typhoid VI Polysaccharide vaccine (Vi vaccine); antibiotic cocktails (AB) of ampicillin, vancomycin, neomycin, and metronidazole; or(probiotics)consisting of Lactobacillus Rhamnosus R0011and L. Acidophilus R0052.While DSS and AB feeding resulted in severe gut pathology characterized by infiltration of inflammatory cells, epithelium shedding, and distortion of paneth cells. Vi vaccine and probiotics feeding resulted in phenotypic improvement of the gut health characterized by epithelial cell proliferation and increased formation of tight junctions between epithelial cells. Interestingly, microarray data showed significant increase in the expression levels of genes regulating cell proliferation and intestinal homeostasis in the gut epithelium of probiotics-and Vi vaccine-fed mice compared to DSS-or AB-fed mice. In addition, expression levels of genes regulating cell death and inflammation were significantly increased in the gut epithelium of DSS- and AB-fed mice. These results suggest that intestinal homeostasis play a pivotal role in maintaining gut health and, subsequently, in protecting host against enteric bacteria and external pathogens infection. Overall design: The design of our study was focused on clarify genotypic and phenotypic changes in mice following oral feeding of various substances which has been shown to differentially affect intestinal homeostasis. Mice were randomly divided into 5 groups according to body weight (n =10 for each group); WT (distilled water), DSS (only 4% DSS in the drinking water), Vi vaccine (Oral feeding with 0.006mg/ 200ul of Vi vaccine diluted in phosphate-buffered saline (PBS)), AB (only AB cocktails in the drinking water) and Probiotics mixture (oral feeding with live 2 × 109 CFU in 200ul PBS).The mice were oral fed with one of the four materials for one day or seven days and then switched to drinking water for 1 day.