Meghan Wymore Brand

The Altered Schaedler Flora: Continued Applications of a Defined Murine Microbial Community

The gastrointestinal (GI) microbiota forms a mutualistic relationship with the host through complex and dynamic interactions. Because of the complexity and interindividual variation of the GI microbiota, investigating how members of the microbiota interact with each other, as well as with the host, is daunting. The altered Schaedler flora (ASF) is a model community of eight microorganisms that was developed by R.P. Orcutt and has been in use since the late 1970s. The eight microorganisms composing the ASF were all derived from mice, can be cultured in vitro, and are stably passed through multiple generations (at least 15 years or more by the authors) in gnotobiotic mice continually bred in isolator facilities. With the limitations associated with conventional, mono- or biassociated, and germfree mice, use of mice colonized with a consortium of known bacteria that naturally inhabit the murine gut offers a powerful system to investigate mechanisms governing host-microbiota relationships, and how members of the GI microbiota interact with one another. The ASF community offers significant advantages to study homeostatic as well as disease-related interactions by taking advantage of a well-defined, limited community of microorganisms. For example, quantification and spatial distribution of individual members, microbial genetic manipulation, genomic-scale analysis, and identification of microorganism-specific host immune responses are all achievable using the ASF model. This review compiles highlights associated with the 37-year history of the ASF, including descriptions of its continued use in biomedical research to elucidate the complexities of host-microbiome interactions in health and disease.

Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice.

Background:Helicobacter bilis infection of C3H/HeN mice harboring the altered Schaedler flora (ASF) triggers progressive immune responsiveness and the development of colitis. We sought to investigate temporal alterations in community structure of a defined (ASF-colonized) microbiota in normal and inflamed murine intestines and to correlate microbiota changes to histopathologic lesions. Methods:The colonic mucosal microbiota of healthy mice and ASF mice colonized with H. bilis for 3, 6, or 12 weeks were investigated by fluorescence in situ hybridization targeting the 16S ribosomal RNA genes of total bacteria, group-specific organisms, and individual ASF bacterial species. Microbial profiling of ASF and H. bilis abundance was performed on cecal contents. Results:Helicobacter bilis–colonized mice developed colitis associated with temporal changes in composition and spatial distribution of the mucosal microbiota. The number of total bacteria, ASF519, and helicobacter-positive bacteria were increased (P < 0.05), whereas ASF360/361-positive bacteria were decreased (P < 0.05) versus controls. Adherent biofilms in colitic mice were most often (P < 0.05) composed of total bacteria, ASF457, and H. bilis. Total numbers of ASF519 and H. bilis bacteria were positively correlated (P = 0.03, r = 0.39 and P < 0.0001, r = 0.73), and total numbers of ASF360/361 bacteria were negatively correlated (P = 0.003, r = −0.53) to histopathologic score. Differences in cecal abundance of ASF members were not observed. Conclusions:Altered community structure with murine colitis is characterized by distinct ASF bacteria that interact with the colonic mucosa, by formation of an isolating interlaced layer, by attachment, or by invasion, and this interaction is differentially expressed over time.

Orally administered extract from Prunella vulgaris attenuates spontaneous colitis in mdr1a(-/-) mice.

AIM To investigate the ability of a Prunella vulgaris (P. vulgaris) ethanolic extract to attenuate spontaneous typhlocolitis in mdr1a(-/-) mice. METHODS Vehicle (5% ethanol) or P. vulgaris ethanolic extract (2.4 mg/d) were administered daily by oral gavage to mdr1a(-/-) or wild type FVB(WT) mice from 6 wk of age up to 20 wk of age. Clinical signs of disease were noted by monitoring weight loss. Mice experiencing weight loss in excess of 15% were removed from the study. At the time mice were removed from the study, blood and colon tissue were collected for analyses that included histological evaluation of lesions, inflammatory cytokine levels, and myeloperoxidase activity. RESULTS Administration of P. vulgaris extracts to mdr1a(-/-) mice delayed onset of colitis and reduced severity of mucosal inflammation when compared to vehicle-treated mdr1a(-/-) mice. Oral administration of the P. vulgaris extract resulted in reduced (P < 0.05) serum levels of IL-10 (4.6 ± 2 vs 19.4 ± 4), CXCL9 (1319.0 ± 277 vs 3901.0 ± 858), and TNFα (9.9 ± 3 vs 14.8 ± 1) as well as reduced gene expression by more than two-fold for Ccl2, Ccl20, Cxcl1, Cxcl9, IL-1α, Mmp10, VCAM-1, ICAM, IL-2, and TNFα in the colonic mucosa of mdr1a(-/-) mice compared to vehicle-treated mdr1a(-/-) mice. Histologically, several microscopic parameters were reduced (P < 0.05) in P. vulgaris-treated mdr1a(-/-) mice, as was myeloperoxidase activity in the colon (2.49 ± 0.16 vs 3.36 ± 0.06, P < 0.05). The numbers of CD4(+) T cells (2031.9 ± 412.1 vs 5054.5 ± 809.5) and germinal center B cells (2749.6 ± 473.7 vs 4934.0 ± 645.9) observed in the cecal tonsils of P. vulgaris-treated mdr1a(-/-) were significantly reduced (P < 0.05) from vehicle-treated mdr1a(-/-) mice. Vehicle-treated mdr1a(-/-) mice were found to produce serum antibodies to antigens derived from members of the intestinal microbiota, indicative of severe colitis and a loss of adaptive tolerance to the members of the microbiota. These serum antibodies were greatly reduced or absent in P. vulgaris-treated mdr1a(-/-) mice. CONCLUSION The anti-inflammatory activity of P. vulgaris ethanolic extract effectively attenuated the severity of intestinal inflammation in mdr1a(-/-) mice.

Pathogenic and non-pathogenic Escherichia coli colonization and host inflammatory response in a defined microbiota mouse model

ABSTRACT Most Escherichia coli strains in the human intestine are harmless. However, enterohemorrhagic E. coli (EHEC) is a foodborne pathogen that causes intestinal disease in humans. Conventionally reared (CONV) mice are inconsistent models for human infections with EHEC because they are often resistant to E. coli colonization, in part due to their gastrointestinal (GI) microbiota. Although antibiotic manipulation of the mouse microbiota has been a common means to overcome colonization resistance, these models have limitations. Currently, there are no licensed treatments for clinical EHEC infections and, thus, new tools to study EHEC colonization need to be developed. Here, we used a defined microbiota mouse model, consisting of the altered Schaedler flora (ASF), to characterize intestinal colonization and compare host responses following colonization with EHEC strain 278F2 or non-pathogenic E. coli strain MG1655. Significantly higher (P<0.05) levels of both strains were found in feces and cecal and colonic contents of C3H/HeN ASF compared to C3H/HeN CONV mice. GI inflammation was significantly elevated (P<0.05) in the cecum of EHEC 278F2-colonized compared to E. coli MG1655-colonized C3H/HeN ASF mice. In addition, EHEC 278F2 differentially modulated inflammatory-associated genes in colonic tissue of C3H/HeN ASF mice compared to E. coli MG1655-colonized mice. This approach allowed for prolonged colonization of the murine GI tract by pathogenic and non-pathogenic E. coli strains, and for evaluation of host inflammatory processes. Overall, this system can be used as a powerful tool for future studies to assess therapeutics, microbe-microbe interactions, and strategies for preventing EHEC infections. Summary: Mice harboring a defined microbiota of the altered Schaedler flora were identified as a reliable tool to assess prolonged Escherichia coli intestinal colonization and activation of the host inflammatory response.

Su1872 Age-Related Influence of Colonization by an AIEC Pathobiont on the Severity of DSS-Induced Colitis

Background:The gastrointestinal microbiota is initially acquired during birth and evolves throughout the postnatal period reaching a mature microbial community (i.e., composition and abundance) over time. In this regard, few studies have evaluated the impact of neonatal colonization by a pathobiont

Mo1776 Increased Severity of DSS-Induced Colitis Following Vertical Transmission of AIEC Pathobiont Associated With Altered Mucosal Homeostasis and Bacterial Community Dynamics

Background:The gastrointestinalmicrobiota is inherited during birth and evolves throughout the postnatal period. Few studies have chronologically evaluated changes in the heritable structure of the microbiota following introduction of a pathobiont. LF82 is an adherent invasive E. coli (AIEC) strain that has been associated with ileal Crohns disease. Gnotobiotic C3H/HeN:TAC mice harboring the altered Schaedler flora (ASF) were utilized as a model to evaluate changes in the microbiota and in the sensitivity to dextran sodium sulfate (DSS)induced colitis following colonization with LF82 by vertical transmission. We hypothesized that ASF mice colonized with LF82 by vertical transmission (F1) would be less susceptible to DSS-induced inflammation compared to ASF mice colonized with LF82 as adults (F0). Methods: Adult ASF mice were colonized with LF82 by oral gavage and bred to produce F1 mice colonized with LF82 from birth. F1 and F0 mice (i.e., colonized with LF82 at 8 weeks of age) were given 2.0 % DSS in drinking water for seven days. At necropsy, colonic tissues, serum, and mesenteric lymph nodes (MLN) were harvested to evaluate the severity of intestinal inflammation and magnitude of immunologic responses. DNA extracted from feces and cecal contents was used for HTP sequencing analysis of microbial abundance. Results: More severe macroscopic and histologic inflammation was present in the F1 + DSS mice when compared to F0 + DSS mice. The mucosal lesions in the F1 + DSS mice were characterized by marked ulceration, inflammatory cell infiltrate, glandular hyperplasia and stromal collapse. Diffuse, transmural mucosal inflammation was observed in most of the F1 + DSS mice. In severely diseased F1 + DSS mice, colitis was accompanied by elevated levels of IFNγ and IL-17 secreted from colonic explants and antigen-stimulated CD4+ T cells recovered from the MLNs. Evaluation of serum for presence of ASF-specific antibody revealed decreased antibody responses in F1 compared to F0 mice. Based on 16s rRNA gene sequence taxonomic profiling, colonization of the ASF mice with LF82 altered the structure of the microbial community as evidenced by significant changes in the abundance of L. murinus (ASF361) and M. schaedleri (ASF457). The abundance of LF82 was increased in the F1 compared to F0 mice and was further increased following DSS treatment. Conclusions: We demonstrated that 1) LF82 persistently colonized ASF mice and was transmissible from dam to offspring, 2) LF82 colonization altered the dynamics of the ASF community, and 3) ASF mice colonized with LF82 from birth develop more severe DSS-induced colitis as compared to mice colonized with LF82 as a young adult. These data suggest that the age at which an individual is colonized by an AIEC pathobiont will differentially impact (i.e., predisposes) the hosts susceptibility to subsequent inflammatory insults.

Mo1708 Serum-Derived Bovine Immunoglobulin/Protein Isolate Attenuated DSSInduced Colitis in a Defined Floral Model

Introduction. A colovesical fistulas present an important clinical problems. They usually occur as a complication of diverticulosis, IBD or malignant process. The pentadecapeptide BPC 157 , in clinical trials for IBD therapy, has already shown the effectiveness in healing colocutaneous fistulas ( J. Pharmacol Sci 2008 ) , gastrocutaneous fistulas ( Dig Dis Sci 2009 ). Therefore we suggest it as a possible therapy of colovesical fistulas. Matherials and methods. Wistar Albino rats were randomly assigned, at 5 cm from anocutanous borderline, with the diameter of 3 mm. The surgical procedure was performed according to rules brought by the Local Ethical Committee. Medication of BPC 157 (10ug/kg, 10ng/kg) was per-orally, in drinkingwater and intraperitoneally, once daily, the first dose immediately after the operation, the last dose at 24 h before sacrifice, at 7, 15, 30 post-operative day, with the biomechanical, functional, macroscopic and microscopic assessment. Results. Biomechanical analysis :we measured pressure of the fistulas rupture for all groups of animals with the system for central venous pressure assesment .In the control group pressures for fistulas rupture were similar for every experimental period : ≤1mL H2O, 1mL±0,3 mL, 2 mL±0,5 ml H2O. In the second group( BPC per-orally in drinking water 10ug/kg and 10ng/kg ) water pressures were 1mL±0,3; 2mL±0,3 and 5mL±0,5 but without fistulas rupture at the and of 30 days experimental period.In the third group( BPC intraperitoneally 10ug/kg and 10ng/kg ) water pressure was increased from 2mL±0,5mL until 5 mL±0,5 at the and of 15 days experimental period. In most animals fistulas were healed at the end of 30 days experimental period. Macroscopic analysis: We measured diameters of fistulas on the colonic and the bladder side.In the control group diameters of fistulas were similar for both sides for all experimental periods : 3mm ±0.5mm on the colon and bladder side,3mm±1mm and 2,5±0,5mm. In the second group fistulas diameter was 3±0,2 mm for both sides ( 7 days ),2mm±0,5 mm for colon and 2,5±0,5 mm for bladder ( 15 days ), 1,5mm±0,5 for colon, 2mm±0,5 mm for bladder side ( 30 days ). In the third group fistulas were reduced : 2,5mm±0,2mm colon, 3mm±0,5 bladder ( 7 days ), 1,5mm±0,5mm colon; 2mm±0,5mm bladder ( 15 days ) and at the end oft he 30 days experimental period in most animals fistulas were healed er reduced diameter at 1mm±0,3mm for both sides.. Conclusion. According to these results the pentadecapeptide BPC 157 could present a new possible pathway in therapy of colovesical fistulas.