Alexandra Proctor

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.

Resistant Starch Alters the Microbiota-Gut Brain Axis: Implications for Dietary Modulation of Behavior

The increasing recognition that the gut microbiota plays a central role in behavior and cognition suggests that the manipulation of microbial taxa through diet may provide a means by which behavior may be altered in a reproducible and consistent manner in order to achieve a beneficial outcome for the host. Resistant starch continues to receive attention as a dietary intervention that can benefit the host through mechanisms that include altering the intestinal microbiota. Given the interest in dietary approaches to improve health, the aim of this study was to investigate whether the use of dietary resistant starch in mice to alter the gut microbiota also results in a change in behavior. Forty-eight 6 week-old male Swiss-Webster mice were randomly assigned to 3 treatment groups (n = 16 per group) and fed either a normal corn starch diet (NCS) or diets rich in resistant starches HA7 diet (HA7) or octenyl-succinate HA7 diet (OS-HA7) for 6 week and monitored for weight, behavior and fecal microbiota composition. Animals fed an HA7 diet displayed comparable weight gain over the feeding period to that recorded for NCS-fed animals while OS-HA7 displayed a lower weight gain as compared to either NCS or HA7 animals (ANOVA p = 0.0001; NCS:HA7 p = 0.244; HA7:OS-HA7 p<0.0001; NCS:OS-HA7 p<0.0001). Analysis of fecal microbiota using 16s rRNA gene taxonomic profiling revealed that each diet corresponded with a unique gut microbiota. The distribution of taxonomic classes was dynamic over the 6 week feeding period for each of the diets. At the end of the feeding periods, the distribution of taxa included statistically significant increases in members of the phylum Proteobacteria in OS-HA7 fed mice, while the Verrucomicrobia increased in HA7 fed mice over that of mice fed OS-HA7. At the class level, members of the class Bacilli decreased in the OS-HA7 fed group, and Actinobacteria, which includes the genus Bifidobacteria, was enriched in the HA7 fed group compared to the control diet. Behavioral analysis revealed that animals demonstrated profound anxiety-like behavior as observed by performance on the elevated-plus maze with time spent by the mice in the open arm (ANOVA p = 0.000; NCS:HA7 p = 0.004; NCS:OS-HA7 p = 1.000; HA7:OS-HA7 p = 0.0001) as well as entries in the open arm (ANOVA p = 0.039; NCS:HA7 p = 0.041; HA7:OS-HA7 p = 0.221; NCS:OS-HA7 p = 1.000). Open-field behavior, a measure of general locomotion and exploration, revealed statistically significant differences between groups in locomotion as a measure of transitions across quadrant boundaries. Additionally, the open-field assay revealed decreased exploration as well as decreased rearing in HA7 and OS-HA7 fed mice demonstrating a consistent pattern of increased anxiety-like behavior among these groups. Critically, behavior was not correlated with weight. These results indicate that diets based on resistant starch can be utilized to produce quantifiable changes in the gut microbiota and should be useful to “dial-in” a specific microbiome that is unique to a particular starch composition. However, undesirable effects can also be associated with resistant starch, including lack of weight gain and increased anxiety-like behaviors. These observations warrant careful consideration when developing diets rich in resistant starch in humans and animal models.

Social Influences on Prevotella and the Gut Microbiome of Young Monkeys

Objective Our aim was to evaluate the bacterial profiles of young monkeys as they were weaned into peer groups with a particular focus on Prevotella, an important taxon in both human and nonhuman primates. The weaning of infants and increased social contact with peers is a developmental stage that is likely to affect the gut microbiome. Methods Gut bacteria were assessed in 63 rhesus monkeys living in social groups comprised of 4 to 7 individuals. Two groups were assessed prospectively on day 1 and 2 weeks after rehousing away from the mother and group formation. Ten additional groups were assessed at 2 weeks after group establishment. Fecal genomic DNA was extracted and 16S ribosomal RNA sequenced by Illumina MiSeq (5 social groups) and 454-amplicon pyrosequencing (7 social groups). Results Combining weaned infants into small social groups led to a microbial convergence by 2 weeks (p < .001). Diversity analyses indicated more similar community structure within peer groups than across groups (p < .01). Prevotella was the predominant taxon, and its abundance differed markedly across individuals. Indices of richness, microbial profiles, and less abundant taxa were all associated with the Prevotella levels. Functional Kyoto Encyclopedia of Genes and Genomes analyses suggested corresponding shifts in metabolic pathways. Conclusions The formation of small groups of young rhesus monkeys was associated with significant shifts in the gut microbiota. The profiles were closely associated with the abundance of Prevotella, a predominant taxon in the rhesus monkey gut. Changes in the structure of the gut microbiome are likely to induce differences in metabolic and physiologic functioning.

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.

Altered Schaedler flora mice: An old defined microbiota animal model to study the new science of microbiota-gut-brain axis

&NA; Despite considerable attention, the mechanisms by which the microbiota affect brain function and host behaviour via the gut‐brain axis remain undefined. Identifying microbe‐specific pathways that influence neuronal function and bi‐directional communication between the gut microbiota and the host central nervous system is challenging due to the extreme microbial diversity in the gut of conventionally‐reared mice. Herein, we describe the use of the altered Schaedler flora (ASF) mouse model as an alternative to conventionally‐reared and germ‐free animals. Colonized with only 8 bacterial species, use of ASF mice greatly simplifies the examination of microbiota‐host interactions. We assessed the extent to which behaviour differed between mice with a limited consortium of bacteria compared with a complex, conventional microbiota. The elevated plus maze and open‐field assays were utilized to assess murine behaviour. Histological analysis of ileum and colon was performed to evaluate intestinal morphology, and 16 s rRNA gene taxonomic profiling was performed to determine host‐stress induced changes in fecal microbial communities. Behavioural and serum corticosterone differences were observed between ASF and conventionally‐reared mice, while no differences were found between the intestinal morphology of these two groups. The stress of the behavioural tests induced significant changes in the ASF fecal microbial community but not in that of the conventionally‐reared mice. In contrast to the conventionally‐reared mice, the results indicated that the ASF mice displayed a marked anxiogenic‐like behaviour. These data indicate that ASF mice represent a unique model to elucidate mechanisms governing microbiota‐gut‐brain communication affecting behaviour.

Low Lactobacilli abundance and polymicrobial diversity in the lower reproductive tract of female rhesus monkeys do not compromise their reproductive success

The lower reproductive tract of nonhuman primates is colonized with a diverse microbiota, resembling bacterial vaginosis (BV), a gynecological condition associated with negative reproductive outcomes in women. Our 4 aims were to: (i) assess the prevalence of low Lactobacilli and a BV‐like profile in female rhesus monkeys; (ii) quantify cytokines in their cervicovaginal fluid (CVF); (iii) examine the composition and structure of their mucosal microbiota with culture‐independent sequencing methods; and (iv) evaluate the potential influence on reproductive success. CVF specimens were obtained from 27 female rhesus monkeys for Grams staining, and to determine acidity (pH), and quantify proinflammatory cytokines. Based on Nugents classification, 40% had a score of 7 or higher, which would be indicative of BV in women. Nugent scores were significantly correlated with the pH of the CVF. Interleukin‐1ß was present at high concentrations, but not further elevated by high Nugent scores. Vaginal swabs were obtained from eight additional females to determine microbial diversity by rRNA gene amplicon sequencing. At the phylum level, the Firmicutes/Bacteroidetes ratio was low. The relative abundance of Lactobacilli was also low (between 3% and 17%), and 11 other genera were present at >1%. However, neither the microbial diversity in the community structure, nor high Nugent scores, was associated with reduced fecundity. Female monkeys provide an opportunity to understand how reproductive success can be sustained in the presence of a diverse polymicrobial community in the reproductive tract.

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.