Exoelectrogenic capacity of host microbiota predicts lymphocyte recruitment to the gut.
Ericsson, A.C., Davis, D.J., Franklin, C.L., and C.E. Hagan. (2015) Exoelectrogenic capacity of host microbiota predicts lymphocyte recruitment to the gut. Physiological Genomics, 47(7): 243-252. PMC4491528
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Electrotaxis, directional cell movement in response to an electric potential, has been demonstrated in a wide range of cell types including lymphocytes. Exoelectrogens, microorganisms capable of generating electrical currents, have been identified in microbial fuel cells. However, no studies have investigated exoelectrogenic microbes in fresh feces, or the effects of an exoelectrogenic microbiota on the host organism. Here we show that commensal gut microbial populations differ in their capacity for electrical current production by exoelectrogens, and that those differences are predictive of increased lymphocyte trafficking to the gut in vivo, despite the lack of increased production of canonical lymphocyte-specific chemokines. Additionally, we demonstrate that the difference in current production between mice purchased from different commercial sources correlates reproducibly with the presence or absence of segmented filamentous bacteria, and while our data do not support a direct role for SFB in ex vivo current production, an exoelectrogenic microbiota can be transferred in vivo via mucosa-associated bacteria present in the ileum. Moreover, we detect upregulation of microbial genes associated with extracellular electron transfer in feces of mice colonized with exoelectrogenic microbiota containing segmented filamentous bacteria. While still correlative, these results suggest a novel means by which the gut microbiota modulates the recruitment of cells of the immune system to the gut.
Manipulating the gut microbiota: methods and challenges.
Ericsson, A.C. and C.L. Franklin. (2015) Manipulating the gut microbiota: methods and challenges. ILAR Journal, 56(2): 205-217. PMC4554251
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Animal models provide a means whereby causal relationships between characteristic differences in the GM and diseases or conditions can be formally tested, using genetically identical animals in highly controlled environments. Clearly, the GM and its interactions with the host and myriad environmental factors are exceedingly complex and it is rare that a single microbial taxon associates with, much less causes, a phenotype with perfect sensitivity and specificity. Moreover, while the exact numbers are the subject of debate, it is well-recognized that only a minority of gut bacteria can be successfully cultured ex vivo. Thus, to perform studies investigating causal roles of the GM in animal model phenotypes, researchers need clever techniques to experimentally manipulate the GM of animals, and several ingenious methods of doing so have been developed, each providing its own type of information and with its own set of advantages and drawbacks. The current review will focus on the various means of experimentally manipulating the GM of research animals, drawing attention to the factors which would aid a researcher in selecting an experimental approach, and with an emphasis on mice and rats, the primary model species used to evaluate the contribution of the GM to a disease phenotype.
Isolation of segmented filamentous bacteria from a complex gut microbiota.
Ericsson, A.C., Turner, G., Montoya, L., Wolfe, A., Meeker, S., Hsu, C., Maggio-Price, L., and C.L. Franklin. (2015) Isolation of segmented filamentous bacteria from a complex gut microbiota. BioTechniques, 59(2): 94-98. PMID: 26260088 (PMCID in process)
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Segmented filamentous bacteria (SFB) modulate the ontogeny of the immune system, and their presence can significantly affect mouse models of disease. Until recently, the inability to culture SFB has made controlled studies of the mechanisms by which SFB exert their influence problematic. Herein, we demonstrate a means of selecting for SFB from a complex microbial mixture, providing researchers a simple and cost-effective means to prepare pure infective inocula for prospective studies, and compare individual isolates of SFB.
Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota.
Ericsson, A.C., Akter, S., Hanson, M., Busi, S.B., Parker, T., Schehr, R., Hankins, M., Ahner, C.E., Davis, J.W., Franklin, C.L., Amos-Landgraf, J., and E.C. Bryda. (2015) Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota. Oncotarget, 6(32):33689-33704. PMID:26378041 (PMCID in process)
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To evaluate the role of qualitatively different but naturally occurring gut microbiota and the relationship with colorectal cancer development, genetically identical embryos from the Polyposis in Rat Colon (Pirc) rat model of colorectal cancer were transferred into recipients of three different genetic backgrounds (F344/NHsd, LEW/SsNHsd, and Crl:SD). Our data show that the gut microbiota varies between rat strains, with LEW/SsNHsd having a greater relative abundance of the bacteria Prevotella copri. Both male and female F344-Pirc rats harboring the Lewis microbiota had decreased tumor burden relative to genetically identical rats harboring F344 or SD microbiota. Significant negative correlations were detected between tumor burden and the relative abundance of specific taxa from samples taken at weaning and shortly thereafter, prior to observable adenoma development. Notably, this naturally occurring variation in the gut microbiota is associated with a significant difference in severity of colorectal cancer, and the abundance of certain taxa is associated with decreased tumor burden.
Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing.
Hart, M.L., Meyer, A., Johnson, P.J., and A.C. Ericsson. (2015) Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing. PLoS One, 10(11): e0143334. PMC4657925
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To determine the influence of extraction technique on the success of PCA amplification and 16S RNA amplicon sequencing, we investigated the use of five common DNA extraction methods on fecal samples from five different species. Our data show that the method of DNA extraction impacts DNA concentration and purity and successful NGS amplification, and influences microbial communities detected in sequencing output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method when designing and interpreting data from cross species studies.
TNFR2 Deficiency Acts in Concert with Gut Microbiota to Precipitate Sex-biased Spontaneous CNS Demyelinating Autoimmune Disease.
Miller, P.G., Bonn, M.B., Franklin, C.L., Ericsson, A.C., and S.C. McKarns. (2015) TNFR2 Deficiency Acts in Concert with Gut Microbiota to Precipitate Sex-biased Spontaneous CNS Demyelinating Autoimmune Disease. Journal of Immunology, 195(10):4668-4684. PMID:26475926 (PMCID in process)
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Tumor necrosis factor alpha (TNF) antagonists are beneficial to patients with inflammatory autoimmune diseases, including Crohn’s disease, rheumatoid arthritis, and ankylosing spondylitis. Paradoxically, anti-TNF therapeutics exacerbate CNS demyelinating autoimmune diseases such as multiple sclerosis (MS), neuromyelitis optica (NMO), and optic neuritis (ON). The underlying mechanisms remain enigmatic. Here, we demonstrate that genetic deficiency of TNF receptor type 2 (TNFR2) in myelin oligodendrocyte glycoprotein (MOG) 35-55–specific TCR (2D2) transgenic mice results in a highly penetrant female-biased spontaneous inflammatory demyelination that histologically resembles autoimmune NMO, also called Devic’s disease. Antibiotic treatment resulted in almost complete abrogation of clinical signs in female mice, and sequencing the V4 region of fecal bacterial 16S rRNA of TNFR2-/- 2D2 and 2D2 mice revealed overlapping, but distinct, gut bacterial communities that associated with sexed-bias autoimmune disease susceptibility.
Composition and predicted metabolic capacity of upper and lower airway microbiota of healthy dogs in relation to the fecal microbiota.
Ericsson, A.C., Personett, A.R., Grobman, M.E., Rindt, H., and C.R. Reinero. (2016) Composition and predicted metabolic capacity of upper and lower airway microbiota of healthy dogs in relation to the fecal microbiota. PLoS One, 11(5): e0154646. PMID:27136381 (PMCID in process)
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The upper and lower airways of healthy humans are reported to harbor stable and consistent bacterial populations, and the composition of these communities is altered in individuals affected with several respiratory diseases. Data regarding the presence of airway microbiota in other animals are scant and a better understanding of the composition and metabolic function of such bacterial populations is essential for the development of novel therapeutic and diagnostic modalities for use in both veterinary and human medicine. Based on targeted next-generation sequencing of feces and samples collected at multiple levels of the airways from 16 healthy female dogs, we demonstrate that canine airways harbor a topographically continuous microbiota with increasing relative abundance of proteobacterial species from the upper to lower airways. The lung-associated microbiota, as assessed via bronchoalveolar lavage fluid (BALF), was the most consistent between dogs and was dominated by three distinct taxa, two of which were resolved to the species level and one to the level of family. The gene content of the nasal, oropharyngeal, and lung-associated microbiota, predicted using the Phylogenetic Investigations into Communities by Reconstruction of Unobserved States (PICRUSt) software, provided information regarding the glyoxylate and citrate cycle metabolic pathways utilized by these bacterial populations to colonize such nutrient-poor, low-throughput environments. These data generated in healthy subjects provide context for future analysis of diseased canine airways. Moreover, as dogs have similar respiratory anatomy, physiology, and immune systems as humans, are exposed to many of the same environmental stimuli, and spontaneously develop similar respiratory diseases, these data support the use of dogs as a model species for prospective studies of the airway microbiota, with findings translatable to the human condition.
16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae.
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Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days post fertilization (dpf), these “conventionalized” larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled “Microbial modulation of behavior and stress responses in zebrafish larvae”.
Microbial modulation of behavior and stress responses in zebrafish larvae.
Davis, D.J., Bryda, E.C., Gillespie, C.H., and A.C. Ericsson. (2016) Microbial modulation of behavior and stress responses in zebrafish larvae. Behavioural Brain Research, 311:219-227. PMID:27217102 (PMCID in progress)
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The influence of the microbiota on behavior and stress responses is poorly understood. Zebrafish larvae have unique characteristics that are advantageous for neuroimmune research, however, they are currently underutilized for such studies. Here, we used germ-free zebrafish to determine the effects of the microbiota on behavior and stress testing. The absence of a microbiota dramatically altered locomotor and anxiety-related behavior. Additionally, characteristic responses to an acute stressor were also obliterated in larvae lacking exposure to microbes. Moreover, these phenomena were not able to be reverted by exposure to exogenous bacterial metabolites introduced via immersion. Lastly, treatment with the probiotic Lactobacillus plantarum was sufficient to attenuate anxiety-related behavior in conventionally-raised zebrafish larvae. These results underscore the importance of the microbiota in communicating to the CNS via the microbiome-gut-brain axis and set a foundation for using zebrafish larvae for neuroimmune research.
Vaccinating with conserved E. coli antigens does not alter the mouse intestinal microbiome.
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Enterotoxigenic Escherichia coli (ETEC) causes diarrheal disease. Antigenic and structural heterogeneity among ETEC colonization factors has complicated vaccine development efforts. Identifying and characterizing conserved ETEC antigens that induce protective immunity is therefore of interest. We previously characterized three proteins (MipA, Skp, and ETEC_2479) that protected mice in an intranasal ETEC challenge model after vaccination. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. While the impact of inactivated viral vaccines and live-attenuated bacterial vaccines on the host microbiota have been examined, the potential impact of using subunit vaccines consisting of antigens that are also encoded by commensal organisms has not been investigated. We addressed this issue by characterizing changes to mouse intestinal microbiomes as a function of vaccination. We failed to observe significant changes to mouse health, to mouse weight gain as a function of time, or to the diversity or richness of mouse intestinal microbiomes, as measured by analyzing alpha- and beta-diversity, as well as overall community structure, before and after vaccination.
Evaluation of fecal microbiota transfer as treatment for post-weaning diarrhea in research-colony puppies.
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Puppies in a research setting frequently have diarrheal disease just prior to weaning or at weaning (approximately 8 to 10 weeks of age). This may be due, in part, to an immature and unstable intestinal microbiota that is permissive to an opportunistic pathogen such as a Coccidian species. The overall objective of this study was to assess whether fecal microbiota transfer (FMT) would result in more rapid transmission of stable maternal microbiota to pups and a lower frequency of post-weaning diarrhea. Puppies were designated by litter as treated (FMT) or sham-treated. The FMT group was administered fecal inoculum for 5 consecutive days during weaning. Diarrhea in both litters was evaluated using the Nestlé Purina Fecal Scoring System for 10 days during the weaning period. Fresh feces were collected from dams and puppies at 3 days prior to weaning and 3, 10, and 24 days post weaning for analysis of the fecal microbiota using 16S rRNA amplicon sequencing. The composition of fecal inoculum refrigerated at temperatures between 3 and 5°C was stable for at least 5 days. No diarrhea was reported in either group during the study period, making comparison of treated and control groups problematic. 16S rRNA gene analysis, however, did reveal microbial variability across time in both. Thus, while neither group appeared to mirror the dam at any of the designated time points during the study, the data provided fundamental and novel information regarding the dynamic instability of the fecal microbiota of puppies following weaning.
Lactobacillus plantarum attenuates anxiety-related behavior and protects against stress-induced dysbiosis in adult zebrafish.
Davis, D.J., Doerr, H.M., Grzelak, A.K., Busi, S.B., Jasarevic, E., Ericsson, A.C., and E.C. Bryda. (2016) Lactobacillus plantarum attenuates anxiety-related behavior and protects against stress-induced dysbiosis in adult zebrafish. Scientific Reports, 6:33726. PMC5027381
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The consumption of probiotics has become increasingly popular as a means to try to improve health and well-being. Not only are probiotics considered beneficial to digestive health, but increasing evidence suggests direct and indirect interactions between gut microbiota (GM) and the central nervous system (CNS). Here, adult zebrafish were supplemented with Lactobacillus plantarum to determine the effects of probiotic treatment on structural and functional changes of the GM, as well as host neurological and behavioral changes. L. plantarum administration altered the β-diversity of the GM while leaving the major core architecture intact. These minor structural changes were accompanied by significant enrichment of several predicted metabolic pathways. In addition to GM modifications, L. plantarum treatment also significantly reduced anxiety-related behavior and altered GABAergic and serotonergic signaling in the brain. Lastly, L. plantarum supplementation provided protection against stress-induced dysbiosis of the GM. These results underscore the influence commensal microbes have on physiological function in the host, and demonstrate bidirectional communication between the GM and the host.
The effect of vendor and genetic background on the fecal microbiota of inbred mice
There is abundant evidence that the gut microbiota of inbred laboratory mice may modulate the phenotype of disease models. It is recognized that the gut microbiota of mice differs dependent on genetic background and commercial source of mice. Using culture-independent methods, we demonstrate significant differences in the richness, diversity, and composition of fecal microbiota in mice purchased from two large commercial vendors. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota.
Segmented filamentous bacteria: commensal microbes with potential effects on research
Segmented filamentous bacteria (SFB) represent long-recognized commensal bacteria capable of modulating mouse models of disease, due largely to effects on the ontogeny of the adaptive immune system. Herein we review the basic biology of SFB and their impact on mouse models of disease.