Progress 10/02/14 to 09/30/19
Outputs
Target Audience:We have not published our results to date, but have submitted one manuscript, have another in preparation (experiments complete) and another awaiting sequencing data. Our target audience would be primarily researchers in the area of metabolism and immunity. However, this information will be pertinent to the average consumer once scientificpublications have been released. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has supported the training of 3 PhD students. These students participated in all aspects of both mouse and pig diet studies. They administered diets (daily feeding and husbandry), weighed the animals, performed back fat measurements on the pigs, collected feces for microbiota measurements, and blood for metabolic measurements. They learned how to process DNA for next generation sequencing and how to measure inflammatory mediatorsin fecal samples. Finally, one of the students has become competent at computationalanalysis ofmetagenomic sequence data. How have the results been disseminated to communities of interest?Three students Keah Higgins, Robert Johnson, and Haley Hallowell attended the Southeastern Regional Meeting of the American Society for Microbiology at Kennesaw State University in November of 2015. Each of them presented posters on specific aspects of this work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have collected fecal microbiota samples from two obesity studies. The first is in a set of 12 pigs and the second is a set of 45 mice. Both studies involved longitudinal collection of feces from animals on different diets. Pigs- Each group of 4 pigs was fed either a standard diet (limited based on weight), fed ad libetum, or fed a high fat diet ad libetum for ~4 months. Wecollected weights, backfat measurements, metabolic measurements, and fecal samples for microbiota measurements. The groups were significantly different each other in weight gain and in fat. However, there were no detectable differences in metabolic readouts such as blood sugar or cholesterol. The DNA samples are still in the sequencing pipeline, awaiting analysis. We plan to complete the analysis this spring and should have a manuscript submitted for publication this summer. Mice- each group of mice were fed either normal chow, a high fat diet, or a high fat diet supplemented with sugar water for 12 weeks. Feces were collected at 0, 2days, 2 weeks 4, 8, and 12 weeks for microbiota analysis. The samples have been collected and analyzed and there are some interesting trends. The mice fed high fat diets (with or without sugar) demonstrated overgrowth of certain bacterial families such as Verucomicrobiaceaeand Enterococcaceae, and losses in others such as Bacteroidacaceae andPrevotellacaceae. There were also several differences in phage abundance that we are now cross-analyzing with the bacterial data to look for predator-prey relationships. This work will be submitted as a collaborative study with Dr. Michael Greene in the late spring of 2017.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Integrative analysis of metabolic phenotype with intestinal bacteria and bacteriophage communities induced by high fat diet. Keah Higgins, Lauren Woodie, Haley Hallowell, Robert Johnson, Michael Greene, and Elizabeth Hiltbold Schwartz; Submitting to Nutrition.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Longitudinal analysis of intestinal microbiome during the development of obesity in a Mangalica pig model. Haley Hallowell, Keah Higgins, Robert Johnson, Terry Brandebourg, and Elizabeth Hiltbold Schwartz. Submitting to PLOS One
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Effects of inflammatory mediators on the dynamics of bacteria-bacteriophage interactions: insights from studies in vivo and in vitro. Keah Higgins, Haley Hallowell, Elizabeth Hiltbold Schwartz. For publication in Microbe
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:This research has been presented to scientific research communities attending two conferences in 2018, Boshell Diabetes Research Day and Southestern Regional Branch meeting of the American Association of Immunology. In attendance at these meetings are academic faculty researchers, post-doctoral researchers, graduate students, and undergraduates involved in research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students, Keah Higgins and Haley Hallowell, as well asseveral undergraduates have directly benefitted from this project. One of the major benefits of this project is the development of bioinformatic analysis tools and expertise by the graduate students in the lab. Keah and Haley have completed several workshops and courses on bioinformatic data analysis and have become quite adept in this capacity. In fact, these students have developed their own computational pipeline for analysis of metagenomic data. This will be incredibly important for the interpretation and subsequentpublication of these data sets. This is a new expertise to my laboratory and one that is incredibly important for our group moving forward. How have the results been disseminated to communities of interest?Keah Higgins and Haley Hallowell, bothPhD candidates,presented co-authored posters at two scientific conferences. Both of these presentations won awards. Effects of the High Fat Western Diet on Microbial and Viral Populations of the Murine Intestinal Microbiome"; Keah Higgins*, Lauren Woodie*, Haley Hallowell*, Michael Greene, Elizabeth Hiltbold Schwartz; Poster Presentationat Boshell Research Day, Boshell Diabetes and Metabolic Diseases Research Program; Auburn University Conference Center, March, 2018; Won 2nd place prize for poster. Poster Presentationat Southeast Regional meeting of the American Society for Microbiology; Georgia Tech Conference Center, 2018; Won 3rd place prize for poster. What do you plan to do during the next reporting period to accomplish the goals?Since the data have been collected on these studies, all that remains is to submit these manuscripts for publication. This will happen in 2019.
Impacts
What was accomplished under these goals?
This project is coming to fruition, having collected the data for three substantialprojects that address these goals: 1) An obesity study in young pigs 2) an obesity study in mice and 3) a gut inflammation study in mice. These projects are now in various stages of analysis and preparation for publication. 1) The obesity study in young pigs follows the changes in the gut microbiome of young mangalica pigs fed either a high fat diet or a standard diet over the course of 14 weeks. These studies were aimed at understanding just how rapidly the microbiome would change in response to diet change and increased weight and fat gain. The animals on the high fat diet gained significantly more weight and back fat, but failed to display any metabolic abnormalities such as insulin resistance or diabetes and had no overt signs of inflammation. However, the constituents of the microbiome changed signifcantly over control as early as 2 weeks after diet change. The early analysis of the microbiome metagenomeindicates that: In terms of bacteria, we saw decreases in the families Prevotellaceae and Streptococcaceae in the High Fat group as compared to the controlgroups. In terms of bacteriophage, we saw fluctuations in individuals who target the generaStreptococcusandStaphylococcusin the High Fat group as compared to control. Specifically in the High Fat group, the genera Sfi21 (Strep), P68 (Strep), and AHJD (Staph) -like viruses are quickly depleted post-diet change, while 1706-like viruses were seen to increase post-diet change.Taken together, our study suggests that changes in microbiome precede changes in metabolism and detectable inflammation during the development of obesity. This study will be submitted for publication in March 2019. 2) Our second study of obesity in mice included three groups of mice fed different diets, standard chow, high fat chow, or high fat chow plus sugar water over 12 weeks. We have completed these studies which include physiological, metabolic, and metagenomic analyses of these animals and have also completed the computational analysis of the microbiomes. We found some very interesting differences induced by the high fat diet not only in the bacterial communities, but also in the bacteriophage that prey on the bacteria. There were significant changes in the weight and fat percentages in all 3 groups as well as demonstrated insulin resistance in the high fat and high fat sugar water groups. The high fat diets also induced an increase in energy expenditure and a loss of circadian changes in energy expenditure. We are in the editing process with this completed manuscript and intend to submit it for publication early in 2019. Notably, this research project has won two awards (2nd and 3rd place) for poster presentations at research conferences this year. 3) Our third project more directly addresses the role of inflammation on the microbiome using a model of chemically-induced inflammation fed in drinking water (Dextran Sulfate Sodium, DSS). There was strong evidence of intestinal inflammation in the DSS-treated mice that resulted in decreased energy expditure and decreased metabolic rate between days 5 and 8. We have recently received the sequencing data on the metagenome of the gut microbiota and it is in the final stages of analysis. We have developed a new pipeline for analysis of this metagenomic data. These results will be prepared for publication likely mid-2019. These data will be the most informative on the direct impact of gut inflammation on microbiome constituents without the added complication of metabolic changes of obesity.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This project will be of interest to scientists in the fields of nutrition, metabolism, microbiome research, and immunology. This will also be of public health relevance to the general public in relation to how different diets contribute to health or dysbiosis of the gut microbiome and how the bacteria in the gut microbiome contribute to the health of the individual. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has been beneficial for training graduate students and undergraduate students in animal husbandry, microbial sequencing, and assessment of phage dynamics in vitro. Most of all however, it has provided a tremendous opportunity for several graduate students to become proficient in bioinformatics and computational analysis. One of my graduate students, Keah Higgins, attended a workshop on microbial metagenomic analysis (EDAME course, held in Michigan, 2016). She has sincecompleted several computational data analysis courses and workshops at Auburn and is now proficient in a variety of advanced statistical methods which she has applied to these data sets. Several of my graduate students are now pursuing proficiency in bioinformatics, and thus it is becoming a strength of my group, one which was previously absent. This will be incredibly important for my research program moving forward as these are the tools with which we must be equipped with to remain on the cutting edge of research. How have the results been disseminated to communities of interest?The results of the current study are being prepared for publication, to be submitted March, 2018. The results of this study were presented by twograduate students at the Boshell Diabetes and Metabolic DiseasesResearch Day at Auburn University March, 2017: Keah Higgins, a PhD candidate,gave an oral presentation entitiled:Effects of the High Fat Western Diet on Microbial and Viral Populations of the Murine Intestinal Microbiome"; Keah Higgins*, Lauren Woodie*, Haley Hallowell*, Michael Greene, Elizabeth Hiltbold Schwartz; Oral Presentation by Keah Higgins, Graduate student at Boshell Research Day, Boshell Diabetes and Metabolic Diseases Research Program; Auburn University Conference Center, March, 2017; Robert Johnson, a PhD student, presented a poster entitled:Quantitating The Metabolic Demand of Infection and Immune Response"; Robert M. Johnson*, Lauren N. Woodie*, Michael W. Greene, Elizabeth H. Schwartz; Poster Presentation by Robert Johnson, Graduate student at Boshell Research Day, Boshell Diabetes and Metabolic Diseases Research Program; Auburn University Conference Center, March, 2017; What do you plan to do during the next reporting period to accomplish the goals?We have a new study underway to more directly address the impact of inflammation on the gut microbiome. We have useda chemically-induced model of colitis and monitor the bacterial and viral communities, again in combination with metabolic phenotyping. We will sequence the microbiome samples we have acquired, perform the computational analysis, then prepare a manuscript of these findings. Two graduate students will also report the results of this study at the national American Society for Microbiology meeting in Atlanta, June, 2018. This work will serve as the framework for acquiring new extramural funding for this project.
Impacts
What was accomplished under these goals?
Impact: The impact of this project is that we will gain an understanding of how diet andinflammation of the intestine regulates the bacterial communities that live in the gut by regulating the viruses that prey on the bacteria. These findings will be instrumental in defining targets for therapies (either nutritional or pharmacological)to regulate specific players that cause intestinal inflammation, so that the bacterial and viral communities in the gut microbiome may return to a healthy state. We have determined that diet-induced changes in the gut microbiomehappen as soon as 2 days after changing to a high fat western diet. One of the first observable changes the microbiome ofanimals fed a high fat diet was a significant increase in both Streptococcus bacteria and in thebacteriophage that prey on these bacteria, p335. In contrast, there was a decrease in Clostridium bacteria and in the phage that prey on these bacteria, phiCD119.Thus, the bacterial and viral communities of the gut microbiome begin to change in concert, well before any symptoms of inflammation. Yet, after 12 weeks on high fat diets (and the associated metabolic changes, wieght gain, insulin resistance), these changes were continued and exacerbated, contributing to dysbiosis compared to chow fed animals. Generally, increases in certain phage populations were also observed as increases in the specific bacterial prey of those phages, suggesting that condiitons that favor the growth of specific bacteria also favor the growth of their phage. Taken together, these findings indicate that a high fat diet is likely to increase the lactic acid fermentation capacity of the gut microbiome (Streptococcus) and to decrease the abundance of certain Clostridia species that both regulate gut homeostasis and produce short chain fatty acids which are beneficial for gut health. Overall, these changes are likely tobe quite detrimental to host health. We have also performed metabolic phenotyping of animals on the chow vs. high fat diets and assessed the relationship between changes in bacterial and viralcommunities and specific features of metabolism. We observed strong positive correlations between the phage p335 and weight, % fat, and energy expenditure, while p335 was negatively correlated with activity. In contrast, phage phiCD119 was negatively correllated with weight, % fat and energy expenditure, but positively correlated with activity. These figures are completed and the manuscript of this study is currently in preparation. We plan to submit this mansucriptto Cell Host and Microbe due to the novelty of the study. Ours is the first comprehensiveexamination of bacteria and phage communities in thegut microbiomein concert with host metabolic phenotyping and thus, should be of broad interest to a large readership.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
The Blonde Mangalica Pig Models the Progressive Nature of Obesity-Induced Metabolic Disease
Morgan M. Roberts, Keah Chambers, Meggan M. Marvin, Jacqueline Tomei, Ashley Landuyt, Julia L. Bartosh, Soren P. Rodning, Chad D. Foradori, Jenna E. Bayne, Herris S. Maxwell, Elizabeth M. Hiltbold, and Terry D. Brandebourg Under review and revision at PLOS One, 2017
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:We have not published out resutls to date, but have submitted one manuscript, have another in preparation and another awaiting sequencing data. Our target audience would be primarily researchers in the area of metabolism and immunity. However, this information will be pertinent to the average consumer once scientific publications have been released. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has supported the training of 3 PhD students. These students participated in all aspects of both mouse and pig diet studies. They administered diets (daily feeding and husbandry), weighed the animals, performed back fat measurements on the pigs, collected feces for microbiota measurements, and blood for metabolic measurements. They learned how to process DNA for next generation sequencing and how to measure inflammatory mediators in fecal samples. Finally, one of hte students has become competent at computational analysis of metagenomic sequence data. How have the results been disseminated to communities of interest?Three students attended the Southeastern Regional Meeting of the American Society for Microbiology at Kennesaw State University in November 2015. Each of them presented posters on specific aspects of this work. What do you plan to do during the next reporting period to accomplish the goals?Continue with the project as described in the methods section.
Impacts
What was accomplished under these goals?
We have collected fecal microara samples from two obesity studies. The first is in a set of 12 pigs and the second is a set of 45 mice. Both studies involved longitudinal collection of feces from animals on different diets. Pigs- Each group of 4 pigs was fed either a standard diet (limited based on weight), fed ad libetum, or fed a high fat diet ad libetum for ~4 months. Wecollected weights, backfat measurements, metabolic measurements, and fecal samples for microbiota measurements. The groups were significantly different each other in weight gain and in fat. However, there were no detectable differences in metabolic readouts such as blood sugar or cholesterol. The DNA samples are still in the sequencing pipeline, awaiting analysis. We plan to complete the analysis this spring and should have a manuscript submitted for publication this summer. Mice- each group of mice were fed either normal chow, a high fat diet, or a high fat diet supplemented with sugar water for 12 weeks. Feces were collected at 0, 2 days and2,4, 8, and 12 weeks for microbiota analysis. The samples have been collected and analyzed and there are some interesting trends. The mice fed high fat diets (with or without sugar) demonstrated overgrowth of certain bacterial families such as Verucomicrobiaceaeand Enterococcaceae, and losses in others such as Bacteroidacaceae andPrevotellacaceae. There were also several differences in phage abundance that we are now cross-analyzing with the bacterial data to look for predator-prey relationships. This work will be submitted as a collaborative study with Dr. Michael Greene in the late spring of 2017.
Publications
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Progress 10/02/14 to 09/30/15
Outputs
Target Audience:Because this has been primarily a data-gathering year, we have not reached our target audiences yet. This should occur in the second year of the project. Changes/Problems:Based on the reviews of my R21 application, we will complete the proposed studies in the pig model, but have already begun the transition of our studies into a mouse model of diet-induced obesity. This model provides the benefit of a variety of reagents available to monitor many aspects of the immune response in the mouse and the potential to modulate that response, whichis not available in the pig. The reviewers of the application felt that mechanistic studies would be important to be competitive for NIH funding. We will be able to achieve these goals in the mouse model. What opportunities for training and professional development has the project provided?Bioinformatics and metagenomics are new areas of research for our laboratory. To enhance our analytical skills in bioinformatics, funds from this project were used to send Keah Chambers, the graduate student on the project, to a week-long immersive workshop on metagenomic analysis run by Michigan State University: Explorations in Data Analyses for Metagenomic Advances in Microbial Ecology(EDAMAME). The workshop was held at the Kellogg Biological Research Station, Hickory Corners, Michigan, Jun 22-29, 2015. Keah learned a great deal from this course and returned with a completely new set of analylitical tools to apply to our data sets. She learned how to use many open source computational tools and gained introductory coding skills. This was an incredibly valuable experience for Keah and for the lab. She will continue to expand her expertise in this area this spring semester by enrolling in two new courses offered the the department of Biological Sciences,Introduction to Bioinformatics and Functional Genomics. The funds from this project enabled meto attend and present our data ata very informative Keytoneconference on Gut Microbiota held in Keystone, CO in March, 2015 entitled"Gut Microbiota Modulation of Host Physiology: The Search for Mechanism". This conference provided an excellent opportunity for me to meet and interact with the leaders in the fieldof host interaction with gut microbiota. I also had the chance to meet several NIH program officers who provided valuable insights on grant submission strategies. These interactions will be extremely valuable to the future of this project, particularly in our ability toreceiveNIH funding. How have the results been disseminated to communities of interest?The results of this project have been presented at 1)onenational conference, and 2) one internal university conference this year. 1. Mangalica pigs as a model for human obesity: metabolic and immunological features interpreted in the context of functional metagenomic analysis of gut microbiotaElizabeth M Hiltbold, Keah Chambers, Ashley Landuyt, Terry Brandebourg; Keystone Symposium on Gut Microbiota Modulation of Host Physiology: Thesearch for Mechanism. Keystone, CO, March, 2015. 2. Bacteriophages as a modulating force in thegut microbiota in the setting of obesity.Elizabeth M Hiltbold, Keah Chambers, Ashley Landuyt, Terry Brandebourg; Auburn University- This is Research Symposium, September, 30, 2105. What do you plan to do during the next reporting period to accomplish the goals?Plans for publications: 1. We plan to submit our nearly complete manuscript detailing our initial observations by February, 2106. 2. The longitudinal analysis of pig obesity developmentwill conclude at the end of January, 2016. At this time, we will submit samples for sequencing, then begin analysis. We expect analysis and illustration of the data to take 3 months, and hope to submit our manuscript on these studies in May of 2016. 3. The mouse studies of diet-induced obesity will conclude in February 2016. With a similar timeline of analysis, but larger groups of animals, we predict to be able to submit a manuscript on this study by July, 2016. Plans for grant submissions: Based on the results of the studies above, I plan to submit one NIH R15 application in October, 2016 and I will also expand the experimental plans to submit an R01 application in September, 2016.
Impacts
What was accomplished under these goals?
Our preliminary studies had shown that obese pigs exhibit enhanced inflammation, poor glucose tolerance, and that their gut microbiota were altered compared to lean pigs. These findings have been compiled for publication and are awaiting final edits from our collaborator. We hope to submit this manuscript early in 2016. The initiation of the new studydescribed in the proposal was delayed by several months due to some difficulties with finding proper housing foranimals based on IACUC requirements. Those issues have been sorted out and we have now started our longitudinal analysis of gut microbiota and inflammation comparing pigs on limited vs. ad-libetum diets. The pigs are now gaining weight at differential rates as would be predicted, and we are able to detect inflammatory mediators in the pig feces. We have isolated microbial DNA from feces monthly, and will send these for metagenomic analysis and phage sequencing upon collection of all of the samples. We expect these studies to yield a strong manuscript (or two) upon completion. Based on our preliminary studies, I submitted an NIH R21 application in February, 2015. The reviews of the proposal were mixed. There was significant interest in our novel observations of phage abundance in obesity and ourhypotheses about the potential roles of phage in the obese gut. However, the reviewers felt that our preliminary data and proposed studies were only observational, and could not address mechanism. Therefore, based on these insights, we have transitioned our studies to a mouse model in which we will be better poised to perform more mechanistic studies with more power to measure inflammation and immunity. We will be able to direclty test the impact of inflammation on phage populations in the gut using a model of chemically-induced inflammation, DSS-colitis. In collaboration with Dr. Michael Greene in Nutrition and Dietetics, we have also initiated a study addressing the role of diet in inflammation and microbiota, looking specifically at phage populations in obesity induced by high fat and sugar water diet. These studies will conclude in spring of 2016 and will yield an exciting manuscript. Based on the results of this study, we will submit a new R15 or R21 application to continue these studies in the mouse model with a likely submission date of October, 2016.
Publications
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