Category: Updates

The first look at Atlantic deep sea scallop bacterial communities in new publication

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Our paper was published in Aquaculture Reports, on identifying the bacteria associated with wild and hatchery-raised Atlantic sea scallop larvae, and the biofilms in larval tanks in a hatchery! For the past few years, I’ve been part of a state-wide collaboration between researchers, industry professionals, and educators all working together to understand and improve Atlantic sea scallop hatchery production. To our knowledge, this is the first study to identify bacteria in Atlantic sea scallops, and even though it was a very small project we hope it will lead to a much larger, mult-year project to investigate this in more detail.

Sarah Hosler presentating a slide of questions about the microbes that associate with scallops
Sarah Hosler, ASM 2022.

Scallops are a diverse animal group of marine bivalve mollusks (family Pectinidae) with global distribution in coastal waters, and Atlantic deep-sea scallops, Placopecten magellanicus, are found along the eastern coast of the United States and Canada. Scallops’ reproductive potential and industry demand make them a prime target for hatchery- and farm-based production, and this has been successfully achieved in bay scallops, but not in sea scallops. Currently, hatcheries collect wild sea scallop adults, or maintain cultured broodstocks, and spawn them in their facilities with the intention of forming a plentiful population to grow to adulthood, spawn, and sell to create a sustainable production cycle while also reducing disruption to the scallops’ natural habitat.

Unfortunately, in sea scallop hatcheries the last two weeks of the larval maturation phase, the veliger-stage, is plagued by large mortality events, going from 60 million sea scallop larvae down to several thousand individuals in a span of 48 hours. Survival of clutches to maturity remains very low, with an industry-standard rate around 1%. This drastic winnowing of larvae reduces the availability of cultured sea scallop spat for farmers, forcing sea scallop farms to rely almost exclusively on sea scallop spat collected from wild populations for stock and is seen as a bottleneck for growth of the industry and achieving sustainable harvests. Hatchery larval die-off is well-demonstrated not to be caused by inadequate diet, lighting, temperature, or atmospheric pressure in aquaculture facilities compared to wild conditions.

This project wanted to know if there was any clue in the bacteria that associate with larvae, or with the tanks they are in. In particular, hactcheries are worried about certain species of bacteria in the genus Vibrio, as they can cause disease to scallops and/or people, but it is tricky to study them because there are many species which do nothing at all. This project is part of another experiment to examine some of the Vibrio we found in tanks.

We sampled from some wild larvae, hatchery larvae, and from tank biofilms to indentify what was there. There were two styles of tank setup, and we collected from used tanks as well as tanks after they had been cleaned and refilled with filtered seawater.

Veliger-stage Atlantic sea scallop larvae (Placopecten magellanicus) were obtained from hatchery tanks in Beals, Maine and from the wild off the coast of Cape Elizabeth, Maine. Swabs of hatchery tank biofilms were collected before and after tank cleaning. Bacterial communities were identified using DNA extraction and 16S rDNA sequencing on wild veligers, hatchery veligers and biofilm swabs. Image created with biorender.

One of the surprising things we found, was that the bacterial communities in biofilms along the sides of larvae tank were more similar to each other (clustering) when samples were collected during the same phase of the lunar cycle. Bacterial richness and community similarity between tank samples fluctuated over the trial in repeated patterns of rise and fall, which showed some correlation to lunar cycle  where richness is high when the moon is about 50% and richness is low during new and full moon phases. This may be a proxy for the effects of spring tides and trends in seawater bacteria and phages which are propagated into hatchery tanks. The number of days since the full moon was significantly correlated with bacterial community richness in tanks: low during the full moon, peaking ~ 21 days after the full moon, and decreasing again at the next full moon.  

Fig. 7. Constrained ordination of bacterial communities in tank samples. Each point represents the bacterial community from one sample. Similarity between samples was calculated using Distance-based Redundancy Analysis (dbDRA), and significant model factors (anova, p < 0.01) are displayed with arrow lengths relative to their importance in the model (f value). The shape of points indicates whether swabbing was either immediately after filtered seawater has been used to fill the tank (cleaned, refilled) or 48 hours after (dirty, drained). Tank setup indicates if water was static, constantly filtered and recirculated in a flow-through system, or setup information was not available (n/a).

These results along with future work, will inform hatcheries on methods that will increase larval survival in these facilities, for example, implementing additional filtering or avoiding seawater collection during spring tides, to reduce certain bacterial taxa of concern or promoting a more diverse microbial community which would compete against pathogens.

Bacterial community trends associated with sea scallop, Placopecten magellanicus, larvae in a hatchery system.

Authors: Suzanne L. Ishaq1*, Sarah Hosler1, Adwoa Dankwa1, Phoebe Jekielek2, Damian C. Brady3, Erin Grey4,5, Hannah Haskell6, Rachel Lasley-Rasher6, Kyle Pepperman7, Jennifer Perry1, Brian Beal8, Timothy J. Bowden1

Affiliations:1 School of Food & Agriculture, University of Maine, Orono ME 044692 Ecology and Environmental Sciences, University of Maine, Orono ME 044733 School of Marine Sciences, Darling Marine Center, University of Maine, Walpole ME 045734 School of Biology and Ecology, University of Maine, Orono ME 044695 Maine Center for Genetics in the Environment, University of Maine, Orono ME 044696 Department of Biological Sciences, University of Southern Maine, Portland ME 041037 Downeast Institute, Beals, ME 046118 Division of Environmental & Biological Sciences, University of Maine at Machias, Machias, ME 04654

Abstract

Atlantic sea scallops, Placopecten magellanicus, are the most economically important marine bivalves along the northeastern coast of North America. Wild harvest landings generate hundreds of millions of dollars, and wild-caught adults and juvenile spat are increasingly being cultured in aquaculture facilities and coastal farms. However, the last two weeks of the larval maturation phase in hatcheries are often plagued by large mortality events. Research into other scallop- and aquacultured-species point to bacterial infections or altered functionality of microbial communities which associate with the host. Despite intense filtering and sterilization of seawater, and changing tank water every 48 hours, harmful microbes can still persist in biofilms and mortality is still high. There are no previous studies of the bacterial communities associated with the biofilms growing in scallop hatchery tanks, nor studies with wild or hatchery sea scallops. We characterized the bacterial communities in veliger-stage wild or hatchery larvae, and tank biofilms using the 16S rDNA gene V3-V4 region sequenced on the Illumina MiSeq platform. Hatchery larvae had lower bacterial richness (number of bacteria taxa present) than the wild larvae and tank biofilms, and hatchery larvae had a similar bacterial community (which taxa were present) to both wild larvae and tank biofilms. Bacterial richness and community similarity between tank samples fluctuated over the trial in repeated patterns of rise and fall, which showed some correlation to lunar cycle that may be a proxy for the effects of spring tides and trends in seawater bacteria and phages which are propagated into hatchery tanks. These results along with future work, will inform hatcheries on methods that will increase larval survival in these facilities, for example, implementing additional filtering or avoiding seawater collection during spring tides, to reduce bacterial taxa of concern or promote a more diverse microbial community which would compete against pathogens.

Acknowledgements

The authors would like to thank the staff at the Downeast Institute for supporting the development and implementation of this project, as well as for financially supporting the DNA sequencing; Meredith White of Mook Sea Farm for sharing her expertise and collecting biofilm samples; the Darling Marine Center for sharing their expertise and collecting biofilm samples; and the Sea Scallop Hatchery Implementation (Hit) Team for their expertise, review of this work, and funding support, who are financially supported by the Atlantic States Marine Fisheries Commission and Michael & Alison Bonney. The authors thank Lilian Nowak for assistance with related lab work to this project, and the Map Top Scholars Program for related financial support. The authors also thank Nate Perry for helping us collect wild scallop larvae. All authors have read and approved the final manuscript. This project was supported by the USDA National Institute of Food and Agriculture through the Maine Agricultural & Forest Experiment Station, Hatch Project Numbers: ME0-22102 (Ishaq), ME0-22309 (Bowden), and ME0-21915 (Perry); as well through NSF #OIA-1849227 to Maine EPSCoR at the University of Maine (Grey). This project was supported by an Integrated Research and Extension Grant from the Maine Food and Agriculture Center, with funding from the Maine Economic Improvement Fund.

Alexis joins the lab again for summer 2023!

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The lab is pelased to welcome back Alexis Kirkendall for the next six weeks to help us with a large-scale culturing project! Alexis is planning to graduate in December, and is in the process of applying to graduate programs here at UMaine so she can join the lab full-time in January!

Alexis Kirkendall

Undergraduate Researcher, Biology, Heidelberg University

Alexis is from Ohio and is majoring in Biology at Heidelberg University. Her research interests are in genetics and she has a love for the fascinating world of microbes. She originally joined the lab through the Summer 2022 REU, and has been working remotely on a few projects in the past year.

Last year, Alexis built skills in microbiology, microscopy, and molecular genetics. This year, she’ll be helping us screen hundreds of bacteria for their ability to grow on certain media and, hopefully, produce anti-inflammatories. This is part of the larger Broccoli Project.

Because of the large number of samples, we are using a robotic liquid handler to dispense culture media and bacterial cells into 96-well plates, that we can later look for growth in. We’ll also be using the anaerobic chamber to grow these bacteria on agar plates to see what their colonies look like, and to grow more of them for additional experiements.

A cartoon of a bacteria eating broccoli and pooping out sulforaphane
Designed by Johanna Holman
Stacks of petri dishes filled with culture media, some of which have bacterial colonies growing on them.
Stacks of petri dishes filled with culture media. The stacks are inside of a vinyl anaerobic chamber, for use with bacterial culturing.
An image of lab equipment inside a microbiology and genetics laboratory.

While visiting the Microbe Musuem in Amsterdam recently, Alexis got me a tardigrade (middle) to join the chalmydia (left) and methicillin-resistant Staphylococcus aureus (right, which she got me last year) in my office!

Upcoming presentations at the 2023 Ecological Society of America annual meeting!

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Ecological Society of America meeting, Aug 6 – 11, 2023, Portland, Oregon

Scallop microbes and sustainable aquaculture: host-microbe dynamics situated in environmental and social context.

Presentation ID: 1372900

Session Information

Session Title: Microbes as Tools to Solve Ecological Problems for All
Session Type: Inspire Session
Date: Thursday August 10, 2023
Session Time: 3:30 PM – 5:00 PM Pacific Time

Authors: Suzanne L. Ishaq1

Affiliations: 1 University of Maine, School of Food and Agriculture, Orono, ME 04469 USA

Atlantic sea scallop (Placopecten magellanicus) is the second largest fishery in Maine, primarily through wild harvest. Farming is a promising way to meet year-round market demands, create jobs, and reduce ecological impacts of harvest, but relies on wild-caught juveniles as larval survival in hatcheries is low for unknown reasons. My collaborative research group explores the role of larval and tank microbiomes in hatcheries compared to wild scallop veligers. In addition to basic and applied microbiome research, the research team meets with industry partners weekly to discuss results, trends, generate real-world-problem-driven project designs, and collaborate on research, education, and student training.

Bacterial community trends associated with sea scallop, Placopecten magellanicus, larvae in a hatchery system.

Poster ID:  1475974
Poster Title: “Bacterial community trends associated with sea scallop, Placopecten magellanicus, larvae in a hatchery system.”

Session Information

Agriculture
Session Date: Tuesday August 8, 2023 
Session Time: 5:00 PM – 6:30 PM Pacific Time

Authors: Suzanne L. Ishaq1*, Sarah Hosler1, Adwoa Dankwa1, Damian C. Brady2, Erin Grey3, Phoebe Jekielek4, Kyle Pepperman5, Jennifer Perry1, Rachel Lasley-Rasher6, Brian Beal3,7, Timothy J. Bowden1

Affiliations: 1 School of Food & Agriculture, University of Maine, Orono ME 04469. 2 School of Marine Sciences, Darling Marine Center, University of Maine. 3 School of Biology and Ecology, University of Maine, Orono ME 04469. 4 Department of Biological Sciences, University of Southern Maine, Portland ME 04103. 5 Downeast Institute, Beals, ME 04611. 6  Ecology and Environmental Sciences, University of Maine, Orono ME 04473. 7 Division of Environmental & Biological Sciences, University of Maine at Machias, Machias, ME 04654

Atlantic sea scallops, Placopecten magellanicus, are the most economically important marine bivalves along the northeastern coast of North America, and wild-caught adults and juvenile spat are increasingly being cultured in aquaculture facilities and coastal farms. While adults can be induced to spawn successfully in hatcheries, the last two weeks of the larval maturation phase are plagued by large mortality events, making production unfeasible. Research into other scallop- and aquacultured-species point to animal loss from bacterial infections or from altered functionality of host-associated microbiota. There are no previous studies of the bacterial communities from biofilms growing in scallop hatchery tanks, nor even host-microbial studies with this species of sea scallops. We identified bacterial communities in veliger-stage wild larvae, hatchery larvae, and tank biofilms, using the V3-V4 region of the 16S rDNA gene, via Illumina MiSeq sequencing. Hatchery larvae had lower bacterial richness (number of bacteria taxa present) than the wild larvae and tank biofilms, and hatchery larvae had a similar bacterial community (which taxa were present) to both wild larvae and tank biofilms. Bacterial richness was not significantly different between tanks which had been occupied by larvae for 48 hours, and those which had just been drained, scrubbed clean, and refilled with filtered seawater. Static-water-flow compared to continuous-water-flow (flow-through) did not generate different levels of bacterial richness overall, and only an equivocal difference when accounting for time as a smoothing feature in the model (GAM, p = 0.04). Bacterial richness and community similarity between tank samples fluctuated over the trial in repeated patterns of rise and fall, which showed some correlation to lunar cycle  where richness is high when the moon is about 50% and richness is low during new and full moon phases. This may be a proxy for the effects of spring tides and trends in seawater bacteria and phages which are propagated into hatchery tanks. The number of days since the full moon was significantly correlated with bacterial community richness in tanks (GAM, p < 0.01): low during the full moon, peaking ~ 21 days after the full moon, and decreasing again at the next full moon.  These results along with future work, will inform hatcheries on methods that will increase larval survival in these facilities, for example, implementing additional filtering or avoiding seawater collection during spring tides, to reduce certain bacterial taxa of concern or promoting a more diverse microbial community which would compete against pathogens.

Giving the keynote presentation at the Boston University Microbiome Day in July!

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I’m honored to be giving the keynote presentation at the Boston University Microbiome Day this July 12! I’ll be sharing my work on microbes and social equity, especially as pertains to health and the environment. You can find event details and registration here. You can also follow the group on Twitter @BuMicrobiome.

TimeEvent
9:00-9:45 AMBreakfast and coffee
9:45-10:00 AMWelcome
Faculty Talks
10:00-10:30 AMDr. Sarah Davies
10:30-11:00 AMDr. Joe Larkin
Keynote Address
11:00AM-12:00 PMDr. Sue Ishaq
12:00PM-1:30 PMLunch and Poster session
Panel Discussion: Translating the Microbiome to Industry
1:30-2:30 PMJennifer Cookson, Andrea Watson and Nili Ostrov:“Translating the Microbiome to Industry”
Student Session Pt 1
2:45-3:05 PMMichael Zulch
3:05-3:35 PMPaul Rousteau
3:35-3:45 PMSession Break
Student Session Pt 2
3:45-4:05 PMCorinne Vietorisz
4:05-4:35 PMMichael Silverstein
5:00 PMSocial Event
 

Upcoming talk at the 9th Southern California Microbiome Symposium in September!

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I’m ecstatic to be heading back to southern California this September to present at the 9th annual Southern California Microbiome Symposium. I’ll be sharing my work on microbes and social equity, especially as pertains to food systems and sustainability. Registration is free, and can be found here.

Happening today: MSE symposium session “Reconsidering ‘One Health’ Through Microbes”

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The Microbes and Social Equity working group, and The University of Maine Institute of Medicine present a virtual symposium on:

“Living in a Microbial World”

June 5 – 9th, 2023.

Format: virtual meeting, Zoom platform.

The full program is here.

Session 1: Reconsidering ‘One Health’ Through Microbes

Monday, June 5th, 11 am – 2:30 pm EST. This event has passed, watch the recorded talks.

Microbes and Social Equity concepts are based on the idea that microbes connect individuals, societies, and ecosystems. One Health & the Environment concepts are based on similar ideas of connectivity. This session will explore the connections between MSE and One Health, how microbiome research connects to One Health, and how we can broaden our own research to include other disciplines. The primary goals for this session are 1) to convene researchers in multiple disciplines and envision ways to work together, and 2) to collaboratively generate definitions of One Health & the Environment with respect to microbiomes.

Hosts and organizers:

Dr. Tiff Mak (they/she), PhD, Postdoctoral Research Fellow at the Novo Nordisk Foundation Center for Biosustainability at DTU. They work at the intersection of Microbial Ecology, Fermentation and Integrated Food Systems, and are interested in community interaction dynamics and relationality, from the scale of the microbial to the planetary.

Dr. Sue Ishaq, PhD, Assistant Professor of Animal and Veterinary Science, School of Food and Agriculture, University of Maine. Animal microbiomes, diet and gut, microbes and social equity.

Speakers, 11~12:00 EDT:

Rob Beiko, PhD

Dr. Rob Beiko, PhD., is a Professor and Head of the Algorithms and Bioinformatics research cluster in the Faculty of Computer Science at Dalhousie University. His research aims to understand microbial diversity and evolution using machine learning, phylogenetics, time-series algorithms, and visualization techniques. His group is developing software tools and pipelines to comprehensively survey genes and mobile genetic elements in bacterial genomes, and understand how these genomes have been shaped by vertical inheritance, recombination, and lateral gene transfer. He is also a co-founder of Dartmouth Ocean Techonlogies, Inc., a developer of environmental DNA sampling devices.

Marta Scaglioni

Dr. Marta Scaglioni, PhD. is a Cultural Anthropologist and holds a PostDoc position at Cà Foscari University of Venice (Italy) within the frame of the ERC Project HealthXCross. She is interested in how microbiome research operates in the African continent and how microbial data, knowledge, and funding travel across national boundaries and across a Global North/Global South axis.

Dr. Lucilla Barchetta, PhD., is a Cultural Anthropologist and PhD in Urban Studies. She currently works as Postdoctoral Fellow within the ERC project Health X Cross based at the University Ca’ Foscari of Venice, where she studies One Health epistemologies and open data governance in multidisciplinary data-centric science and collaboration.

Break, ~12:05 – 12:20 EDT

Panel Discussion, 12:20~13:00 EDT:

  • Need for interdisciplinarity and collaboration, with collection and ontological credit
  • Narrative on One Health, and thinking about other definitions of health

Break, 3:00 – 13:15 EDT

Breakout room discussions, 13:15 ~ 14:30 EDT:

  1. Microbes in One Health research
  1. Defining One Health/Conservation
  1. Teaching microbes + One Health

Related to this session, here are recorded talks from previous MSE events:

Recording available from my recent talk on broccoli sprouts, microbes, and social equity

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I was recently in Boston, MA to present some of my collaborative research on broccoli sprouts, gut microbes, and social equity, to the Harvard Chan Medical School NIEHS Center for Environmental Health. You can watch the recording of that presentation, as well as their full speaker series, here.

A cartoon of brightly colored, smiling bacteria.

Ishaq, S. “Microbes and Social Equity: what is it and how do we do it?” Harvard Chan-NIEHS Center for Environmental Health’s “Environmental Health in Action” colloquium series. Boston, MA, May 10, 2023. (invited)

A logo that says "The Broccoli Project"
Designed by Johanna Holman.

If you’d like more info on this ongoing research, you can check out:

Lola passes her comprehensive exam!

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Congratulations to Lola Holcomb, for passing the graduate comprehensive exam!! She now goes from being a PhD student to a PhD candidate, and for the next few years will focus on developing her own research designs and writing her thesis.

Lola standing in front of her poster which displays the results of her research on gut microbes.
Portrait of Lola Holcomb, wearing a block sweater on a beach at sunset

The exam was set by her PhD program in the Graduate School of Biomedical Sciences and Engineering, and involved writing a 6-page research proposal over the past two months, and on the day of, giving a 1-hour presentation and answering questions from her committee for up to two hours. The focus of the proposal and the presentation are a hypothetical experiment she designs on a topic that is similar to her existing research but not directly related. In that way, you can test a student’s ability to translate their existing knowledge and fact-finding skills to a totally new area and see how well they can reason through a new problem. The committee will ask students to explain their thought process, methods, and how they will assess the progress on the hypothetical project. It’s a long and arduous process, but Lola’s depth of knowledge and ability to problem-solve helped her pass with ease!

Paper by former colleagues published on rainbow Trout (Oncorhynchus mykiss) midgut and hindgut microbiomes using whole shotgun metagenomics

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As a postdoc at Montana State University in 2015/2016 with Carl Yeoman’s lab, I consulted on a project led by then-PhD student Lola Betiku on a metagenomics dataset from two locations from the digestive tract of trout. Lola is now an Assistant Professor at Florida A&M University, and has been kind enough to continue working on this project to get it published. It was just accepted in the journal Aquaculture Reports!

Citation

Betiku, O., Yeoman, C., Gaylord, T.G., Ishaq, S., Duff, G., Sealey, W. 2023. Evidence of a Divided Nutritive Function in The Rainbow Trout (Oncorhynchus mykiss) Mid- and Hind-Gut Microbiomes by Whole Shotgun Metagenomic Approach. Aquaculture Reports 30: 101601.

Highlights

  • •Animal and plant protein diets were fed to rainbow trout in a commercial setting.
  • •Shotgun metagenomic analysis of the mid-GIT and hind-GIT was carried out.
  • •Diets influenced microbial compositions in the two GIT sections.
  • •Animal protein-based diet provided metabolites for microbial protein fermentation.
  • •Plant-based diet enhanced amino acid catabolism in the mid-GIT section.

Abstract

The nutritive role and ecology of gut-dwelling microbes in rainbow trout remain enigmatic. To improve our understanding of the rainbow trout gastrointestinal tract (GIT) microbiome, we performed whole shotgun metagenomic analyses on the assembled contigs from luminal contents from both mid- and hind-GIT regions for taxonomic and functional classifications of fish-fed animal and plant protein dietary sources. Our study revealed that trout respond well to the two diets containing animal and plant protein sources when supplemented with essential amino acids to meet the requirements of the fish. Microbes present were predominantly bacteria (89.9%) and mainly of the phyla Tenericutes, Firmicutes, Fusobacteria, and Proteobacteria. Eukaryotic (8.8%) microbes were mainly from phyla Ascomycota and Basidiomycota, while Archaea (<1%) were also present and predominantly from the phylum Euryarchaeota. Comparisons of genus-level classifications and functional profiles revealed compositional differences in these GIT locations that appear modulated by differences in the dietary treatments. The functional analysis provided evidence of amino acid biosynthesis/catabolism and methane production in the mid-GIT, while in the hind-GIT, proteolytic hydrolysis and butyrate metabolism were expressed in the trout fed with plant protein diet. The animal protein-based diet provided metabolites for microbial protein fermentation in the hind-GIT. Our report highlights and identifies the potential nutritive contributions of GIT microbes to trout and a potentially crucial functional division along the GIT. Finally, the plant-based diet enhanced amino acid catabolism in the midgut section, while the hindgut section supports evidence of methanogen fermentation.

Upcoming presentations at the American Society for Nutrition conference

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The Ishaq Lab will be presenting at a few research conferences this summer, with a few more in the works for the fall.

Broccoli sprouts in a tray

American Society for Nutrition meeting, July 22-25, 2023, Boston, Massachusetts

Steamed broccoli sprouts alleviate gut inflammation and retain gut microbiota against DSS-induced dysbiosis.

Poster, abstract P20-022-23, July 23

Authors: Johanna M. Holman*1, Lola Holcomb2, Louisa Colucci3, Dorien Baudewyns4, Joe Balkan5, Grace Chen6, Peter L. Moses7,8, Gary M. Mawe7, Tao Zhang9, Yanyan Li1, Suzanne L. Ishaq1

Affiliations: 1 School of Food and Agriculture, University of Maine, 2 Graduate School of Biomedical Sciences and Engineering, University of Maine, 3 Department of Biology, Husson University, 5 Department of Chemical and Biological Engineering, Tufts University, 6 Department of Internal Medicine, University of Michigan Medical School, 7 Departments of Neurological Sciences and of Medicine, Larner College of Medicine, University of Vermont, 8 Finch Therapeutics, 9 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University.

Objectives: Inflammatory Bowel Diseases (IBD) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective, affordable, and safe for managing symptoms. Ongoing research has identified inactive compounds in broccoli sprouts, like glucoraphanin, and that mammalian gut microbiota play a role in metabolizing it to the anti-inflammatory sulforaphane. The objectives were to identify biogeographic location of participating microbiota and correlate that to health outcomes. 

Methods: We fed specific pathogen free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet, and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 40-day experiment to simulate chronic, relapsing ulcerative colitis. We monitored body weight, fecal characteristics, fecal lipocalin, and sequenced bacterial communities from the contents and mucosa in the jejunum, cecum, and colon. 

Results: Mice fed the broccoli sprout diet while receiving DSS performed better than mice fed the control diet while receiving DSS for all disease parameters, including significantly more weight gain (2-way ANOVA, p < 0.05), lower Disease Activity Index scores (2-way ANOVA, p < 0.001), and higher bacterial richness in all gut locations (linear regression model, p < 0.01 for all locations measured). Bacterial communities were assorted by gut location except in the mice receiving the control diet and DSS treatment (Beta-diversity, ANOVA, p < 0.05 for each). Importantly, our results suggested that broccoli sprout feeding completely abrogated the effects of DSS on gut microbiota, as bacterial communities were similar between mice receiving broccoli sprouts with and without DSS. 

Conclusions: Spatially resolved microbial communities provide greater insight when investigating host-microbe interactions. Here, we show that a 10% broccoli sprout diet protects mice from the negative effects of dextran sodium sulfate induced colitis, that colitis erases biogeographical patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. 

Funding Sources: This work was funded by the NIH, USDA, NSF NRT, and UMaine GSBSE.

 

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.

Poster, abstract P20-021-23, July 23

Authors: Lola Holcomb*1, Johanna Holman2, Molly Hurd3, Brigitte Lavoie3, Louisa Colucci4, Gary M. Mawe3, Peter L. Moses3,5, Emma Perry6, Allesandra Stratigakis7, Tao Zhang7, Grace Chen8, Suzanne L. Ishaq1, Yanyan Li1

1 Graduate School of Biomedical Sciences and Engineering, University of Maine, 2 School of Food and Agriculture, University of Maine, 3 Larner College of Medicine, University of Vermont, 4 Department of Biology, Husson University, 5 Finch Therapeutics, 6 Electron Microscopy Laboratory, University of Maine, 7 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, 8 Department of Internal Medicine, University of Michigan Medical School.

Objectives: Inflammatory Bowel Diseases (IBD) are chronic conditions characterized by inflammation of the gastrointestinal (GI) tract that burden daily life, result in complications, and disrupt the gut microbiome. Many studies on diet and IBD in mice use an ulcerative colitis model, despite the availability of an immune-modulated Crohn’s Disease model. The objective of this study was to establish IL-10 deficient mice as a model for studying the role of dietary broccoli and broccoli bioactives in reducing inflammation, modifying the immune response, and supporting GI tract microbial systems. 

Methods: Interleukin-10-knockout (IL-10-ko) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed either a control diet or one containing 10% raw broccoli sprouts. Diets began 7 days prior to inoculation with Helicobacter hepaticus, which triggers Crohn’s-like symptoms in these immune-impaired mice, and ran for 2 additional weeks. 

Results: Broccoli sprouts decreased (p < 0.05), fecal lipocalin (LCN2), a biomarker for intestinal inflammation, and fecal blood, diarrhea, and overall Disease Activity Index. Sprouts increased gut microbiota richness, especially in younger mice (p < 0.004), and recruited different communities in the gut (B-diversity, ANOVA, p < 0.001), especially in the colon (B-diversity, ANOVA, p = 0.03). The control group had greater prevalence and abundance of otherwise commensal bacteria which trigger inflammation in the IL-10-ko mice. Helicobacter was within the top-5 most prevalent core genera for the control group, but was not within the top-5 for the broccoli group. Disease parameters and microbiota changes were more significant in younger mice receiving broccoli.

Conclusions : A diet containing 10% raw broccoli sprouts may be protective against negative disease characteristics of Helicobacter-induced enterocolitis in IL-10-ko mice, and younger age is the most significant factor (relative to diet and anatomical location) in driving gut bacterial community richness and similarity. The broccoli diet contributes to prevalence and abundance of bacterial genera that potentially metabolize dietary compounds to anti-inflammatory metabolites in the gut, are bacteriostatic against pathogens, and may ease disease severity.

Funding Sources: This work was funded by the NIH, USDA, NSF NRT, and UMaine GSBSE.

Establishing Growth Curve Assays for Bacterial Glucosinolate Metabolism: A Study Protocol

Poster, abstract P22-030-23, July 23

Marissa Kinney*1, Ryan Wijayanayake1, Johanna Holman1, Timothy Hunt2, Benjamin Hunt 2, Tao Zhang3, Grace Chen4, Yanyan Li1 , Suzanne L. Ishaq1

1School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469; 2 School of Biology and Ecology, University of Maine, Orono, Maine, USA 04469; 3 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790; 4 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109 

Objective: Inflammatory bowel diseases (IBD) cause dysfunction of the gastrointestinal (GI) tract and can result in hospitalization, suffering and disruption to overall health. Recent work has demonstrated the anti-inflammatory capacity of a broccoli sprout-diet in artificially-induced GI inflammation in pathogen free C57BL/6 mice. Microbiota samples obtained from the GI tract of these mice will be used to study the presence and activity of broccoli glucosinolate hydrolysis to create microbial-sourced bioactives, to further understand the relationship between broccoli-diets and inflammation reduction. It is imperative to validate or replicate qPCR protocols which have been established for glucosinolate metabolism in Bacteroides thetaiotaomicron (B. theta), in other bacterial species. Additionally, this project will focus on developing new growth curve assays for glucosinolate metabolism, as these methods are lacking in published literature. 

Methods: Pathogen free C57BL/6 mice were given dextran sodium sulfate (DSS) into their drinking water to create a disease profile similar in development and morphology to human ulcerative colitis (UC), a type of IBD. DSS and a steamed broccoli sprout diet were administered. Samples were taken from the digesta, jejunum, cecum, and colon of mice fed broccoli diets. About 806 bacterial isolates will be grown up/cultured anaerobically on minimal/selective media containing glucosinolate-related compounds (glucoraphanin, sinigrin) to determine hydrolysis activity via spectroscopy to measure optical density of growth in competent isolates. Successful isolates will be further analyzed with LC/MS to confirm production of bioactive products, and with qPCR using the B. theta positive control genome to help identify gene targets (α-1,6-mannanase, glycosyl hydrolase, nicotinamide-dependent oxidoreductase, and transcriptional regulator protein) for glucosinolate conversion in isolates. 

Results  N/A

Conclusions: In the initial study from which these samples are sourced, mice fed a broccoli diet had less inflammation than those fed a control diet and DSS, and had higher bacterial diversity in their gut. We expect that bacteria isolated from the GI of broccoli-fed mice will contain more glucosinolate-metabolizing genes.

Funding: NIH (Li and Ishaq) and USDA (Li). 

Broadening Perspectives by Situating Nutrition Education in Broader Social Contexts: A Study Protocol

Poster, abstract number 1490287, July 23

Authors: Ashley Toney*1, Patricia Wolf*2, Sue Ishaq*3

Affiliations: 1Dept. of Cardiovascular and Metabolic Sciences, Cleveland Clinic Lerner Research Institute, 2 Department of Nutrition Science, Purdue University, 3 School of Food and Agriculture, University of Maine

Objectives: The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Given the complexity and nuance of evidence-based nutrition delivery, MSE sought to provide a conceptual structure. The goals are 1) convene diverse researchers, educators, learners, and practitioners, 2) publish and present evidence-based information within socio-cultural contexts, and 3) teach audiences to define ways for integrating equity into their work.

Methods: MSE hosted a 14-week speaker series in 2021, 2022, and 2023, with researchers from various disciplines (e.g., nutrition, gut microbes, food security). The series lead into a 5-day symposium of speakers and guided discussions to generate co-written documents identifying research needs and resources. We use targeted and non-targeted event promotion to attract audiences, and reach/impact is evaluated through registration, attendance, social media views, or attendee feedback. The series provides learning sessions that build concepts over time, guiding attendees past the conceptual roadblock of being new to interdisciplinary research and grappling with grand challenges. This extended period to learn stimulates participation in activity-based collaboration during symposia, even from new members or students. 2023 symposium attendees will be surveyed on their impressions on the event, how it impacted their perspective on experimental design, whether attendees started with the series and followed up with the symposium, and whether having the preface of the series aided in being able to create actionable outputs in a guided co-working session.

Results: n/a

Conclusions: Benefits include nuanced knowledge/perspective sharing, establishing and nurturing interdisciplinary collaboration, and sparking conversations on critical topics in policy, sustainability, host and microbial metabolism, etc. In the last 3 years, MSE published 4 papers together, with ~ 50 independent papers from members. Event recordings are used as curriculum in nutrition, microbiology, and pre-med biology.

Funding Sources: NIH/NIDDK, Allen Foundation (Ishaq); National Dairy Council (Toney)