Performing Department
(N/A)
Non Technical Summary
Edible plants host abundant microorganisms, many of which can survive transit through the mammalian gut and may even be metabolically active. Previous studies have quantified microbial abundance on commercial produce during harvest, storage, transportation, and at point of sale, but limited data exists at the point of consumption, particularly for garden-fresh produce. Garden-fresh and store-bought produce likely harbor distinct microbial communities, due to differences in handling and processing over the supply chain. Since garden-fresh produce typically undergo minimal disinfection procedures, they may yet host a broad diversity of soil- and plant-associated microbiota. Exposure to some of these environmental microorganisms has been linked with beneficial health effects, however, other microorganisms contaminating fruits and vegetables may be pathogenic.This project aims to 1) characterize microbial communities of garden-fresh and store-bought produce and 2) compare effects of consuming garden-fresh versus store-bought produce on gut microbiome diversity and composition. It will do so by recruiting 20 healthy adult gardeners to participate in a two-arm crossover diet trial comparing consumption of USDA-recommended daily amounts of raw fruits and vegetables sourced either from their own garden (Arm 1) or the same fruits and vegetables from a supermarket (Arm 2). Subjects will provide fruit/vegetable and fecal samples daily during each arm of the trial. Shotgun metagenomic sequencing will be used to characterize diversity and composition of microbial communities inhabiting garden-fresh vs store-bought produce and changes in the human gut microbiome during each arm of the trial.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
The goal of this Seed Grant, therefore, is to generate preliminary data needed to support a full-scale proposal to examine fruit and vegetable microbiomes across a spectrum of supply chain systems (e.g., garden, CSA/farmers market, supermarket) and to assess their potential to impact human health via the microbiome-gut-brain axis. To realize this goal, this proposal aims to achieve:Objective 1. Characterize microbial communities of garden-fresh and store-bought produce (representing opposite ends of the supply chain spectrum).Objective 2. Compare effects of consuming garden-fresh versus store-bought produce on gut microbiome diversity and composition in a healthy adult population of Master Gardeners (MGs).
Project Methods
Dietary crossover trialParticipant recruitment. To be eligible for the study, prospective participants must be healthy adults who currently garden for food, either at home or in community gardens, and agree to consume USDA-recommended amounts of fruits (2 cups/day) and vegetables (2.5 cups/day) for two 1-week periods during the dietary trial. No prospective participants will be excluded on the basis of sex/gender, race, ethnicity, or other demographic attribute. Recruitment channels will include webinars, Master Gardener (MG) listserv emails, and blog and social media postings. An informational website and sign-up portal will be maintained, where prospective volunteers can access a brief screening survey to confirm their eligibility and enroll in the study. All study details will be thoroughly described in the informed consent materials and prospective volunteers will have opportunities to have their questions answered via phone, email, or virtual meeting. Signed consent forms will be obtained from all subjects prior to their participation in the study. Minimal risk to subjects stemming from participation in the study is anticipated. The condition of interest is regular consumption of fresh garden fruits and vegetables, which all eligible subjects are presumed to do as a matter of habit.Intervention. The dietary trial comprises a two-arm randomized crossover design where study participants (n=20) will consume USDA-recommended amounts of fresh fruits and vegetables sourced either fresh from their own garden or from a local supermarket. Participants will spend one week in each treatment arm with a one-week washout period between arms, similar to methods used in other humandiet-microbiome studies. During each arm of the trial, participants will be asked to collect produce and stool samples on a daily basis, maintain a detailed log of daily food intake, and complete a survey with questions drawn from the American Gut Project. Using their food intake log as reference, participants will match fruit and vegetable types as closely as possible between the two trial arms.Participants will be provided with OMNIgene · GUT stool sample collection kits, including detailed printed instructions, to facilitate recovery of adequate high-quality fecal material. The OMNIgene · GUT collection kit stabilizes stool samples at ambient temperatures for extended periods, allowing for study participants to collect all samples and mail them together to the UO BioBE laboratory. For produce samples, participants will combine small samples from all fruits and vegetables they consume in a single resealable plastic bag for each intervention day (days 3-9) of each trial arm. To ensure successful sample collection by the participants, the PD will create online instructional videos and lead an interactive 1-hour training workshop. Similar approaches have been very successful in other citizen science microbiome studies, such as the American Gut Project, Wild Life of Our Homes, and the PD's recentNIFA Postdoctoral Fellowship project.Microbial characterization and data analysis techniques. After receiving samples from study participants, produce samples will be homogenizedand both produce and stool samples will be stored at -80 degrees Celsiusuntil further processing. All samples will be prepared for shotgun metagenomic sequenc- ing using Qiagen DNeasy PowerSoil Kits to extract DNA and Nextera DNA Prep Kits to prepare sample libraries, including negative and positive control samples. During all laboratory procedures, samples will be randomized to minimize batch effects due to extraction, library prep kits, or sequencing runs. Samples will be sequenced to a depth of 20-25 million reads/sample on the NovaSeq 6000 platform (PE 2 × 150) at the UO Genomic Core & Cell Characterization Facility.All bioinformatics processing, statistical analysis, and data visualization will be performed in the R statistical computing environment. Prior to downstream testing and visualization, low-quality reads andpotential laboratory contaminants will be identified and removed, as will samples that do not meet a specified abundance threshold. Rarefaction analysis will be employed to assess microbial alpha diversity (as a whole and for each domain--archaea, bacteria, fungi, virus--individually), followed by analysis of variance (ANOVA) to test for differences across groups while controlling for individual variation in baseline diversity. Permutational multivariate analysis of variance (PERMANOVA) and differential abundance analysis will be used to test for differences in beta diversity and identify individual microbial taxa responsible for driving compositional variation. All work will be thoroughly documented using literate programming to maintain lab notebooks, perform bioinformatics and data analysis, and prepare manuscripts and presentations; source code will be made freely available on Github or other repository.