Source: UNIVERSITY OF NEBRASKA submitted to
ASSESSMENT OF ENVIRONMENTAL IMPACTS AND ECOLOGICAL CONSEQUENCES ASSOCIATED WITH GENETICALLY ENGINEERED MICROBES IN LIVESTOCK PRODUCTION SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1033241
Grant No.
2024-33522-43699
Cumulative Award Amt.
$649,929.00
Proposal No.
2024-03808
Multistate No.
(N/A)
Project Start Date
Sep 1, 2024
Project End Date
Aug 31, 2028
Grant Year
2024
Program Code
[HX]- Biotechnology Risk Assessment
Project Director
Fernando, S.
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
This proposal aligns with the program focused on "Methods to monitor and understand the dispersal of GE organisms" and investigates the survival and fitness of the GE organisms compared to the non-GE counterpart across different environments and hosts. With tools for genome engineering and advances in synthetic biology, attempts have been made to engineer better probiotics with desired characteristics and functionalities. Such probiotics with enhanced functional features can be used to improve animal health and productivity as microbes play an important role in nutrient utilization and immune modulation. To date, systematic analysis of feeding genetically engineered microorganisms to livestock and how engineered microbes persist, integrate, and stabilize within the gut microbiome and associated environments in the short and long term has not been performed. The proposed experiments will directly address this knowledge gap by 1) examining ecological changes in the animal's fecal and oral microbiome in response to feeding a genetically engineered organism; 2) evaluate changes in environmental microbiomes associated with soil; 3) evaluate long and short-term stability and persistence of the genetically engineered microbe in the animal gut and gene flow to other microbes; 4) transmission of the genetically engineered microbe between animals. As such, this study will develop critical and timely information to help evaluate the long and short-term risks associated with utilizing genetically engineered microbes in livestock production and will help regulatory agencies make science-based informed decisions on utilizing genetically engineered microbes in livestock production.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053510110050%
3113599107050%
Goals / Objectives
Research Objective 1: Determine the effects of feeding a genetically engineered organism to swine on the ecology of the microbial community composition and function and the stability and persistence of the genetically modified organisms in the gastrointestinal tract, saliva, and soil in the long and short-term using longitudinal sampling. Specifically, we will evaluate changes in the microbial community composition and function in the gastrointestinal tract of genetically engineered Bacillus pumilus fed pigs and pigs fed the unmodified wild type GRAS approved Bacillus pumilus strain at nursery stage of the production cycle using fecal, oral, and soil samples using 16S rDNA and shotgun metagenome sequencing. The soil experiments will be performed in microcosms inoculated with feces from pigs fed the engineered and wild type Bacillus pumilus strains. Additionally, stability and persistence of the genetically engineered and non-engineered strains will be evaluated by feeding the engineered or non-engineered strain for 28 days and then stop feeding both strains and evaluating the persistence of the strains over the next 28 days in the fecal, oral and soil samples. Soil samples from the microcosms will be collected up to a year after initial mixing to evaluate long-term effects and persistence of the engineered microbe and gene flow. The resulting information will provide critical information into the persistence, spread, and frequency of the engineered organism and its impact on the animal and the associated environments.Research Objective 2: Determine movement of the genetically engineered organism from animal to animal. To this end, we will comingle animals that have been fed the genetically engineered organism with animals fed the GRAS approved wild type B. pumilus strain and monitor the abundance, persistence, and stability of the genetically engineered strain in the animals that were not fed the genetically engineered organism using 16S rDNA and shotgun metagenome sequencing.Research Objective 3: Evaluate the movement of the engineered gene from the B. pumilis species to other bacterial species in the, fecal, oral, and soil microbiomes. Specifically, we will use emulsion, paired isolation and concentration PCR (epicPCR) to link phylogenetic markers to the engineered gene, followed by genomic library preparation to identify which bacterial species are associated with the gene engineered to monitor the potential of the engineered gene to move between bacterial species.
Project Methods
We will test the hypothesis that utilizing genetically engineered microbes as probiotics or DFMs will provide unique opportunities to improve livestock health and performance while reducing antibiotic use. However, an improved understanding of the risks associated with introducing genetically engineered microbes into livestock production systems with respect to persistence, spread and the frequency of genetically engineered microorganisms in the animal and in the environment is critically needed to assess applicability and risks associated with utilization of genetically engineered microbes in the livestock industry. We further hypothesize that studies to uncover the risks associated with utilizing genetically engineered microbes as probiotics will help regulatory agencies make science-based informed decisions with respect to introduction of genetically engineered microbes.Our innovative approach integrates quantitative microbiome analysis as phenotyping tools to capture the effect of utilizing genetically engineered microbes on the host animal and the environment by investigating the ecology, persistence, and transmission of genetically modified microbes and genes. Combined with the powerful new methodologies available for sequencing and bioinformatics analyses, this proposal allows the investigation of the ecology, diversity, abundance, and distribution of genetically engineered microbes and its application in the livestock industry to provide science-based information to make informed decisions regarding the use of genetically engineered microbes. Research Objective 1: Determine the effects of feeding a genetically engineered organism to swine on the ecology of the microbial community composition and function and the stability and persistence of the genetically modified organisms in the gastrointestinal tract, saliva, and soil in the long and short-term using longitudinal sampling. Specifically, we will evaluate changes in the microbial community composition and function in the gastrointestinal tract of genetically engineered Bacillus pumilus fed pigs and pigs fed the unmodified wild type GRAS approved Bacillus pumilus strain at nursery stage of the production cycle using fecal, oral, and soil samples using 16S rDNA and shotgun metagenome sequencing. The soil experiments will be performed in microcosms inoculated with feces from pigs fed the engineered and wild type Bacillus pumilus strains. Additionally, stability and persistence of the genetically engineered and non-engineered strains will be evaluated by feeding the engineered or non-engineered strain for 28 days and then stop feeding both strains and evaluating the persistence of the strains over the next 28 days in the fecal, oral and soil samples. Soil samples from the microcosms will be collected up to a year after initial mixing to evaluate long-term effects and persistence of the engineered microbe and gene flow. Sequencing of the 16S rRNA gene and shotgun metagenome sequencing of the microbiome will provide information into community compositional changes and functional changes that occur during and after feeding genetically engineered microbes compared to a wild type microbe. The study will be carried out at the UNL Life science Annex in a controlled environment to mitigate the genetically engineered organisms from being released to the environment.Research Objective 2 - Determine movement of the genetically engineered organism from animal to animal. To this end, we will comingle animals that have been fed the genetically engineered organism with animals fed the GRAS approved B. pumilus strain and monitor the abundance, persistence and stability of the genetically engineered strain in the animals that were not fed the genetically engineered organism using 16S rDNA and shotgun metagenome sequencing.A key objective of risk assessment of genetically engineered microbes in the livestock industry is to monitor movement of the genetically engineered organisms from animal to animal, as this may have profound impacts on animal and human health. Information describing the spread of genetically engineered organisms from animal to animal can inform the potential risk associated with movement of genetically engineered organisms and their persistence and distribution. Determine movement of the genetically engineered organism from animal to animal - As described in objective one after 24 days of feeding either the genetically engineered or wild type B. pumilus strain, six animals from each treatment group will be co-mingled in 3 new pens that has not previously being exposed either B. pumilus strain (4 animals/pen). Baseline fecal and oral fluid samples will be collected from each animal and pen floor fecal samples will be collected from the new pen floor at 14-day intervals as described . Samples will also be collected from the remaining animals that are no co-mingled but that are not fed the probiotic to evaluate persistence of the genetically engineered microbes after removal of the organism from the diet. Microbial community analysis and functional analysis will be performed as described in objective 1 to evaluate if the genetically engineered microbe is easily transmissible to other animals.Research Objective 3 - Evaluate the movement of the engineered gene from the B. pumilis species to other bacterial species in the fecal, oral and soil microbiomes. Specifically, we will use emulsion, paired isolation and concentration PCR (epicPCR) to link phylogenetic markers to the engineered gene followed by genomic library preparation to identify which bacterial species are associated with the gene engineered to monitor the potential of the engineered gene to move between bacterial species.EpicPCR for identifying engineered gene-organisms associations - EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a relatively new approach to link any gene of interest to 16S rRNA gene. Essentially it helps link function to taxonomy. We will utilize this approach to link the presence of genetically engineered gene to different organisms in the fecal, oral and soil microbial species to identify the movement of the genetically engineered gene into other microbial species. The epicPCR will be performed to detect associations between the engineered gene and the taxa as described previously (68, 69).Data analysis of Fusion PCR products - EpicPCR sequence analysis will be performed as described previously (71). Briefly, Quality filtering will be performed as described above for 16S community data to identify the taxonomic distribution of species carrying the bacteriocin1 gene. After quality filtering reads will be split into 16S rRNA gene and the bacteriocin1 gene sequences using Prinseq (72). As described above, ASVs will be picked based on both the 16S rRNA gene and the variation in the bacteriocin1 gene sequence. Raw fastq files will be processed using the DADA2 pipeline to delineate amplicon sequence variants (ASVs). Taxonomy will be assigned using the SILVA ribosomal RNA database (50, 51). By comparing the taxonomic group with the bacteriocin gene presence and abundance, the distribution of the engineered bacteriocin gene will be determined and compared to baseline samples.