Progress 04/01/21 to 03/31/23
Outputs
Target Audience:The target audience for this project include faculty, students, and fresh produce growers. Changes/Problems:The research was delayed since USDA-ARS-NEA-BARC and Ezbiome facilities were closed some part of 2021 due to COVID-19. What opportunities for training and professional development has the project provided?The specific focus of the training was on metagenomics and bioinformatics, which provided the PI with the necessary tools to investigate how surface water, including pond and creek irrigation water, influences the composition of bacterial communities on the surfaces of produce. By applying metagenomics and bioinformatics techniques, the PI obtained more comprehensive answers regarding the characterization of microbial profiles in produce farms. How have the results been disseminated to communities of interest?Antimicrobial resistant bacteria in irrigation water used by small scale growers were disseminated to faculty, extension agents, and students. Overall, the dissemination of information on bacteria and antimicrobial resistant bacteria in irrigation water to faculty, extension agents, students, and other stakeholders plays a crucial role in promoting awareness, education, and informed decision-making. By reaching out to these audiences, the knowledge generated from the research can have a positive impact on the agricultural community, public health, and the overall sustainability of food production systems. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Train the PI in metagenomics and bioinformatics: To conduct professional development on metagenomics and bioinformatics, the PI planted lettuce (var. Coastal star)and irrigated growing plants with water from creeks, streams, pond water, and municipal water. The PI received training on the principles and applications of microbial 16S sequencing, metagenomics, and bioinformatics. Through the study, antibiotic resistant genes (ARGs) were exhibited in water, soil, and growing lettuce plants. The presence of antibiotic resistance genes (ARGs) in irrigation water, soil, and lettuce leaves suggests that these environments can be contaminated with antibiotic-resistant bacteria (ARB) and ARGs. The study highlights the potential risk of using contaminated water for irrigation in agricultural areas. Irrigation water from shallow streams and small rivers is commonly used in agriculture due to its availability and convenience. However, these water sources are susceptible to contamination from various sources, including fecal matter and sewage. In this study, the identified ARGs in the irrigation water, such as aac(6')-I, aadA2, bla genes (bla2a, blaACT, blaFIA, blaFRI, blaOXA, blaPER), and blasé, indicated that irrigation water was contaminated with antimicrobial resistant bacteria.Similarly, the soil samples showed the presence of several ARGs, including qacA, qacE, sat2, str, sul1, sul2, tet genes (tet(30), tet(Q), tet(S), tet(T), tetB(P)), van genes (van, vanS-O, vanS-Pt), vat(E), vga(A)), aph(3')-IIIa, vanXY-C, tet(33), erm(33), and blaAIM, among others. These ARGs suggest the potential for antibiotic resistance development in the soil microbial community. Furthermore, the lettuce plants irrigated with creek water, streams, and ponds carried ARGs such as aadA9, qacE, tet(W), tetA(P), nimC, vanTc, aad9, bla genes (blaSHV, blaTEM), cat-TC, cfxA, cfxA6, cmlB1, eat(A), floR2, mcr genes (mcr-10, mcr-3, mcr-7), nimJ, and others. This finding indicates that the irrigation water can contribute to the transmission of ARB and ARGs to fresh produce. The study highlights the importance of periodically testing irrigation water to ensure its safety and to prevent the spread of pathogenic bacteria and antibiotic resistance. Growers should be educated about the potential risks associated with using contaminated water for irrigation. Additionally, the use of metagenomic approaches, as employed in this study, can be valuable in assessing the quality of freshwater used in agricultural ecosystems. In conclusion, the presence of ARGs in irrigation water, soil, and lettuce samples emphasizes the need for proper water management and hygiene practices in agricultural settings to minimize the spread of antibiotic resistance and protect public health. These findings suggest that irrigation water used in agricultural practices may serve as a significant reservoir of antimicrobial-resistant bacteria. The presence of antibiotic resistance in water sources raises concerns about potential transmission to produce, highlighting the importance of understanding and managing antimicrobial resistance in agricultural settings to maintain food safety and public health. Training through webinar series: Three webinar series on metagenomics and bioinformatics were conducted by Ezbiome Inc. at Tennessee State University. This aimed to provide comprehensive training on the entire workflow of metagenomics, including wet lab procedures and post-sequencing bioinformatic and statistical analysis. The webinars covered a wide range of topics, enabling the Principal Investigator (PI) and students to gain proficiency in metagenomic research and analysis. Here is an overview of the topics covered: metagenomic study design and power calculation: sample collection, storage, and transportations, sample processing, nucleic acid extraction, and quality assessment, sequencing library preparation, quality assessment, and sequencing, rationale for sequencing parameters, introduction to metagenomics data analysis, tools for comparative metagenomic and statistical analysis, and submitting metagenomic data to public repositories. Upon completion of this training, the PI students , and other faculty were enhanced with the necessary skills and knowledge to conduct metagenomics wet lab procedures and bioinformatics analysis in their laboratory at Tennessee State University. They are also equipped to train students, technicians, and researchers at TSU, enabling them to contribute to food safety research and further expand the capabilities of metagenomic research at the institution. This project also provided valuable experiential learning opportunities for two graduate students. They were actively involved in various aspects of the research, including the cultivation and care of plants until the harvesting period. This hands-on experience allowed them to gain practical knowledge and skills related to plant cultivation in the context of the project. In addition to plant care, the students were trained on the extraction of DNA from lettuce, water, and soil samples for metagenomic analysis. Furthermore, the students were provided with comprehensive training on the entire workflow of metagenomics wet lab procedures through a series of three webinars. By participating in these experiential learning opportunities and comprehensive training, the graduate students gained practical skills in both wet lab techniques and bioinformatic analysis related to metagenomics. This will not only contribute to their own professional development but also equip them with the necessary knowledge and expertise to contribute to future research projects and collaborations in the field of metagenomics. Objective 2: Incorporate metagenomics and bioinformatics in food science courses in TSU. Microbiology and Food Plant Sanitation courses at TSU were revised to include a metagenomics and Computational Bioinformatics. Overall, the revision of these courses to include metagenomics and Computational Bioinformatics is a forward-looking decision that equips students with the knowledge and skills necessary for success in the twenty-first century STEM workforce. By integrating these topics, the courses empower students to embrace emerging technologies and contribute to advancements in food microbiology and food safety research. Graduates with proficiency in metagenomics and Computational Bioinformatics have a competitive edge in the job market. This training enhanced PI's knowledge, skills, and abilities, enabling them to gain experience in current analytic technologies and innovative technologies relevant to food safety research. The PI gained insights into how surface water affects the microbial composition on produce surfaces. This knowledge is valuable for understanding the potential sources of microbial contamination in agricultural settings and identifying strategies to enhance food safety measures. Overall, the training in metagenomics and bioinformatics provided the PI with the necessary tools and expertise to conduct research on microbial communities in the context of food safety. It expanded their capabilities to employ cutting-edge analytical technologies and innovative approaches, enabling a deeper understanding of microbial profiles in produce farms and their relationship with irrigation water sources. Objective 3: Create long-term collaboration between TSU, USDA-ARS-NEA-BARC and Laboratories. The PI established collaboration with scientists from USDA and EzBiome Inc. This collaboration facilitated the exchange of knowledge, resources, and expertise, leading to impactful research outcomes and advancements in the field of agricultural science. Additionally, the collaboration, enhanced understanding of research methodologies, and fostered long-term partnerships.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Agnes Kilonzo-Nthenge, Abdullah Ibn Mafiz, and Tobenna Aniume. 2022. Antimicrobial Resistant Escherichia coli and Enterococcus Spp. in Irrigation Water: Ponds, Creeks, Farms, and Streams in Small-Scale Produce Farms. P1-128 A, July 31 August 3 | David L. Lawrence Convention Center, Pittsburgh, Pennsylvania
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Pittsburgh, Pennsylvania
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Microbial Diversity and Antibiotic-Resistant Genes in Different Sources of Irrigation Water (Creeks, Ponds, Streams) in Small-Scale produce Farms " is under preparation for publication.
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience for this project include faculty, students, and fresh produce growers. Changes/Problems: Due to COVID-19 challenges, the travel (Beltsville, MD) (Ezbiome lab) for PI training on metagenomics analysis has been delayed. What opportunities for training and professional development has the project provided?The PI has been introduced to some of workflow on metagenomics wet lab procedures. How have the results been disseminated to communities of interest?The results have not being disseminated yet. What do you plan to do during the next reporting period to accomplish the goals?The PI will be trained more on the entire workflow of metagenomics wet lab procedures as well as post sequencing bioinformatics and statistical analysis using an intuitive, easy to use, cloud based metagenomics platform that can be operated without any prior skill of programming.
Impacts
What was accomplished under these goals?
Foodborne illnesses attributed to consumption of field-grown produce, mainly leafy greens, have increased, resulting in significant challenge to public health and multiple disease outbreaks worldwide. Producing and delivering safe fresh produce on a sustainable scale is essential to the maintenance of public confidence in the supply chain and industry. There is a necessity for more and better trained scientists to engage in cutting edge produce safety research that contributes to knowledge-transfer to produce growers. The first step to understanding the public health risk of microorganisms on fresh vegetables is to identify and describe microbial communities in farming environment. The overall goal of this project is to train metagenomics, a fundamental tool in food safety that allows the detection, identification and characterization of inclusive range of pathogens in foods to faculty and students at Tennessee State University (TSU). Metagenomics and computational bioinformatics will be applied to enhance food safety research at TSU. Metagenomics approaches will be used by TSU scientist to determine and describe microbial taxonomic and functional diversity in various environments including agricultural soils, as well as waters used for irrigation in produce farms. Training in metagenomics and bioinformatics enables the investigation of how surface water including pond and creek irrigation water influence the composition of bacterial communities on the surfaces of produce. Application of metagenomics and bioinformatics provides more answers pertaining to characterization of microbial profiles in produce farms. Better-trained food safety scientists and students at TSU will engage in cutting edge produce safety research and outreach that contributes to knowledge transfer to produce growers; hence safe produce to consumers. Data on research findings will be used to educate producers, our stakeholders to make informed decisions and as a result reduce the spread of foodborne illnesses and increase capital gains. Additionally, this project will integrate metagenomics and bioinformatics in food safety research, training of undergraduate and graduate students with concentration in the Animal and Food Science, in the College of Agriculture at Tennessee State University. Development of protocol: Methods on isolation and characterization of bacteria from irrigation water and lettuce plants were developed. These methods were used to isolate bacteria from creek, pond, well, and municipal irrigation water and lettuce irrigated with these different water sources. Growing and Harvesting of Lettuce: Summer Crisp Lettuce seedlings were planted in 16 blocks with different treatments. Standard compost (5 g/plant) was applied uniformly in all blocks. One-month old seedlings were planted in the field with a 5 ft row to row and 1 ft plant to plant spacing in each block. Field were scouted weekly for any insect pest and diseases occurrences. Creek and pond irrigation water was used for irrigating the lettuce until harvest. Water from the creek, well, and pond was used for drip irrigation to grow letuce. Visits were made to collect water from farms twice in a week to irrigate lettuce grown in TSU research farm. Water from the creek and pond was used for drip irrigation and rain-style sprinkler to simulate rain and associated splash. During this reporting period, two graduate students were engaged: 1. Planting and harvesting of the lettuce plants; 2.Experiential learning opportunity; extracted DNA from lettuce, water, and soil for metagenomics study.
Publications
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