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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety & Quality Research » Research » Research Project #433482

Research Project: Investigation of Nutrient Enrichment as the Main Cause of Antimicrobial Resistance Occurrence in Livestock Environments

Location: Meat Safety & Quality Research

Project Number: 3040-42000-018-07-T
Project Type: Trust Fund Cooperative Agreement

Start Date: Sep 1, 2017
End Date: Aug 31, 2018

The goal of this experiment is to use sterile nutrients to enrich the soil bacterial population to levels observed in cattle feedlot environments and observe the effects on the AMR bacterial population through culture based methods, using E. coli and enterococci as indicators of the native bacterial populations in the soil, and metagenomic approaches. Specific objectives: A. Determine the effect of enrichment on the levels of native antimicrobial resistant bacteria in soil using culture-based methods. B. Determine the effect of enrichment on the diversity and levels of native antimicrobial resistance genes in soil using metagenomic and quantitative PCR methods. C. Compare prevalence and levels of AMR bacterial populations and ARG from enrichments plots with those from feedlot environments.

Specific Experiment Procedure: Enrichment plots. Green space located at USMARC will be the site where plots will be developed. This space has had little to no impact by antimicrobials or manure through direct application or from runoff for at least 15 years. The population of soil bacteria in this space should be ideal for this experiment, as it has been minimally influenced by agricultural or urban practices. The four plots will be developed by tilling areas of 6’ x 14’. Each will be protected from wildlife by installing fencing adequate to exclude deer and rabbits around each of the plots. Within each plot three enrichment sites (2’ x 2’) will be designated. For Phase I, one enrichment site will receive sterile bacterial growth media (2x tryptic soy broth [2x TSB]). Another enrichment site will receive sterile water, while the final enrichment site receives no treatment. For Phase II, each of the three enrichment sites will receive nutrient media to encourage specific bacterial types. New plots will be used for Phase II. Sample collection and treatment. Following tilling of the 6’ x 14’ plots, samples will be collected from each enrichment site following the sampling schedule below. Immediately prior to all sample collections, the soil in each enrichment plot will be homogenized with the use of a “Garden Claw”. Homogenization will consist of a thorough mixing of the top 3 to 6 inches of soil within each enrichment plot. To prevent cross-contamination, Garden Claws will be wiped clean, sanitized with 70% ethanol, and wiped dry between enrichment sites. Samples will be collected with a gloved hand used to grab small portions of surface material and placing them into a ziplock bag. To prevent cross-contamination, clean gloves will be used for each sample. Separate disposable shoe covers will be used for each enrichment plot during sample collection, and homogenization and application of treatments. The final sample weight will be approximately 100 g. Following each sampling, 1.5 L of treatment (either 2x TSB or water) will be uniformly added to the enrichment plot. Enrichment plots will be homogenized and treatments added on Mondays, Wednesdays and Fridays of each week to simulate the continuous manure excretion of cattle. The volume of treatment added is based on the amount of manure (27 kg) produced by a 450 kg animal per day in a feedlot setting. Ninety percent of manure is liquid, hence 24 L of liquid manure is spread over 200 ft2 (typical space requirement from FASS Guidelines). This would equal 3.5 L over a 4-ft2 area per week. Sampling schedule. TSB or water will be added every Monday, Wednesday, and Friday. Samples will be collected each Monday. Sample analysis. All samples will be processed using microbiology culture-based methods for multiple AMR organisms. In addition, DNA will be isolated from select samples for qPCR and metagenomic analysis of antimicrobial resistance genes.