Manure is a commonly used soil fertilizer, but there are concerns that this practice could affect the spread of antibiotic resistance genes (ARGs) from farm to fork. A microcosm-scale study evaluated the effect of prior antibiotic use (manure-based soil amendments generated from dairy and beef cattle with or without antibiotic administration), composting, and soil type on the quantity of ARGs and the microbial community composition of dairy and beef manure applied soil. ARGs were analyzed through novel metagenomic techniques and quantitative polymerase chain reaction of sul1, tet(W), and 16S rRNA gene, while the microbial community composition was determined via 16S rRNA amplicon sequencing. The results indicated that while prior antibiotic administration elevated the relative abundance of ARGs and changed the microbial community of raw manure applied soils, composting reduced this effect. However, compost applied soils still had a higher relative abundance of ARGs than the unamended soils and occasionally soil applied with raw manure of untreated cattle. Soil type may be a mediating factor as there were differences observed between the three soil types (sandy loam, silty clay loam, and silty loam) with sandy loam amended soils often having the least attenuation of ARGs. As the relative abundance of ARGs was still elevated and the microbial community composition still significantly different from the unamended soils after 120 days, these results suggest that 120 days is not a long enough waiting period between biological soil amendments and crop harvest for ARG dissipation. / Master of Science / Antibiotics are lifesaving drugs that kill infection-causing bacteria. However, bacteria are living organisms and can adapt to stresses, such as antibiotics. When antibiotics are used, not all of the targeted bacteria are necessarily killed, and populations of resistant bacteria can survive. Resistant bacteria can not only continue to grow, but can also share their resistance capabilities with other unrelated bacteria through the transfer of antibiotic resistance genes (ARGs). ARGs are segments of DNA encoding mechanisms for the bacteria to survive antibiotic attack, such as pumping antibiotics outside of the cell or strengthening the cell wall so antibiotics cannot enter. The transfer of ARGs to human pathogens is of utmost concern, as it can cause once treatable diseases to turn deadly. Antibiotics are thus a double-edged sword because they can save lives on one hand, while their overuse or misuse can undermine their effectiveness by increasing antibiotic resistance. In the U.S. and many other countries, the biggest user of antibiotics is the livestock industry. Thus, there is growing interest in possible routes by which antibiotic resistance can spread from agriculture to humans. While some previous work has been done on direct contact with animals and meat products, less attention has been paid to the potential role raw produce grown in soils fertilized with manure-based amendments. This study thus sought to determine which factors impact ARG levels in soil. Questions of interest included: What is the effect of composting raw manure prior to soil application? Does prior treatment of cattle with antibiotics matter? Does the soil type influence the levels of ARGs? Do the ARG composition and microbial community composition respond similarly to such factors? These and other questions were evaluated in a controlled environment by simulating amended field conditions in small glass jars (microcosms) containing mixtures of different soils and manure-based amendments. Three different soils were amended with one of the following manure-based amendments: raw manure from antibiotic administered cattle, composted manure of antibiotic administered cattle, raw manure from cattle not given antibiotics, composted manure of untreated cattle, and no amendment. This experimental setup was done in duplicate, one for treatments from dairy cows and one for the beef steer treatments. The experiment lasted 120 days, as that is a current standard for how long organic farmers must wait between manure application and crop harvest. Samples were taken throughout the 120-day experiment, and the quantity of targeted ARGs was determined by analyzing the DNA through qPCR, while the overall ARG profile was studied using a new tool, called metagenomics. To identify the kinds of bacteria present in the samples (microbial community composition), the 16S rRNA gene, which is a universal gene in organisms, was targeted and sequenced via amplicon sequencing. The results of these analyses indicated that administering antibiotics to cattle and then subsequently amending soil with their manure was associated with the highest levels of ARGs compared to the other treatments, but composting reduced the effect of prior antibiotic use. Depending on the ARG, composting decreased ARG levels relative to the other treatments, but in some instances, it increased ARGs compared to soils with raw manure of untreated cattle. Even after composting, there were still higher levels of ARGs in the soil than unamended soils. Different soil types did react differently to the amendments, but more research is needed. All of the treatments resulted in different changes to the microbial community composition and did not return to the unamended soil’s community structure even after 120 days. Overall, based on these results, ARGs and the microbial community do not return to the initial condition within 120 days, which is a recommended wait time between amendment and harvest, while composting and soil type appear to be mediating factors. Additional research is needed to further advance understanding of potential mitigation options and to benchmark them to defined and measureable risk endpoints.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/86672 |
| Date | 20 July 2017 |
| Creators | Pankow, Christine Ann |
| Contributors | Civil and Environmental Engineering, Pruden, Amy, Xia, Kang, He, Zhen |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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