Antibiotic resistance is a growing human health threat, making infections more difficult to treat and increasing fatalities from and cost of treatment of associated diseases. The rise of multidrug resistant pathogens threatens a return to the pre-antibiotic era where even the most common infections may be impossible to treat. It is estimated that the majority of global antibiotic use, and use in the U.S., is dedicated towards livestock, where they are used to promote growth, treat, or prevent disease. Given that exposure to antibiotics selects for antibiotic resistant bacteria (ARBs) and can stimulate the horizontal transfer of their associated antibiotic resistance genes (ARGs), it is important to examine livestock operations as a reservoir of resistance. Correspondingly, there is growing interest in identifying how agricultural practices can limit the potential for spread of antibiotic resistance through the "farm to fork continuum," starting with antibiotic use practices, manure management and land application and ending with the spread of ARBs and ARGs present onto edible crops and serving as a route of exposure to consumers. This study focused specifically on the effect of composting on the prevalence of ARBs and ARGs in cattle manure. Three composting trials were performed: small-scale, heat-controlled, and large-scale. The small-scale composting trial compared dairy and beef manures, with or without antibiotic treatment (treated beef cattle received chlortetracycline, sulfamethazine, and tylosin while treated dairy cattle received cephapirin and pirlimycin), subject to either static or turned composting. The heat-controlled composting trial examined only dairy manure, with or without antibiotic treatment, subject to static composting, but using external heat tape applied to the composting tumblers to extend the duration of the thermophilic (>55°C) temperature range. The large-scale composting trial examined dairy manure, with or without antibiotic treatment, subject to static composting at a much larger scale that is more realistic to typical farm practices. Samples were analyzed to assess phenotypic resistance using the Kirby Bauer disk diffusion method and by diluting and plating onto antibiotic-supplemented agar. Genetic markers of resistance were also assessed using quantitative polymerase chain reaction (qPCR) to quantify sul1 and tet(W) ARGs; metagenomic DNA sequencing and analysis were also performed to assess and compare total ARG abundance and types across all samples. Results indicate that composting can enrich indicators of phenotypic and genetic resistance traits to certain antibiotics, but that most ARGs are successfully attenuated during composting, as evidenced by the metagenomic sequencing. Maintaining thermophilic composting temperatures for adequate time is necessary for the effective elimination of enteric bacteria. This study suggests that indicator bacteria that survive composting tend to be more resistant than those in the original raw manure; however, extending the thermophilic stage of composting, as was done in the heat-controlled trial, can reduce target indicator bacteria below detection limits. Of the two ARGs specifically quantified via qPCR, prior administration of antibiotics to cattle only had a significant impact on tet(W). There was not an obvious difference in the final antibiotic resistance profiles in the finished beef versus dairy manure composts according to metagenomics analysis. Based on these results, composting is promising as a method of attenuating ARGs, but further research is necessary to examine in depth all of the complex interactions that occur during the composting process to maximize performance. If not applied appropriately, e.g., if time and temperature guidelines are not enforced, then there is potential that composting could exacerbate the spread of certain types of antibiotic resistance. / Master of Science / Antibiotics are drugs that are used to treat bacterial infections by killing the bacteria that cause the infection. Bacterial infections now exist that are resistant to several antibiotics; which are extremely difficult and costly to treat. Many antibiotics are used in the agriculture industry where they are used to promote growth, treat, or prevent disease in livestock animals. The antibiotics may then cause an increase in antibiotic resistance in bacteria by encouraging changes to the DNA of the bacteria which allow them to survive in the presence of antibiotics that would normally kill them. These DNA segments are called antibiotic resistance genes. Once developed, bacteria can share resistance genes among themselves, allowing for single bacteria that can resist several types of antibiotics. For this reason, it is important to see if it is possible to prevent the spread of antibiotic resistance from animal agriculture to people. One way that people could be affected would be if produce were exposed to resistant bacteria when grown in soil that had been fertilized with manure or compost. This study looks at the impact of composting on the presence and amount of antibiotic resistance genes in composted cattle manure. Three composting trials were performed: small-scale, heat-controlled, and large-scale. The small-scale composting trial compared dairy and beef manures, with or without antibiotic treatment, with or without regular turning during composting. The heat-controlled composting trial examined only dairy manure, with or without antibiotic treatment, without regular turning during composting, but using external heat to maintain high temperatures. The large-scale composting trial examined dairy manure, with or without antibiotic treatment, without regular turning during composting, but at a larger scale that is more realistic to how composting is actually performed on farms. Antibiotic resistance of compost bacteria was tested by growing bacteria on nutrient-dense plates containing antibiotic disks and measuring how much each antibiotic prevented the growth of the bacteria, in terms of the diameter about each disk where bacteria did not grow. Individual target resistance genes were measured throughout the study by using a method called qPCR. Metagenomic analysis was performed to identify all of the genes, especially resistance genes, in each of the samples. Results v show that composting may increase antibiotic resistance in bacteria that survive the composting process, but that most resistance genes are themselves reduced. The key to successful composting is maintaining high temperatures for as long as possible; this is necessary to kill off infectious bacteria. Extending the high temperature (>55°C) phase of composting is a potential method for improving the effectiveness of composting in eliminating pathogens and destroying resistance genes. Results were not significantly affected by whether antibiotics were given to the cattle and were not different between dairy or beef cattle. Based on these results, composting is a promising method of reducing resistance genes in composted manure, but further research is necessary to maximize performance. If not performed correctly, composting could have the opposite effect and be detrimental.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/74952 |
| Date | 06 February 2017 |
| Creators | Williams, Robert Kyle |
| Contributors | Civil and Environmental Engineering, Pruden, Amy, Boardman, Gregory D., Krometis, Leigh-Anne H. |
| 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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