HEATED RESISTANCE: THERMAL TREATMENT TECHNOLOGY MITIGATION OF BIOLOGICAL WASTES’ ANTIBIOTIC RESISTANCE AND GENE MOBILITY IN WASTE SYSTEMS.

Abstract

The burgeoning global threat of antimicrobial resistance (AMR) has policy makers,veterinarians, farmers, and physicians re-evaluating antibiotic stewardship. Worldwide, millions of people are affected by multidrug resistant bacteria. Human and animal waste are primary transporters of antibiotics, antibiotic resistant bacteria (ARB), and antibiotic resistant genes (ARGs) through rural and urban systems. Resistance within biological waste, as it moves through the landscape and sanitary/manure infrastructure to adjacent natural systems, is yet to be fully understood. The various environmental conditions, bacterial composition, and genetic factors result in highly complex interdependent relationships that influence the occurrence and dissemination of ARB and ARGs. Understanding the fate of ARB and movement of ARGs is critical to evaluating environmental and anthropogenic impact. Agricultural systems and wastewater treatment plants are target locations for quantifying connections between clinical and animal antibiotic use and environmental AMR. Waste management techniques/technologies, such as compositing and anerobic digestion (AD), have been shown to be effective in combating AMR. Studies have highlighted temperature as a key environmental determinant that could influence antibiotic degradation, ARG, and ARB abundance. The proposed research examines advanced heat-based manure and wastewater technological capacity for AMR reduction, while measuring treatment impact on ARG dissemination. Focusing on the reduction of AMR within biological waste treatment and the distribution of AMR factors into the environment. A key metric to understanding AMR is accurate detection and quantitation of antibiotic concentration within manure and other biosolid waste products. The first phase of this dissertation research focused on the development of a liquid chromatography in tandem with mass spectrometry (LC-MS/MS) method for detecting multi-class antibiotics residuals in various manure substrates. To combat the challenges of manure heterogeneity, this work focused on novel extraction methodology to achieve higher recovery of tetracyclines, macrolides, sulfonamides, and beta lactams simultaneously in a complex manure matrix. The method includes a two-step, liquid-solid extraction using 10 mL of 0.1 M EDTA-McIlviane buffer followed by 10 mL of methanol. Reporting total antibiotic recoveries of 67–131% for tetracyclines, 56% for sulfonamide, 49–53% for macrolides, and 1.3–66% for β-lactams. This method is novel in its application to four different manure substrate and utilization for waste risk assessment. This developed method was used for antibiotic quantification throughout the three thermal treatment studies to determine antibiotic concentrations, degradation, and monitor agricultural contributions to environmental AMR. The following research extensively focused on the evaluation of three advanced, high temperature waste treatment technologies on the mitigation of antibiotic resistance factors, including a composting rotary drum bedding recovery unit (BRU), thermophilic (55°C) and mesophilic (35°C) AD, and thermal hydrolysis pretreatment to reduce antibiotics, ARGs, and ARB. The assessment of environmental components, such as metals, bacterial community, and nutrient composition, are also included to determine any relational trends. The BRU study was conducted as a mass balance analysis to highlight antibiotics, ARGs and ARB partitioning within the BRU system. Dairy manure samples were collected over 24-hour period as the manure was treated with a solid-liquid separator producing two streams of substrates (liquids and separated solid), with the separated solid fraction continuing to the high temperature BRU processing. This study generated a mass flow analysis of manure and partitioning of antibiotic resistance factors throughout the manure treatment system. The study indicated that most of the manure mass containing the AMR factors goes untreated following solid-liquid separation, with 95% of the mass pumped to a storage lagoon and 5% proceeding to BRU processing. The removal of antibiotic residuals during BRU processes was insignificant, yet the BRS processing was 100% effective in removing the ARB examined. Five (Intl1, sul1, tetQ, tetX and tetM) of the eight ARGs were found to have significant reduction (>95%) following the thermophilic rotary drum composting portion of the BRU system. While the three other ARGs (tetW, ermB and bla2) remained constant despite treatment. An AD experiment was implemented as lab-scale destructive assay, highlighting antibiotic removal at two temperature and over time. This destructive batch assay used 18 sets of triplicate AD reactors filled with antibiotic spiked dairy manure and incubated under anerobic conditions at 35°C or 55°C for 43 days. Triplicates bottles destructively sampled at six time points (Day 0, 3, 9, 21, 36, and 43) to generate a degradation curve. The antibiotic erythromycin was more efficiently degraded under mesophilic conditions, with 100% removal by Day 36 compared to 97% reduction for thermophilic conditions during the 43-day digestion period. Though the higher temperature conditions proved better for oxytetracycline degradation, with 66% removal compared to only 22% removal for mesophilic conditions. ARG removal was dependent on the bacterial community, as the different conditions selected for various bacteria. While both conditions proved to be effective in reducing most of the ARGs (4-5 out of 8 genes tested), enrichment of other resistance genes was also documented. The tetW gene was found to increase >81% for both digester temperatures, highlighting the variety of bacteria harboring resistance genes and their varied responses to environmental conditions. The ermB genes was found to be located on the intl1 mobile genetic element and likely resided within bacteria that was not heat tolerant. This study highlighted the role of residential digester bacteria in harboring and potentially transferring resistance genes. The thermal hydrolysis (THP) technology ability to extensively lysis substrates was examined with subsequent AD for its impact on reducing antibiotic resistance factors. Comparative analysis of THP processing on spiked diary manure and wastewater biosolids followed by mesophilic digestion at 35°C was conducted to document substrate response to the treatment. AD was conducted as a destructive assay for 30 days with a 4-point sample curve (Day 0, 10, 20 and 30). This study can be found in the appendix. In addition to the lab and field work described above, this body of research also included a review and a proposed communication model for antibiotic resistance education for the general public. Lay audiences’ exposure and understanding of complex natural issues, such as AMR and climate change, are essential to behavioral changes and potentially legislative actions. By surveying and evaluating various aspects of scientific communication, this research empathized five rhetorical elements of storytelling shown to influence audience reception to scientific messaging. Communication techniques, such as narrative structure, normalization of the subject using human scaling, non-agentive language, trusted experts for message delivery, and future simulation, were all analyzed and reviewed for their effectiveness and incorporated into a mock model for presenting information about AMR. Bridging gaps between research institutions and the public is key to generating more inclusive spaces for innovation and mitigating issues interwoven within the built and natural environment.