Evaluating Microbial and Molecular Mechanisms that Affect Changes in Antibiotic Resistance during Anaerobic Digestion of Manure

由于广泛使用兽用抗生素，畜牧生产和畜牧业被认为是产生和排放耐抗生素细菌（ARB）的主要原因。抗生素耐药基因（ARG）是编码多种耐药机制的遗传单位，由于它们通常位于易于在微生物物种之间转移的移动遗传元件（MGE）中，因此在抗生素耐药性的传播中起着重要作用。因此，不直接接触抗生素的细菌也容易获得抗生素耐药机制。由于使用动物粪便作为土壤肥料是一种常见的农业做法，人们越来越关注农业领域和周围水道中ARB和ARG的扩散，以及最终对公众和生态系统健康造成的后果。解决这一问题的一种替代方案是设计粪肥处理工艺，确保在ARB和ARG到达环境之前减少它们。厌氧消化装置正在加拿大各地迅速扩展（在农场或更大的区域工厂），以满足对可持续废物管理和可再生能源日益增长的需求。我们建议以粪便厌氧消化为模型，更好地了解参与ARG进化的微生物和分子机制。制定通过粪便处理减少ARB和ARG的有效战略将为建立和运行厌氧消化系统提供额外的激励，这将增强加拿大农业的长期可持续性和复原力。本研究计划的具体目标是：1)确定不同规模、运营和粪肥管理系统的奶牛场的原始和处理粪肥中ARGs的存在、水平和流行程度；2)评估不同操作参数对厌氧消化池中抵抗组（总ARG）和可移动组（总MGE）分布和微生物种群动态的长期影响；3)通过使用宏基因组学和生物信息学工具以及生物统计学，确定负责携带和传播ARG的特定微生物群；4)通过比较其在其他厌氧消化应用（如城市污水处理和工业废水处理）中的存在和流行（共发生），验证潜在的ARG微生物群宿主；5)开发并验证一种计算算法，以估计与未经处理和处理的动物粪便中ARG、MGE和微生物群落存在相关的潜在风险，该算法可用于样品和工艺之间的系统比较。该研究项目将提供生物处理、废物处理和宏基因组学方面的多学科培训，并为加拿大不断发展的生物经济提供高技能专业人员（2名博士、2名硕士、5名理学士），加拿大公司报告了严重的劳动力和技能短缺。

Livestock production and animal husbandry are considered key contributors responsible for generating and emitting antibiotic-resistant bacteria (ARB) due to the extensive use of veterinary antibiotics. Antibiotic resistance genes (ARG), the genetic units encoding the diverse mechanisms of resistance, play an important role in disseminating antibiotic resistance because they are usually located in mobile genetic elements (MGE) that are easily transferred between microbial species. Therefore, bacteria that are not directly exposed to antibiotics are also susceptible to acquiring antibiotic resistance mechanisms. Since the application of animal manures as soil fertilizers is a common agricultural practice, there is increasing concern about the proliferation of ARB and ARG in agricultural fields and surrounding waterways and ultimately the consequences this will have on public and ecosystem health. One alternative to address this problem is to design manure treatment processes that ensure the reduction of ARB and ARG before they reach the environment. Anaerobic digester installations are readily expanding across Canada (at farm level or larger regional plants) to meet the growing demand in sustainable waste management and renewable energy. We propose using manure anaerobic digestion as a model to better understand microbial and molecular mechanisms involved in the evolution of ARG. Developing effective strategies for ARB and ARG mitigation through manure treatment will provide an additional incentive to build and operate anaerobic digestion systems, which will enhance the long-term sustainability and resilience of Canadian agriculture. The specific objectives of this research program are to: 1) Determine the presence, levels, and prevalence of ARGs in raw and treated manures in dairy farms with different sizes, operations, and manure management systems; 2) Assess the long-term impact of different operational parameters on the resistome (total ARG) and mobilome (total MGE) profiles and microbial population dynamics in anaerobic digesters; 3) Identify specific microbial groups responsible for carrying and spreading ARG, through the use of metagenomic and bioinformatic tools and biostatistics; 4) Validate potential microbial group hosts of ARG by comparing their presence and prevalence (co-occurrence) in other anaerobic digestion applications, such as in municipal wastewater treatment and industrial wastewater treatment; 5) Develop and validate a computational algorithm to estimate the potential risk associated with the presence of ARG, MGE, and microbial communities in raw and treated animal manures that can be used for the systematic comparison between samples and processes. This research program will provide multidisciplinary training in bioprocessing, waste treatment, and metagenomics, and supply highly skilled professionals (2 PhD, 2 MSc, 5 BSc) to Canada's growing bio-economy, where companies reported significant labour and skill shortages.