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

由于兽用抗生素的广泛使用，畜牧业被认为是产生和排放抗生素耐药细菌(ARB)的关键贡献者。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可移动遗传元件(MGE)中，因此在传播抗生素耐药性方面发挥着重要作用。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ARB和ARG在农田和周围水道中的扩散，以及最终对公众和生态系统健康的影响。解决这一问题的一个替代方案是设计粪便处理工艺，确保在ARB和ARG到达环境之前减少它们。厌氧消化池的安装正在加拿大各地迅速扩大(在农场或更大的地区性工厂)，以满足在可持续废物管理和可再生能源方面日益增长的需求。我们建议使用粪便厌氧消化作为一个模型，以更好地了解ARG演变过程中涉及的微生物和分子机制。制定通过粪便处理缓解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弹性。这项研究的具体目标是：1)确定不同规模、操作和粪便管理系统的奶牛场生粪便和处理粪便中ARG的存在、水平和流行率；2)评估不同操作参数对厌氧消化器中耐药组(总ARG)和流动组(总MGE)谱和微生物种群动态的长期影响；3)通过使用元基因组学和生物信息学工具和生物统计学，确定负责携带和传播ARG的特定微生物群；4)通过比较ARG在其他厌氧消化应用中的存在和流行(共生)来验证ARG的潜在微生物组宿主；5)开发和验证一种计算算法，以估计与生和处理的动物粪便中ARG、MGE和微生物群落的存在相关的潜在风险，该算法可用于样品和过程之间的系统比较。这项研究计划将提供生物处理、废物处理和元基因组学方面的多学科培训，并为加拿大不断增长的生物经济提供高技能的专业人员(2名博士、2名理学硕士、5名理学学士)，这些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.