Tavares Rafael D S, Rodrigues Elsa T, Tacão Marta, Henriques Isabel
Department of Life Sciences, Centre for Functional Ecology, Associate Laboratory TERRA, Faculty of Sciences and Technology, University of Coimbra, 3000-456, Coimbra, Portugal; Centre for Environmental and Marine Studies (CESAM) and Department of Biology, University of Aveiro, 3810-193, Aveiro, Portugal.
Department of Life Sciences, Centre for Functional Ecology, Associate Laboratory TERRA, Faculty of Sciences and Technology, University of Coimbra, 3000-456, Coimbra, Portugal.
Water Res. 2025 Sep 1;283:123718. doi: 10.1016/j.watres.2025.123718. Epub 2025 May 8.
Wastewater is a hotspot for the spread and evolution of antimicrobial resistance (AMR), with integrons playing a key role in the dissemination of AMR. However, there is limited information available regarding the integron pool of wastewater environments. The present study aims to characterize the integron pool in outflow secondary-treated municipal wastewater samples collected weekly over the course of a year (n = 52 samples) and to identify the risks associated with the carriage of clinically relevant antimicrobial resistance gene cassettes (ARGCs). Integron arrays were amplified with the primer combinations intI-R/3'CS and intI3F/class3R. PacBio long-read sequencing was performed on 12 monthly pooled samples per primer pair. The analysis yielded 283,579 integron reads, capturing an estimated 10%-20% of the total integron pool, with the most abundant arrays successfully recovered. Most identified GCs had no known function (33.3% to 74.5% depending on integron class). Regarding GCs with known function, AMR was the most prevalent function in class 1 integrons. Class 3 integrons primarily encoded defence mechanisms and amino acid transport and metabolism, while cassettes associated with replication, recombination and repair were abundant in integrons of unknown class. The prevalence of ARGCs for class 1, 3 and integrons of unknown class was 61.7%, 19.2% and 9.6%, respectively. Most ARGCs (from the 146 identified) encoded resistance to aminoglycosides and beta-lactams, the most abundant being aadA10, aac(6')-IIc and bla. Most beta-lactamase-encoding GCs (BLGCs) identified encoded enzymes with carbapenemase and/or extended-spectrum activity, with class 1 and class 3 integrons being relevant reservoirs of BLGCs. GCs encoding proteins involved in oxidative stress repair, as well as in other functions, were also identified and might promote co-selection of ARGCs. Large variation of the integron arrays and the GC pool was observed between seasons and pooled monthly samples, with resistance genotypes such as macrolide and phenicol resistance differentially abundant across seasons. Overall, these findings show that secondary-treated wastewater is a reservoir of highly diverse class 1 and class 3 resistance integrons, some encoding critically relevant resistance mechanisms (e.g., carbapenemases).
废水是抗生素耐药性（AMR）传播和演变的热点，整合子在AMR传播中起关键作用。然而，关于废水环境中整合子库的信息有限。本研究旨在描述一年中每周采集的流出二级处理城市废水样本（n = 52个样本）中的整合子库，并确定携带临床相关抗生素耐药基因盒（ARGCs）的相关风险。使用引物组合intI-R/3'CS和intI3F/class3R扩增整合子阵列。对每个引物对的12个月度混合样本进行PacBio长读长测序。分析产生了283,579条整合子读数，估计捕获了总整合子库的10%-20%，最丰富的阵列被成功回收。大多数鉴定出的基因盒没有已知功能（根据整合子类别为33.3%至74.5%）。对于具有已知功能的基因盒，AMR是1类整合子中最普遍的功能。3类整合子主要编码防御机制以及氨基酸转运和代谢，而与复制、重组和修复相关的基因盒在未知类别的整合子中丰富。1类、3类和未知类整合子的ARGCs流行率分别为61.7%、19.2%和9.6%。大多数ARGCs（来自146个鉴定出的）编码对氨基糖苷类和β-内酰胺类的耐药性，最丰富的是aadA10、aac(6')-IIc和bla。大多数鉴定出的编码β-内酰胺酶的基因盒（BLGCs）编码具有碳青霉烯酶和/或超广谱活性的酶，1类和3类整合子是BLGCs的相关储存库。还鉴定出了编码参与氧化应激修复以及其他功能的蛋白质的基因盒，它们可能促进ARGCs的共选择。在季节和月度混合样本之间观察到整合子阵列和基因盒库的巨大差异，大环内酯类和氯霉素耐药性等耐药基因型在不同季节的丰度不同。总体而言，这些发现表明，二级处理后的废水是高度多样化的1类和3类耐药整合子的储存库，其中一些编码关键的相关耐药机制（如碳青霉烯酶）。
