Environmental Microbiology Group, Department of Microbiology and Virology, Faculty of Biology, University of Havana, Havana, Cuba.
Microbiology and Environmental Technologies Section, Water Research Institute, University of Granada, Granada, Spain.
Microb Ecol. 2024 May 15;87(1):71. doi: 10.1007/s00248-024-02385-0.
The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, bla, bla, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.
几种环境中抗生素耐药菌（ARB）的高流行率是一个令人担忧的问题，威胁着人类健康。特别是，由于高度抗生素浓缩废水的管理或处理效率低下，污水处理厂（WWTP）成为 ARB 传播到受纳水体的重要贡献者。因此，开发能够正确监测这些重要治疗化合物（抗生素耐药基因，ARGs）编码抗性的分子工具至关重要。为了准确量化 ARGs，需要支持良好引物设计和验证方案的灵敏而稳健的 qPCR 检测。在这项研究中，选择了 11 个相关的 ARGs 作为靶标，包括 aadA 和 aadB（对氨基糖苷类药物的抗性）；ampC、bla、bla 和 mecA（对β-内酰胺类药物的抗性）；dfrA1（对 trimethoprim 的抗性）；ermB（对大环内酯类药物的抗性）；fosA（对 fosfomycin 的抗性）；qnrS（对喹诺酮类药物的抗性）；和 tetA(A)（对 tetracyclines 的抗性）。新引物组的计算机设计是基于目标 ARG （同源性等级> 70%）在京都基因与基因组百科全书（KEGG）数据库中的所有序列比对进行的，允许对 ARGs 的生物多样性进行更高的覆盖，而不是迄今为止描述的几种引物的覆盖。在从与污水处理相关的自然和工程环境中采集的六个样本中验证了新分子工具的适当设计和性能。优化 qPCR 检测的特点是高扩增效率（> 90%）、标准曲线的良好线性（R> 0.980）、实验之间的重复性和再现性以及宽线性动态范围。本文描述的新引物组和方法是 WWTP 和相关环境中通过 qPCR 升级对目标 ARGs 丰度和出现进行监测的有价值的工具。
