摘要
      背景证明抗生素抗性基因（ARGs）和可移动遗传元件（MGE）在复杂环境基质中的转移、动力学和调节在实验上仍然具有挑战性，有许多重要的悬而未决的问题，如ARGs在自然界中的转移和传播是如何发生的以及在哪里发生的。在自然环境和微生物群落中，携带ARGs的MGE转移的程度和条件在很大程度上仍未被探索。生物膜是一种结构，包括高密度多物种细菌群落，嵌入自产的胞外聚合物（EPS）中，构成促进基因转移的基质，细菌对压力和抗生素表现出高耐受性。在这项研究中，我们采用了一种采样和分析方法，可以对原位和重组的体外医院和城市污水（WW）生物膜进行表型和基因组分析。为了评估医院和城市污水处理生物膜在污水处理系统中有效传播ARGs的潜力，我们探索了生物膜基质中的EPS，并通过元转录组学评估了耐药组（ARGs）和迁移组（MGE）的表达。
结果我们首先表明，a）EPS的组成因其生长环境（原位和体外）和取样来源（医院和城市污水）而异，b）少量的环丙沙星会影响EPS的组成。接下来，元转录组学方法表明，a）对来自医院WW的生物膜添加少量环丙沙星后，ARGs和MGE的表达增加，但对来自城市WW的不添加；b）携带单个或多个ARGs的特异性质粒的表达根据生物膜的WW来源而变化。当相同的质粒在城市和医院WW生物膜中表达时，它们携带并表达不同的ARG。
结论我们表明，医院和城市污水塑造了环境生物膜的结构和活性抗性，并证实医院污水是AMR传播和选择的重要热点。医院和城市生物膜对抗生素压力的不同反应，加上生物膜结构的差异，有助于描述医院和城市WW生物膜的不同特征，突出了耐药性及其在环境生物膜及其周围生态系统中的表达之间的关系。
Abstract
Background Demonstration of the transfer, dynamics, and regulation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in a complex environmental matrix is yet experimentally challenging, with many essential open questions such as how and where transfer and dissemination of ARGs happens in nature. The extent and conditions of MGEs transfer that carry ARGs is still largely unexplored in natural environments and microbial communities. Biofilms are structures that include high density multi-species bacterial communities embedded in self-produced extracellular polymeric substances (EPS) constituting a matrix that facilitates gene transfer and where bacteria exhibit high tolerance to stress and to antibiotics. In this study we implemented a sampling and analysis approach that allows phenotypic and genomic analyses of in situ and reconstituted in vitro hospital and urban wastewater (WW) biofilms. To assess the potential of hospital and urban WW biofilms to efficiently disseminate ARGs in the WW system, we explored the EPS within the biofilm matrix and assessed the expression of the resistome (ARGs) and mobilome (MGEs) by metatranscriptomics.

Results We first showed that a) the composition of EPS differs depending on their growth environment (in situ and in vitro) and their sampling origin (hospital vs urban WW) and that b) a low amount of ciprofloxacin impacted the composition of the EPS. Next, the metatranscriptomic approach showed that a) expression of ARGs and MGEs increase upon adding a low amount of ciprofloxacin for biofilms from hospital WW but not for those from urban WW and b) that expression of specific plasmids that carry individual or multiple ARGs varies depending on the WW origins of the biofilms. When the same plasmids were expressed in both, urban and hospital WW biofilms, they carried and expressed different ARGs.

Conclusion We show that hospital and urban wastewaters shape the structure and active resistome of environmental biofilms, and we confirmed that hospital WW is an important hot spot for the dissemination and selection of AMR. The different responses to antibiotic pressure in hospital vs urban biofilms, coupled with differences in biofilm structure helps delineate distinct characteristics of hospital and urban WW biofilms highlighting the relationships between the resistome and its expression in environmental biofilms and their surrounding ecosystems.

https://www.biorxiv.org/content/10.1101/2023.01.19.524754v3.abstract