摘要

       水环境是许多抗生素耐药基因（ARGs）的贮存库之一。鱼类作为重要的水生动物，具有理想的肠道生态位，可以从水环境中摄取抗药性细菌。鱼的肠道将是一个适合夫妻基因转移的环境，包括那些编码抗生素抗性的环境。然而，很少有人知道摄入的ARGs或抗生素耐药菌（ARB）对肠道菌群的影响。本文应用培养方法、qPCR、核分子遗传标记和16srDNA扩增子测序技术，建立了斑马鱼ARG转移的质粒介导模型。此外，我们还研究了携带编码ARGs的自转移质粒的供体被引入到水族馆后，ARGs在斑马鱼肠道微生物群落中的传播情况。平均而言，15%的粪便细菌通过RP4介导的配偶转移获得ARGs。后肠是支持ARG传播的最重要的肠区，供体和跨连接细胞的浓度几乎是其他肠段的25倍。此外，在配偶转移最活跃的后肠，RP4质粒调控基因trbBp和trfAp的mRNA表达显著上调。外源细菌似乎通过增加大肠杆菌和类杆菌的种类来改变细菌群落，同时与对照组相比减少气单胞菌。我们鉴定了转接剂的组成以及可培养和不可培养细菌的丰度（后者占总转接剂的90.4%-97.2%）。我们的研究表明，水生动物内脏有助于ARGs在水环境中的传播。

       The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid-mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self-transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4-mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%-97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.

        https://onlinelibrary.wiley.com/doi/abs/10.1111/mec.14255