Background

The anti-diabetic pharmaceutical metformin is frequently analysed in the aquatic environment. Its impact on the fish microbiome is studied to get a deeper knowledge about the consequence of the metformin presence in river systems. Gut microbiome analyses were performed on larval brown trout with metformin including environmental concentrations. Therefore, the fish were exposed to metformin in concentrations of 0, 1, 10, 100, and 1000 µg/L. Especially, the lower metformin concentrations were measured in river waters containing percentages of conditioned wastewater from municipal wastewater treatment plants.

Results

Two complementary molecular biological methods for population analysis targeting the 16S rRNA gene regions V1–V3, i.e.: (1) 16S amplicon sequencing and (2) polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE). Both analyses demonstrated significant microbiome alterations even at low metformin concentrations being analysed in German rivers. The amplicon sequencing revealed the most distinct shifts in the Firmicutes phylum, or more specifically, within the Bacillales order, which were most affected by metformin exposure. Within the Bacillales order, the Planococcaceae family, which is described to provide essential amino acids for the fish, completely disappeared after metformin treatment. Conversely, the percentage of other bacteria, such as Staphylococcaceae, increased after exposure to metformin. Similarity profiles of the microbiomes could be generated using the Sørensen index calculation after PCR-DGGE analyses and confirmed shifts in the composition of the brown trout intestinal microbiome after metformin exposures. In vitro gene expression analyses of virulence factors from fish pathogens, previously identified in the fish microbiomes DNA extracts, were conducted in the presence or absence of environmentally relevant concentrations. Here, marker genes of Enterococcus faecium, Enterococcus faecalis, and Aeromonas hydrophila were detected and quantified via PCR approaches, firstly. An increased expression of the species-specific virulence genes was observed after normalisation with control data and ribosomal housekeeping genes.

Conclusion

Environmentally relevant concentrations of metformin can alter the composition in gut microbiome of brown trout in different ways. Both, the metformin-induced expression of virulence genes in fish pathogens in vitro and the impact of metformin on the microbiome composition in vivo in larval brown trout open the discussion about a possible long-term effect on the vitality, growth, and development in more mature brown trouts.

中文翻译：

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抗糖尿病药物二甲双胍对幼鱼褐鳟肠道微生物组的影响

背景

在水生环境中经常分析抗糖尿病药物二甲双胍。研究了其对鱼类微生物组的影响，以更深入地了解二甲双胍在河流系统中的存在后果。在含有环境浓度的二甲双胍上对幼虫褐鳟进行肠道微生物组分析。因此，鱼暴露于二甲双胍的浓度分别为0、1、10、100和1000 µg / L。特别是，在含有市政污水处理厂处理后废水百分比的河水中测量出较低的二甲双胍浓度。

结果

针对16S rRNA基因区域V 1至V 3的两种互补分子生物学方法，即：（1）16S扩增子测序和（2）聚合酶链反应（PCR）结合变性梯度凝胶电泳（DGGE）。两项分析均表明，即使在德国河流中分析的二甲双胍浓度较低时，微生物组也会发生显着变化。扩增子测序揭示在最明显的变化厚壁菌门门，或更具体地，内芽孢杆菌顺序，将其最受二甲双胍曝光。在芽胞菌纲内，平球菌科二甲双胍处理后，该家族被描述为鱼类提供了必需氨基酸，该家族完全消失了。相反，暴露于二甲双胍后，其他细菌（如葡萄球菌）的百分比增加。在PCR-DGGE分析后使用Sørensen指数计算可以得出微生物组的相似性，并确认二甲双胍暴露后棕鳟肠道微生物组的组成发生了变化。在有或没有环境相关浓度的情况下，对先前在鱼类微生物群系DNA提取物中鉴定出的鱼类病原体的致病因子进行了体外基因表达分析。在这里，粪肠球菌，粪肠球菌和首先通过PCR方法检测并定量了嗜水气单胞菌。用对照数据和核糖体管家基因标准化后，观察到物种特异性毒力基因的表达增加。

结论

与环境有关的二甲双胍浓度可以不同方式改变褐鳟肠道微生物组的组成。二甲双胍诱导的鱼类致病菌体外毒力基因表达以及二甲双胍对幼体褐鳟鱼体内微生物组组成的影响，开启了关于可能长期影响其活力，生长和发育的讨论。成熟的褐鳟。