Seven wastewater treatment plants (WWTPs) with different population equivalents and catchment areas were screened for the prevalence of the colistin resistance gene mcr-1 mediating resistance against last resort antibiotic polymyxin E. The abundance of the plasmid-associated mcr-1 gene in total microbial populations during water treatment processes was quantitatively analyzed by qPCR analyses. The presence of the colistin resistance gene was documented for all of the influent wastewater samples of the seven WWTPs. In some cases the mcr-1 resistance gene was also detected in effluent samples of the WWTPs after conventional treatment reaching the aquatic environment. In addition to the occurrence of mcr-1 gene, CTX-M-32, blaTEM, CTX-M, tetM, CMY-2, and ermB genes coding for clinically relevant antibiotic resistances were quantified in higher abundances in all WWTPs effluents. In parallel, the abundances of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli were quantified via qPCR using specific taxonomic gene markers which were detected in all influent and effluent wastewaters in significant densities. Hence, opportunistic pathogens and clinically relevant antibiotic resistance genes in wastewaters of the analyzed WWTPs bear a risk of dissemination to the aquatic environment. Since many of the antibiotic resistance gene are associated with mobile genetic elements horizontal gene transfer during wastewater treatment can't be excluded.
BackgroundDue to the rising number of type 2 diabetes patients, the antidiabetic drug, metformin is currently among those pharmaceuticals with the highest consumption rates worldwide. Via sewage-treatment plants, metformin enters surface waters where it is frequently detected in low concentrations (µg/L). Since possible adverse effects of this substance in aquatic organisms have been insufficiently explored to date, the aim of this study was to investigate the impact of metformin on health and development in brown trout (Salmo trutta f. fario) and its microbiome.ResultsBrown trout embryos were exposed to 0, 1, 10, 100 and 1000 µg/L metformin over a period from 48 days post fertilisation (dpf) until 8 weeks post-yolk sac consumption at 7 °C (156 dpf) and 11 °C (143 dpf). Chemical analyses in tissues of exposed fish showed the concentration-dependent presence of metformin in the larvae. Mortality, embryonic development, body length, liver tissue integrity, stress protein levels and swimming behaviour were not influenced. However, compared to the controls, the amount of hepatic glycogen was higher in larvae exposed to metformin, especially in fish exposed to the lowest metformin concentration of 1 µg/L, which is environmentally relevant. At higher metformin concentrations, the glycogen content in the liver showed a high variability, especially for larvae exposed to 1000 µg/L metformin. Furthermore, the body weight of fish exposed to 10 and 100 µg/L metformin at 7 °C and to 1 µg/L metformin at 11 °C was decreased compared with the respective controls. The results of the microbiome analyses indicated a shift in the bacteria distribution in fish exposed to 1 and 10 µg/L metformin at 7 °C and to 100 µg/L metformin at 11 °C, leading to an increase of Proteobacteria and a reduction of Firmicutes and Actinobacteria.ConclusionsOverall, weight reduction and the increased glycogen content belong to the described pharmaceutical effects of the drug in humans, but this study showed that they also occur in brown trout larvae. The impact of a shift in the intestinal microbiome caused by metformin on the immune system and vitality of the host organism should be the subject of further research before assessing the environmental relevance of the pharmaceutical.Electronic supplementary materialThe online version of this article (10.1186/s12302-018-0179-4) contains supplementary material, which is available to authorized users.
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 speciesspecific 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.
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