The impact of the anti-diabetic drug metformin on the intestinal microbiome of larval brown trout (Salmo trutta f. fario)

Abstract

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.

Chronic Embryo‐Larval Exposure of Fathead Minnows to the Pharmaceutical Drug Metformin: Survival, Growth, and Microbiome Responses

“…There was no effect of metformin exposure on the community (beta diversity) or alpha diversity of the fathead minnow larvae microbiome examined in the present study, and these results contrast with other studies on metformin and other waterborne contaminants. Brown trout larvae exposed to 1 and 1000 µg/L of metformin had an increase in bacterial alpha diversity of their gut, whereas those exposed to 10 and 100 µg/L of metformin had decreased alpha diversity (observed species, Chao1, and Shannon) compared to controls; however, similar to our study these changes were not significant (Rogall et al 2020 ). Beta diversity of larval zebrafish was altered after 20‐d exposure to arsenic (larval microbiome; Dahan et al 2018 ) and after 7 d of exposure to triclosan (gut microbiome [Narrowe et al 2015 ]); communities clustered by low and high exposure in the latter.…”

“…It is possible that differences in the methods used in the aforementioned studies contributed to differences in alpha diversity results. For example, brown trout larvae were 156 d old and exposed to metformin for 108 d (Rogall et al 2020), zebrafish larvae exposed to arsenic were sampled at 20 dpf, and zebrafish larvae exposed to triclosan were sampled 9 wk posthatch (Narrowe et al 2015), whereas larvae in the present study were sampled at 16 dph. Alpha diversity of the microbiome differs with age in grass carp (Li et al 2017); thus, it is possible that age differences may be a factor in the variable results among studies.…”

“…With increasing concentrations of metformin in aquatic ecosystems, the effects on the fish microbiome are largely unknown. Although the mechanism of action for metformin (and other contaminants of concern) on the fish microbiome is unknown, metformin has been shown to alter the diversity and composition of bacteria in larval fish (Jacob et al 2018 ; Rogall et al 2020 ), providing the basis for our study.…”

“…The gut content microbiome of adult fish (wild brown trout, Salmo trutta ; common carp, Cyprinus carpio ; and rainbow darter, Etheostoma caeruleum ) downstream of MMWE outfalls (containing mixtures of contaminants) had both decreased and increased alpha diversity, increased bacteria associated with MWWEs, and increased abundance of Proteobacteria compared to upstream fish; such changes have been linked to dysbiosis (microbial imbalance) and altered health outcomes (Giang et al 2018 ; Sakalli et al 2018 ; Restivo et al 2020 ). Metformin affects the intestinal microbiome of larval brown trout (Jacob et al 2018 ; Rogall et al 2020 ), and those exposed to metformin (up to 1000 µg/L) had shifts in Firmicutes, with the families Planococcacea disappearing and Staphlococcaceae increasing compared to controls (Rogall et al 2020 ). Zebrafish larvae exposed to arsenic had distinct beta diversity and decreased alpha diversity, as well as altered abundances of amplicon sequence variants (ASVs), in their full‐body microbiome.…”

Transition towards sustainable pharmacy? The influence of public debates on policy responses to pharmaceutical contaminants in water

“…There is growing evidence that pharmaceutical residues in surface waters cause adverse effects in aquatic life [5,17,36,69,74]. Pharmaceuticals have been designed as biologically active agents to cure diseases and to manipulate biological processes within living organisms.…”