Exploring the effect of wastewater discharge on the antibiotic resistance prevalence and microbial community composition in aquatic ecosystems

The rapid spread of antibiotic resistance is a major global health concern, jeopardizing the successful treatment of bacterial infections. Natural environments are potential hotspots for the emergence and spread of antibiotic resistance genes (ARGs). Among these potential hotspots, aquatic ecosystems are of particular concern, as they receive wastewater containing antibiotic-resistant bacteria and ARGs originating from both human and animal sources. Several key questions remain to be addressed. What is the fate of ARGs in receiving water bodies? What are implications of environmental ARGs for human health? How does wastewater discharge impacts aquatic microbial communities with regard to the overall ecosystem well-being? The objective of this work was to investigate the impact of wastewater, seasonal variations, and the riverine compartment on the prevalence of selected ARGs and the composition of natural microbial communities in a near-pristine river, and to specifically assess the effect of antibiotics on riverine microbial communities. Quantitative real-time PCR was used to monitor the abundance of three indicator ARGs (sul1 and sul2, conferring resistance against sulfonamide antibiotics, and intI1, a marker for anthropogenic pollution) upstream and downstream from a wastewater treatment plant (WWTP). Furthermore, the impact of WWTP effluent on the riverine microbial community was examined through 16S rRNA amplicon sequencing. Wastewater was the main source of all three target genes and significantly altered the microbial community in the river. The surface water compartment served as a dissemination route for ARGs, with increased prevalence even 13 km downstream of the WWTP, particularly during the summer season when the proportion of wastewater in the river was high. Notably, riverbed biofilms served as a local reservoir for ARGs only at the discharge point, with little abundance of target genes further downstream. The sulfonamide antibiotic sulfamethoxazole (SMX) was persistent in both near-pristine and wastewater-impacted river water when introduced at a concentration of 12.5 µg/L, but had neglectable effects on the microbial community diversity. Interestingly, concentrations as high as 100 µg/L SMX induced a short-term increase in microbial activity in both surface water and biofilm compartment, as revealed by bulk and nanoscale measurements. Altogether, this work underscores the fundamental role of wastewater treatment in combating the environmental dissemination of antibiotic resistance.:Summary 1 Zusammenfassung 5 1 Introduction 9 1.1 Rundown of the global antibiotic resistance crisis 9 1.1.1 History of antibiotics 9 1.1.2 Emergence of antibiotic resistance 9 1.1.3 Integrons as vehicles for antibiotic resistance 10 1.1.4 Risks related to environmental antibiotic resistance 12 1.2 Fate of antibiotic resistance genes in the aquatic environment 14 1.2.1 Genetic indicators for antibiotic resistance 14 1.2.2 River surface water compartment as dissemination route for antibiotic resistance 15 1.2.3 River biofilm compartment as reservoir for antibiotic resistance 17 1.3 Impact of antibiotics on aquatic microbial communities 18 1.4 Fate and effect of sulfamethoxazole in surface waters 20 2 Scope of the thesis 22 3 Main findings and scientific implications 24 3.1 Fate of antibiotic resistance genes after wastewater discharge into a near-pristine river 24 3.1.1 Wastewater is the primary source for aquatic antibiotic resistance 24 3.1.2 Drought increases the antibiotic resistance prevalence in surface waters 25 3.1.3 Riverbed biofilms serve as local reservoirs for antibiotic resistance genes 26 3.2 Anthropogenic pollution is the key driver for microbial community alteration 26 3.3 Sulfamethoxazole increases the microbial activity of aquatic microbial communities 27 4 Conclusions and future perspective 29 5 References 31 6 Publications 43 6.1 Publication 1 43 6.2 Publication 2 56 6.3 Publication 3 69 7 Appendix 94 7.1 Declaration of independent work 94 7.2 List of publications and conference contributions 95 7.2.1 Publications 95 7.2.2 Conference contributions 96 7.3 Contribution of Co-authors 97 7.4 Curriculum vitae 101 7.5 Acknowledgements 104 7.6 Supplementary Material 105 7.6.1 Supplementary Material for Publication 1 105 7.6.2 Supplementary Material for Publication 2 118 7.6.3 Supplementary Material for Publication 3 12