A resistome survey across hundreds of freshwater bacterial communities reveals the impacts of veterinary and human antibiotics use.

Our decreasing ability to fight bacterial infections is a major health concern. It is arising due to the evolution of antimicrobial resistance (AMR) in response to the mis- and overuse of antibiotics in both human and veterinary medicine. Lakes integrate watershed processes and thus may act as receptors and reservoirs of antibiotic resistance genes (ARGs) introduced into the watershed by human activities. The resistome - the diversity of ARGs - under varying anthropogenic watershed pressures has been previously studied either focused on few select genes or few lakes. Here, we link the resistome of ~350 lakes sampled across Canada to human watershed activity, trophic status, as well as point sources of ARG pollution including wastewater treatment plants and hospitals in the watershed. A high percentage of the resistance genes detected was either unimpacted by human activity or highly prevalent in pristine lakes, highlighting the role of AMR in microbial ecology in aquatic systems, as well as a pool of genes available for potential horizontal gene transfer to pathogenic species. Nonetheless, watershed agricultural and pasture area significantly impacted the resistome. Moreover, the number of hospitals and the population density in a watershed, the volume of wastewater entering the lake, as well as the fraction of manure applied in the watershed as fertilizer significantly impacted ARG diversity. Together, these findings indicate that lake resistomes are regularly stocked with resistance genes evolved in the context of both veterinary and human antibiotics use and represent reservoirs of ARGs that require further monitoring.

A Glyphosate-Based Herbicide Cross-Selects for Antibiotic Resistance Genes in Bacterioplankton Communities.

Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria. IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Widespread agrochemicals differentially affect zooplankton biomass and community structure.

Anthropogenic environmental change is causing habitat deterioration at unprecedented rates in freshwater ecosystems. Despite increasing more rapidly than many other agents of global change, synthetic chemical pollution-including agrochemicals such as pesticides-has received relatively little attention in freshwater community and ecosystem ecology. Determining the combined effects of multiple agrochemicals on complex biological systems remains a major challenge, requiring a cross-field integration of ecology and ecotoxicology. Using a large-scale array of experimental ponds, we investigated the response of zooplankton community properties (biomass, composition, and diversity metrics) to the individual and joint presence of three globally widespread agrochemicals: the herbicide glyphosate, the neonicotinoid insecticide imidacloprid, and nutrient fertilizers. We tracked temporal variation in zooplankton biomass and community structure along single and combined pesticide gradients (each spanning eight levels), under low (mesotrophic) and high (eutrophic) nutrient-enriched conditions, and quantified (1) response threshold concentrations, (2) agrochemical interactions, and (3) community resistance and recovery. We found that the biomass of major zooplankton groups differed in their sensitivity to pesticides: ≥0.3 mg/L glyphosate elicited long-lasting declines in rotifer communities, both pesticides impaired copepods (≥3 µg/L imidacloprid and ≥5.5 mg/L glyphosate), whereas some cladocerans were highly tolerant to pesticide contamination. Strong interactive effects of pesticides were only recorded in ponds treated with the combination of the highest doses. Overall, glyphosate was the most influential driver of aggregate community properties of zooplankton, with biomass and community structure responding rapidly but recovering unequally over time. Total community biomass showed little resistance when first exposed to glyphosate, but rapidly recovered and even increased with glyphosate concentration over time; in contrast, taxon richness decreased in more contaminated ponds but failed to recover. Our results indicate that the biomass of tolerant taxa compensated for the loss of sensitive species after the first exposure, conferring greater community resistance upon a subsequent contamination event; a case of pollution-induced community tolerance in freshwater animals. These findings suggest that zooplankton biomass may be more resilient to agrochemical pollution than community structure; yet all community properties measured in this study were affected at glyphosate concentrations below common water quality guidelines in North America.

Resistance, resilience, and functional redundancy of freshwater bacterioplankton communities facing a gradient of agricultural stressors in a mesocosm experiment.

Agricultural pollution with fertilizers and pesticides is a common disturbance to freshwater biodiversity. Bacterioplankton communities are at the base of aquatic food webs, but their responses to these potentially interacting stressors are rarely explored. To test the extent of resistance and resilience in bacterioplankton communities faced with agricultural stressors, we exposed freshwater mesocosms to single and combined gradients of two commonly used pesticides: the herbicide glyphosate (0-15 mg/L) and the neonicotinoid insecticide imidacloprid (0-60 µg/L), in high or low nutrient backgrounds. Over the 43-day experiment, we tracked variation in bacterial density with flow cytometry, carbon substrate use with Biolog EcoPlates, and taxonomic diversity and composition with environmental 16S rRNA gene amplicon sequencing. We show that only glyphosate (at the highest dose, 15 mg/L), but not imidacloprid, nutrients, or their interactions measurably changed community structure, favouring members of the Proteobacteria including the genus Agrobacterium. However, no change in carbon substrate use was detected throughout, suggesting functional redundancy despite taxonomic changes. We further show that communities are resilient at broad, but not fine taxonomic levels: 24 days after glyphosate application the precise amplicon sequence variants do not return, and tend to be replaced by phylogenetically close taxa. We conclude that high doses of glyphosate - but still within commonly acceptable regulatory guidelines - alter freshwater bacterioplankton by favouring a subset of higher taxonomic units (i.e., genus to phylum) that transiently thrive in the presence of glyphosate. Longer-term impacts of glyphosate at finer taxonomic resolution merit further investigation.