Biogeographical insights into the aquatic metal and antibiotic resistome

Abstract

Antibiotic resistance in environmental microbiota is a naturally occurring, yet looming threat to global society and is estimated to contribute to over 10 million deaths annually by 2050. While human-derived contamination from industrial activity and wastewater worsens this, other environmental stressors like heavy metals and the genes conferring their resistance (MRGs) within microbes may enhance antibiotic resistance genes (ARGs). Ultimately, this leads to their persistence due to shared export mechanisms and coupled transcriptional regulation. However, the occurrence and relative abundance of MRGs and ARGs remains poorly understood across aquatic ecosystems and geographies. Here, we hypothesized that estuary ecosystems and highly industrialized countries would harbor the most abundant and diverse repertoire of ARGs and MRGs within its waters, showing ecosystem and geographic differences in resistome composition that could reflect the extent of human contamination within. 57 aquatic shotgun sequencing projects were downloaded from MG-RAST and the SRA spanning global waters and their nucleotide sequences annotated using the two-step ARGs-oap pipeline built from the Structured Antibiotic Resistance Gene Database (SARG), the Bacterial Metals and Biocides Database (BacMet), and a custom mobile genetic element (MGE) database curated to include plasmid, transposase, and integrase sequences. To determine the most abundant and frequently co-occurring ARG and MRG types, the relative abundances were stratified and plotted by ecosystem, country, continent, and hemisphere, while pairwise spearman correlation matrices were constructed to assess co-occurrence patterns and the results visualized with network analysis. Lastly, co-resistance was studied further within the environmental isolate Arthrobacter aurescens TC1 using chromium-enriched and marine salt antibiotic Kirby-Bauer disk diffusion assays. It was found that estuaries were more likely to have higher abundances of resistance genes for tetracycline resistance compared to marine environments, but held elevated concentrations of zinc, chromium, iron, arsenic and lead compared to freshwater and marine environments. Kenya carried an elevated abundance of tetracycline and sulfonamide resistance genes compared to both the industrialized nations of the USA and China, but no differences in MRG abundance were observed. Network analysis revealed that some multidrug efflux genes were highly correlated to MRGs for copper resistance, but genes conferring resistance to biocides and multi-metals may play an undermined role within aquatic ecosystems due to their prominence. Laboratory studies revealed that low concentrations of Cr(VI) under estuarine salinity conditions improved the resistance of known metal-resistant A. aurescens TC1 to many antibiotics, including bacitracin. These results suggest that estuaries and freshwater environments are more at-risk environments for ARG/MRG co-occurrence compared to marine systems, and Lake Victoria in Kenya is at a risk for ARG/MRG co-occurrence and possibly co-resistance, highlighting it as a promising location for future studies.