Investigation of cyanobacteria-hosted antibiotic resistance genes in cyanoHAB-impacted drinking water sources

Freshwater cyanobacterial blooms (cyanoHABs) are expanding across the world, and their frequency and severity are becoming more intense due to prevalent eutrophication and a changing climate. Traditionally, the concerns about cyanoHABs have mainly focused on cyanotoxins in water. CyanoHABs are also hypothesized to play a role in the antibiotic resistome, but whether cyanobacteria host clinically relevant antibiotic resistant genes (ARGs) in the environment is largely unknown. To investigate this emerging issue, we examined whether cyanobacteria host ARGs within the broader microbiome context. We looked for the presence of cyanobacteria-hosted ARGs using shotgun metagenomic sequencing of drinking water source samples collected during the bloom season (summer and fall) from Lake Erie and Grand Lake St. Marys (GLSM). ARGs were annotated using DeepARG and Resistance Gene Identifier (RGI). Cyanobacteria were annotated to host genes conferring putative antibiotic resistance, including efflux pumps qac/EmrE, vatB, van genes, and an OXA homolog. A maximum likelihood tree with cyanobacteria and OXA reference sequences showed OXA-like homology across multiple families of cyanobacteria. Most cyanobacteria sequences clustered in a large clade with ybxI, suggesting very limited or negligible class-D beta-lactamase activity, but a small subset formed a clade with OXA-2 and OXA-46. While those hits suggest potential resistance to clinical antibiotics, overall cyanobacteria were not found to host ARGs conferring resistance to drugs of last resort in these samples. Additionally, BLAST searches of the cyanobacteria ARG contigs and coding sequences resulted in top hits for cyanobacteria, further supporting that annotated genes are likely intrinsic rather than acquired. rpoB2 and arlR ARG annotations appear to be spurious hits on housekeeping genes, which demonstrates the need to verify automated ARG annotation tool results. Selected cyanotoxins, cyanobacteria, and ARGs were also chosen for quantification. We found high levels of Microcystis in Lake Erie as well as Planktothrix and microcystin concentrations in GLSM, supporting previous trends in these water bodies. This study takes a novel approach, pairing the issues of cyanoHABs and ARGs together in two drinking water sources. In a changing climate, drinking water treatment strategies should consider the treatment and public health implications of multiple contaminants.