Extreme copper tolerance and genomic adaptation in early-stage marine biofilms.

Biofouling, the accumulation of unwanted organisms on submerged assets, presents an ongoing challenge within the maritime industry. Conventional methods to inhibit growth rely on copper-laden (≤75% copper compounds) antifouling coatings. However, the efficacy of these coatings is declining due to the broad emergence of copper-tolerant species. Here, we investigated the mechanisms underlying this decline in antifouling performance. Polyvinyl chloride (PVC) panels coated with a commercial copper ablative paint were deployed in the Chesapeake Bay to collect early-stage biofilms; uncoated PVC panels were deployed as an inert control group. Community profiles of the harvested biofilms were obtained via 16S amplicon sequencing of the V4 and V5 regions. To elucidate genomics information about specific bacteria, primary bacterial colonizers were isolated on marine agar plates and identified through 16S amplicon sequencing and whole genome sequencing. The isolates were subsequently assessed for copper tolerance using copper sulfate (CuSO4) in both marine agar and broth assays. While bacterial tolerance declined with increasing CuSO4 concentrations on agar, tolerance was significantly greater in liquid media and minimum inhibitory concentrations were equal to or above 5 mM CuSO4. Genomic annotation confirmed that this high phenotypic tolerance was driven by active genetic resistance mechanisms. Every isolate harbored at least three copper-related genes (e.g., copA, cueO, and copZ), with highly adaptable species carrying a full spectrum of resistance genes. Ultimately, these findings highlight how the accumulation of metal resistance genes allows bacteria to colonize metal-rich surfaces, paving the way for subsequent macrofouling and coating failure.