Heavy metals promote the formation of multidrug-tolerant Staphylococcus aureus and Escherichia coli persisters

Bacterial persisters are dormant phenotypic variants that are tolerant to antibiotics, contributing to treatment failure and the emergence of antimicrobial resistance. Although the formation of persisters has been extensively studied in regards to bacterial infections and treatment, such as antibiotic exposure or intracellular survival within macrophages, the role of environmental stressors in persister formation remains largely unexplored. In this study, we investigate the role of environmental heavy metals, specifically arsenic (As), cadmium (Cd), and mercury (Hg), in promoting persister cell formation in Staphylococcus aureus and Escherichia coli. Log-phase cultures were exposed to heavy metals (5 mM As, 1.25 mM Cd, 4 µM Hg for S. aureus; 12.5 mM As, 2 mM Cd, and 15 µM Hg for E. coli) for 0.5 h to induce persister cells. We observed that exposure to these metals induced persister cell formation, confirmed by intracellular ATP levels through microscopy and luciferase assays, as well as by reactive oxygen species (ROS) levels using carboxy-H2DCFDA. Short-term heavy metal exposure strongly depleted intracellular ATP while generating ROS. Moreover, we observed enhanced expression of genes involved in the SOS response, including recA, umuC, dinB, rexA, rexB, sulA, rpoS, and soxR, as measured by qPCR. This response was likely induced by elevated ROS levels following heavy metal exposure. Furthermore, we demonstrate that heavy metal-induced bacterial persisters exhibited a substantially increased emergence of antibiotic resistance, as shown by ciprofloxacin resistance developing in the presence of heavy metals. Therefore, our results clearly demonstrate that heavy metals can induce persister cells by depleting cellular ATP and generating ROS, and these bacterial responses to heavy metals substantially contribute to antibiotic resistance. These findings highlight the intricate relationship between environmental heavy metals, bacterial persister formation, and antibiotic resistance, emphasizing the need for a "One Health" strategy to address the growing antibiotic resistance crisis.