Reactive oxygen species as quantitative indicator of antibiotic resistance in environmental bacteria

The global spread of antibiotic-resistant bacteria poses threat to public health and environmental safety. Rapid quantification of antibiotic resistance in environmental bacteria is crucial for mitigating resistance development. In this study, an innovative reactive oxygen species-based antibiotic susceptibility testing method (ROS-AST) was developed, leveraging the newly discovered temporal variations and dose-response relationship of ROS in bacteria under antibiotic stress. Specifically, bacteria exposed to antibiotics at varied concentrations generate different levels of intracellular ROS. The antibiotic concentration resulting in the maximum ROS accumulation (around 3 h) closely correlated with the minimal inhibitory concentration (MIC). Therefore, ROS-AST allows rapid MIC estimation and antibiotic resistance risk assessment within 4 h, faster than the microbroth dilution method (≥16 h). It achieved high accuracy (7.23 %±13.43 %) across 3 classes of antibiotics and 26 environmental bacteria, with MICs ranging from 0.03 to 128 µg/mL. ROS-AST also facilitated a semi-quantitative assessment of antibiotic resistance in microbial communities from water and soil, yielding results consistent with the disk diffusion method, where wastewater communities exhibited the highest resistance. In a wastewater-isolated Escherichia coli exposed to prolonged low-level kanamycin, ROS accumulation in the first six days coincided with reversible resistance, which was considered as a window period for controlling resistance. Transcriptomics further elucidated the mechanisms underlying ROS dynamics, including stress responses (reversible evolution) and enhanced reductive processes to eliminate ROS (irreversible evolution). This study highlights the potentials of ROS-AST in indicating antibiotic resistance and resistance reversibility in environmental bacteria, offering insights into antibiotic resistance mitigation in the environment.