Hierarchical activation of resistance genes under tetracyclines selective pressure in complex microbial community

The pervasive use of antibiotics exerts selective pressure in both natural and anthropogenic environments, driving the propagation and evolution of antibiotic resistance genes (ARGs) in microbial communities. Understanding the succession of resistome under varying antibiotic stresses is crucial for mitigating the spread of ARGs. This study investigates the succession of resistome under exposure to four structurally different tetracyclines (TC) across concentrations ranging from environmental to clinical levels. A clear hierarchical activation of ARGs was observed, starting with the upregulation of multidrug and TC-specific efflux pump genes, followed by those involved in TC inactivation and ribosomal protection. By identifying the specific thresholds of transcriptional onset times and critical TC concentration ranges that triggered ARG abundance increases, it was found that all ARGs as a whole did not significantly increase when TC concentrations were maintained below 10^-5 of the initial minimum inhibitory concentration (MIC₀) within 2 h. Similarly, high-risk TC resistance genes do not proliferate when TC concentrations were kept below 10^-3 × MIC₀ within 24 h. These findings provide quantifiable benchmarks for concentration-time thresholds that can inform the establishment of environmental discharge limits and guide the implementation of targeted treatment technologies to mitigate ARG dissemination.