Fast Evolution of SOS-Independent Multi-Drug Resistance in Bacteria

The killing mechanism of many antibiotics involves the induction of DNA damage, either directly or indirectly, which activates the SOS response. RecA, the master regulator of the SOS response, has shown to play a central role in the evolution of resistance to fluoroquinolones, even after short-term exposure. While this paradigm is well established for DNA-damaging antibiotics, it remains unclear whether β-lactams elicit similar resistance dynamics or depend on RecA and SOS-mediated mechanisms. In this study, we observed a rapid and stable evolution of β-lactam resistance (20-fold MIC increase within 8 hours) inEscherichia colilacking RecA after a single exposure to ampicillin. Contrary to expectation, this resistance emerged through an SOS-independent mechanism involving two distinct evolutionary forces: increased mutational supply and antibiotic-driven selection. Specifically, we found that RecA deletion impaired DNA repair and downregulated base excision repair pathways, while concurrently repressing the transcription of antioxidative defence genes. This dual impairment led to excessive accumulation of reactive oxygen species (ROS), which in turn promoted the emergence of resistance-conferring mutations. While ampicillin treatment did not alter survival, it selectively enriched for rare mutants arising in the RecA-deficient and ROS-elevated background. Collectively, our findings demonstrate that this oxidative environment, together with compromised DNA repair capacity, increases genetic instability and creates a selective landscape favouring the expansion of resistant clones. These results highlight the repair-redox axis as a key determinant of bacterial evolvability under antimicrobial stress.