Chloramphenicol stress triggers oxidative adaptation in Acinetobacter baumannii ATCC19606 devoid of RND efflux pumps...

Efflux pumps play a key role in both intrinsic and acquired antibiotic resistance in Acinetobacter baumannii, yet their broader physiological roles remain unclear. Here, we investigated the transcriptomic and phenotypic responses of A. baumannii ATCC19606 mutants lacking the efflux pumps AdeAB, AdeIJ, or CraA under chloramphenicol stress. The deletion of craA resulted in a 32-fold reduction in chloramphenicol MIC, while ΔadeIJ showed a modest 4-fold decrease, and ΔadeAB had no effect on chloramphenicol susceptibility. Transcriptomic profiling revealed minimal alterations in ΔcraA, but notable transcriptional reprogramming in ΔadeAB and ΔadeIJ, including upregulation of ribosomal genes, iron-sulfur cluster biogenesis, aromatic compound catabolism, and amino acid transport systems. Under chloramphenicol stress, ΔadeAB exhibited metabolic remodeling, activating oxidative stress defenses and protein quality control pathways while repressing type VI secretion. Both ΔadeIJ and wild-type strains upregulated arginine and glutamate metabolism, likely contributing to redox balance. Phenotypically, the ΔadeIJ strain showed elevated basal and H₂O₂-induced ROS levels, as well as heightened sensitivity to nitrosative stress and Cu²⁺, which may suggest a role for AdeIJ in oxidative and metal stress responses. Despite increased adeAB and craA expression under chloramphenicol stress, only craA deletion significantly impaired resistance, highlighting its dominant role in chloramphenicol efflux. Collectively, our findings reveal efflux pump-specific and strain-specific adaptations to antibiotic stress, positioning CraA as a major determinant of chloramphenicol resistance, while RND transporters such as AdeABC and AdeIJK may also be associated with broader stress adaptation processes, including redox and metal homeostasis, in A. baumannii.IMPORTANCEEfflux pumps are key drivers of multidrug resistance in Acinetobacter baumannii, yet their broader roles in stress adaptation remain insufficiently understood. Here, we show that the loss of major efflux systems reshapes the transcriptomic and metabolic landscape under chloramphenicol stress, a condition that also imposes oxidative stress. In particular, the RND efflux pump AdeIJK may be linked to alterations in cellular responses to oxidative, nitrosative, and metal stress, although the mechanistic basis of this interplay remains to be further investigated. Overall, these findings suggest that efflux pumps may contribute to bacterial resilience beyond drug resistance, providing additional insight into efflux system hierarchy and functional redundancy in A. baumannii, and may inform future studies aimed at understanding persistence and efflux-mediated multidrug resistance mechanisms.