ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health threat with few effective treatment options remaining. Cefiderocol, a last-resort siderophore-cephalosporin antibiotic, exploits bacterial iron transport systems via TonB-dependent receptors (TBDRs) to gain cellular entry. However, treatment failures and the emergence of resistance highlight concerns with in vivo efficacy. In this study, we report an unanticipated cefiderocol resistance mechanism where vitamin B12, a micronutrient supplement, modulates cefiderocol susceptibility. Our work revealed that vitamin B12 (methylcobalamin) affects and interacts with TBDRs and other metabolic and adaptation processes that contribute to increased cefiderocol MIC levels and the emergence of persistence phenotypes. We demonstrate that vitamin B12 supplementation elicits strain-specific transcriptomic responses in the AB5075 and AMA17 CRAB strains, characterized by the downregulation of genes encoding siderophore-mediated iron acquisition functions, stress responses, metabolic reprogramming, and biofilm biogenesis. Structural modeling and molecular docking reveal overlapping binding sites for vitamin B12 and cefiderocol within TBDRs such as CirA and PirA, suggesting competitive inhibition. Additionally, vitamin B12 exposure increases cefiderocol MICs across a panel of A. baumannii clinical and reference strains, enhances survival in time-kill assays, and promotes the emergence of small-colony variants displaying persistent phenotypes. Notably, this effect is stable, dose dependent, and further enhanced in the presence of host-derived fluids. These findings describe a previously unrecognized host–pathogen–drug interaction with potential clinical implications, suggesting that vitamin B12 exposure could contribute to cefiderocol treatment failure. Our results underscore the urgent need to consider the potential contribution of vitamin supplements to antimicrobial therapy and management strategies for CRAB infections. IMPORTANCE Cefiderocol, a last-line antibiotic for treating carbapenem-resistant Acinetobacter baumannii (CRAB) infections, uses iron-uptake receptors to enter bacterial cells. Our work demonstrates that vitamin B12, a common supplement in outpatients and hospitalized adults, can antagonize cefiderocol by affecting TonB-dependent receptor expression and competing at shared entry sites. As a result, cefiderocol MICs are raised, thus promoting persistent small-colony variants. This dose-dependent, strain-specific effect is amplified by host fluids, revealing a clinically plausible pathway leading to treatment failure that current susceptibility testing assays do not consider. Recognizing vitamin B12 exposure and incorporating physiological B12/iron conditions into antimicrobial susceptibility testing and models could improve decision-making for treatment regimens. More broadly, our findings highlight nutrient–antibiotic interactions as overlooked drivers of CRAB’s resistance and persistence.
Mezcord et al. (2026) studied this question.