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 some of the problems with its in vivo activity. In this study, we report an unanticipated cefiderocol resistance mechanism, where vitamin B12, a commonly used micronutrient supplement, modulates cefiderocol susceptibility. 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 CRAB 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 key 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.

Files Description: ---- raw_seq_ecoli.tar.gz: FASTQ files of E.coli K12 NDM5 wild-type strain (ECNDM51_S95_R1_001.fastq.gz ECNDM51_S95_R2_001.fastq.gz) and E.coli K12 NDM5 IHC strain (IHC1: ECNDM5IHCA2_S96_R1_001.fastq.gz, ECNDM5IHCA2_S96_R2_001.fastq.gz; IHC2:ECNDM5IHCB2_S97_R1_001.fastq.gz, ECNDM5IHCB2_S97_R2_001.fastq.gz) . ---- PDB files: E. coli: btuB3 , A. baumannii: btuB3 , pirA , piuA , btuB2 , cirA K. pneumoniae: cirA ---- PDB files complex with FDC: E. coli: btuB3, A. baumannii: btuB3, pirA, piuA, btuB2, cirA K. pneumoniae: cirA ---- PDB files complex with VitB-12: E. coli: btuB3, A. baumannii: btuB3, pirA, piuA, btuB2, cirA K. pneumoniae: cirA ---- PDB files complex with VitB-12 and FDC: E. coli: btuB3, A. baumannii: btuB3, pirA, piuA, btuB2, cirA K. pneumoniae: cirA