Nutrient-Driven Small-Colony Variants as an Adaptive Outcome in Carbapenem-Resistant Acinetobacter baumannii

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a serious clinical threat due to its remarkable adaptability and emerging resistance to antibiotics of last resort such as cefiderocol. There is growing evidence that host-mimicking nutrient conditions can significantly modulate antibiotic susceptibility, but the contribution of micronutrients to persistence-associated phenotypes remains unclear. Methods: We investigated the contribution of vitamin B12 as a driver of small colony variants (SCV) and altered antibiotic response in CRAB. Using AB5075 and CDC AR Bank #0033 (M27835) as model strains, we combined static time-to-kill assays, whole-genome sequencing, transcriptional profiling, and phenotypic analysis to define the genetic and physiological basis of vitamin B12-associated SCVs selected in the presence or absence of cefiderocol. Results: Exposure to vitamin B12 promoted the stable emergence of SCVs characterized by reduced growth, altered cefiderocol killing kinetics, and persistence-like behavior. SCVs showed incomplete bactericidal activity and regrowth during cefiderocol exposure across multiple concentrations, a phenotype reproduced in genetically distinct CRAB backgrounds. Whole-genome sequencing identified mutations affecting pathways linked to envelope structure, nutrient transport, and energy homeostasis, including alterations in wecC, aromatic amino acid transporters, ion antiport systems, and siderophore-related genes. SCVs showed altered aminoglycoside killing dynamics, moderate changes in susceptibility profiles, and remodeling of capsule architecture and biofilm-associated traits. Transcriptional analyses revealed coordinated reprogramming of iron acquisition systems, together with differential regulation of vitamin B12 uptake, resistance determinants, efflux mechanisms, and energy metabolism genes. Combined exposure to vitamin B12 and cefiderocol further selected SCV-like derivatives harboring mutations in key siderophore receptors and transcriptional regulators, supporting a nutrient-drug interaction that leads to adaptative antibiotic resistance. Conclusions: These findings further support our observations, in which vitamin B12 is an environmental signal that drives the emergence of SCV and persistence-associated phenotypes, leading to altered cefiderocol activity in CRAB. Our data highlight nutrient availability as a critical modulator of antibiotic efficacy and reveal SCV formation as an adaptive outcome linking micronutrient sensing, TonB-dependent transport, and antibiotic uptake.