Abstact

Intrinsic Acinetobacter-derived cephalosporinases (ADCs) in Acinetobacter baumannii are AmpC-type beta-lactamases that confer resistance to beta-lactam agents, typically through insertion sequence (IS) element-driven overexpression. However, the contribution of ADCs to resistance against advanced beta-lactam agents has not been systematically investigated. Given the increasing clinical use of these agents, including cefiderocol, we analyzed the diversity and function of ADC variants in a global collection of carbapenem-resistant A. baumannii (CRAb) isolates. We identified 52 distinct ADC variants in a collection of 428 CRAb clinical isolates from the United States. Among these, variants carrying both a valine substitution at position 292 in the R2 loop and an alanine duplication (ADUP) in the Omega loop consistently conferred stable resistance to cefiderocol. Biochemical and crystallographic analyses demonstrated that ADC-227, which harbors a V292W substitution with an ADUP at position 218a in the Omega loop, exhibits enhanced catalytic efficiencies for ceftazidime and cefiderocol and moderately reduced inhibition by avibactam, leading to resistance not only to cefiderocol but also to ceftazidime-avibactam. Structural studies revealed conformational flexibility of the R2 loop, allowing dynamic accommodation of substrates. Collectively, the findings identify Val292, in combination with an ADUP in the Omega loop, as a mutational "hot spot" for ADCs evolution that may undermine the efficacy of newer beta-lactams, including cefiderocol. These results underscore the need for ongoing molecular surveillance of A. baumannii isolates to detect and track the emergence of such variants in clinical settings.IMPORTANCECarbapenem-resistant Acinetobacter baumannii (CRAb) has been designated as a critical priority pathogen by the World Health Organization (WHO). Cefiderocol has been introduced as a novel therapy against CRAb; however, recent clinical data highlight concerning treatment failures and excess mortality. Understanding resistance mechanisms is therefore essential to preserve the clinical utility of this agent. This study addresses a critical knowledge gap by investigating the role of intrinsic Acinetobacter-derived cephalosporinases (ADCs), which are ubiquitous in A. baumannii and diverse in sequence. By defining specific mutational patterns that endanger cefiderocol activity, this work highlights how chromosomally encoded enzymes can evolve to erode the effectiveness of newer beta-lactams such as cefiderocol. These insights underscore the importance of integrating molecular surveillance into clinical practice and antimicrobial stewardship to ensure timely detection of emerging resistance in clinical A. baumannii isolates, ultimately informing treatment strategies and guiding future drug development.