PiuA promoter mutations confer cefiderocol resistance in Acinetobacter baumannii: from in vitro evolution to global genomic screening.

INTRODUCTION: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a global health threat due to limited therapeutic options. Cefiderocol, a novel siderophore-conjugated cephalosporin, demonstrates potent in vitro activity against a variety of Gram-negative bacteria, including CRAB. However, cefiderocol's recent introduction into clinical practice means that while several resistance mechanisms have been identified, their full characterization remains incomplete, and novel mechanisms are likely to emerge as clinical use expands. METHODS: In this study, in vitro evolution experiments were performed under cefiderocol pressure using a clinical CRAB isolate as the parental strain to investigate cefiderocol resistance mechanisms. RESULTS: Genomic comparison and mutant reconstruction revealed that mutations in pbp1b (G271R) and the intergenic region upstream of piuA (-6_-8del) conferred a 4-fold increase in cefiderocol MIC. In silico predictions and EMSA confirmed that the intergenic mutation occurred within the piuA promoter region. qRT-PCR analysis indicated that the piuA promoter mutation significantly reduced piuA expression. This downregulation of piuA expression impaired cefiderocol transport, leading to reduced susceptibility to cefiderocol. A search across publicly deposited genome sequences identified ten piuA promoter mutations in A. baumannii. Among these, the--6_-8del was the most commonly observed variant, which was primarily associated with ST2Pas/ST457Oxf, ST2Pas/ST208Oxf, and ST499Pas. Functional characterization of the--6_-8del variant and three additional promoter mutations (-18del, A-17T&-18del, A-17T&-14del) confirmed suppression of piuA expression and reduction of cefiderocol susceptibility. DISCUSSION: Combining in vitro evolution with publicly available A. baumannii genome sequences, this study demonstrates promoter-mediated piuA downregulation as a cefiderocol resistance mechanism in A. baumannii, providing a genomic target for predicting cefiderocol resistance.