Carbapenem-resistant Gram-negative bacteria exhibiting clinically undetected cefiderocol heteroresistance leads to treatment failure in a murine model of infection

Hongwei Liu¹Peng Zhou¹Peng Ma^1*Yaqin Liu¹Yingfeng Zhang²Qiwei Li³Lingqing Xu¹Wenchang Yuan⁴Weiguo Yin^1*Linhai Li^1*Yang Lu^1*

• ¹The Affiliated Qingyuan Hospital (Qingyuan People's Hospital),Guangzhou Medical University, Qingyuan, China
• ²Department of Public Health Laboratory Sciences, School of Public Health, Hengyang Medical College, University of South China, Hengyang, Hunan Province, China
• ³Department of Laboratory Medicine, The Second Affiliated Hospital, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China
• ⁴KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou, Guangdong Province, China

ABSTRACTCarbapenem-resistant (CR) Gram-negative pathogens, prioritized by the WHO as critical threats, face limited therapeutic options, with cefiderocol (CFD) emerging as a promising siderophore cephalosporin. This study investigated the prevalence, clinical impact, and genetic mechanisms of cefiderocol heteroresistance (CFD-HR) in 407 CR and ESBL-producing clinical isolates from China, where CFD remains unapproved. Population analysis profiles (PAPs) revealed CFD-HR rates of 17.4% (16/92) in carbapenem-resistant A. baumannii (CRAB), 27.9% (24/86) in carbapenem-resistant P. aeruginosa (CRPA), 23.8% (10/42) in carbapenem-resistant E.coli(CRE), and ≤10% (1/10 in P. aeruginosa extended-spectrum β-lactamase (ESBL), 8/177 in E. coli ESBL). Although 72.9% (43/59) of HR isolates were classified as CFD-susceptible by disk diffusion, time-kill assays showed that 66.7% (4/6) of HR strains required ≥8 mg/L CFD (vs. 4 mg/L for non-HR) to prevent regrowth. In a murine peritonitis model, CFD achieved 100% (3/3) survival in non-HR infections but only 16.7% (1/6) in HR-infected mice, directly linking HR to in vivo treatment failure. Whole-genome sequencing identified transient genetic alterations in HR subpopulations, including sitABCD duplications (CRE), oprD mutations (CRAB), and vgrG SNPs (CRPA), which reverted post-antibiotic withdrawal. Fitness cost assays revealed unstable growth deficits in 33.3% (2/6) of HR subpopulations, correlating with genetic instability. These findings highlight the clinical significance of CFD-HR, even in susceptible isolates, and underscore the need for improved diagnostic methods to detect HR and monitor cross-resistance, offering critical insights for regions transitioning to CFD use.