Characterization of Cefiderocol Heteroresistance in Carbapenem-Resistant Acinetobacter Baumannii (CRAB) Following Exposure to Human Pleural Fluid (HPF)

Abstract

Objectives: Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates are one of the most difficult pathogens to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 indicated for the treatment of infections due to CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported higher than all-cause mortality rate in patients treated with cefiderocol. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population seemingly isogenic. Recent results have shown that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here we describe molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Methods: Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were subjected to whole genome sequencing and transcriptional analysis to identify mutations and expression changes associated with cefiderocol heteroresistance. The impact of these mutations on the pharmacodynamic activity of cefiderocol was assessed via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Results: Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses showed significant modifications in levels of expression of genes associated with these functions. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes’ expression was reduced. One of the mutations common to both heteroresistant strains mapped within pipA, a gene associated with iron homeostasis in other species. Static time-kill assays showed that supplementing cation-adjusted Mueller-Hinton broth with human serum albumin, the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. Conclusions: We conclude that exposing CRAB to fluids containing high HSA facilitates the rise of heteroresistance associated with point mutations and upregulation of genes coding for β-lactamases and biofilm formation.