Establishing the mechanistic basis of action for novel siderophore-based antibiotics in gram-negative bacteria

Abstract

The spread of antimicrobial resistance (AMR) remains one of the most pressing contemporary threats to global human and agricultural health. The lack of novel antimicrobial agents has left healthcare professionals with a dwindling number of treatment options and the drug discovery pipeline for such compounds does not meet the demand. As such, the World Health Organisation (WHO) has deemed pathogens such as K. pneumoniae a critical priority for the research and development of novel antibiotics.

Iron is an essential micronutrient for bacterial metabolic processes and must be sequestered in restrictive environments, thus bacteria synthesise and secrete small molecules with extremely high affinities for iron(III) called siderophores. Siderophores must be transported via energy-dependent receptors known as iron-regulated outer-membrane proteins (IROMPs), and both are highly expressed in iron-restricted in vivo conditions due to de-repression by the Ferric uptake regulator (Fur). Cefiderocol is a novel siderophore-cephalosporin and the first siderophore-antibiotic conjugate to be approved for clinical use. It has been demonstrated to be effective against Gram-negative bacteria compared to other cephalosporins due to an iron-binding moiety and enhanced mechanism of cell entry via IROMPs, notably catechol receptors CirA and Fiu.

In this work, we aimed to determine how mutations following selection alter cefiderocol uptake and the strain-wide nature of cefiderocol entry and susceptibility in K. pneumoniae, and how regulation of iron homeostasis influences cefiderocol efficacy. We then leveraged our findings to assess cefiderocol susceptibility at the population level using comparative genomics. The frequency of cefiderocol resistance in first-step selection mutants was ~10-fold lower than the comparator antibiotic ceftazidime, with cefiderocol MICs increasing 2-fold – 32-fold. Mutants acquired non-synonymous mutations in EnvZ, BaeS, RamR and Fiu, then after a second round of selection, CpxA and ExbB or BaeS/EnvZ double mutations. BaeS and EnvZ mutants had increased OmpK36 protein levels and a significant mean log2-fold reduction in cirA and fiu mRNA expression, and we hypothesised that increased passive diffusion of iron via OmpK36 resulted in Fur-mediated repression of CirA and Fiu, restrict-ing cefiderocol uptake. Cefiderocol selection in a CirA-truncated strain resulted in a 100-fold higher mutation frequency than the wild-type and mutants harboured mutations in PiuC.

We determined cefiderocol susceptibility across 45 diverse clinical K. pneumoniae iso-lates and found MDR-associated strains harbouring carbapenemases had the highest cefiderocol MICs, and while some strains produced significantly more siderophores, there were no significant differences in cirA or fiu mRNA levels. Additionally, decreasing iron-acquisition by virulence plasmid curing had no impact on cefiderocol susceptibility.

While the complete Fur regulon has not been elucidated in K. pneumoniae, an in silico screen revealed putative Fur binding sites upstream of all siderophore biosynthesis loci and both cirA and fiu. Deletion of the Fur ORF resulted in cefiderocol hyper-susceptibility, yet had no effect on ceftazidime susceptibility, and we found that Ecl8Δfur had a significant log2-fold increase in expression of cirA and fiu.

Following comparative genomic analysis of 1000 public K. pneumoniae genomes, we re-ported plasmid-borne OXA-family, NDM-family and KPC-family enzymes in >50%, <10% and ~25% of strains respectively. We also analysed the genomes of 50 K. pneumoniae isolates from the SIDERO-WT studies with reduced cefiderocol susceptibility. The MLST types of these isolates were all associated with MDR outbreak clones except atypical ST23 clones, and all strains harboured at least one ESBL gene, carbapenemase gene or combination of the two. All genomes analysed harboured intact CirA and Fiu ORFs and known mutations in EnvZ, BaeS, TonB, ExbB, PiuC and CpxA were not detected.

Taken together, cefiderocol is an effective novel therapy for MDR K. pneumoniae, with a low resistance frequency. Mutations in the two-component systems result in the down regulation of the portals of entry used by cefiderocol but reduce overall fitness and are not common in clinical strains. Strains that are less susceptible to cefiderocol are associated with carriage of a combination of carbapenemases and ESBLs, yet cefiderocol is more stable against these enzymes than comparative cephalosporins and has proven to be a primary therapeutic agent for problematic clinical Gram-negative pathogens.