New Delhi Metallo-Beta-Lactamase Facilitates the Emergence of Cefiderocol Resistance in Enterobacter cloacae

Abstract

Cefiderocol is a promising novel siderophore cephalosporin for the treatment of multidrug-resistant Gram-negative bacilli and with stability against degradation by metallo-β-lactamases. Nonetheless, the emergence of cefiderocol in metallo-β-lactamase-producing Enterobacterales during therapy has been reported on more than one occasion. To understand the underlying mechanisms and factors facilitating the resistance development, we conducted an in vitro evolution experiment using clinical E. cloacae isolates via serial passaging under cefiderocol pressure. In this study, we showed that the presence of the New Delhi metallo-β-lactamase (NDM) facilitates the emergence of resistance via nonsynonymous mutations of the CirA catecholate siderophore receptor. Inhibition of metallo-β-lactamase activity using dipicolinic acid prevented the emergence of cefiderocol-resistant mutants successfully. This finding implies that caution should be taken when using cefiderocol for the treatment of infections caused by metallo-β-lactamase-producing bacteria.

Keywords: Enterobacter cloacae; Enterobacterales; New Delhi metallo-β-lactamase; antibiotic resistance; carbapenem resistance; cefiderocol; cefiderocol resistance; drug resistance mechanisms.

FIG 1

Emergence of cefiderocol resistance in serial passaging under cefiderocol pressure. (a) Serial passaging of clinical E. cloacae in increasing cefiderocol concentrations (ranged from 0.5 to 128 mg/liter) in cation-adjusted Mueller-Hinton broth (CA-MHB). After initial growth in liquid culture without antibiotics, E. cloacae were transferred to fresh CA-MHB in a 1:50 dilution factor containing cefiderocol and repeated after overnight incubation at 37°C with increasing cefiderocol concentrations. (b) Disk diffusion of liquid culture revealed the emergence of smaller colonies in the zone of inhibition after reaching cefiderocol concentrations >8 mg/liter. In high cefiderocol concentrations (>64 mg/liter), no zone of inhibition was visible indicating the development of high-level resistance toward this substance. Variations in colony color were caused by various lighting conditions. The zone of inhibition of the disk diffusion was provided in millimeters. FDC=cefiderocol.

FIG 2

Variability of MICs and mutation frequency. (a) MICs of 10 randomly selected E. cloacae isolates growing in various cefiderocol concentrations (ranged 0.5 to 128 mg/liter) in a serial passage experimental revealed the development of heterogeneous phenotypic resistance toward cefiderocol. Bars in shades of gray indicate phenotypic susceptibility and bars in shades of red indicate MIC >4 mg/liter. (b) Mutation frequency at 4× and 10× the MIC for etcl_1 and KE5351. (c) Growth under antibiotic pressure (4× and 10× MIC) was measured by optical density at 600 nm (OD₆₀₀). Data presented for (b) and (c) were collective data from 3 independent experiments. Where applicable, statistical analysis was performed using a two-way ANOVA. ***, P < 0.001.

FIG 3

Heterogeneous mutations in the catecholate siderophore receptor gene cirA confer phenotypic resistance. (a) Alignment of the cirA gene between the cefiderocol-susceptible parental etcl_1 isolate and randomly picked resistant mutants (MIC >128 mg/liter) of etcl_1 (mut1 to mut5) revealed heterogeneous nonsynonymous alterations in the cirA gene of all resistant isolates. For the isolate KE5351, all resistant mutants (KE5351 mut1 to mut5) exhibited identical IS5-like transposon insertion leading to a truncation of the CirA protein. (b) Changes in the cefiderocol MIC associated with cirA mutations in the presence and absence of metallo-β-lactamase inhibitor, pyridine-2,6-dicarboxylic acid (DPA), measured at OD₆₀₀ and displayed as a heat map. The acquisition of cirA mutation in the mutants generated by serial passaging led to an increase of the cefiderocol MIC from 4 mg/liter to >128 mg/liter (the highest concentration shown is 32 mg/liter). The cefiderocol MIC of isolates with cirA mutations increased from 0.25 mg/liter to 4 mg/liter independent of the metallo-β-lactamase activity. The MICs are indicated by “#”. The “POS” column indicates the growth control without antibiotics.

FIG 4

Inhibition of metallo-β-lactamase activity by pyridine-2,6-dicarboxylic acid prevents the emergence of cefiderocol resistance. (a) The influence of initial bacterial density (in CFU/mL) on bacterial growth under cefiderocol selection pressure (4× and 10× MIC of the respective isolate). Bacterial growth was quantified by a photometer (OD₆₀₀). An initial inoculum of ≥10⁶ CFU/mL leads to bacterial growth under cefiderocol pressure consistently (in 3 out of 3 independent experiments). An initial inoculum of <10⁶ CFU/mL led to random emergence of cefiderocol resistance (1 out of 3 independent experiments from different inoculum). (b) The supplementation with 100 mg/liter pyridine-2,6-dicarboxylic acid (DPA) to inhibit the metallo-β-lactamase activity prevented the growth of resistant mutants under cefiderocol pressure.