Abstract Background Cefiderocol (FDC) is a novel cephalosporin with a siderophore-mimicking moiety that favors uptake into the periplasm through iron transporters. FDC was reported as the only cephalosporin refractory to hydrolysis by metallo-β-Lactamases (MBLs). However, resistance to FDC has been reported due to substitutions in the iron transporter coupled to the expression of the MBLs NDM-1 and NDM-5. In contrast, resistance emergence involving other MBLs such as VIM or IMP are not known. We aimed to identify the interaction of FDC with NDM and other MBLs.Table 1.Kinetic parameters of FDC hydrolysis by different MBLsNDM variants show considerable activity in FDC hydrolysis. In contrast, VIM-2 and IMP-1 show poor activity. Methods MICs values of FDC against Escherichia coli DH10B cells expressing different MBLs cloned into a pHSG298 vector were determined by the microdilution method in iron-depleted cation-adjusted Mueller-Hinton broth. NDM-1, NDM-5, IMP-1 and VIM-2 were expressed in E. coli BL21(DE3) pLysS and purified by affinity chromatography. Steady state catalytic parameters were determined by initial rate measurements measured in HEPES buffer, pH 7.5 at 25°C. NMR spectra were run in a 700 MHz Bruker spectrometer. Stopped-flow data were acquired in an Applied Photophysics spectrophotometer. Mechanism of MBL-mediated resistance to FDC Under high levels of NDM expression, the enzymatic activity counter the cefiderocol action, confering bacterial resistance against this antibiotic. Results MIC values of FDC in E. coli cells expressing NDM variants were 2 μg/ml, similar to NDM-1. Steady-state kinetic measurements showed that NDM-1 and NDM-5 hydrolyze FDC efficiently, with kcat/KM values of 1.9 and 1.2 x10^6 M-1s-1, respectively. In contrast, IMP-1 and VIM-2 displayed poor catalytic efficiencies (Table 1). Electrospray ionization mass spectrometry (ESI-MS) revealed that FDC forms an adduct with MBLs, which is significantly more stable in the case of IMP-1 and VIM-2. Stopped flow experiments under pre-steady state conditions show that both IMP-1 and VIM-2 form a stable adduct with FDC, that results in enzyme inhibition. Instead, NDM-1 is poorly inhibited since the formed adduct is less stable. NMR experiments confirmed the formation of an hydrolysis product and adduct. Conclusion Our results indicate that NDM-1 and NDM-5 hydrolyze FDC efficiently, in contrast to the poor activity of IMP-1 and VIM-2. These findings support the observation that clinical resistance is linked to overexpression of NDM variants and not to IMP-1 and VIM-2. This may guide the use of FDC against a specific group of MBLs and justifies pairing with an MBL inhibitor. Disclosures All Authors: No reported disclosures
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