Metallo-β-lactamase resistance in Enterobacteriaceae is an artefact of currently utilized antimicrobial susceptibility testing methods

Abstract

Background MBLs are a major contributor to β-lactam resistance when tested using CAMHB. Despite in vitro resistance, positive outcomes have been reported in MBL-infected patients following carbapenem treatment. The impact of physiological zinc concentrations on this in vitro–in vivo MBL discordance warrants investigation. Objectives To evaluate meropenem in vitro activity against MBL-producing Enterobacteriaceae in zinc-depleted broth (Chelex-CAMHB, EDTA-CAMHB) and assess meropenem efficacy in murine infection models. Methods Neutropenic mice received a meropenem human-simulated regimen of 2 g q8h or levofloxacin 750 mg q24h (for model validation). Zinc concentrations were determined in conventional CAMHB, zinc-depleted CAMHB and epithelial lining fluid (ELF) of lung-infected mice. Results All MBL-producing isolates (NDM, n = 25; VIM, n = 3; IMP, n = 2) examined were meropenem resistant in CAMHB and susceptible in zinc-depleted CAMHB (5- to 11-fold reduction), with zinc depletion having no impact on levofloxacin MICs. Zinc concentrations (mean ± SD) in CAMHB were 0.959 ± 0.038 mg/L and in both zinc-depleted CAMHB and ELF were <0.002 mg/L. In vivo, levofloxacin displayed predictable efficacy consistent with its phenotypic profile, while meropenem produced >1 log unit bacterial killing despite in vitro resistance in conventional CAMHB. Conclusions Results indicate that meropenem in vivo efficacy is best represented by the pharmacodynamic profile generated using MICs determined in zinc-depleted media for MBL-producing Enterobacteriaceae. These translational data suggest that the use of conventional CAMHB for MBL susceptibility testing is inappropriate in distinguishing meaningful in vivo resistance given that zinc concentrations are supraphysiological in conventional CAMHB and negligible at infection sites.

Therapeutic Options for Metallo-β-Lactamase-Producing Enterobacterales

“…MBL enzymes can readily hydrolyze carbapenems in vitro, yet some data suggests that their ability to cause carbapenem resistance is an artifact of the current testing modalities that utilize media with supraphysiologic zinc concentrations. 93 Since zinc is required at the active site of the enzyme, the quantity of zinc at the site of infection could impact the function of the enzyme and also the rate of antibiotic hydrolysis. However, the data are inconclusive.…”

“…Asempa et al found that meropenem against a panel of NDM-, VIM-, and IMP-producing Enterobacterales appeared resistant in vitro but generated >1 log bacterial killing in murine infection models. 93 They showed that the meropenem in vivo activity better correlated with MICs performed in zinc-depleted media, where the isolates appeared susceptible to carbapenems, than in traditional cation adjusted Mueller Hinton broth.…”

Antibiotics and Innate Immunity: A Cooperative Effort Towards the Successful Treatment of Infections

“…However, these organisms are susceptible to carbapenems in vivo, where nutritional immunity restricts zinc concentrations to much lower levels. Removal of zinc from MHB broth more reliably predicts in vivo activity of carbapenems against these organisms [ 16 ]. Rigid continuation of AST paradigms utilizing only standard bacteriological media overlooks potential useful activities of the currently available antibiotics against multidrug-resistant bacteria [ 14 ], but it also hinders the transition of pharmacodynamic analysis of innate immunity synergy with antibiotics to a more clinical mainstream.…”

Activities of Cefiderocol with Simulated Human Plasma Concentrations against Carbapenem-Resistant Gram-Negative Bacilli in an In Vitro Chemostat Model

“…The purpose of this study is to evaluate the efficacy of cefiderocol under simulated human plasma concentrations over 24 h against clinical strains with a cefiderocol MIC around the breakpoint in a one-compartment in vitro chemostat model. MBL-producing strains were also included in this study because of the limited treatment options for such bacteria, and difficulties in evaluation by animal models have been reported due to the in vitro-in vivo discordance against MBL producers caused by the poor activity of MBL in animal models ( 7 ). Overall, this chemostat model is expected to provide useful information for treatment against isolates with cefiderocol MICs of 0.5 to 4 μg/ml, including MBL producers.…”