Effect of in vitro Zinc Depletion on Carbapenemase Activity in Enterobacterales Clinical Isolates

Abstract

Introduction: Metallo-β-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE) represent a serious clinical challenge due to limited treatment options, necessitating accurate testing to identify effective treatments and combat antibiotic resistance. Supraphysiological zinc levels in media can lead to skewed resistance profiles in MBL- producing CRE, complicating treatment decisions. This study investigates the effect of zinc depletion on carbapenem resistance. Methods: Thirty MBL-producing CRE isolates (NDM, n=15; VIM, n=15) underwent testing before and after zinc depletion using broth microdilution, disk diffusion, and gradient diffusion testing. Zinc depletion from media was achieved through ethylenediaminetetraacetic acid (EDTA) addition. Results: All isolates were resistant to at least one carbapenem by broth microdilution—100% (30/30) to imipenem and 93% (28/30) to meropenem—with both imipenem and meropenem having an MIC50 of 32μg/ml and MIC90 > 64μg/ml. Zinc depletion resulted in a notable shift towards susceptibility (p < 0.0001). MIC50 values dropped to 0.25μg/ml for imipenem and < 0.12μg/ml for meropenem, with MIC90 decreasing to 4μg/ml and 8μg/ml, respectively. NDM- producing isolates exhibited a higher MIC50 (> 64μg/ml) compared to VIM-producing isolates (8-16 μg/ml). Cohen's Kappa analysis showed substantial agreement (k = 0.63) between broth microdilution and gradient diffusion strip test in zinc-depleted media. Conclusion: In MBL-producing Enterobacterales, zinc exacerbates in vitro carbapenem resistance, highlighting the potential for optimising antibiotic susceptibility testing by reducing zinc levels in media. While further clinical data would be required to support changes in testing protocols, lowered zinc levels may contribute to more accurate treatment decisions and expand carbapenem use in certain cases. These findings are valuable for improving CRE diagnostics and therapeutics, aiding in more effective antimicrobial stewardship.