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Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli

ABSTRACT: The emergence of drug-resistant Enterobacteriaceae carrying plasmid-mediated β-lactamase genes has become a significant threat to public health. Organisms in the Enterobacteriaceae family containing New Delhi metallo-β-lactamase‑1 (NDM-1) and its variants, which are capable of hydrolyzing nearly all β-lactam antibacterial agents including carbapenems, are referred to as superbugs and distributed worldwide. Despite efforts over the past decade, the discovery of an NDM-1 inhibitor that can reach the clinic remains a challenge. Here, we identified oxidized glutathione (GSSG) as a metabolic biomarker for blaNDM-1 using a non-targeted metabolomics approach and demonstrated that GSSG supplementation could restore carbapenem susceptibility in Escherichia coli carrying blaNDM-1 in vitro and in vivo. We showed that exogenous GSSG promotes the bactericidal effects of carbapenems by interfering with intracellular redox homeostasis and inhibiting the expression of NDM-1 in drug-resistant E. coli. This study establishes a metabolomics-based strategy to potentiate metabolism-dependent antibiotic efficacy for the treatment of antibiotic-resistant bacteria.

SUBMITTER: Dong-Yang Ye

PROVIDER: S-SCDT-10_1038-S44321-024-00061-X | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundAntibiotics exert an outstanding selective pressure on bacteria, forcing their chromosomal gene mutations and drug resistance genes to spread. The objective of this study is to evaluate the expression of the New Delhi Metallo-β-Lactamase-1 gene (blaNDM-1) in the clinical isolate (Klebsiella pneumoniae TH-P12158), transformant strains Escherichia coli BL21 (DE3)-blaNDM-1, and Escherichia coli DH5α- blaNDM-1 when exposed to imipenem.Methodsβ-Lactamase genes (blaSHV, blaTEM-1, blaCTX-M-9, blaIMP, blaNDM-1, blaKPC, blaOXA, blaGES, and blaDHA) from randomly selected carbapenems-sensitive K.pneumoniae (n = 20) and E.coli (n = 20) strains were amplified by PCR. The recombinant plasmid of pET-28a harboring blaNDM-1 was transformed into E.coli BL21 (DE3) and E.coli DH5α by electroporation. The resistance phenotype and higher blaNDM-1 expression in K.pneumoniae TH-P12158, transformant E.coli BL21 (DE3)-blaNDM-1, and E.coli DH5α-blaNDM-1 were observed when exposed to imipenem with grade increasing, decreasing, and canceling doses, respectively.ResultsAfter being exposed to different doses of imipenem, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of antimicrobial drugs and blaNDM-1 expression of strains increased, which was positively correlated with doses of imipenem. On the contrary, with the decrease or cancellation of imipenem doses, the blaNDM-1 expression was deteriorated, while the MIC and MBC values remained relatively stable. These results demonstrated that low doses of imipenem (˂MIC) could press blaNDM-1 positive strains producing stable drug resistance memory and altered blaNDM-1 expression.ConclusionsLow doses of imipenem could press blaNDM-1 positive strains producing sustained resistance memory and altered blaNDM-1 expression. In particular, the positive correlation between the resistance genes expression and antibiotics exposure shows promising guiding significance for clinical medication.

Project description:The worldwide dissemination of carbapenem-resistant Enterobacteriaceae (CRE) poses a critical human health issue by limiting the range of antibiotics that are usable in the treatment of common bacterial infections. Along with CRE, carbapenem heteroresistance has disseminated worldwide, which is described as different levels of carbapenem resistance within a seemingly isogenic bacterial population. Unstable carbapenem resistance will likely lead to unexpected treatment failure due to the enhanced resistance after initiation of treatment, contradicting antimicrobial susceptibility test results. Porin mutation and tandem amplification of the carbapenemase gene have been reported as mechanisms underlying enhanced carbapenem resistance. In this study, we identified multimerization of plasmids carrying carbapenemase genes, by using Southern blotting, whole-genome sequencing, and quantitative PCR (qPCR) analysis for the CRE isolates obtained in our previous surveillance in Osaka, Japan. Plasmids harboring a carbapenemase gene were multimerized by recA, likely through recombination at two consecutive sets of transposase genes of the IS91 family, thereby producing various plasmids of discrete sizes in a single bacterial cell of an Escherichia coli isolate. This multimerization resulted in increased copy numbers of carbapenemase genes, leading to enhanced gene transcription as well as carbapenem resistance. Prior exposure to meropenem further increased the copy number of carbapenemase genes, readily resulting in enhancement of carbapenem resistance. This mechanism may lead to clinical treatment failure by sifting antimicrobial resistance after the treatment initiation. IMPORTANCE We demonstrated the multimerization of plasmids harboring carbapenemase genes, and multimeric plasmids of various discrete sizes existed in a host bacterial cell of Escherichia coli. Plasmid multimerization along with increased copy numbers of carbapenemase genes resulted in enhanced carbapenemase resistance, which was readily accelerated by an overnight preexposure to meropenem. This mechanism may lead to treatment failure in clinical settings after the initiation of antimicrobial therapy.

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Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli

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