Copy number of the blaNDM-1 gene (New Delhi metallo-β-lactamase-1) and meropenem susceptibility in Enterobacterales: Consideration for treatment with meropenem?

Abstract

Some Enterobacterales carrying blaNDM were still susceptible to meropenem, exhibiting meropenem minimum inhibitory concentration (MIC) values below the clinical breakpoint. As this phenomenon is a rare observation, this study investigated the underlying regulatory mechanisms contributing to the low MIC values and evaluated the efficacy of meropenem as a therapeutic preference for such isolates. Six clinical blaNDM-positive Enterobacterales exhibited meropenem MICs between 0.5-2.0 mg/L. Successful transmission of blaNDM into recipient E. coli J53 indicated its presence on plasmids. Conventional Sanger and whole-genome sequencing revealed that no truncation or deletion was observed within blaNDM or its core promoter region up to 120 bp upstream. Quantitative real-time PCR of blaNDM showed significantly lower expression levels of blaNDM, i.e., 0.2-0.5 in study isolates, compared to a clinical blaNDM-possessing isolate (expression=1) exhibiting a high meropenem MIC value (64 mg/L). Droplet digital PCR (ddPCR) further demonstrated that these isolates carried a lower copy number of the blaNDM gene, compared to the blaNDM-possessing isolate with high meropenem MIC (64 mg/L). Furthermore, the in vitro time-kill assay exhibited bactericidal efficacy of meropenem >3 log10 decrease in cfu/mL over the 24 hours from the starting inoculum. Administration of meropenem in in vivo neutropenic mice produced a 4-log cfu reduction compared to untreated controls at the start of dosing. Our data suggest that a reduction in blaNDM gene dosage, reflecting lower expression of blaNDM, correlates with lower meropenem MIC values. Meropenem is effective against these isolates when the meropenem MICs for the isolates are 0.5-2.0 mg/L. ImportanceThe production of NDM by Enterobacterales poses one of the most urgent global public health threats due to its plasmid-mediated spread, leading to treatment failure by exhibiting high MICs against a broad-spectrum of antibiotics. However, in rare instances, blaNDM-1-positive clinical Enterobacterales with unexpectedly low meropenem MICs (0.5-2.0 mg/L) raise a further question of reconsidering meropenem as a potential therapeutic option in these specific cases. This study identified that low MICs were associated with reduced expression and lower copy number of the plasmid-mediated blaNDM gene, despite intact promoter regions. Both in vitro and in vivo assays validated the bactericidal activity of meropenem against these isolates. The study raised an argument about the potential limitations of relying solely on genotypic detection of resistance genes for therapeutic decision-making, which emphasises the need for further investigation. As a safe medication, repurposing meropenem for neonatal treatment may eliminate the need for other toxic drugs.