A Natural Novel Mutation in the blaNDM-5 Promoter Reducing Carbapenems Resistance in a Clinical Escherichia coli Strain

The worldwide emergence of New Delhi metallo-b-lactamase (NDM) is of great concern. NDM could hydrolyze almost all b-lactam antibiotics, compromising antimicrobial treatments for patients with severe infection (1). Few NDM inhibitors are effective for treating infection caused by carbapenem-resistant bacteria, and no promising compounds have been developed in the process as future NDM inhibitors (2). To the best of our knowledge, no report of a naturally inactivated blaNDM gene has been described so far. In this study, we identified a novel mutation in the promoter of IncX3-plasmid-carrying blaNDM-5 that leads to the reduction of carbapenems resistance in a clinical Escherichia coli strain. The E. coli isolate Z503 was cultured from a fecal sample of a female patient in Zhejiang in 2018, using enrichment media supplemented with meropenem (0.25 mg/mL), and further confirmed by 16S rRNA gene sequencing. The presence of gene blaNDM-5 was screened by PCR, as described previously (3). NDM-producing Escherichia coli strains are usually associated with medium to high MIC for carbapenems. Interestingly, strain Z503 shows low resistance to meropenem (MIC = 1 mg/mL) and imipenem (MIC = 2 mg/mL), with MICs that remained within the susceptibility range. Whole-genome sequencing (WGS) of Z503 was performed using Illumina Hiseq2500 platform (Bionova) and Nanopore MinION sequencing platform (Oxford Nanopore Technologies). The whole-genome sequencing data were analyzed using multiple programs, including Unicycler (assembly), Prokka (annotation), and SRST2 (antibiotic resistance gene analysis) (3–5). WGS revealed that E. coli Z503 harbored a 4.27-Mb circular chromosome and four plasmids, including IncX3 (;46 kb), IncFIB (;47 kb), IncFII (;83 kb), and colRNA (;5 kb) type plasmids. The blaNDM-5 gene was located on an IncX3 type plasmid, namely, p503-IncX3 (;46 kb), which showed 98.61% identity to plasmid p3R-IncX3 (a blaNDM-5-carrying IncX3plasmid identified in our previous study) (accession number CP049352). In addition to blaNDM-5, blaTEM-98 (extended-spectrum b-lactamase [ESBL]-encoding gene), ampC1 and mrdA (b-lactam resistance genes), and 8 other antimicrobial resistant genes were identified in E. coli Z503. To investigate the conjugation efficiency of the p503-IncX3 plasmid, we mixed donor stain Z503 and recipient J53 at a ratio of 1:3 on LB agar and cultured them overnight. The mixtures were plated on LB agar containing meropenem (0.25 mg/mL) and sodium azide (150 mg/mL). The recombinant strains were confirmed by PCR-based screening. The plasmid p503-IncX3 could be successfully transferred at a high frequency (2.16 3.0) 1021, and the meropenem MIC of transconjugants was only 0.5mg/mL. The blaNDM-containing sequences identified share two common features. The upstream of blaNDM is always the insertion sequence ISAba125, while the downstream is always the bleomycin-resistant gene bleMBL (1). The insertion sequence ISAba125 plays a key role in the mobilization of NDM (6, 7). A group of genes were located downstream of bleMBL, including trpF, dsbC, and cutA (1). Similar genetic context was found in E. coli Z503 in this study, with 99.96% homology to the corresponding sequences in plasmid p3R-IncX3 and its similar variants, which has been widely reported worldwide (Fig. 1a). Notably, a single Editor Aude A. Ferran, INTHERES Copyright © 2022 Li et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Shaolin Wang, shaolinwang@cau.edu. cn. The authors declare no conflict of interest. Published 9 February 2022