Phenotypic and genomic characterization of NDM-producing Escherichia coli colonizing the pediatric gut in Shenzhen, China.

Carbapenem-resistant Enterobacterales, driven by New Delhi metallo-β-lactamases (NDMs), severely restrict treatment options and are increasingly detected outside hospitals. Yet, integrated genomic-phenotypic data on NDM-positive Escherichia coli colonizing the pediatric gut in China remain scarce. Antimicrobial susceptibility and blaNDM conjugation were tested. Whole-genome sequencing (WGS) profiled the resistome, virulome, and plasmid replicon types; multilocus sequence typing (MLST), core-gene MLST, and fimH alleles were analyzed. We assessed biofilm-forming capacity, screened gene-biofilm associations using Benjamini-Hochberg false discovery rate, and measured cytotoxicity and siderophore activity. Twenty-five NDM-positive E. coli were isolated from 1,189 children stool specimens. ST48 (ST10 complex) was the most frequent sequence type (6/25, 24%); fimH41 was the most common fimH allele (4/25, 16%). All isolates were resistant to AMC, TZP, CXM, FOX, CAZ, CRO, CSL, ETP, and IMP, while none was resistant to tigecycline. All isolates transferred blaNDM by conjugation (frequency 1.0 × 10⁻⁴ to 9.0 × 10⁻⁴). WGS identified blaNDM-5 as predominant (24/25, 96%), with one blaNDM-1 (1/25, 4%); all isolates harbored multiple virulence and resistance genes. IncFIB (19/25, 76%) was the most common plasmid replicon. All isolates formed biofilms, including three strong producers; an exploratory screen suggested a positive association between espL4 carriage and biofilm formation ability. All isolates increased LDH release from NCM460 cells versus the ATCC 25922 control (low cytotoxicity), without significant differences among isolates; all isolates exhibited siderophore activity as determined by Chrome Azurol S assays. NDM-positive E. coli colonizing children in this study were dominated by blaNDM-5, frequently carried IncFIB plasmids, and readily transferred blaNDM. All isolates showed biofilm, siderophore activity, and epithelial cytotoxicity, with espL4 positively associated with biofilm.IMPORTANCEThis study provides an integrated genomic and phenotypic characterization of pediatric gut-derived New Delhi metallo-β-lactamase (NDM)-positive Escherichia coli isolates. These strains were dominated by blaNDM-5, carried extensive resistomes, and maintained a conserved set of virulence determinants that support colonization and persistence. The frequent presence of poly-replicon plasmids, particularly IncF-type backbones, underscores their high potential for resistance dissemination. Functionally, all isolates exhibited biofilm formation, siderophore activity, and epithelial cytotoxicity, with a hypothesis-generating signal linking espL4 to enhanced biofilm production. Together, these findings demonstrate that children can harbor high-risk, multidrug-resistant NDM-producing E. coli in their gut and emphasize the need for continued genomic surveillance and mechanistic studies to guide infection control and therapeutic strategies.