Phenotypic and genomic characterization of NDM-producing <i>Escherichia coli</i> 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 <i>Escherichia coli</i> colonizing the pediatric gut in China remain scarce. Antimicrobial susceptibility and <i>bla</i>NDM conjugation were tested. Whole-genome sequencing (WGS) profiled the resistome, virulome, and plasmid replicon types; multilocus sequence typing (MLST), core-gene MLST, and <i>fimH</i> 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 <i>E. coli</i> were isolated from 1,189 children stool specimens. ST48 (ST10 complex) was the most frequent sequence type (6/25, 24%); <i>fimH</i>41 was the most common <i>fimH</i> 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 <i>bla</i>NDM by conjugation (frequency 1.0 × 10⁻⁴ to 9.0 × 10⁻⁴). WGS identified <i>bla</i>NDM-5 as predominant (24/25, 96%), with one <i>bla</i>NDM-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 <i>espL4</i> 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 <i>E. coli</i> colonizing children in this study were dominated by <i>bla</i>NDM-5, frequently carried IncFIB plasmids, and readily transferred <i>bla</i>NDM. 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 <i>Escherichia coli</i> isolates. These strains were dominated by <i>bla</i>NDM-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 <i>espL4</i> to enhanced biofilm production. Together, these findings demonstrate that children can harbor high-risk, multidrug-resistant NDM-producing <i>E. coli</i> in their gut and emphasize the need for continued genomic surveillance and mechanistic studies to guide infection control and therapeutic strategies.