Emergence of a KL239-OCL6-ST63 Carbapenem-Resistant Acinetobacter pittii Strain, Co-carrying blaNDM-1 and blaOXA-500

To characterize the genomic features, antimicrobial resistance mechanisms, and biological characteristics of a carbapenem-resistant Acinetobacter pittii strain co-harboring plasmid-borne bla NDM-1 and chromosomally located bla OXA-500 . An A. pittii strain (L802) was isolated from an intestinal sample of a diarrhea outpatient in Hangzhou, Zhejiang Province, China. Whole-genome sequencing was performed using Illumina and Oxford Nanopore platforms, followed by comprehensive bioinformatics analysis. The localization of bla NDM-1 was determined by S1-PFGE and Southern blotting. Horizontal gene transfer potential was evaluated by conjugation and electrotransformation assays. Antimicrobial susceptibility testing, biofilm formation assays, virulence evaluation using a Galleria mellonella infection model, scanning electron microscopy, phylogenetic analysis, and RT-qPCR analysis of resistance gene expression under carbapenem induction were conducted. Strain L802 was identified as A. pittii ST63 and exhibited high-level resistance to carbapenems and multiple cephalosporins, while remaining susceptible to polymyxin B and tigecycline. Whole-genome analysis revealed a 3.86 Mb circular chromosome and four plasmids. The bla NDM-1 gene was located on a ~ 41 kb IncR-type plasmid (pL802-NDM-1) together with aph(3′)-VI , sharing 99–100% sequence identity with plasmids from diverse Enterobacteriaceae species. Conjugation assays failed to yield transconjugants; however, electrotransformation confirmed that the bla NDM-1 -carrying plasmid could be introduced into Escherichia coli DH5α under laboratory conditions. Importantly, bla OXA-500 was located on the chromosome, representing a rare genetic configuration that may contribute to enhanced stability compared with plasmid-borne resistance genes. Phenotypic assays showed weak biofilm formation and low virulence in the Galleria mellonella model. Phylogenetic analysis indicated that L802 clustered closely with other A. pittii strains isolated in China, suggesting possible regional dissemination. This study reports, for the first time in Zhejiang, China, an A. pittii strain co-harboring plasmid-borne bla NDM-1 and chromosomally located bla OXA-500 . The coexistence of mobile and chromosomally encoded carbapenemase genes highlights a concerning resistance strategy and underscores the need for continuous surveillance and infection control measures against emerging multidrug-resistant Acinetobacter species.