Genomic Epidemiology of NDM-1 Carbapenemase-Producing Acinetobacter spp. from Hospital Wastewater in Shenzhen, China

Background: Hospital wastewater (HWW) is a critical reservoir for carbapenem-resistant Gram-negative bacteria. Methods: Between November 2024 and August 2025, sixty 24 h composite wastewater samples were collected from five tertiary hospitals. Of the 244 carbapenem-resistant isolates recovered, 34 blaNDM-1-positive Acinetobacter isolates were subjected to phenotypic, genotypic, and plasmid analyses. Results: Eleven species were identified among the 34 carbapenem-resistant Acinetobacter isolates, predominantly non-baumannii Acinetobacter (NBA). All isolates were carbapenem-resistant (34/34, 100%) with high-level MICs (meropenem MIC50/90, 32/64 mg/L; imipenem MIC50/90, >128/>128 mg/L); 21% (7/34) of isolates were resistant to colistin, and resistance to ceftazidime, cefepime, and trimethoprim-sulfamethoxazole was 100%, 94%, and 76%, respectively. Core-genome SNP analysis revealed highly similar isolates across hospitals within the same season (1-2 SNPs) or within the same hospital across seasons (19 SNPs). Genomic analysis showed that blaNDM-1 was present in all isolates (34/34, 100%), with plasmid carriage in 85.3% (29/34); blaOXA-58 co-occurred in 62.1% (18/29), mainly on Rep_3 plasmids (19/29), especially R3-T28 (15/29) that frequently carried blaOXA-58 (10/15). Two unclassified plasmids co-harboring blaNDM-1 and blaOXA-23 were detected in Acinetobacter tandoii isolates. The blaNDM-1 gene was embedded in a conserved Tn125-like structures with variable flanks. Conclusions: Overall, carbapenem-resistant Acinetobacter from hospital wastewater frequently carried Rep_3 plasmid-borne blaNDM-1, especially R3-T28 and often co-occurring with blaOXA-58, within a conserved Tn125-like core structures. These findings highlight HWW as a potential hotspot for dissemination of carbapenem resistance and support routine genomic surveillance under a One Health framework.