Abstract
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 bla(NDM-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 MIC(50/90), 32/64 mg/L; imipenem MIC(50/90), >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 bla(NDM-1) was present in all isolates (34/34, 100%), with plasmid carriage in 85.3% (29/34); bla(OXA-58) co-occurred in 62.1% (18/29), mainly on Rep_3 plasmids (19/29), especially R3-T28 (15/29) that frequently carried bla(OXA-58) (10/15). Two unclassified plasmids co-harboring bla(NDM-1) and bla(OXA-23) were detected in Acinetobacter tandoii isolates. The bla(NDM-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 bla(NDM-1), especially R3-T28 and often co-occurring with bla(OXA-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.