Genomic landscape of bla NDM-1 - and bla OXA-181 -carrying Citrobacter portucalensis sequence type 151 strains from hospital wastewater in Ghana

ABSTRACT Carbapenemases represent one of the major driving forces of antimicrobial resistance (AMR), which has been recognized as a significant public health concern. Additionally, hospital wastewater (HWW) serves as a critical hotspot for the spread of antibiotic-resistant bacteria (ARB) and genes (ARGs). In this study, we report the isolation of difficult-to-identify Citrobacter portucalensis strains that harbor bla NDM-1 and bla OXA-181 from hospital wastewater in Ghana. Wastewater was collected at the Effia Nkwanta Regional Hospital of Ghana, and carbapenemase producers were screened using selective media and multiplex PCR. Antimicrobial susceptibility testing, conjugation, and whole-genome sequencing were performed on the isolates. Two strains (HW7-CARBA and HW8-CARBA) isolated from hospital wastewater were initially identified as Citrobacter freundii but confirmed as C. portucalensis by whole-genome sequencing. These strains were assigned to ST151 and harbored ARGs consistent with their multidrug-resistant phenotype. The bla NDM-1 gene was located on the ~45.0 kb IncX3 plasmid (pHW7-CARBA-NDM-1), which was transferable in the conjugation assay, while the bla OXA-181 gene was bracketed with IS 26 on the ~90.0 kb nonconjugative ColKP3 plasmid (pHW8-CARBA-OXA-181). pHW7-CARBA-NDM-1 showed close phylogenetic similarity to a plasmid from China, whereas pHW8-CARBA-OXA-181 was closely related to the bla OXA-181 plasmid derived from Escherichia coli isolated from the stool sample of a pediatric patient with diarrhea in Ghana. Consequently, HWW serves as a hotspot for ARB and ARGs, increasing the risk of AMR proliferation. Addressing these challenges is essential for protecting public health and contributing to global efforts to preserve antibiotic efficacy. Recommendations include implementing comprehensive AMR-focused regulatory frameworks. IMPORTANCE The operations of hospitals generate wastewater that serves as a critical hotspot for antibiotic-resistant bacteria and genes. Many hospitals discharge untreated wastewater, leading to the widespread occurrence of carbapenemase-producing Enterobacteriaceae , which presents significant challenges for environmental health. The significance of our research lies in determining the genomic landscape of antibiotic-resistant genes in the less-explored Citrobacter portucalensis isolated from hospital wastewater. This research will contribute to preventing the spread of environmental resistance, implementing antimicrobial resistance monitoring standards, and protecting vulnerable populations from exposure.