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Background: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla SFO-1 , bla NDM-1 , and bla KPC-2 ., Methods: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids., Results: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla SFO-1 , bla NDM-1 , and bla KPC-2 on plasmids of various sizes. The uncommon ESBL gene bla SFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS 26-fosA3 -IS 26-ampR-bla SFO-1 -IS 26 . The bla NDM-1 was found on an IncX3 plasmid, coexisting with bla SHV-12 , displaying the sequence IS 5 -IS 3000 -IS 3000 -Tn 2-bla NDM-1 - ble-trpF-dsbD-cutA-gros-groL , lacking IS Aa125 . The bla KPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn 2-tnpR -IS Kpn27-bla KPC-2 -IS Kpn6-korC . Conjugation assays confirmed the transferability of both bla NDM-1 and bla KPC-2 ., Conclusion: We discovered the coexistence of bla SFO-1 , bla NDM-1 , and bla KPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Guo et al.)

Abstract: The emergence of the plasmid-borne colistin-resistant gene ( mcr-1 ) poses a great threat to human health. What is worse, the recent observations of the co-existence of mcr-1 with other antimicrobial resistance genes in some bacteria cause further concern. Here, we present the first report of a wild Escherichia coli strain that co-carries an mcr-1 encoding phage-like IncY plasmid (pR15_MCR-1) and a bla NDM-5 encoding IncX3 plasmid (pR15_NDM-5) from a pharmaceutical industry, wastewater treatment plant, in China. This study highlights the spreading of E. coli carrying both mcr-1 and bla NDM-5 genes in the pharmaceutical industry., Importance: Escherichia coli strains that carry both mcr-1 and bla NDM-5 genes are of great health concern and are already found in humans and animals worldwide, yet there is a paucity of observations of this resistant strain in the environment. Here we present the first isolation of an E. coli strain (R15) that co-carries mcr-1 and bla NDM-5 genes from a wastewater treatment plant in China. Whole-genome sequencing indicated that R15 harbored two plasmids, pR15_MCR-1 and pR15_NDM-5, that carry mcr-1 and bla NDM-5 , respectively. The observation of this wild-derived E. coli strain that carries mcr-1 and bla NDM-5 genes simultaneously calls for the urgency to improve monitoring and reducing its further spreading., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Han et al.)