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Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.

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Klebsiella michiganensis is a recently emerging human pathogen causing nosocomial infections. This study aimed to characterize the complete genome sequence of a clinical Klebsiella michiganensis strain KMIB106 which exhibited extensive drug-resistance. The whole genome of the strain was sequenced using PacBio RS III systems and Illumina Nextseq 500. Annotation, transposable elements and resistance gene identification were analyzed by RAST, prokka and Plasmid Finder, respectively. According to the results, KMIB106 was resistant to multiple antimicrobials, including carbapenems, but it remained susceptible to aztreonam. The genome of KMIB106 consisted of a single chromosome and three predicted plasmids. Importantly, a novel KPC plasmid pB106-1 was found to carry the array of resistance genes in a highly different order in its variable regions, including mphA, msrE, mphE, ARR-3, addA16, sul1, dfrA27, tetD and fosA3. Plasmid pB106-2 is a typical IncFII plasmid with no resistant gene. Plasmid pB106-IMP consists of the IncN and IncX3 backbones, and two resistance genes, blaIMP-4 and blaSHV-12, were identified. Our study for the first time reported an extensively drug-resistant Klebsiella michiganensis strain recovered from a child with a respiratory infection in Southern China, which carries three mega plasmids, with pB106-1 firstly identified to carry an array of resistance genes in a distinctive order, and pB106-IMP identified as a novel IncN-IncX3 cointegrate plasmid harboring two resistance genes blaIMP-4 and blaSHV-12.

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Colistin is considered as an antibiotic of 'last resort' for the treatment of lethal infections caused by carbapenem-resistant Enterobacterales (CRE), dissemination of plasmid-borne colistin resistance gene mcr-1, particularly into CRE, resulting in the emergence of strains that approach pan-resistance. A wide variety of plasmid types have been reported for carrying mcr-1. Among which, large IncHI2-type plasmids were multidrug-resistant (MDR) plasmids harbored multiple resistance determinants in addition to mcr-1. Herein, we characterized a novel hybrid IncHI2-like mcr-1-bearing plasmid in an NDM-7-producing ST167 Escherichia coli strain EC15-50 of clinical origin. Antimicrobial susceptibility testing showed E. coli EC15-50 exhibited an extensively drug-resistant (XDR) profile that only susceptible to amikacin and tigecycline. S1-PFGE, Southern hybridization and Whole-genome Sequencing (WGS) analysis identified a 46,161 bp bla NDM-7-harboring IncX3 plasmid pEC50-NDM7 and a 350,179 bp mcr-1-bearing IncHI2/HI2A/N/FII/FIA plasmid pEC15-MCR-50 in E. coli EC15-50. Sequence detail analysis revealed the type IV coupling protein (T4CP) gene was absent on pEC15-MCR-50, explaining that pEC15-MCR-50 was a non-conjugative plasmid. Comparative genetic analysis indicated the hybrid plasmid pEC15-MCR-50 was probably originated from pXGE1mcr-like IncHI2/HI2A/N plasmid and pSJ_94-like IncFII/FIA plasmid, and generated as a result of a replicative transposition process mediated by IS26. Currently, the prevalent mcr-1-carrying IncHI2 plasmids were rarely reported to be fused with other plasmids. The identification of the novel hybrid plasmid pEC15-MCR-50 in this study highlighted the importance of close surveillance for the emergence and dissemination of such fusion MDR plasmids, particularly in NDM-producing Enterobacterales.

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Cointegrate/hybrid plasmids combine the genetic elements of two or more plasmids and generally carry abundant antimicrobial resistance determinants. Hence, the spread of cointegrate plasmids will accelerate the transmission of AMR genes. To evaluate the transmission risk caused by cointegrate plasmids, we investigated the structural diversity, fitness cost, and stability of a cointegrate plasmid in Klebsiella pneumoniae YZ6 and Escherichia coli EC600. The cointegrate plasmid pSL131_IncA/C_IncX3 was from a clinical Salmonella Lomita strain. After transferring the plasmid into E. coli EC600 by conjugation, we observed plasmids with different structures, including a full-length original plasmid and two truncated versions. By contrast, DNA fragment deletion and blaCTX-M-14 gene insertion in the plasmid were detected in a transconjugant derived from K. pneumoniae YZ6. These results suggest that the structure of the plasmid was unstable during conjugation. Furthermore, both the full-length plasmid in EC600 and the structurally reorganized plasmid in YZ6 imposed a fitness cost on the bacterial host and enhanced biofilm formation ability. Serial passaging in antibiotic-free medium resulted in a rapid decline of the plasmid in YZ6. However, the stability of the structurally reorganized plasmid in YZ6 was improved via serial passaging in antibiotic-containing medium. SNP calling revealed that mutations of the outer membrane porin may play an essential role in this process. These findings indicate that structural versatility could contribute to the dissemination of cointegrate plasmids. Although the plasmid incurred a fitness cost in other Enterobacteriaceae species, positive selection could alleviate the adverse effects.

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To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, bla CTX-M-55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A-:B- plasmid pS13-2, respectively, fused with IncFI:A-:B- plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A-:B- plasmid and IncFI:A-:B- plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.

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This study aimed to characterise the molecular events underlying formation and evolution of a cointegrate plasmid harbouring blaNDM-1 and blaCMY-2 in a clinical Salmonella Lomita (S. Lomita) strain. The Salmonella strain SL131 was found to harbour two multidrug resistant (MDR) plasmids. One plasmid, pSL131_IncHI2, is a typical IncHI2 plasmid containing blaOXA-1, catB3, arr-3, sul1, qnrB4 and blaDHA-1 in a complex class 1 integron. The other plasmid, pSL131_IncA/C-IncX3, is a blaNDM-1-bearing cointegrate plasmid consisting of IncX3 and IncA/C backbones, the formation of which is mediated by IS26. Stability assay showed that the cointegrate plasmid was highly stable in its natural host - S. Lomita - but would readily resolve into single plasmids upon conjugation, during which the IncX3 blaNDM-1-bearing plasmid could be transferred to Escherichia coli strain EC600. Plasmid evolution through integration of two or more MDR plasmids would not only expand the resistance profile of the resultant plasmid, but also broaden the host spectrum of such resistance-encoding mobile elements. Better understanding of the underlying and triggering mechanisms of cointegration may facilitate development of intervention measures to curb formation and dissemination of such elements.

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