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Extended-spectrum-β-lactamases (ESBLs)-producing Escherichia coli conferred resistance to most β-lactams, except for carbapenems. To date, the transmission mechanism of bla CTX-M , as the most common ESBLs subtype, in E. coli has received sustained attention around the worldwide, but the research on the pathogenicity of bla CTX-M -bearing E. coli is still scarce. The aims of this study were to discern the spread characteristics of ColV (encoding colicin V) plasmids in bla CTX-M -positive E. coli. The multi-drug resistance traits, phylogroups, and ColV plasmid profilings were screened in 76 bla CTX-M -positive E. coli. Thereafter, the genetic profiles of E. coli G12 and GZM7 were determined by whole genome sequencing, conjugation and S1-pulsed-field gel electrophoresis. The median lethal dose was analyzed in E. coli G12 and TG12A, the ColV-plasmid transconjugant of G12. Of all 76 bla CTX-M -bearing E. coli, 67.11% exhibited resistance to at least 2 drugs in addition to ceftiofur, 14.47% carried ColV-positive plasmids, and 53.95% were phylogroup C. Further studies demonstrated that the bla CTX-M -bearing E. coli G12 was assigned to the predominant lineage O78:H4-ST117 of phylogroup G. In addition, its ColV-positive plasmid simultaneously carried multiple resistance genes, and could be independently transferred to confer partial pathogenicity on its host by plasmid mating. E. coli GZM7 was O53:H9-ST23 of phylogroup C, which belonged to another representative lineage of APEC (avian pathogenic E. coli). Its ColV-positive plasmid could complete conjugation with the help of the other coexisting-resistance conjugative plasmid, although it failed to transfer alone. Our findings highlight the flexibly horizontal transfer of ColV plasmids along with multidrug-resistant genes among bla CTX-M -bearing E. coli poses a threat to poultry health and food safety, which contributes to elucidate the concept of "One Health" and deserves particular concern., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Two diverse conjugative plasmids can interact within bacterial cells. However, to the best of our knowledge, the interaction between bla CTX-M -carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600 mcr-1 -carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600 FII , C600 I2 , and C600 FII+I2 of E. coli C600 and J53 FII , J53 I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried FII+I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried bla CTX-M and mcr-1 genes, although the molecular mechanisms of their coresidence warrant further study. IMPORTANCE More and more Enterobacteriaceae , which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of bla CTX-M and mcr-1 , which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of bla CTX-M -bearing IncFII and mcr-1 -bearing IncI2 in clinical E. coli, which will pose a serious threat to clinical therapy and public health.

The aims of this study were to elucidate the role of IS 1294 in plasmid reorganization and to analyze biological characteristics of cointegrates derived from different daughter plasmids. The genetic profiles of plasmids in Escherichia coli strain C21 and its transconjugants were characterized by conjugation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern hybridization, whole-genome sequencing (WGS) analysis, and PCR. The traits of cointegrates were characterized by conjugation and stability assays. bla -bearing IncI2 pC21-1 and nonresistant IncI1 pC21-3, as conjugative helper plasmids, were fused with nonconjugative CTX-M-55 -bearing IncI2 pC21-1 and nonresistant IncI1 pC21-3, as conjugative helper plasmids, were fused with nonconjugative rmtB -bearing IncN-X1 pC21-2, generating cointegrates pC21-F1 and pC21-F2. Similarly, pC21-1 and pC21-3 were fused with nonconjugative IncF33:A-:B- pHB37-2 from another E. coli strain to generate cointegrates pC21-F3 and pC21-F4 under experimental conditions. Four cointegrates were further conjugated into the E. coli strain J53 recipient at high conjugation frequencies, ranging from 2.8 × 10 -3 to 3.2 × 10 -2 . The formation of pC21-F1 and pC21-F4 was the result of host- and IS 1294- mediated reactions and occurred at high fusion frequencies of 9.9 × 10 -4 , respectively. Knockout of RecA resulted in a 100-fold decrease in the frequency of plasmid reorganization. The phenomenon of cointegrate pC21-F2 and its daughter plasmids coexisting in transconjugants was detected for the first time in plasmid stability experiments. IS -4 , respectively. Knockout of RecA resulted in a 100-fold decrease in the frequency of plasmid reorganization. The phenomenon of cointegrate pC21-F2 and its daughter plasmids coexisting in transconjugants was detected for the first time in plasmid stability experiments. IS 26 - orf - oqxAB -mediated fusion between plasmids with different replicons. This study provides insight into the formation and evolution of cointegrate plasmids under different drug selection pressures, which can promote the dissemination of MDR plasmids. 1294 The increasing resistance to β-lactams and aminoglycoside antibiotics, mainly due to extended-spectrum β-lactamases (ESBLs) and 16S rRNA methylase genes, is becoming a serious problem in Gram-negative bacteria. Plasmids, as the vehicles for resistance gene capture and horizontal gene transfer, serve a key role in terms of antibiotic resistance emergence and transmission. IS IMPORTANCE , present in many antibiotic-resistant plasmids from Gram-negative bacteria, plays a critical role in the spread, clustering, and reorganization of resistance determinant-encoding plasmids and in plasmid reorganization through replicative transposition mechanisms and homologous recombination. However, the role of IS 26 , present in many MDR plasmids, in the formation of cointegrates remains unclear. Here, we investigated experimentally the intermolecular recombination of IS 1294 , present in many MDR plasmids, in the formation of cointegrates remains unclear. Here, we investigated experimentally the intermolecular recombination of IS 1294 , which occurred with high frequencies and led to the formation of conjugative MDR cointegrates and facilitated the cotransfer of bla CTX-M-55 and rmtB -driven cointegration of plasmids.1294 in the formation of cointegrates and the common features of IS 1294 -driven cointegration of plasmids.

Objectives: To characterize the complete sequences of four plasmids in MCR-1-producing clinical Escherichia coli strain D72, and to depict the formation mechanism and characteristics of the cointegrate plasmid derived from the pD72-mcr1 and pD72-F33 plasmids., Methods: The genetic profiles of plasmids in strain D72 and its transconjugant were determined by conjugation, S1-PFGE, Southern hybridization, WGS analysis and PCR. Plasmid sequences were analysed with bioinformatic tools. The traits of the fusion plasmid were characterized by cointegration, stability and conjugation assays., Results: Strain D72, belonging to ST1114, contained four plasmids, including mcr-1-carrying pD72-mcr1, blaCTX-M-55-carrying pD72-F33, blaTEM-238-bearing pD72-IncP and pD72-IncX1 carrying aph(3')-Ia, qnrS2 and floR. A single plasmid, pD72C, in the transconjugant was found to be larger than any plasmid in the original strain D72. Sequence analysis showed that pD72C was the fusion product of pD72-mcr1 and pD72-F33, and the recombinant event involved an intermolecular replicative mechanism. Plasmid fusion occurred at a frequency of 1.75 × 10-4 cointegrates per transconjugant. The fusion plasmid presented a high stability and conjugation frequency of 8.00 × 10-3., Conclusions: To our knowledge, this is the first report of the IS26-mediated fusion of an IncN1-F33:A-:B- plasmid and an mcr-1-carrying phage-like plasmid, providing evidence for the important role of IS26 in the recombination of plasmids. The biological advantages of the fusion plasmid indicated that the fusion event presumably plays a potential role in the dissemination of mcr-1., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This study was undertaken to discern the transmission characteristics of mcr-1 and bla CTX-M-type in one multidrug-resistant Escherichia coli LWY24 from chicken in China. The genetic profiles of LWY24 isolate were determined by conjugation, S1-pulsed-field gel electrophoresis, southern blot hybridization, and whole genome sequencing analysis. Meanwhile, co-transfer of plasmids in LWY24 isolate was screened by dual conjugation assays. The LWY24 isolate was identified as ST93, and harbored 3 conjugative plasmids, pLWY24J-3 (bla CTX-M-55 -bearing IncFⅡ), pLWY24J- mcr-1 (mcr-1 -carrying IncI2), and pLWY24J-4 (non-resistance-conferring IncI1), and one nonconjugative plasmid pLWY24 (bla CTX-M-14 -containing IncHI2/IncHI2A). Numerous resistance genes, insertion sequences (especially IS 26), and transposons were found in the 4 plasmids, suggesting that horizontal transmission have occurred by plasmid mating, homologous recombination, and transpositions. Under the selection pressure of cefotaxime and colistin or cefotaxime alone, the mcr-1 -bearing plasmid and the bla CTX-M-55 -harboring plasmid could be co-transferred at a similar frequency, with 8.00 × 10−4 or 9.00 × 10−4 transconjugants per donor cell, respectively. The specific shufflon region in mcr-1 -encoding plasmid could generate up to 6 diverse PilV structures, which may further accelerate the horizontal transfer of plasmid. In conclusion, the transmission characteristics of mcr-1 and bla CTX-M-type in LWY24 isolate could due to clonal spread of ST93, selective pressure of cefotaxime, IS 26 -mediate homologous recombination and transposition, and the specific shufflon region. [ABSTRACT FROM AUTHOR]