A #MDR #Plasmid pIMP26, Carrying blaIMP-26, fosA5, blaDHA-1, and qnrB4 in #Enterobacter cloacae (Sci Rep., abstract)

[Source: Scientific Reports, full page: (LINK). Abstract, edited.]

Article | OPEN | Published: 15 July 2019

A Multidrug Resistance Plasmid pIMP26, Carrying blaIMP-26, fosA5, blaDHA-1, and qnrB4 in Enterobacter cloacae

Su Wang,  Kaixin Zhou, Shuzhen Xiao, Lianyan Xie, Feifei Gu, Xinxin Li, Yuxing Ni, Jingyong Sun & Lizhong Han

Scientific Reports, volume 9, Article number: 10212 (2019)



IMP-26 was a rare IMP variant with more carbapenem-hydrolyzing activities, which was increasingly reported now in China. This study characterized a transferable multidrug resistance plasmid harboring blaIMP-26 from one Enterobacter cloacae bloodstream isolate in Shanghai and investigated the genetic environment of resistance genes. The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using broth microdilution method, Etest and PCR. The plasmid was analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis and hybridization. Whole genome sequencing and sequence analysis was conducted for further investigation of the plasmid. E. cloacae RJ702, belonging to ST528 and carrying blaIMP-26, blaDHA-1, qnrB4 and fosA5, was resistant to almost all β-lactams, but susceptible to quinolones and tigecycline. The transconjugant inherited the multidrug resistance. The resistance genes were located on a 329,420-bp IncHI2 conjugative plasmid pIMP26 (ST1 subtype), which contained trhK/trhV, tra, parA and stbA family operon. The blaIMP-26 was arranged following intI1. The blaDHA-1 and qnrB4cluster was the downstream of ISCR1, same as that in p505108-MDR. The fosA5 cassette was mediated by IS4. This was the first report on complete nucleotide of a blaIMP-26-carrying plasmid in E. cloacae in China. Plasmid pIMP26 hosted high phylogenetic mosaicism, transferability and plasticity.

Keywords: Antibiotics; Drugs Resistance; Carbapenem; Beta-lactams; Enterobacter cloacae; Shanghai; China; Quinolones; Tigecycline.



Optical #DNA #Mapping Combined with #Cas9-Targeted #Resistance #Gene #Identification for Rapid #Tracking of Resistance #Plasmids in a #NICU #Outbreak (mBio, abstract)

[Source: mBio, full page: (LINK). Abstract, edited.]

Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak

Santosh K. Bikkarolla, Viveka Nordberg, Fredrika Rajer, Vilhelm Müller, Muhammad Humaun Kabir, Sriram KK, Albertas Dvirnas, Tobias Ambjörnsson, Christian G. Giske, Lars Navér,Linus Sandegren, Fredrik Westerlund

Spyros Pournaras, Invited Editor, Karen Bush, Editor

DOI: 10.1128/mBio.00347-19



The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.


This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

Keywords: Antibiotics; Drugs Resistance; Enterobacteriaceae; Beta-lactams; Nosocomial Outbreaks; Diagnostic tests.


#Structural bases for F #plasmid #conjugation and F pilus #biogenesis in #Escherichia coli (Proc Natl Acad Sci USA, abstract)

[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]

Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

Bo Hu, Pratick Khara, and Peter J. Christie

PNAS first published June 25, 2019 / DOI: https://doi.org/10.1073/pnas.1904428116

Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved June 5, 2019 (received for review March 14, 2019)



Bacterial “sex,” or conjugation, is a central mechanism underlying the proliferation of antibiotic resistance. Despite the discovery of conjugation by F plasmids in Escherichia coliover seven decades ago, we have only now visualized the F-encoded transfer channel and F pilus-associated platforms in the E. coli cell envelope by cryoelectron tomography. The channel supports plasmid transfer or assembly of F pili, which remarkably upon synthesis are deposited onto alternative basal structures around the cell surface. The F plasmid transfer system is a paradigm for the bacterial type IV secretion system (T4SS) superfamily. Consequently, the F-encoded structures are broadly informative of mechanisms underlying the biogenesis and function of type IV secretion machines and associated conjugative pili.



Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or “mating” channel, the channel–pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

cryoelectron tomography – DNA conjugation – type IV secretion – pilus – protein transport



1 To whom correspondence may be addressed. Email: bo.hu@uth.tmc.edu or peter.j.christie@uth.tmc.edu.

Author contributions: B.H. and P.J.C. designed research; B.H. and P.K. performed research; B.H., P.K., and P.J.C. analyzed data; and P.J.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Density maps and coordinate data that support the F-encoded channel structures determined by cryoelectron tomography have been deposited in The Electron Microscopy Data Bank (EMDB), https://www.ebi.ac.uk/pdbe/emdb (entry nos. EMD-9344 and EMD-9347).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904428116/-/DCSupplemental.

Published under the PNAS license.

Keywords: Enterobacteriaceae; Plasmids.


#mcr-9, an inducible #gene encoding an acquired phosphoethanolamine transferase in #Escherichia coli, and its origin (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in Escherichia coli, and its origin

Nicolas Kieffer, Guilhem Royer, Jean-Winoc Decousser, Anne-Sophie Bourrel, Mattia Palmieri, Jose-Manuel Ortiz De La Rosa, Hervé Jacquier, Erick Denamur, Patrice Nordmann,Laurent Poirel

DOI: 10.1128/AAC.00965-19



The plasmid-located mcr-9 gene encoding a putative phosphoethanolamine transferase was identified in a colistin-resistant human fecal Escherichia coli belonging to a very rare phylogroup D-ST69-O15:H6 clone. This MCR-9 protein shares 33-65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1- to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a similar function as MCR-1 by adding a phosphoethanolamine group to the lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once cloned and produced in an E. coli K-12 derived strain. Nevertheless, we showed here that sub-inhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally-encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate quite silently and remaining undetected unless induced by colistin.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; MCR9; Colistin; E. Coli.


IncI1 ST3 and IncI1 ST7 #plasmids from CTX-M-1-producing #Escherichia coli obtained from #patients with #bloodstream infections are closely related to plasmids from E. coli of #animal origin (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin

Adam Valcek, Louise Roer, Søren Overballe-Petersen, Frank Hansen, Valeria Bortolaia, Pimlapas Leekitcharoenphon, Helle B Korsgaard, Anne Mette Seyfarth, Rene S Hendriksen, Henrik Hasman, Anette M Hammerum

Journal of Antimicrobial Chemotherapy, dkz199, https://doi.org/10.1093/jac/dkz199

Published: 14 May 2019




Fully sequenced IncI1 plasmids obtained from CTX-M-1-producing Escherichia coli of human and animal origin were compared.


Twelve E. coli isolates sharing identical ESBL genes and plasmid multilocus STs sequenced on Illumina and MinION platforms were obtained from the Danish antimicrobial resistance surveillance programme, DANMAP. After de novoassembly, the sequences of plasmids harbouring blaCTX-M-1 were manually curated and ORFs annotated. Within-group comparisons were performed separately for the IncI1 ST3 plasmid type and the IncI1 ST7 plasmid type. The IncI1 ST3 plasmid group was obtained from 10 E. coli isolates (2 from patients with bloodstream infections, 6 from food and 2 from animals). The IncI1 ST7 plasmids originated from E. coli isolates obtained from a patient with bloodstream infection and from a pig. Sequences of IncI1 ST3 and IncI1 ST7 plasmids harbouring blaCTX-M-1 with determined origin were retrieved from GenBank and used for comparison within the respective group.


The 10 IncI1 ST3 blaCTX-M-1 plasmids were highly similar in structure and organization with only minor plasmid rearrangements and differences in the variable region. The IncI1 ST7 blaCTX-M-1 plasmids also showed high similarity in structure and organization. The high level of similarity was also observed when including plasmids from E. coli of animal origin from Australia, Switzerland, the Netherlands and France.


This study shows broad spread of a very successful CTX-M-1-producing IncI1 type plasmid among E. coli of both human and animal origin.

Topic: plasmids – drug resistance, microbial – food – genes – ichthyosis, x-linked – sequence tagged sites – escherichia coli – sodium thiosulfate – bloodstream infections – genbank


© 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 article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Antibiotics; Drugs Resistance; E. Coli; Bacteremia; Pigs; Human; Plasmids.


Functional characterization of a Miniature Inverted Transposable Element at the origin of #mcr-5 gene acquisition in #Escherichia coli (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Functional characterization of a Miniature Inverted Transposable Element at the origin of mcr-5 gene acquisition in Escherichia coli

Nicolas Kieffer, Patrice Nordmann, Yves Millemann, Laurent Poirel

DOI: 10.1128/AAC.00559-19



Plasmid-mediated colistin resistance of the MCR type is a growing concern in Enterobacteriaceae since it has been described worldwide either in humans and in animals. Here we identified a series of MCR-producing Escherichia coli isolates, corresponding to two different clones (respectively represented by isolates PS1 and PS8b) producing MCR-1 and MCR-5, respectively, from pig fecal samples in France. Plasmid analysis showed that the plasmid carrying the mcr-1 gene (pPS1) possesses an IncHI2 backbone whereas the mcr-5gene was carried onto a 6,268 bp non-typeable, non self-conjugative plasmid (pPS8b). Detailed analysis of plasmid pPS8b revealed a 3,803 bp-long cassette containing the mcr-5 gene that was bracketed by two inverted-repeat sequences (IRs) with 5-bp long direct repeats at each extremity, similarly to an insertion sequence, but with the exception that no transposase gene was identified within this cassette. By performing in-vitro transposition experiments, we showed that the mcr-5 cassette could be mobilized by the TnAs1 transposase provided in-trans, displaying a similar mobilization mechanism as miniature inverted repeat transposable elements (MITEs).

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Colistin; E. Coli; MCR5; Plasmids.


Identification of Novel Mobilized #Colistin #Resistance #Gene #mcr9 in a #MDR, Colistin-Susceptible #Salmonella enterica Serotype #Typhimurium Isolate (mBio, abstract)

[Source: mBio, full page: (LINK). Abstract, edited.]

Identification of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate

Laura M. Carroll, Ahmed Gaballa, Claudia Guldimann, Genevieve Sullivan, Lory O. Henderson, Martin Wiedmann

Mark S. Turner, Editor

DOI: 10.1128/mBio.00853-19



Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcrhomologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcrhomologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S. Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coliNEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance.



Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a “highest priority critically important antimicrobial for human medicine” (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

Keywords: Antibiotics; Drugs Resistance; Enterobacteriaceae; Salmonella enterica; MCR9; MCR3; Colistin; USA; Plasmids.