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      PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms

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          Abstract

          ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

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          Most cited references 55

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          Plasmids and the spread of resistance.

          Plasmids represent one of the most difficult challenge for counteracting the dissemination of antimicrobial resistance. They contribute to the spread of relevant resistance determinants, promoting horizontal gene transfer among unrelated bacteria. Undistinguishable plasmids were identified in unrelated bacterial strains isolated at huge geographically distant area, with no apparent epidemiological links. These plasmids belong to families that are largely prevalent in naturally occurring bacteria, usually carry multiple physically linked genetic determinants, conferring resistance to different classes of antibiotics simultaneously. Plasmids also harbour virulence factors and addiction systems, promoting their stability and maintenance in the bacterial host, in different environmental conditions. The characteristics of the most successful plasmids that were at the origin of the spread of carbapenemase, expanded-spectrum β-lactamase, and plasmid-mediated quinolone resistance genes are discussed in this review. Copyright © 2013 Elsevier GmbH. All rights reserved.
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            The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair.

            Various mechanisms exist that enable bacteria to resist bacteriophage infection. Resistance strategies include the abortive infection (Abi) systems, which promote cell death and limit phage replication within a bacterial population. A highly effective 2-gene Abi system from the phytopathogen Erwinia carotovora subspecies atroseptica, designated ToxIN, is described. The ToxIN Abi system also functions as a toxin-antitoxin (TA) pair, with ToxN inhibiting bacterial growth and the tandemly repeated ToxI RNA antitoxin counteracting the toxicity. TA modules are currently divided into 2 classes, protein and RNA antisense. We provide evidence that ToxIN defines an entirely new TA class that functions via a novel protein-RNA mechanism, with analogous systems present in diverse bacteria. Despite the debated role of TA systems, we demonstrate that ToxIN provides viral resistance in a range of bacterial genera against multiple phages. This is the first demonstration of a novel mechanistic class of TA systems and of an Abi system functioning in different bacterial genera, both with implications for the dynamics of phage-bacterial interactions.
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              The diversity of conjugative relaxases and its application in plasmid classification.

              Bacterial conjugation is an efficient and sophisticated mechanism of DNA transfer among bacteria. While mobilizable plasmids only encode a minimal MOB machinery that allows them to be transported by other plasmids, conjugative plasmids encode a complete set of transfer genes (MOB1T4SS). The only essential ingredient of the MOB machinery is the relaxase, the protein that initiates and terminates conjugative DNA processing. In this review we compared the sequences and properties of the relaxase proteins contained in gene sequence databases. Proteins were arranged in families and phylogenetic trees constructed from the family alignments. This allowed the classification of conjugative transfer systems in six MOB families:MOB(F), MOB(H), MOB(Q), MOB(C), MOB(P) and MOB(V). The main characteristics of each family were reviewed. The phylogenetic relationships of the coupling proteins were also analysed and resulted in phylogenies congruent to those of the cognate relaxases. We propose that the sequences of plasmid relaxases can be used for plasmid classification. We hope our effort will provide researchers with a useful tool for further mining and analysing the plasmid universe both experimentally and in silico.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                16 March 2018
                2018
                : 9
                Affiliations
                1Departamento de Sanidad Animal and Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Facultad de Veterinaria, Universidad Complutense de Madrid , Madrid, Spain
                2Departamento de Microbiología, Facultad de Veterinaria, Universidad Alfonso X el Sabio , Madrid, Spain
                3Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences , Lucknow, India
                4Servicio de Microbiología Hospital Universitario Ramón y Cajal, Instituto de Investigación Sanitaria (IRYCIS) , Madrid, Spain
                Author notes

                Edited by: Manuel Espinosa, Centro de Investigaciones Biológicas (CSIC), Spain

                Reviewed by: Fabián Lorenzo, Universidad de La Laguna, Spain; Raul Fernandez-Lopez, University of Cantabria, Spain; Ellen Lorraine Zechner, University of Graz, Austria; Antonio Juárez, Universitat de Barcelona, Spain

                *Correspondence: Bruno Gonzalez-Zorn bgzorn@ 123456ucm.es

                This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology

                †Present Address: Alfonso Santos-Lopez, Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, United States

                Article
                10.3389/fmicb.2018.00469
                5864857
                Copyright © 2018 Ares-Arroyo, Bernabe-Balas, Santos-Lopez, Baquero, Prasad, Cid, Martin-Espada, San Millan and Gonzalez-Zorn.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 66, Pages: 15, Words: 9677
                Funding
                Funded by: Universidad Complutense de Madrid 10.13039/501100002911
                Categories
                Microbiology
                Original Research

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