title: High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4.
abstract: Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid's host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA Institute for Integrative Biology of the Cell (I2BC), CNRS, CEA, Université Paris-Sud, Gif-sur-Yvette 91198, France yoshiharu.yamaichi@cgm.cnrs-gif.fr.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Yamaichi
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Chao
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo160-8582, Japan.
authors.forename: Jumpei
authors.initials: J
authors.lastname: Sasabe
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Lars
authors.initials: L
authors.lastname: Clark
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Brigid M
authors.initials: BM
authors.lastname: Davis
authors.affiliation: Earth-Life Science Institute, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Information, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mori
authors.affiliation: Earth-Life Science Institute, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan Department of Biological Information, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Kurokawa
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA Howard Hughes Medical Institute, Boston, MA 02115, USA.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Waldor
mesh_terms: D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D022761:Gene Transfer, Horizontal; D005798:Genes, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements