A culture-independent method for studying transfer of IncI1 plasmids from wild-type Escherichia coli in complex microbial communities

Research output: Contribution to journalJournal article – Annual report year: 2018Researchpeer-review

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  • Author: Anjum, Mehreen

    University of Copenhagen, Denmark

  • Author: Madsen, Jonas Stenløkke

    University of Copenhagen, Denmark

  • Author: Espinosa-Gongora, Carmen

    University of Copenhagen, Denmark

  • Author: Jana, Bimal

    University of Copenhagen, Denmark

  • Author: Wiese, Maria

    University of Copenhagen, Denmark

  • Author: Nielsen, Dennis Sandris

    University of Copenhagen, Denmark

  • Author: Sørensen, Søren Johannes

    University of Copenhagen, Denmark

  • Author: Moodley, Arshnee

    University of Copenhagen, Denmark

  • Author: Bortolaia, Valeria

    Research group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kemitorvet, 2800, Kgs. Lyngby, Denmark

  • Author: Guardabassi, Luca

    University of Copenhagen, Denmark

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IncI1 plasmids play a central role in the transfer of antimicrobial resistance genes among Enterobacteriaceae in animals and humans. Knowledge on the dynamics of IncI1 plasmid transfer is limited, mainly due to lack of culture-independent methods that can quantify donor strain survival and plasmid transfer in complex microbial communities. The aim of this study was to develop a culture-independent method to study the dynamics of IncI1 plasmids transfer by fluorescence-activated cell sorting. We genetically modified three wild-type Escherichia coli of animal (n = 2) and human (n = 1) origin carrying blaCMY-2 or blaCTX-M-1 on two epidemic IncI1 plasmids (pST12 and pST7). Non-coding regions on the chromosome and on the IncI1 plasmid of each strain were tagged with mCherry (red) and GFPmut3 (green) fluorescent proteins, respectively, using lambda recombineering. A gene cassette expressing mCherry and lacIq was inserted into the chromosome, whereas the plasmid was marked with a GFPmut3 cassette with LacIq repressible promoter. Therefore, gfpmut3 was repressed in donor strains but expressed in recipient strains acquiring the plasmids. We demonstrated that genetic engineering of the strains did not affect the growth rate and plasmid transfer-ability in filter and broth matings. A proof-of-concept experiment using the CoMiniGut, an in vitro model of the colon, proved the validity of our method for studying the survival of wild-type E. coli and horizontal transfer of IncI1 plasmids under different pH and oxygen conditions. The dual-labeling method by fluorescent proteins is useful to determine persistence of exogenous E. coli and transfer dynamics of IncI1 plasmids in microbial communities.
Original languageEnglish
JournalJournal of Microbiological Methods
Volume152
Pages (from-to)18-26
ISSN0167-7012
DOIs
Publication statusPublished - 2018
CitationsWeb of Science® Times Cited: No match on DOI

    Research areas

  • Horizontal gene transfer, Antimicrobial resistance, Enterobacteriaceae, ESBL

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