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

Anjum, M; Madsen, J S; Espinosa-Gongora, C; Jana, B; Wiese, M; Nielsen, D S; Sørensen, S J; Moodley, A; Bortolaia, V; Guardabassi, L


M Anjum

J S Madsen

C Espinosa-Gongora

B Jana

M Wiese

D S Nielsen

S J Sørensen

A Moodley

V Bortolaia

L Guardabassi


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.


Anjum, M., Madsen, J. S., Espinosa-Gongora, C., Jana, B., Wiese, M., Nielsen, D. S., …Guardabassi, L. (2018). A culture-independent method for studying transfer of IncI1 plasmids from wild-type Escherichia coli in complex microbial communities. Journal of Microbiological Methods, 152, 18-26.

Journal Article Type Article
Acceptance Date Jul 16, 2018
Publication Date Sep 1, 2018
Deposit Date Jun 6, 2019
Publicly Available Date Jul 19, 2019
Print ISSN 0167-7012
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 152
Pages 18-26
Public URL


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