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

Abstract

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 bla or bla 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 lacI was inserted into the chromosome, whereas the plasmid was marked with a GFPmut3 cassette with LacI 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.

Fate of CMY-2-Encoding Plasmids Introduced into the Human Fecal Microbiota by Exogenous Escherichia coli

“…The plasmid transfer efficiency in human fecal microbiota was likely similar for both strains. Indeed, in the in vitro experiments with lab strain, both strains had a transfer efficiency of 10 Ϫ5 transconjugants per donor cell (12). The plasmid transfer thus was not dependent on the concentration of the exogenous strain but on transconjugant survival and secondary transfer.…”

“…Strains and media. The two genetically modified human and poultry meat E. coli strains used in this study (C20-GM and 1061-1-GM, respectively) were constructed and validated previously (12). The strains were typed as ST155 (C20-GM) and ST10 (1061-1-GM) by MLST and harbor bla CMY-2 on IncI1 plasmids of sequence type 12 (pST12) sharing 99% nucleotide identity over 97% of the length of the plasmid (European Nucleotide Archive accession number PRJEB9625) (5).…”

“…The strains were typed as ST155 (C20-GM) and ST10 (1061-1-GM) by MLST and harbor bla CMY-2 on IncI1 plasmids of sequence type 12 (pST12) sharing 99% nucleotide identity over 97% of the length of the plasmid (European Nucleotide Archive accession number PRJEB9625) (5). The strains were genetically modified by inserting an mCherry fluorescent marker (red) into the pseudogene ybeM on the chromosome and a GFP fluorescent marker (green) into a noncoding region on the IncI1 plasmid (12).…”

“…All colonies were observed with confocal microscopy to detect green and red fluorescence. The green colonies were subjected to PCR targeting the region on the plasmid where the GFPmut3 cassette was inserted using the primers Fwd pC20/1061-1 confirm and Rev pC20/1061-1 confirm (12). All PCR-positive colonies were identified to species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (bioMérieux, France).…”

IncHI1A plasmids potentially facilitate a horizontal flow of antibiotic resistance genes to pathogens in microbial communities of urban residential sewage

“…In this study, we investigated the host range, including the initial host range stages, and transfer efficiency of the HI1A plasmid R27 in the microbial community of urban residential sewage collected at three WWTPs located in urban areas of southern Sweden. We utilized a dual fluorescent reporter gene platform, which previously has been used to examine transfer of various plasmid groups, including IncI1 (Anjum et al, 2018(Anjum et al, , 2019 and P1 (Jacquiod et al, 2017;Klümper et al, 2015;Li et al, 2018;Musovic et al, 2014;Pinilla-Redondo et al, n.d.). This platform is based on a plasmid-encoded green fluorescent protein (gfp ) gene under the control of a lacI q repressible promoter.…”