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

Publication date: Available online 18 July 2018Source: Journal of Microbiological MethodsAuthor(s): Mehreen Anjum, Jonas Stenløkke Madsen, Carmen Espinosa-Gongora, Bimal Jana, Maria Wiese, Dennis Sandris Nielsen, Søren Johannes Sørensen, Arshnee Moodley, Valeria Bortolaia, Luca GuardabassiAbstractIncI1 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 ...
Source: Journal of Microbiological Methods - Category: Microbiology Source Type: research