Abstract
Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors—active partitioning, toxin-antitoxin and conjugational transfer—are all involved in the prevalence of pESBL in the E. coli population.
Highlights
Commensal, harmless bacteria can turn into pathogens by acquiring virulence factors
Transposon-insertion sequencing (Tnseq) experiments comparing two transposon insertion libraries were designed to identify pESBL maintenance loci, where inactivation by transposon insertion would result in plasmid loss
Virulence plasmids can be fully or partially responsible for the pathogenicity of the host pathogen, while Multidrug resistance (MDR) plasmids can confer on host cells a great fitness advantage when they are challenged by corresponding antibiotics
Summary
Harmless bacteria can turn into pathogens by acquiring virulence factors. Bacteria can gain resistance to certain antibacterial agents, together with or independently of virulence factors. Virulence/resistance genes can move from one cell to the other via mobile genetic elements (MGEs) such as transposons, prophages, integrative and conjugative elements (ICEs) and plasmids. Plasmids that are in the cytoplasm replicate and segregate independently to the host chromosome, and so are in danger of being lost at every cell division cycle. In the outbreak of enterohemorrhagic/enteroaggregative Escherichia coli in Germany in 2011, the causative strain encoded Shiga-toxin, the key virulence factor for diarrhea, on a prophage integrated into the chromosome. It commonly harbored two plasmids, pAA and pESBL. We were able to capture mis-segregation as well as conjugational transfer events under the microscope
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