The Evolution of Plasmids Carrying Multiple Resistance Genes: The Role of Segregation, Transposition, and Homologous Recombination

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Abstract
We used a mathematical model and experiments with laboratory populations of Escherichia coli to examine conditions for the evolution of plasmids conferring resistance to multiple antibiotics. In our model and its experimental analogue, two different resistance genes are initially carried on separate plasmids, and recombination between the plasmids can generate a third element that carries both resistances. The environment is such that only cells carrying the two resistance genes can replicate. Our simulations predict that under these conditions, populations carrying a single plasmid with both resistance genes increase in frequency at a rate approximately equal to that at which the two-plasmid state decays by vegetative segregation. When the segregation rate is high, the pace of evolution is nearly independent of the rates of recombination and plasmid transfer or of whether the antibiotic is bacteriostatic or bacteriocidal. In the main, our experimental results are consistent with these predictions. When the complementary genes were borne on incompatible plasmids and segregation occurred at a high rate (0.4 h-1), stable inheritance of the resistance genes evolved in less than 150 generation. In the majority of cases, the stable state was achieved by the evolution of a single trasmissible plasmid carrying both resistance genes. Variation in rates of conjugation, recombination, and fitness had little or no effect on the rate at which the stable state evolved. When the resistance genes were borne on compatible plasmids and segregation rates were low (0.01 h-1 or lower), there was no evidence for an evolutionary change of the plasmids, although genetic variants were probably generated. Not all of the experimental results were anticipated from our theoretical analysis. In some cases, stable inheritance of the complementary resistance genes was obtained by the movement of one gene to the chromosome and the persistence of the other gene on an unaltered plasmid. In a few cases, we could not determine the molecular basis of evolution.