The Role of Clonal Interference in the Evolutionary Dynamics of Plasmid-host Adaptation

Unlabelled: Promiscuous plasmids replicate in a wide range of bacteria and therefore play a key role in the dissemination of various host-beneficial traits, including antibiotic resistance. Despite the medical relevance, little is known about the evolutionary dynamics through which drug resistance plasmids adapt to new hosts and thereby persist in the absence of antibiotics. We previously showed that the incompatibility group P-1 (IncP-1) minireplicon pMS0506 drastically improved its stability in novel host Shewanella oneidensis MR-1 after 1,000 generations under antibiotic selection for the plasmid. The only mutations found were those affecting the N terminus of the plasmid replication initiation protein TrfA1. Our aim in this study was to gain insight into the dynamics of plasmid evolution. Changes in stability and genotype frequencies of pMS0506 were monitored in evolving populations of MR-1 (pMS0506). Genotypes were determined by sequencing trfA1 amplicons from individual clones and by 454 pyrosequencing of whole plasmids from entire populations. Stability of pMS0506 drastically improved by generation 200. Many evolved plasmid genotypes with point mutations as well as in-frame and frameshift deletions and duplications in trfA1 were observed in all lineages with both sequencing methods. Strikingly, multiple genotypes were simultaneously present at high frequencies (>10%) in each population. Their relative abundances changed over time, but after 1,000 generations only one or two genotypes dominated the populations. This suggests that hosts with different plasmid genotypes were competing with each other, thus affecting the evolutionary trajectory. Plasmids can thus rapidly improve their stability, and clonal interference plays a significant role in plasmid-host adaptation dynamics.

Importance: Promiscuous plasmids play an important role in the spread of antibiotic resistance and many other traits between closely and distantly related bacteria. However, little is known about the dynamics by which these broad-host-range antibiotic resistance plasmids adapt to novel bacteria and thereby become more persistent, even in the absence of antibiotics. In this study, we show that after no more than 200 generations of growth in the presence of antibiotics, a plasmid that was initially poorly maintained in a novel bacterial host evolved to become drastically more persistent in the absence of antibiotics. In each of the evolving populations, an unexpectedly large number of bacterial variants arose with distinct mutations in the plasmid's replication initiation protein. Our results suggest that clonal interference, characterized by competition between variant clones in a population, plays a major role in the evolution of the persistence of drug resistance.