Estimating the rate of plasmid transfer with an adapted Luria–Delbrück fluctuation analysis and a case study on the evolution of plasmid transfer rate

To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need a reliable estimate of their rate of transfer between bacterial cells. Accurate estimates of plasmid transfer have remained elusive given biological and experimental complexity. Current methods to measure transfer rate can be confounded by many factors, such as differences in growth rates between plasmid-containing and plasmid-free cells. However, one of the most problematic factors involves situations where the transfer occurs between different strains or species and the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias transfer estimates, thereby limiting our capabilities for measuring transfer within diverse microbial communities. We develop a novel low-density method (“LDM”) for measuring transfer rate, inspired by the classic fluctuation analysis of Luria and Delbrück. Our new approach embraces the stochasticity of conjugation, which departs in important ways from the current deterministic population dynamic methods. In addition, the LDM overcomes obstacles of traditional methods by allowing different growth and transfer rates for each population within the assay. Using stochastic simulations, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to other commonly used methods. Lastly, we implement the LDM to estimate transfer on an ancestral and evolved plasmid-host pair, in which plasmid-host co-evolution increased the persistence of an IncP-1β conjugative plasmid in its Escherichia coli host. Our method revealed the increased persistence can be at least partially explained by an increase in transfer rate after plasmid-host coevolution. ### Competing Interest Statement The authors have declared no competing interest.