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Evolution of plasmid-mediated resistance in Pseudomonas aeruginosa

Final Report Summary - PLASREVO (Evolution of plasmid-mediated resistance in Pseudomonas aeruginosa)

Overview of the results
Plasmids play a key role in bacterial evolution by allowing bacteria to adapt to novel environmental pressures; for example, the rapid evolution of antibiotic resistance in many pathogenic bacteria has been driven by plasmids. However, it remains challenging to understand how plasmids can persist in bacterial populations over the long term, and this has been termed the “plasmid paradox” [1]. This paradox arises because positive selection for plasmid-encoded traits is likely to be transient, but plasmids impose a fitness burden on their hosts, and can be lost at cell division. Classical models predict that horizontal gene transfer is required to maintain plasmids [2,3], but recent analyses of bacterial genomes have shown that almost half of plasmids are non-transmissible [4].
First year [5]
In our first year, we use a combination of experimental and bioinformatic methods to study how epistatic interactions between plasmids contribute to plasmid persistence. First, we demonstrate that positive epistasis between co-infecting plasmids in Pseudomonas aeruginosa minimizes the cost associated with plasmid carriage and increases plasmid stability at a population level using experimental evolution. Second, we demonstrate that the patterns of distribution of plasmids across all sequenced bacterial genomes are consistent with the hypothesis that positive epistasis maintains plasmids in environmental and clinical populations. We anticipate that this study will be of broad interest to researchers who are interested in microbial evolutionary biology and plasmid biology because it provides a novel, and potentially very general, solution to the plasmid paradox and it helps to explain why antibiotic resistance is rarely lost after antibiotic use is discontinued in pathogen populations [6].
Second year [7]
How can we explain the maintenance of non-transmissible plasmids? In this work, we address this question using a combination of experimental evolution, mathematical modelling and whole genome sequencing. We show that a costly and highly unstable plasmid, pNUK73, can be rapidly stabilized in populations of the pathogenic bacterium P. aeruginosa by a combination of compensatory adaptation and positive selection. Compensatory adaptation by P. aeruginosa recovers the cost associated with plasmid carriage, but compensation alone is not sufficient to maintain the plasmid as a result of segregational loss of plasmids. Positive selection mediated by exposure to antibiotics is necessary to increase plasmid frequency and offset the effects of segregational loss. Crucially, we find that feedback occurs between these processes. Positive selection increases the efficacy of selection for compensatory adaptation by increasing the population size of plasmid-bearing lineages. Compensatory adaptation, in turn, increases the effect of positive selection on plasmid stability by slowing the rate at which the plasmid is lost between episodes of positive selection. Therefore, we argue that it is the interaction between compensatory adaptation and positive selection that helps to stabilize non-conjugative plasmids in bacterial populations.
Conclusions and the socio-economic impacts of the project
Antibiotic resistance is currently one of the major threats to human health in developed countries [8,9] and plasmids play a major role in the spread of antibiotic resistance determinants among bacterial pathogens. The results obtained in this project help to understand the ecological and evolutionary bases of the maintenance of plasmid-mediated antibiotic resistance in bacterial populations, which is essential to develop new strategies to combat this threat.

1. Harrison E, Brockhurst MA (2012) Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends Microbiol 20: 262-267.
2. Stewart FM, Levin BR (1977) The Population Biology of Bacterial Plasmids: A PRIORI Conditions for the Existence of Conjugationally Transmitted Factors. Genetics 87: 209-228.
3. Bergstrom CT, Lipsitch M, Levin BR (2000) Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155: 1505-1519.
4. Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EP, de la Cruz F (2010) Mobility of plasmids. Microbiol Mol Biol Rev 74: 434-452.
5. San Millan A, Heilbron K, MacLean RC (2014) Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations. ISME J 8: 601-612.
6. Andersson DI, Hughes D (2010) Antibiotic resistance and its cost: is it possible to reverse resistance? Nat Rev Microbiol 8: 260-271.
7. San Millan A, Peña-Miller R, Toll-Riera M, Halbert ZV, McLean AR, et al. (2014) Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids. Nature Communications.
8. eCDC (2013) European Center for Disease Prevention and Control. Annual Epidemiological Report 2012.
9. CDC (2013) Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States.

Alvaro San Millan
Marie Curie Research Fellow
Department of Zoology
University of Oxford
South Parks Road