Final Report Summary - EMBCC (Evolution of mutator bacteria in a community context) Mutators (bacteria with increased mutation rates) have been found among natural populations of bacteria at a frequency much higher than expected and are a major cause of severe, antibiotic-resistant nosocomial infections. For this reason, the role of mutator cells in adaptive evolution has been a subject of considerable biological and medical interest. To date, the benefits and costs of increased mutation rates have been extensively investigated in the laboratory and a number of excellent studies clearly indicate that the occurrence of mutators in bacterial populations results in a higher probability of generating advantageous mutations allowing faster adaptation to new and/or changing environments. However, experimental work to date has largely ignored a crucial feature of the environment: the natural microbial community. In natural environments, individual organisms do not live in isolation but rather form a complex system of inter-species interactions that shape the structure of the community and play a crucial role in ecological and evolutionary processes. Despite their relevance, it is currently unclear whether the presence of natural competitors selects for or against elevated mutation rates. The main aim of the Marie Curie IIF-project EMBCC is to investigate for the first time the role of natural microbial community in the selection of mutation rates in the human-pathogenic bacterium, Pseudomonas aeruginosa, under two different environments: soil and cystic fibrosis (CF) context. EMBCC provides a unique opportunity to bridge the gap between microbial ecology and clinical microbiology with the help of evolutionary theory to increase our understanding of the fate of microbial mutators within communities in natural environments.To achieve its objectives EMBCC has produced research results of high quality that have been presented in top international conferences and have been published or will be submitted for publication in prestigious international journals:(i) Competition experiments in soil microcosms were carried out with the goal of determining the fitness of P. aeruginosa mutator strain in soil in the presence and absence of the natural microbial community. The results obtained showed that mutator bacteria exhibited a fitness cost in soil under both conditions, indicating that regardless of the complexity of the microbial community, soil as it is a hostile environment for selecting high mutation rates, and thus not the proper context to study the role of competitors in the evolution of mutation rates. For this reason, we continue to study this question in the CF context. (ii) Using an in vitro model of artificial CF communities, we have found that P. aeruginosa mutator increase in frequency and outcompete the non-mutator strain only in the absence of the microbial community. In the “community” treatments the proportion of mutator and non-mutators remain equal. Mutators strains showed higher fitness in the absence than in the presence of competitors. This suggests that the selection and fixation of P. aeruginosa mutators is constrained by the presence of the microbial community in artificial CF communities. By performing competition experiments of evolved non-mutator strains against the ancestral, we found that the presence of the microbial community reduces the availability of beneficial mutations and thus suppress the hitchhiking of mutators in these populations. (iii) In addition to in vitro experiments, a survey was conducted to investigate whether there is a link between the bacterial community diversity and P. aeruginosa mutation frequency in natural CF communities. This was analyzed at 28 independent sputum samples collected from chronic infected patients at the Rigshospitalet, Denmark. P. aeruginosa average mutation frequency, the prevalence of P. aeruginosa (proportion of P. aeruginosa reads) and the microbial community diversity (Shannon index) was determined for each sputum sample. Correlation analysis indicated that P. aeruginosa populations displaying high mutation frequencies were those where P. aeruginosa is predominant in the CF community. That is, the more prevalent P. aeruginosa is in the CF natural community the higher the probability of mutators to increase in frequency. Taken together, these results demonstrate for the first time that the fate of P. aeruginosa mutators is influenced by the biological interactions ongoing in CF microbial communities. (iv) To gain insight into the genetic bases of the response to the selection imposed by interspecific competition in the CF context, deep genome sequencing of 12 P. aeruginosa populations that evolved in vitro with or without the microbial community and of the 28 natural P. aeruginosa populations isolated from the CF sputum samples was performed. DNA isolation, library preparation and Illumina sequencing has been just completed. In the begging of 2016 three months, the bioinformatics analysis and SNPs identification will be performed. (v) The exhaustive sampling of CF sputum samples and the metagenomic analysis of P. aeruginosa CF populations have resulted in collaborations with another metagenomic project directed to investigate the structure and diversity of Pseudomonas aeruginosa populations in CF patients carried out by Lea Madsen, a PhD student at Prof Molin´s laboratory. A manuscript of this work is in preparation and will be submitted by the end of September 2015. (vi) In the process of carrying out the soil mutator experiments, the social evolution of siderophore production in this natural context was investigated for the first time. This is because there is a clear link between mutators and social evolution (Harrison & Buckling, 2005, Current Biology). These results demonstrate for the first time that siderophore-mediated cooperation is import in soil, and has resulted in a first author publication in the prestigious journal Biology Letters.• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)At the moment, there is a good understanding of the evolutionary forces (hitchhike with beneficial mutations, population size, deleterious mutation accumulation, clonal interference, adaptive landscape, antagonistic coevolution, etc.) that may select for or against mutators in clonal bacterial populations. However, these efforts have focused on pure test-tube laboratory environments. What has been largely ignored is the important role that the natural microbial community could play in mutator evolutionary dynamics. The expected final result of the project is to bridge the gap between microbial ecology and clinical microbiology with the help of evolutionary theory to increase our understanding of the fate of microbial mutators within communities in natural environments. Particularly, the results obtained here could have significant medical impacts since they could provide an additional element to combat CF chronic infections: the natural microbial community. As mentioned above, CF natural microbial community would select against clones with an elevated mutation rate, and hence antibiotic resistance; thus, maintaining a proper natural microbial community may have clinical benefits and could be a good alternative for the prevention of mutator fixation and the emergence of antibiotic-resistance bacteria. In addition, the research results of EMBCC facilitated the launch of one new project aimed at investigating the role of the natural microbial community in the evolution of P. aeruginosa adaptive traits, other than mutation rates, (antibiotic resistance, production of exoproducts, virulence, etc) needed for the establishment and the maintenance of chronic infections.Socioeconomic impacts*Knowledge transfer to University level through co-supervising two undergraduate students. *Knowledge transfer to European level through new research collaboration with University of Liverpool and Danish Technical University.*Knowledge transfer to global level through new research collaboration with University of Cordoba, Argentina. *The research results of EMBCC facilitated the launch of two new projects aimed to investigate the evolution of P. aeruginosa as a whole in a community context.
