Bacterial adaption to novel selection pressures

The rates and trajectories of bacterial evolution are determined both by microbial factors and environmental parameters. In this thesis I have investigated how bacterial mutation rates and selection pressure affect the rate and extent of adaptation to novel environments. Bacterial strains with increased mutation rates have been found at high frequencies among pathogenic bacteria. We collected natural isolates of E. coli in three different European countries and determined the mutation frequency to rifampicin resistance in these strains. In this collection we found an enrichment of weak mutator strains and furthermore, weak mutators were more common among clinical isolates than among normal flora bacteria. Pathogenic bacteria experience a rapidly changing environment in its host and this has been suggested as one explanation for the high frequencies of mutator strains found among clinical isolates. We found that mutation supply rate was limiting for bacterial adaptation in a pathogenesis model where S. typhimurium was evolved in mice. Here, the rate of adaptation could be increased by either increasing population size or mutation rate. An increased mutation rate however, often comes at a cost. We could detect a decreased fitness of evolved mutator populations in second unselected environments due to accumulation of deleterious mutations. The course of bacterial evolution is determined by how selection and genetic drift sort among genetic variation. Antibiotic resistance development is to a large degree influenced by strong selective pressures. Resistance mutations usually confer a fitness cost to the resistant bacteria in terms of reduced growth rates and /or virulence. We showed that resistance to the two antibiotics fosfomycin and actinonin generally confers heavy fitness costs on the resistant bacteria under several different growth conditions. Using mathematical modeling we demonstrated that the fitness costs associated with fosfomycin resistance significantly reduced the probability of resistance development during antibiotic therapy. The biological cost of fosfomycin resistance is therefore suggested to be a significant contributor to the low level of clinical resistance observed for this antibiotic. For resistance to develop clinically the possibility to genetically compensate for the fitness costs of antibiotic resistance is of importance. We showed that the severe fitness cost of actinonin resistance can be compensated for by acquisition of both intragenic and extragenic compensatory mutations. Among the extragenic compensatory mutations we identified tRNA gene amplifications resulting in over-production of a limiting component for bacterial growth in the resistant strains. Evolution towards reduced bacterial genome size is associated with an intracellular life-style that is characterized by small bacterial population sizes and relaxed selection pressures. We set up an experimental system to study the process of genome shrinkage in real time where we mimicked the characteristics of an intracellular life-style. We could observe a rapid initial rate of DNA loss where large deletions removed substantial amounts of DNA in single events. The data agrees well with the observation that genome reduction in bacteria with small genomes initially was a rapid process mediated via large deletions. RecA functions have been suggested to be important for the decrease in genome size of small genomes. We could however not detect any dependence on RecA mediated functions for the deletion formation process in our experimental set-up. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text by their roman numerals. I. Maria-Rosario Baquero, Annika I. Nilsson, Maria del Carmen Turrientes, Dorthe Sandvang, Juan Carols Galan, Jose Luis Martinez, Niels Frimodt-Moller, Fernando Baquero and Dan I. Andersson Polymorphic Mutation Frequencies in Escherichia coli: Emergence of Weak Mutators in Clinical Isolates. Journal of Bacteriology. 186(16): 5538-5542. 2004 II. Annika I. Nilsson, Elisabeth Kugelberg, Otto G. Berg and Dan I. Andersson Experimental Adaptation of Salmonella typhimurium to Mice. Genetics. 168: 1119-1130. 2004 III. Annika I. Nilsson, Otto G. Berg, Olle Aspevall, Gunnar Kahlmeter and Dan I. Andersson Biological Costs and Mechanisms of Fosfomycin Resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy. 47(9): 2850-2858. 2003. IV. Annika I. Nilsson, Anna Kanth and Dan I. Andersson Reducing the Cost of Antibiotic Resistance by Selective Gene Amplifications. Manuscript. V. Annika I. Nilsson, Sanna Koskiniemi, Sofia Eriksson, Elisabeth Kugelberg, Jay C. D. Hinton and Dan I. Andersson Experimental Evolution to Reduce Bacterial Genome Size. Manuscript.