There is urgent need to develop novel treatment strategies to reduce antimicrobial resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases susceptibility to other drugs, might enable selection against resistance during treatment. However, the success of this approach would depend on the conservation of CS networks across genetically diverse bacterial strains. Here, we examine CS conservation across diverse Escherichia coli strains isolated from urinary tract infections. We determine collateral susceptibilities of mutants resistant to relevant antimicrobials against 16 antibiotics. Multivariate statistical analyses show that resistance mechanisms, in particular efflux-related mutations, as well as the relative fitness of resistant strains, are principal contributors to collateral responses. Moreover, collateral responses shift the mutant selection window, suggesting that CS-informed therapies may affect evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-informed therapy and further suggest that rapid detection of resistance mechanisms is important to accurately predict collateral responses.

The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Natural transformation in bacteria facilitates the uptake and genomic integration of exogenous DNA. This allows horizontal exchange of adaptive traits not easily achieved by point mutations, and has a major role in the acquisition of adaptive traits exemplified by antibiotic resistance determinants and vaccination escape. Mechanisms of DNA uptake and genomic integration are well described for several naturally transformable bacterial species; however, the selective forces responsible for its evolution and maintenance are still controversial. In this study we evolved transformation-proficient and -deficient Acinetobacter baylyi for 175 days in serial transfer cultures where stress was included. We found that natural transformation-proficient populations adapted better to active growth and early stationary phase. This advantage was offset by the reduced performance in the late stationary/death phase. We demonstrate fitness trade-offs between adaptation to active growth and survival in stationary/death phase caused by antagonistic pleiotropy. The presented data suggest that the widely held assumption that recombination speeds up adaptation by rapid accumulation of multiple adaptive mutations in the same genetic background is not sufficient to fully account for the maintenance of natural transformation in bacteria.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made towards resolving this “plasmid paradox” but mainly in a non-clinical context. Here we propose an additional explanation for the maintenance of multidrug resistance (MDR) plasmids in clinical Escherichia coli strains. After co-evolving two MDR plasmids encoding last resort carbapenem resistance with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (Carbon Catabolite Repression) and ArcAB (Aerobic Respiration Control) pleiotropically improved the maintenance of both plasmids. Mechanistically, a net downregulation of plasmid gene expression reduced the fitness cost. Our results suggest that global chromosomal transcriptional re-wiring during bacterial niche-adaptation may facilitate plasmid maintenance.

Dissemination of bacterial clones carrying plasmid-mediated resistance genes is a major factor contributing to the increasing prevalence of antibiotic resistance. Understanding the evolution of successful clones and the association to mobile resistance elements are therefore crucial. In this study, we determined the sequence of a 145 kb IncC multi-drug resistance plasmid (pK71-77-1-NDM), harbouring resistance genes to last-resort antibiotics including carbapenems. We show that the plasmid is able to transfer into a range of genetically diverse clinical Escherichia coli strains and that the fitness cost imposed on the host is often low. Moreover, the plasmid is stably maintained under non-selective conditions across different genetic backgrounds. However, we also observed a lower conjugation frequency and higher fitness cost in the E. coli sequence type (ST) 73 background, which could partially explain why this clone is associated with a lower level of antibiotic resistance than other E. coli clones. This is supported by a bioinformatical analysis showing that the ST73 background harbours plasmids less frequently than the other studied E. coli STs. Studying the evolution of antibiotic resistance in a clinical context and in diverse genetic backgrounds improves our understanding of the variability in plasmid-host associations.

Transposons are genetic elements that change their intracellular genomic position by transposition and are spread horizontally between bacteria when located on plasmids. It was recently discovered that transposition from fully heterologous DNA also occurs in the course of natural transformation. Here, we characterize the molecular details and constraints of this process using the replicative transposon Tn1 and the naturally competent bacterium Acinetobacter baylyi . We find that chromosomal insertion of Tn1 by transposition occurs at low but detectable frequencies and preferably around the A. baylyi terminus of replication. We show that Tn1 transposition is facilitated by transient expression of the transposase and resolvase encoded by the donor DNA. RecA protein is essential for the formation of a circular, double-stranded cytoplasmic intermediate from incoming donor DNA, and RecO is beneficial but not essential in this process. Absence of the recipient RecBCD nuclease stabilizes the double-stranded intermediate. Based on these results, we suggest a mechanistic model for transposition during natural transformation.

The persistence of plasmids in bacterial populations represents a puzzling evolutionary problem with serious clinical implications due to their role in the ongoing antibiotic resistance crisis. Recently, major advancements have been made towards resolving this 'plasmid paradox' but mainly in a non-clinical context. Here we propose an additional explanation for the maintenance of multidrug resistance (MDR) plasmids in clinical Escherichia coli strains. After co-evolving two MDR plasmids encoding last resort carbapenem resistance with an extraintestinal pathogenic E. coli strain, we observed that chromosomal media adaptive mutations in the global regulatory systems CCR (Carbon Catabolite Repression) and ArcAB (Aerobic Respiration Control) pleiotropically mitigated the costs of both plasmids. Mechanistically, cost reductions were due to a net downregulation of plasmid gene expression. Our results suggest that global chromosomal transcriptional re-wiring during bacterial niche-adaptation may facilitate plasmid maintenance.

24There is urgent need to develop novel treatment strategies to reduce antimicrobial 25 resistance. Collateral sensitivity (CS), where resistance to one antimicrobial increases 26 susceptibility to other drugs, is a uniquely promising strategy that enables selection 27 against resistance during treatment. However, using CS-informed therapy depends on 28 conserved CS networks across genetically diverse bacterial strains. We examined CS 29 conservation in 10 clinical strains of E. coli resistant to four clinically relevant 30 antibiotics. Collateral susceptibilities of these 40 resistant mutants were then determined 31 against a panel of 16 antibiotics. Multivariate statistical analyses demonstrate that 32 resistance mechanisms, in particular efflux-related mutations, as well as relative fitness 33were principal contributors to collateral changes. Moreover, collateral responses shifted 34 the mutant selection window suggesting that CS-informed therapies could affect 35 evolutionary trajectories of antimicrobial resistance. Our data allow optimism for CS-36 informed therapy and further suggest that early detection of resistance mechanisms is 37 important to accurately predict collateral antimicrobial responses. 38 39 Keywords 40 Collateral sensitivity, cross-resistance, antimicrobial resistance, fitness cost of resistance, 41 selection inversion 42 43 44The evolution and increasing frequency of antimicrobial resistance (AMR) in bacterial 45 populations is driven by the consumption and misuse of antimicrobials in human 46 medicine, agriculture, and the environment (1-3). Historically, the threat of AMR was 47 overcome by using novel antimicrobials with unique drug targets. However, the 48 discovery rate of new antimicrobial agents has dwindled (4-6) and severely lags behind 49 the rate of AMR evolution. While concerted scientific, corporate and political focus is 50 needed to recover antimicrobial pipelines (7-9), there is an urgent need for alternative 51 strategies that prolong the efficacy of existing antimicrobials and prevent or slow the 52 emergence, spread and persistence of AMR. Current global efforts to improve 53 antimicrobial stewardship largely focus on awareness and reducing overall consumption 54 (8, 10-12). While these efforts will affect the evolution and spread of AMR, mounting 55 evidence suggests that these changes alone will not lead to large-scale reductions in the 56 occurrence of AMR (13-17). 5758 Several recent studies have examined novel treatment strategies using multiple 59 antimicrobials that could reduce the rate of resistance emergence and even reverse pre-60 existing AMR. These approaches, collectively termed "selection inversion" strategies, 61 refer to cases where resistance becomes costly in the presence of other antimicrobial 62 agents (18). Among the most promising of these strategies are those based on a 63 phenomenon first reported in 1952 termed "collateral sensitivity" (CS), where resistance 64 to one antimicrobial simultaneously increases the susceptibility to another (19). CS...