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Scientific abstract Antibiotic resistance, especially in Gram-negative pathogens, represents a substantial clinical and financial burden to our society. The presented work investigated mechanisms and evolutionary dynamics that promote the emergence and maintenance of resistance in bacteria. In the first study, conserved collateral susceptibility changes were identified across resistant uropathogenic Escherichia coli, which included also changes towards increased sensitivity of these isolates to certain antibiotics. This so-called collateral sensitivity potentiated the effect of antibiotics and prevented the selection of resistant isolates compared to wildtype strains. The mechanism and fitness cost of resistance were important predictors of collateral responses in resistant bacteria, and their rapid clinical identification could inform future evolution-based infection treatment. The second study demonstrated the potential of a transposable element (Tn1) to spread antibiotic resistance during natural transformation of Acinetobacter baylyi. In the course of this horizontal gene transfer mechanism, Tn1-containing DNA entered the bacterial cell, and specific host, as well as transposon proteins, facilitated Tn1-insertion into the recipient chromosome. A mechanistic model of transposition-mediated natural transformation from a circular, cytoplasmic intermediate is presented. In the third study, uropathogenic E. coli improved its permissiveness towards two unrelated multidrug resistance plasmids while adapting to a new environmental niche. Mutations in the CCR and ArcAB regulatory systems resulted in transcriptional downregulation of plasmid genes and explained plasmid cost reduction. The presented evolutionary dynamics improve our understanding of successful associations between bacterial pathogens and resistance plasmids and provide a novel solution to the so-called ‘plasmid paradox’. In a broader perspective, the findings of this thesis advanced our knowledge on the selection, spread and maintenance of antibiotic resistance in bacteria, which is important to counteract its evolution.

Antimikrobiell resistens (AMR) utgjør en av de tre viktigste helseproblemene i verden i dag, og kan medføre alvorlige konsekvenser ifølge Verdens helseorganisasjon (WHO). Resistensutviklingen fører til terapisvikt mot infeksjonssykdommer som tidligere lot seg behandle av antimikrobielle midler og flere millioner menneskeliv kan gå tapt, hvis ikke tiltak iverksettes for å redusere forekomsten av AMR. Utviklingen av resistens inkluderer en av de mest kritiske karbapenem-resistente bakterier, Acinetobacter baumannii. Evnen til å kunne erverve og uttrykke AMR, samt eksepsjonell overlevelse på både biotiske og abiotiske overflater, gjør at A. baumannii grupperes som en av de seks hyppigst forekomne multiresistente (MDR) bakteriene i verden i dag, også kjent som ESKAPE patogener. Formålet med oppgaven var å undersøke om gener involvert i c-di-GMP signalering kunne være koblet til resistens mot selekterte antimikrobielle midler i A. baumannii. Dette skulle gjøres ved å screene ekspresjonen av målgener ved eksponering for sub-MIC nivåer av utvalgte antibiotika ved hjelp av RT-qPCR, samt ved screening for endring i resistensmønster hos et sett transposon insersjonsmutanter i c-di-GMP relaterte gener (cdg) i den multiresistente stammen A. baumannii AB5075-UW relativt til villtype, ved MIC-analyse. I første del av oppgaven ble det etablert MIC-screening oppsett for A. baumannii i laboratoriet, først ved å bruke den apatogene A. baylyi ADP-1, og så en ikke MDR-stamme, A. baumannii ATCC 17978. Av tekniske årsaker kunne ekspresjonsnivået av cdg-genene ikke bestemmes, og forsøket bør gjentas. MIC-screeningen viste at insersjonsmutant i genet ABUW_2255 var minimum fire ganger mer følsom mot amikacin, relativt til villtype. ABUW_2255 koder for et protein med PilZ-domene, et domene som klassisk er kjent å kunne binde c-di-GMP og fungere som effektorproteiner som responderer på endringer i c-di-GMP nivåer i cellen under intracellulær signalisering. Dette studiet utgjør et av de første som viser effekt av c-di-GMP relaterte gener på antimikrobiell resistens. Genet for det ortologe pilZ-domene proteinet er i P. aeruginosa identifisert som essensielt for type 4 pilus dannelse og twitching motilitet, dette antas også for pilZ-domene proteinet i A. baumannii. Ytterligere studier kreves for å forklare de molekylære mekanismene for hvordan ABUW_2255 påvirker resistens mot amikacin, og for å kartlegge funksjonen av proteinet.

Background At present time, antimicrobial resistance is emerging more rapidly than the development of novel antimicrobials, presenting a serious threat to how we prevent and treat infectious diseases. Several treatment strategies to counteract this development have been proposed, among these is the use of collateral sensitivity in clinical treatment. The ability to predict collateral sensitivity and cross-resistance effects is essential to exploiting this concept. In this study, we aimed to investigate the patterns of collateral sensitivity and cross-resistance in ciprofloxacin resistant isolates carrying gyrA and parC mutations. Method Ciprofloxacin resistant isolates were evolved from three clinical E. coli strain using static and dynamic selection methods. Isolates were selected based on identified mutations and level of ciprofloxacin resistance measured with diffusion gradient strips. DNA sequencing was used to detect mutations in gyrA and parC. Resistant isolates carrying at least one gyrA and parC mutation were characterized by IC90 assays with micro-broth dilutions of six unrelated antimicrobial agents. The observed collateral sensitivity and cross-resistance effects were displayed in a heat map. Results Various non-synonymous point mutations in gyrA and parC were identified in several of the generated ciprofloxacin resistant isolates. These mutants displayed collateral sensitivity and cross-resistance to several unrelated antimicrobials. Collateral sensitivity to gentamicin and trimethoprim was observed in the majority isolates. Cross-resistance effects were found in several mutants, specifically to ceftazidime, chloramphenicol and colistin. Conclusion Our findings suggest that ciprofloxacin resistant mutants with gyrA and parC mutations display a clear tendency of collateral sensitivity to gentamicin, an effect which potentially can be exploited in future treatment. However, we propose further investigation into specific point mutations within these genes, to better understand the observed variations in collateral sensitivity and cross-resistance.

The increase in antimicrobial resistance in bacteria is a serious problem in public heath limiting the efficacy of available antimicrobials for treatment of infections. E. coli is the most frequent cause of uncomplicated urinary tract infections. Ciprofloxacin is an important antimicrobial for treatment of uncomplicated urinary tract infections. Unfortunately, resistance to ciprofloxacin is commonly found in the clinics and confers cross-resistance to other antimicrobials. Recent studies on a previously discovered phenomenon known as collateral sensitivity, the opposite of cross-resistance, where resistance to one antimicrobial confers increased sensitivity to others. Recently, some have suggested that using collateral sensitivity to choose the order of antimicrobial used can beneficially slow the progress of antimicrobial resistance during drug cycling. Therefore, we aim to study the collateral sensitivity and cross-resistance profiles across different clinical isolates of E. coli from urinary tract infections. In this study we generated 10 ciprofloxacin resistant mutants and tested their susceptibility to 8 different antimicrobials. Our results show that E. coli resistant to ciprofloxacin above clinical breakpoints are cross-resistant to many other clinically-relevant antimicrobials, such as mecillinam, trimethoprim, nitrofurantoin, chloramphenicol, and ceftazidime. Our results also showed that ciprofloxacin resistant mutants are collaterally sensitive to gentamicin. This study provides important data on cross-resistance and collateral sensitivity in a collection of clinical E. coli isolates resistant to ciprofloxacin. Since it is important to confirm previous findings on laboratory strains with clinical isolates, hopefully these findings will add to growing data on collateral sensitivity and inform future drug cycling treatment strategies to combat antimicrobial resistance.

Because of an increasing development of antimicrobial resistance, treatment options for bacterial infectious diseases are narrowed. A strategy to reduce the evolution of resistance is to take advantage of antimicrobial collateral networks. In this thesis, we explored these collateral networks with a focus on antimicrobials used in Norway for the treatment of cystitis. Cystitis is the most common outcome of urinary tract infection. Five clinical strains from a collection of pan-susceptible Escherichia coli (E. coli) isolated from uncomplicated urinary tract infection (ECO•SENS) were used. Spontaneous, single step mutants selected on trimethoprim, ciprofloxacin and nitrofurantoin were generated and their susceptibility determined by E-test. They were confirmed to belong to E. coli species by performing random amplified polymorphism DNA polymerase chain reaction (RAPD PCR). The susceptibility of the mutants was thereafter tested towards seven different antimicrobials. These results were compared to the ancestral strains’ susceptibility profiles to the same antimicrobials. This method represented a scenario where the susceptibility of an isolate is decreased towards trimethoprim, ciprofloxacin or nitrofurantoin due to a spontaneous mutation, but at the same time, the mutants’ susceptibility can increase or decrease for a different antimicrobial. The collateral networks of mutants with decreased susceptibility to three antimicrobials were visualized in heat maps, where blue color displays collateral sensitivity and red color indicates drug combinations that show collateral resistance. The use of drug combinations that show collateral resistance should be avoided and instead, the use of drug combinations that display collateral sensitivity should be applied to hinder the increase of antimicrobial resistance. The presented data suggest that acquisition of trimethoprim resistance simultaneously increases susceptibility to aminoglycosides and mecillinam. Moreover, we provide evidence that the temporal order of antimicrobial consumption can be optimized for resistance to trimethoprim and that the use of nitrofurantoin may be carried out with caution.

Resistance towards antimicrobial agents is an increasing medical problem nowadays caused by the elimination of susceptible bacteria leaving only the resistant ones to survive and evolve. Evolution through mutations that lead to antibiotic resistance in bacteria increases the diversity of bacterial genomes. Under antibiotic treatment, these mutations might be advantageous for the bacteria, but disadvantage in the absence of antibiotic due to fitness cost. By understanding, how mutations can have an impact on the fitness cost for individual resistance determinants and the interactions that occur within one genetic background can foster our understanding of epistasis. The importance of understanding epistatic interactions between genetic determinants that are responsible for a resistant phenotype can be essential in the treatment of bacterial infections with antimicrobial drugs. In this study, mutants of clinical Escherichia coli strains with reduced susceptibility towards trimethoprim and/or ciprofloxacin were generated to test whether epistatic interactions in certain combinations of mutants existed. Growth curve measurements were used to calculate the relative generation time as a measure of fitness. The result revealed that the generation time of double mutant TP+CIP22 was reduced with 7 minutes compared to the single mutant TP22, resulting in a potentially positive epistatic interaction. However, a negative epistatic interaction may have occurred due to a decrease in minimal inhibitory concentration (MIC) determined for trimethoprim in all double mutants (TP+CIP) compared to the respective single mutants (TP). The results provided here promote our understanding of epistatic interactions in bacteria. Hopefully, together with findings of further experiments, they will be implemented in innovative guidelines for antibiotic treatment.

Høye resistensfrekvenser i bakteriepopulasjoner, kombinert med en nedgang i antall nye antibiotikalanseringer og nye strukturer under utprøvning, gjør undersøkelser av forutsetningene for reverserbarheten av resistens mer aktuelt. I tillegg til generelle likheter blant ulike antibiotika i prosessene som styrer reversering, vil det også for nye antibiotika være viktig å skaffe redskaper som kan hjelpe med å forutsi mulige resistensmekanismer og spesifikke faktorer for reversering. Biologiske kostnader for resistens, i form av redusert fitness er avgjørende for reverseringshastighet, og ulike perspektiver på nivå av seleksjon fra molekylærgenetikk og bakteriepopulasjoner til farmakoepidemiologi på et samfunnsnivå, kan belyse flere sider ved resistensproblematikken. Kvantitative beregninger er nødvendig for å kunne optimalisere gode retningslinjer for antibiotikabruk, som kan minimere utvikling og fremskynde reversering av resistens. Sentrale faktorer involvert i reversering av resistens, er seleksjonspresset bruken av antibiotika utøver, mekanismer for samseleksjon av resistensdeterminanter, rater for horisontal genoverføring, de relative fitnessforskjellene mellom resistente og sensitive populasjoner, og evnen til adaptiv kompensasjon som minimerer den biologiske kostnaden av resistens. Andre muligheter for økt reversering som ikke innebærer endringer i antibiotikabruk kan forventes å komme fra til dels nye strategier, utviklet som adjuvans til konvensjonelle antibiotika eller som kan mer indirekte redusere behovet for antibiotikaterapi.

The emergence and increase of antimicrobial resistance among bacterial populations has raised interest in the factor that contributes to this situation. Mobile genetic elements and in particular integrons have been found to play a role in the spread of resistance genes due to their ability to capture and integrate one or more gene cassettes by site-specific recombination. The genes can be integrated within the same attachment sites resulting in the formation of composite clusters of antibiotic resistance genes that facilitate multiple resistance phenotypes. The aim of this study was to test the hypotheses that stated that the widespread dispersal of integrons in gram-negative bacteria is due to (1) low if any biological cost associated with harboring a class 1 integrons, (2) integron encoded cassettes are stably maintained in the absence of antimicrobial selection. The class 1 integron was obtained from the clinical strains A.baumannii Ab64 and Ab65 FFC and integrated into A.baylyi ADP1, an integron-free strain, by natural transformation. The fitness cost of three different strains was determined in pair wise competitions with the otherwise isogenic model organism ADP1. The strain Ab64.T1b that contained the newly acquired integron was found to have a fitness cost of 7% and stabile gene cassettes. The biological cost of the integrase was assessed with an integrase knock out strain Ab64.T1b int::cat and was observed to be 2% where as the fitness cost associated with harboring the gene cassettes nptII and sacB inserted in the selectively neutral locus was found to be zero. The stability of the integron encoded cassettes was tested in a 10 day experiment that corresponded to approximately 70 generation and it was found that the gene cassettes in the integron where 100% stable.