In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels. / AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien. / The National Research Foundation (NRF) / South African Medical Research Council (MRC) / Harry Crossley Foundation

Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Evidence suggests that biological mechanisms, such as energy dependant efflux pumps, in addition to the rpoB gene mutations, define the level of rifampicin (RIF) resistance in drug resistant Mycobacterium tuberculosis (M. tuberculosis) strains with similar genetic backgrounds. Additionally, proteomic studies showed up-regulation of components of F1F0-ATP synthase enzyme, encoded by the atpBEFHAGDC operon which is responsible for ATP production, in response to RIF. The hypothesis of the current study is that the exposure of a multi-drug resistant (MDR) M. tuberculosis strain to RIF leads to the initiation of a signalling cascade of events. This results in the increased expression of F1F0-ATP synthase leading to an increase in energy production and subsequent activation of efflux pumps. RIF will thus be actively extruded from the cell, increasing the level of RIF resistance. This study aims to identify genetic regions responsible for the regulation of expression of the atpBEFHAGDC operon. Additionally we aim to identify other novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. A specialised reporter vector was constructed to monitor the expression of atpBEFHAGDC, with the use of a fluorescent protein. Subsequently a library of random knockouts was created by transposon mutagenesis in order to identify possible regulators, as well as novel mechanisms contributing to RIF resistance. Two hypothetical proteins, Rv2005c and Rv2417c, were identified in M. tuberculosis transposon mutants showing decreased fluorescence correlating to decreased expression of atpBEFHAGDC. Rv2005c encodes a universal stress protein, suggesting its potential role in a signalling cascade initiated upon RIF exposure. In a model pathway of regulation we propose that the product of Rv2005c is responsible for releasing a repressor protein, Rv1049, thereby stimulating a cascade of signalling events resulting in the up-regulation of atpBEFHAGDC. This increase in ATP production thereby fuels the extrusion of RIF from the cell via efflux pumps. In addition, it was found that disruptions in Rv2524c (fatty acid synthase), Rv1048c (hypothetical protein) and Rv3163c (probable conserved secreted protein) resulted in an increase in the level of RIF resistance in a RIF resistant clinical isolate. Interestingly, Rv2524c also showed to have a potential role in regulation of atpBEFHAGDC, whereby it ensures the repression of atpBEFHAGDC. Another gene identified to be involved in the increase in RIF resistance, Rv0260c, is annotated as a possible transcriptional regulator. This study was successful in identifying possible regulatory proteins involved in regulation of the F1F0-ATP synthase in response to RIF, and highlights the complexity of the regulatory events that occur in response to RIF in a MDR M. tuberculosis strain. This study was also successful in identifying candidates for functional analysis to determine novel mechanisms contributing to the level of RIF resistance in M. tuberculosis. Together these findings demonstrate that RIF resistance in M. tuberculosis is more complex than originally thought. Considering that anti-Tuberculosis (TB) drug TMC207 targets the F1F0-ATP synthase, a key enzyme in the production of energy in mycobacteria, the newly identified regulatory genes of F1F0-ATP synthase may represent ideal targets for novel anti-TB drug design. / AFRIKAANSE OPSOMMING: Huidige dogma toon dat mutasies in die rpoB geen vir rifampicin (RIF) weerstandigheid in Mycobacterium tuberculosis (M. tuberculosis) verantwoordelik is. Onlangs is egter bevind dat ander biologiese meganismes, soos energie afhanklike membraanpompe, saam met mutasies in hierdie geen, die verskillende vlakke van RIF weerstandigheid in M. tuberculosis isolate met soortgelyke genetiese agtergrond kan verklaar. Addisionele proteïen studies het gewys dat die komponente van die F1F0-ATP sintase ensiem, wat verantwoordelik vir ATP sintese is en gekodeer word deur die atpBEFHAGDC operon, opgereguleer word na RIF blootstelling. Die hipotese van hierdie studie is dat blootstelling van ‘n multi-middelweerstandige M. tuberculosis isolaat aan RIF aanleiding sal gee tot ʼn aanvanklike sein wat dan verskeie ander biologiese paaie sal aanskakel. Hierdie gebeure sal dan lei tot ‘n verhoging in geenuitdrukking van die F1F0-ATP sintase operon met gevolglike verhoging in energie produksie, wat uiteindelik energie afhanklike membraan pompe sal aanskakel. Die aktiewe uitpomp van RIF uit die sel sal dan ʼn verhoging in die vlak van RIF weerstandigheid veroorsaak. Die eerste doel van hierdie studie is om genetiese areas te identifiseer wat verantwoordelik is vir die regulering van geenuitdrukking van die atpBEFHAGDC operon. Die tweede doel is om nuwe meganismes te identifiseer wat verskille in die vlakke van RIF weerstandigheid in verskillende nou verwante kliniese isolate sal verklaar. ʼn Gespesialiseerde vektor wat die geenuitdrukking van die atpBEFHAGDC operon sal monitor is suksesvol ontwikkel met die gebruik van ʼn fluoresserende proteïen. Daarna is van die transposon mutagenese metode gebruik gemaak om ʼn biblioteek van ewekansige geenuitlatings te maak en hierdie biblioteek is dan gebruik om nuwe meganismes van RIF weerstandigheid te ondersoek. Hierdie studie het twee hipotetiese proteïene, Rv2005c en Rv2417c, in M. tuberculosis transposon mutante geïdentifiseer wat verantwoordelik is vir verlaagde fluoressensie. Dit korreleer met die verwagte verlaagde geenuitdrukking van atpBEFHAGDC. Die geen Rv2005c kodeer vir ʼn universele spanningsproteïen en die resultaat voorspel dat Rv2005c ʼn potensiële rol het om ʼn netwerk van seine in die bakterium aan te skakel direk na blootstelling aan RIF. In ʼn voorgestelde model van regulerende paaie voorspel ons dat die produk van Rv2005c verantwoordelik is vir die vrystelling van ʼn onderdrukker proteïen, Rv1049. Dit lei dan tot die stimulering van ʼn netwerk van intrasellulêre seine wat aanleiding gee tot die opregulering van atpBEFHAFDC. Die opregulering van atpBEFHAFDC sal dan aanleiding gee tot ʼn verhoging in ATP produksie wat die uitpomp van RIF uit die sel sal versnel met die gebruik van energie afhanklike membraan pompe. Dit is verder gevind dat uitskakeling van die gene Rv2524c (vetsuur sintase), Rv1048c (hipotetiese proteïen) en Rv3163c (moontlike konserwatiewe uitskei proteïen) aanleiding gegee het tot die verhoging in die vlakke van RIF weerstandigheid in ʼn RIF weerstandige kliniese isolaat. In die studie is ook bewys dat Rv2524c ʼn potensiële rol in die regulering van atpBEFHAGDC het deurdat dit die onderdrukking van atpBEFHAGDC verseker. Rv0260c is voorheen gelys as ʼn moontlike transkripsionele reguleerder wat betrokke is by die verhoging van RIF weerstandigheid. Hierdie studie was suksesvol in die identifisering van moontlike gene en proteïne wat betrokke is in die regulering van die F1F0-ATP sintase in reaksie tot RIF blootstelling. Dit beklemtoon die kompleksiteit van die regulerende gebeurtenisse wat plaasvind in reaksie tot RIF blootstelling in ʼn multi-middelweerstandige M. tuberculosis isolaat. Verder was daar suksesvol kandidaat gene en ʼn reguleerder geïdentifiseer wat in toekomstige studies ondersoek kan word vir hulle funksionele bydrae om nuwe meganismes te vind wat die varierende vlakke van RIF weerstandigheid in M. tuberculosis sal verklaar. Opsommend demonstreer hierdie studie dat RIF weerstandigheid meer kompleks is as wat voorheen aangeneem was. Die nuwe baie belowende teen-Tuberkulose middel, TMC207, se aanslag is gemik op die belangrike ensiem (F1F0-ATP sintase) wat in hierdie studie ondersoek was. Dus kan nuut geïdentifiseerde proteïene wat betrokke is by die regulering van hierdie ensiem beskou word as ideale kandidate vir die ontwikkeling van nuwe teen -Tuberkulosemiddels. / The National Research Foundation and the Department of Biomedical Sciences

Antibiotic resistance in bacteria is acquired through spontaneous chromosomal mutations or horizontal gene transfer. In the absence of antibiotics, resistant mutants generally show reduced fitness due to compromised growth rate, competitive ability and virulence compared to their antibiotic-sensitive ancestors. The focus of my research is to dissect the molecular underpinnings of the variations in the fitness cost of chromosomal antibiotic resistance using a systems-level approach. From an evolutionary perspective, my research aims are to understand how the fitness cost influences adaptation in resistant populations in an antibiotic-free environment. Using rifampicin resistance in Pseudomonas aeruginosa as a model, my work shows that most of the variation in the fitness cost of rifampicin resistance can be attributed to the direct effect of rifampicin resistance mutations on transcriptional efficiency. Through RNA-Seq transcriptome profiling, I demonstrate that global changes in gene expression levels associated with resistance mutations are surprisingly subtle, suggesting that the transcriptional regulatory network of P. aeruginosa is robust against compromised transcriptional efficiency. Using experimental evolution and whole-genome sequencing, my work reveals a systematic difference in the genetic basis of adaptation in mutants that were propagated in the absence of antibiotics. During compensatory adaptation, resistant mutants can recover the fitness cost of resistance by fixing second-site mutations that directly offset the deleterious effects of resistance mutations. Amongst resistant mutant populations with low fitness costs, general adaptation limits compensatory adaptation, which is most likely to be due to the rarity of compensatory mutations and clonal interference. Far from being the most ubiquitous mechanism in the evolution of resistance, compensatory adaptation is the exception that is more likely to be observed in resistant mutants with high fitness costs. In addition, I applied key elements of the integrative experimental approach developed in this work to dissect the molecular basis of the fitness cost associated with carriage of the pNUK73 small plasmid in P. aeruginosa, which carries the rep gene encoding a plasmid replication protein. My results confirmed that rep expression generates a significant fitness cost in P. aeruginosa and demonstrate how the molecular origins of the fitness cost of resistance can be dissected in a different biological context.