Die genomische und transkriptomische Landschaft der klinischen Isolate von Escherichia coli und Pseudomonas aeruginosa

Large amounts of genomic data have been obtained due to the rapid advances in DNA sequencing technology. With efficient computational platforms, these data can provide many possibilities to improve our knowledge on species evolution and their genetic makeup. The general interest of this thesis is to facilitate studies on important biological questions by attaining the relevant information from transcriptomic and genomic data. The aims of my thesis were i) to develop the pan-genome based RNA-Seq data analysis pipeline in order to analyze ex vivo gene expression profiles of uro-pathogenic Escherichia coli isolates and ii) to create the consensus sequence of the Pseudomonas aeruginosa core genome in order to identify single nucleotide polymorphisms (SNPs) at high accuracy and to find the patho-adaptive mutations in P. aeruginosa clinical isolates. To address these aims I developed and used the pan-genome of E. coli in order to map and analyze the RNA-Seq reads that were associated with an acute urinary tract infection. Whereas the in vivo gene expression profiles of the majority of genes were conserved among the 21 E. coli strains, the specific gene expression profiles of the accessory genome were diverse and reflected phylogenetic relationships. In addition to that, whole genome sequencing data was used to gain insights into the genetic variations of 99 clinical P. aeruginosa isolates. I created the consensus sequence for every core gene based on the most frequent nucleotide. I used it as reference for the identification of SNPs across all clinical isolates. The identified SNPs were classified into clonal-specific, single and phylogenetically independent SNPs. The majority of the SNPs were clonal-dependent and single SNPs. However, I identified a large set of 2,252 genes which had one or more phylogenetically independent non-synonymous mutation. Moreover, the ratio of dN/dS on 3,814 genes revealed that the core genome is not under selection pressure. In summary, this thesis explores pan-genome-based as well as consensus sequence-based approaches on transcriptomic and genomic sequencing data of clinical isolates of E. coli and P. aeruginosa respectively. The results of the thesis contributed to understanding of sequence variations that are selected in the environment of the human host and lead to bacterial adaptation and pathogenicity. This is not only important for the basic scientific research, but also to understand the link between diversity and community structure and function.Aufgrund der schnellen Fortschritte in der DNA-Sequenzierungstechnologie wurden große Mengen genomischer Daten erhalten. Mit effizienten Rechenplattformen können diese Daten viele Möglichkeiten bieten, unser Wissen über die Evolution von Arten und ihren genetischen Aufbau zu verbessern. Das allgemeine Interesse dieser Arbeit ist es, Studien zu wichtigen biologischen Fragen zu ermöglichen, indem relevante Informationen aus transkriptomischen und genomischen Daten gewonnen werden. Ziel meiner Dissertation war es, i) die auf dem Pan-Genom basierende RNA-Seq Datenanalyse Pipeline zu entwickeln, um ex vivo-Genexpressionsprofile von uropathogenen Escherichia coli-Isolaten zu analysieren und ii) die Konsensussequenz des Pseudomonas aeruginosa-Kerns zu erstellen Genom, um einzelne Nukleotidpolymorphismen (SNPs) mit hoher Genauigkeit zu identifizieren und die pathoadaptiven Mutationen in klinischen P. aeruginosa-Isolaten zu finden. Um diese Ziele zu erreichen, entwickelte und verwendete ich das Pan-Genom von E. coli, um die RNA-Seq-Reads abzubilden und zu analysieren, die mit einer akuten Harnwegsinfektion assoziiert waren. Während die in vivo-Genexpressionsprofile der meisten Gene unter den 21 E. coli-Stämmen konserviert waren, waren die spezifischen Genexpressionsprofile des akzessorischen Genoms unterschiedlich und spiegelten phylogenetische Beziehungen wider. Darüber hinaus wurden Daten zur vollständigen Genomsequenzierung verwendet, um Einblicke in die genetischen Variationen von 99 klinischen P. aeruginosa-Isolaten zu erhalten. Ich erstellte die Konsensussequenz für jedes Kerngen basierend auf dem häufigsten Nukleotid. Ich habe es als Referenz für die Identifizierung von SNPs in allen klinischen Isolaten verwendet. Die identifizierten SNPs wurden in klonenspezifische, einzelne und phylogenetisch unabhängige SNPs eingeteilt. Die Mehrheit der SNPs waren klonabhängige und einzelne SNPs. Ich identifizierte jedoch eine große Menge von 2.252 Genen, die eine oder mehrere phylogenetisch unabhängige, nicht synonyme Mutationen aufwiesen. Darüber hinaus zeigte das Verhältnis von dN / dS bei 3.814 Genen, dass das Kerngenom nicht unter Selektionsdruck steht. Zusammenfassend werden in dieser Dissertation Pan-Genom-basierte sowie Consensus-Sequenz-basierte Ansätze zur Transkriptom- und Genom-Sequenzierung von klinischen Isolaten von E. coli bzw. P. aeruginosa untersucht. Die Ergebnisse der Dissertation trugen zum Verständnis von Sequenzvariationen bei, die in der Umgebung des menschlichen Wirts selektiert werden und zu einer bakteriellen Anpassung und Pathogenität führen. Dies ist nicht nur für die wissenschaftliche Grundlagenforschung wichtig, sondern auch, um den Zusammenhang zwischen Vielfalt und Struktur und Funktion der Gemeinschaft zu verstehen

Core regulon of the global anaerobic regulator Anr targets central metabolism functions in Pseudomonas species

A comparative genome analysis of the global anaerobic regulator Anr regulon in five species of Pseudomonas with different life style was performed. Expression of this regulator was detected in all analyzed Pseudomonas. The predicted Anr regulon (pan-regulon) consisted of 253 genes. However, only 11 Anr-boxes located upstream of qor/hemF, hemN, cioA/PA3931, azu, rpsL, gltP, orthologous to PA2867, cspD, tyrZ, slyD and oprG, were common to all species. Whole genome in silico prediction of metabolic pathways identified genes belonging to heme biosynthesis, cytochromes and Entner-Doudoroff pathway as members of Anr regulon in all strains. Extending genome analysis to 28 Pseudomonas spp. spanning all phylogenetic groups showed Anr-boxes conservation in genes related to these functions. When present, genes related to anaerobic metabolism were predicted to hold Anr-boxes. Focused on the genomes of eight P. aeruginosa isolates of diverse origins, we observed a conserved regulon, sharing nearly 80% of the genes, indicating its key role in this opportunistic pathogen. The results suggest that the core Anr regulon comprises genes involved in central metabolism and aerobic electron transport chain, whereas those genes related to anaerobic metabolism and other functions constitute the accessory Anr-regulon, thereby differentially contributing to the ecological fitness of each Pseudomonas species.Fil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Lujan, Adela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Pardo, Agustin Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Ibarra, José Gervasio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Smania, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: López, Nancy I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

The genomic basis of rapid adaptation to antibiotic combination therapy in Pseudomonas aeruginosa

Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sublethal doses of distinct antibiotic combinations and corresponding single-drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps are the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness toward sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future

Compendium-Wide Analysis of Pseudomonas aeruginosa Core and Accessory Genes Reveals Transcriptional Patterns across Strains PAO1 and PA14.

Pseudomonas aeruginosa is an opportunistic pathogen that causes difficult-to-treat infections. Two well-studied divergent P. aeruginosa strain types, PAO1 and PA14, have significant genomic heterogeneity, including diverse accessory genes present in only some strains. Genome content comparisons find core genes that are conserved across both PAO1 and PA14 strains and accessory genes that are present in only a subset of PAO1 and PA14 strains. Here, we use recently assembled transcriptome compendia of publicly available P. aeruginosa RNA sequencing (RNA-seq) samples to create two smaller compendia consisting of only strain PAO1 or strain PA14 samples with each aligned to their cognate reference genome. We confirmed strain annotations and identified other samples for inclusion by assessing each sample\u27s median expression of PAO1-only or PA14-only accessory genes. We then compared the patterns of core gene expression in each strain. To do so, we developed a method by which we analyzed genes in terms of which genes showed similar expression patterns across strain types. We found that some core genes had consistent correlated expression patterns across both compendia, while others were less stable in an interstrain comparison. For each accessory gene, we also determined core genes with correlated expression patterns. We found that stable core genes had fewer coexpressed neighbors that were accessory genes. Overall, this approach for analyzing expression patterns across strain types can be extended to other groups of genes, like phage genes, or applied for analyzing patterns beyond groups of strains, such as samples with different traits, to reveal a deeper understanding of regulation

Phenotypic and transcriptomic analyses of seven clinical Stenotrophomonas maltophilia isolates identify a small set of shared and commonly regulated genes involved in the biofilm lifestyle

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings. IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms

Big Data For Microorganisms: Computational Approaches Leveraging Large-Scale Microbial Transcriptomic Compendia

Genome-wide transcriptomics data captures the molecular state of microorganisms – the expression patterns of genes in response to some condition or stimuli. With advancements in high-throughput sequencing technologies, there are thousands of microbial transcription profiles publicly available. Consequently, this data has been collected and integrated to form transcriptomic compendia, which are collections of diverse gene expression experiments. These compendia were found to be a valuable resource for studying systems level biology and hypothesis generation. We describe the construction, benefits and challenges in creating microbial transcriptomic compendia in Chapter 1. One challenge for compendia, which integrates across many different experiments, is batch effects, which are technical sources of variability that can disrupt the detection of underlying biological signals of interest. In Chapter 2, we use a generative neural network to simulate gene expression compendia with varying amounts of technical variability and assess the ability to detect the underlying biological structure in the data after noise was added and then after batch correction was applied. We define a set of principles for how batch correction should be used in the context of these large-scale compendia. In Chapter 3 and 4 we introduce computational approaches to use compendia to improve the analysis of individual experiments and analysis of genomic patterns respectively. In Chapter 3, we develop a portable framework to distinguish between common and context specific transcriptional signals using a compendium to autogenerate a null set of expression changes. This approach allows researchers to put gene expression changes from their individual experiment of interest into the context of existing compendia of experiments. In Chapter 4 we develop an approach to examine the effect of different Pseudomonas aeruginosa genomes, using two dominant strain types, on transcriptional profiles in order to understand how traits manifest. This genome-wide approach reveals a more complete picture of how different genomes affect expression, which mediates different traits present. Overall, these compendia provide a valuable resource that computational tools can leverage to extract patterns and inform research directions

Metabolome-based studies of virulence factors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen and an important causative agent of potentially life-threatening nosocomial infections in predisposed patients. The Gram-negative bacterium produces a large and diverse repertoire of small-molecule secondary metabolites that serve as regulators and effectors of its virulence. In this study, a range of mass spectrometry-based bacterial metabolomics approaches was used to investigate these small-molecule virulence factors and their interplay with pseudomonal metabolism as well as with phenotypic traits related to virulence. The groundwork was laid by exploring the metabolite inventory of P. aeruginosa and improving the coverage of its metabolome by the application of a custom software named CluMSID, that clusters analytes based on similarities of their MS² spectra. CluMSID led to the annotation of, i.a., 27 novel members of the class of alkylquinolone quorum sensing signalling molecules, which represent crucial players in the highly complex network that regulates pseudomonal virulence. The tool was developed towards a versatile and user-friendly R package hosted on Bioconductor, whose functionalities and benefits are described in detail. The new findings on the alkylquinolone chemodiversity led to further studies with a mechanistic focus that probed the substrate specificity of the enzyme complex PqsBC. It was demonstrated that PqsBC accepts different medium-chain acyl-coenzyme A substrates for the condensation with 2-aminobenzoylacetate and thereby produces alkylquinolones with various side chain lengths, whose distribution is a function of substrate specificity and substrate availability. Moreover, it was shown that PqsBC also synthesises alkylquinolones with unsaturated side chains. The focus was further broadened from metabolite and pathway-centred questions to a more global perspective on pseudomonal virulence and metabolism, which directed attention at PrmC, an enzyme with a partially unknown function indispensable for in vivo virulence. An untargeted metabolomics experiment yielded insights into the role of PrmC and its influence on the pseudomonal endo- and exometabolome. Finally, clinical P. aeruginosa strains with different virulence phenotypes were examined by untargeted metabolomics in order to disclose metabolic variation and interconnections between virulence and metabolism. The analysis resulted in the discovery of a putative virulence biomarker and enabled the construction of a random forest classification model for certain virulence phenotypes based only on metabolomics data. In summary, this study demonstrated the potential of metabolomics for the investigation of P. aeruginosa virulence factors and thereby contributed towards the comprehension of the complex interplay of metabolism and virulence in this important pathogen.Pseudomonas aeruginosa ist ein wichtiger opportunistischer Erreger potenziell lebensbedrohlicher nosokomialer Infektionen bei prädisponierten Patienten. Das Gram-negative Bakterium produziert ein vielfältiges Repertoire an niedermolekularen Sekundärmetaboliten, die als Regulatoren und Effektoren seiner Virulenz dienen. In dieser Studie wurde eine Reihe von Massenspektrometrie-basierten Ansätzen der bakteriellen Metabolomik verwendet, um diese niedermolekularen Virulenzfaktoren und ihre Wechselwirkungen mit dem pseudomonalen Metabolismus sowie mit virulenzassoziierten phänotypischen Merkmalen zu untersuchen. Die Grundlage bilden die Untersuchung des Metaboliteninventars von P. aeruginosa und die Verbesserung der analytischen Abdeckung des Metaboloms durch die Anwendung einer selbstentwickelten Software namens CluMSID, die MS²-Spektren nach Ähnlichkeit clustert. CluMSID führte zur Annotation von u.a. 27 neuen Mitgliedern der Klasse der Alkylchinolone, die als Quorum-Sensing-Signalmoleküle entscheidende Akteure im hochkomplexen Netzwerk der Virulenzregulation darstellen. Das Tool wurde zu einem R-Paket entwickelt, das auf Bioconductor verfügbar ist und dessen Funktionalitäten und Vorteile ausführlich beschrieben werden. Die neuen Erkenntnisse über die Chemodiversität der Alkylchinolone führten zu weiteren Studien mit mechanistischem Schwerpunkt, die die Substratspezifität des Enzymkomplexes PqsBC untersuchten. Es wurde nachgewiesen, dass PqsBC verschiedene mittelkettige Acyl-Coenzym-A-Substrate für die Kondensation mit 2-Aminobenzoylacetat akzeptiert und dadurch Alkylchinolone mit verschiedenen Seitenkettenlängen produziert, deren Verteilung eine Funktion der Substratspezifität und der Substratverfügbarkeit ist. Zudem konnte gezeigt werden, dass PqsBC auch Alkylchinolone mit ungesättigten Seitenketten synthetisiert. Im Weiteren wurde der Fokus von Metaboliten- und Stoffwechselweg-zentrierten Fragen hin zu einer globaleren Perspektive der pseudomonalen Virulenz und des Metabolismus erweitert, was die Aufmerksamkeit auf PrmC lenkte, ein Enzym mit teilweise unbekannter, für die in vivo-Virulenz unverzichtbarer Funktion. Ein globales Metabolomik-Experiment lieferte Einblicke in die Rolle von PrmC und seinen Einfluss auf das pseudomonale Endo- und Exometabolom. Schließlich wurden klinische P. aeruginosa-Stämme mit unterschiedlichen Virulenzphänotypen mittels ungerichteter Metabolomik untersucht, um metabolische Variationen und Zusammenhänge zwischen Virulenz und Metabolismus aufzudecken. Die Analyse resultierte in der Entdeckung eines putativen Virulenzbiomarkers und ermöglichte die Konstruktion eines Random-Forest-Klassifikationsmodells für bestimmte Virulenzphänotypen, das nur auf Metabolomik-Daten basiert. Zusammenfassend hat diese Studie das Potenzial der Metabolomik für die Untersuchung der Virulenzfaktoren von P. aeruginosa aufgezeigt und damit zum Verständnis des komplexen Zusammenspiels von Metabolismus und Virulenz bei diesem wichtigen Pathogen beigetragen

Repetitive Exposure to Bacteriophage Cocktails against Pseudomonas aeruginosa or Escherichia coli Provokes Marginal Humoral Immunity in Naïve Mice

Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either Pseudomonas aeruginosa or Escherichia coli—two VAP-associated pathogens—in naïve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in naïve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections

Understanding evolution to tackle antibiotic resistance in Pseudomonas aeruginosa

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 04-11-2022Antibiotic resistance (AR) constitutes a major public health concern, which has been aggravated in recent decades due to the emergence and spread of multidrug-resistant microorganisms, especially Gram-negative bacteria. Among them, Pseudomonas aeruginosa stands out; it is an opportunistic pathogen, widely distributed in nature, that frequently infects hospitalized patients and presents low susceptibility to many antimicrobials, as well as an overwhelming capacity to develop AR via mutation, mainly during chronic infections. Hence, novel treatment strategies are needed to deal with the infections caused by this bacterium. Collateral sensitivity, whereby acquiring resistance to one drug increases susceptibility to a second drug, is an evolutionary trade-off that may be exploited for treating bacterial infections by the combination or sequential use of drugs' pairs. This application is only possible if those collateral sensitivity phenotypes are conserved within different genetic contexts, environments and situations; robust collateral sensitivity events were searched for during this thesis. We determined that tobramycin, tigecycline and ceftazidime resistance acquisition in P. aeruginosa is associated with a robust fosfomycin collateral sensitivity and ascertained the mechanism responsible for this event. Further, we observed that ciprofloxacin exposure selects distinct mutations in different genetic backgrounds of P. aeruginosa, all of them leading to a robust tobramycin and aztreonam collateral sensitivity, and we proposed tobramycin-ciprofloxacin and ciprofloxacin-aztreonam combinations as promising therapies against infections caused by this bacterium. We also determined that media composition and nutrients’ availability constrain the pathways towards tobramycin, ceftazidime and ceftazidime-avibactam resistance in P. aeruginosa, but fosfomycin collateral sensitivity associated with ceftazidime resistance robustly emerges when P. aeruginosa evolves in different media mimicking those that can be encountered during infection. The compensation of fitness costs associated with the acquisition of AR in the absence of selective pressure could cause a decline of AR, which may also be used for designing therapeutic strategies considering those specific antibiotics whose resistance is robustly unstable in absence of selection. In this thesis, we observed that compensatory evolution of fitness costs associated with ceftazidime resistance in P. aeruginosa leads to a ceftazidime resistance decline in distinct genetic backgrounds, both in antibiotic-free and in sublethal tobramycin environments. The alternation of ceftazidime with drug restriction periods or the switch back to ceftazidime after a ceftazidime-tobramycin alternation may be feasible therapeutic approaches against P. aeruginosa infections. For its part, AR may be transiently induced by some conditions encountered by bacteria during infection, compromising the antibiotic treatments. In this thesis we identified dequalinium chloride, procaine and atropine, which can be present in P. aeruginosa site infections, as inducers of the expression of MexCD-OprJ efflux pump encoding genes, hence transiently increasing ciprofloxacin resistance of this bacterium. Finally, by further studying efflux pumps regulation and considering their ancestral function, we determined that the identification of compounds which are both substrates and inducers of efflux pumps of P. aeruginosa constitutes an effective strategy for finding molecules that reduce the virulence potential of this pathogen. Overall, the results of this thesis allow us to propose novel treatment strategies against P. aeruginosa infections, based on the identification of novel drugs and on the rational use of the antibiotics that we already have, as well as to better understand AR evolutio

Understanding the intraspecies genetic and phenotypic diversity of the clover symbiont Rhizobium leguminosarum

Rhizobia are agriculturally important bacteria capable of forming symbiosis with legumes and fixing atmospheric nitrogen which sustainably improves plant productivity and soil fertility. The Rhizobium leguminosarum species complex is highly genetically diverse and contains five genetically distinct genospecies. Significant phenotypic diversity is also displayed within Rhizobium leguminosarum; however, no phenotypes are genospecies-exclusive. The importance of the broad genetic diversity of Rhizobium leguminosarum and its influence on phenotypic diversity and rhizosphere-associated interactions are unclear. In this thesis, Rhizobium leguminosarum symbiovar trifolii (Rlt) intraspecies diversity was investigated by assessing the genetic and phenotypic variation of white clover nodule Rlt from agricultural field managements across Europe. This thesis identified that the significant genetic diversity of Rlt can manifest in substantial transcriptional and phenotypic variation across strains, and this diversity can influence plant-mediated symbiont selectivity and competitive strain interactions. A novel multiplexed high-throughput amplicon sequencing approach, MAUI-seq, was developed to improve detection of chimeras and other erroneous sequences for confident determination of intraspecies diversity from environmental samples. Using this method, significant Rlt nodule population diversity was identified between clover genotypes due to the combined effects of plant-host filtering and geospatial variation in allele frequencies of individual genes. Investigation of multiple Rlt strain transcriptomes demonstrated that genospecies displayed differences in core genome expression which was associated with phenotypic growth traits and putative differences in bacterial metabolism. Genomic and transcriptomic variation was utilised to identify transcriptional units conserved across strains. Pairwise growth competition experiments between Rlt strains further showed that significant competitive variation is evident and potentially associated with genospecies differences. This research demonstrates that utilising multiple strains can aid identification of species-specific traits by considering the representative variation within a species. The work presented here has laid the groundwork for future investigation into the implications of intraspecies diversity for symbiotic effectiveness in the rhizobia-legume symbiosis