Cystic fibrosis (CF) is the most common fatal genetic disorder that afflicts young Canadians. The major cause of morbidity and mortality in patients with CF is chronic pulmonary infection with the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. Once established, P. aeruginosa lung infections cannot be cleared despite sustained and aggressive antimicrobial therapy. Treatment failure of P. aeruginosa lung infections is caused by a combination of antibiotic resistance and tolerance mechanisms. Antibiotic resistance is mainly mediated by multidrug efflux pumps such as MexAB-OprM. Antibiotic tolerance has been attributed to biofilms and to nutrient starvation. In this thesis, I present an analysis of three transcriptional regulators (PA3225, RpoS, and RpoN) and their contributions to resistance and tolerance in P. aeruginosa. PA3225 is a transcriptional regulator that I initially identified as a candidate regulator of a type VI secretion system (T6SS) that had been previously implicated in biofilm tolerance. While a ΔPA3225 deletion mutant did not, unfortunately, have dysregulated expression of the T6SS, I fortuitously discovered that the mutant displayed increased resistance to various antibiotics from different functional classes. I linked the increased antibiotic resistance of ΔPA3225 to upregulation of MexAB-OprM and provided evidence that PA3225 may be a direct repressor of mexAB-oprM. Next, I sought to identify a transcriptional regulator of ndvB, which is another gene that plays a role in biofilm tolerance. I found that the stationary phase sigma factor, RpoS, was essential for expression of ndvB in stationary phase and biofilm cells. Moreover, RpoS was important for tolerance of stationary phase cells to tobramycin (TOB), an aminoglycoside antibiotic that is used to treat CF patients. In recent years, several groups have sought to identify novel treatments to combat antibiotic tolerance in P. aeruginosa. A popular strategy is metabolic potentiation, which involves co-administration of an antibiotic with a metabolite to reverse tolerance due to nutrient starvation. For example, one group found that fumarate (FUM) combined with TOB (TOB+FUM) was highly effective at killing tolerant P. aeruginosa. FUM uptake depends on C4-dicarboyxlate transporters, which are transcriptionally regulated by the alternative sigma factor, RpoN. Importantly, rpoN loss-of-function mutations are a recognised mechanism of pathoadaptation in CF clinical isolates. I demonstrated that TOB+FUM was unable to kill ΔrpoN stationary phase and biofilm cells due to loss of FUM uptake and that rpoN alleles from CF clinical isolates were unable to complement the ΔrpoN mutant. These findings could have important implications for TOB+FUM as a treatment modality in CF patients with a high burden of rpoN mutants. Overall, my work has provided interesting and, in the case of RpoN, clinically relevant insights into the regulatory networks that determine antibiotic susceptibility in P. aeruginosa.

The historical notion of a microbial population has been of a clonal population of identical swimming planktonic cells in a laboratory flask. As the field has advanced, we have grown to appreciate the immense diversity in microbial behaviors, from their propensity to grow in dense surface-attached communities as a biofilm, to the consequences of social dilemmas between cells, to their ability to form spores able to survive nearly any environmental insult. However, the historically biased view of the clonal microbial population still persists – even when a rare phenotype is investigated, the focus simply shifts to that narrower focal population - and this bias can lead to some of the broader questions relating to the consequences of phenotypic diversity within populations to be overlooked. This work seeks to address this gap by investigating the evolutionary causes and consequences of phenotypic heterogeneity, with a focus on clinically relevant phenotypes. We first develop and experimentally validate a theoretical model describing the evolution of a microbial population faced with a trade-off between survival and fecundity phenotypes (e.g. biofilm and planktonic cells), which suggests that simultaneous investment in both types maximizes lineage fitness in heterogeneous environments. This model helps to inform the experimental studies in the following chapters. We find that biofilm-mediated phenotypic resistance to antibiotics is evolutionarily labile, and responsive to antibiotic dose and whether biofilm or planktonic cells are passaged. We also show that persistence in E. coli is age-independent, supporting the current hypothesis of stochastic metabolic fluctuations as the cause of this rare phenotype. Finally, we explore phenotypic variation across a library of natural isolates of P. aeruginosa, and find few organizing principles among key phenotypes related to virulence. Together these results suggest that phenotypic heterogeneity is a crucial component in the ecology and evolution of microbial populations, and directly affects pressing applied concerns such as the antibiotic resistance crisis.

Pseudomonas aeruginosa é um importante patógeno humano oportunista capaz de causar severas infecções em pacientes imunodeprimidos e em pacientes que apresentam fibrose cística. Para desencadear uma resposta efetiva contra a infecção pela P. aeruginosa é necessário uma primeira linha de reconhecimento desta bactéria pelo sistema imunológico inato. Apesar do desencadeamento da resposta imune inata ser benéfica no controle da infecção pela P. aeruginosa, esta resposta deve ser controlada. Estudos recentes têm começado a esclarecer como os miRNAs desempenham papéis fundamentais na regulação de processos como infecção, resposta imune e inflamação participando da modulação da resposta imune. Dada a relevância da bactéria P. aeruginosa nos processos infecciosos em humanos e estimulados pela necessidade de desvendar os mecanismos de regulação da reposta imune contra esta bactéria, o objetivo deste trabalho foi avaliar o papel do mmu-miR-155-5p e mmu-miR-146b-5p expressos em camundongos infectados pelas cepas ATCC 27853 e PA14 da bactéria P. aeruginosa. Para avaliar o nível de expressão dos mmu-miR-155-5p e mmu-miR-146b-5p e das citocinas IL-1β, IL-6 e IL-12 in vitro, o RNA total foi extraído de células Raw 264.7, macrófagos derivados da medula óssea (BMDM) e de células dendríticas derivadas da medula óssea (BMDC) para análise de PCR em tempo real. Após a infecção das células Raw 264.7, BMDMs e BMDCs foi possível observar que a expressão dos miRNAs e das citocinas ocorreu de forma dependente em cada tipo celular infectado. Para as análises in vivo camundongos C57BL/6 foram infectados via intratraqueal com as cepas ATCC 27853 e PA14 de P. aeruginosa para análises de UFCs, RT-qPCR, histopatologia e estereologia. Apenas a cepa PA14 foi recuperada no pulmão e baço dos animais, onde não foi observado variação na expressão do mmu-miR-155-5p nem aumento significativo de uma série de citocinas efetoras. As análises histopatológicas demostraram intenso processo inflamatório difuso apresentando número maior de células inflamatórias, diminuição no número de alvéolos e do volume da estrela quando comparados aos animais não infectados e animais infectados pela cepa avirulenta. No pulmão dos animais infectados com a cepa ATCC 27853, observou-se aumento de expressão de mmu-miR-155-5p e mmu-miR-146b-5p, das citocinas inflamatórias. A identificação das redes de regulação dos miRNAs em estudo mostrou importantes alvos diretos e indiretos associados à resposta imune inata que podem estar comprometidas durante a expressão diferencial de mmu-miR-155-5p e mmu-miR-146b-5p em favorecimento ou em detrimento da resolução da infecção pela P. aeruginosa. Os resultados obtidos até o momento permitem sugerir que a infecção pela P. aeruginosa exerce uma modulação na expressão de miRNAs e consequentemente na resposta imune contra esta bactéria e que a virulência de diferentes cepas de P. aeruginosa influenciam na expressão dos miRNAs mmu-miR-155-5p e mmu-miR-146b-5p, de citocinas pró-inflamatórias e na patologia. Mais estudos são necessários para desvendar os mecanismos pelos quais cepas virulentas da P. aeruginosa conseguem subverter a resposta imune e garantir a sua replicação no hospedeiro. / Pseudomonas aeruginosa is an important opportunistic human pathogen capable of causing severe infections in immunocompromised patients and in patients with cystic fibrosis. To trigger an effective response against infection by P. aeruginosa is required a first line of recognition of this bacterium by the innate immune system. Although the innate immune response is beneficial in the control of P. aeruginosa infection, this response should be controlled. Recent studies have begun to clarify how miRNAs play key roles in regulating processes such as infection, immune response and inflammation by participating in the immune response modulation. Due to the importance of the P. aeruginosa bacterium in infectious processes in humans and stimulated by the need to uncover the mechanisms of regulation of the immune response against this bacterium, the objective of this study was to evaluate the role of mmu-miR-155-5p and mmu-miR-146b -5p expressed in mice infected with strains ATCC 27853 and PA14 of the bacterium P. aeruginosa. To assess the level of expression of mmu-miR-155-5p and mmu-miR-146b-5p and the cytokines IL-1β, IL-6 and IL-12 in vitro, the total RNA was extracted from Raw 264.7 cells, bone marrow derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) for real-time PCR analysis. After infection of the Raw 264.7, BMDMs and BMDCs cells, it was possible to observe that the expression of miRNAs and cytokines occurred in a dependent manner in each infected cell type. For the in vivo analyzes C57BL / 6 mice were infected intratracheally with the strains ATCC 27853 and PA14 of P. aeruginosa for analysis of CFU, RT-qPCR, histopathology and stereology. Only the PA14 strain was recovered in the lungs and spleens of the animals, where no variation in the expression of mmu-miR-155-5p or a significant increase in a series of effector cytokines was observed. Histopathological analyzes demonstrated an intense diffuse inflammatory process, presenting a larger number of inflammatory cells, a decrease in the number of alveoli and the volume of the star when compared to uninfected animals and animals infected by the avirulent strain. In the lung of animals infected with ATCC 27853 strain, increased expression of mmu-miR-155-5p and mmu-miR-146b-5p, of inflammatory cytokines was observed. The identification of regulatory networks of the miRNAs under study showed important direct and indirect targets associated with the innate immune response that may be compromised during the differential expression of mmu-miR-155-5p and mmu-miR-146b-5p in favor or detriment of P. aeruginosa infection. The results obtained to date suggest that the infection by P. aeruginosa exerts a modulation in the expression of miRNAs and consequently in the immune response against this bacterium and that the virulence of different strains of P. aeruginosa influence the expression of miRNAs mmu-miR- 155-5p and mmu-miR-146b-5p, of proinflammatory cytokines and in pathology. Further studies are needed to uncover the mechanisms by which virulent P. aeruginosa strains can subvert the immune response and ensure replication in its host. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

Pseudomonas aeruginosa is extremely harmful to immunocompromised individuals. An atomic force microscope was used to measure the surface forces of this bacteria’s exopolymers. These forces were characterized with the AdG force model, which is a function of brush length, probe radius, temperature, separation distance and an indefinite density variable, s. This last parameter could represent the root spacing or mesh spacing of the exopolymers. This study aims to clarify s by obtaining force values as a function of temperature. The data suggest that s represents the mesh spacing. If s is the root spacing it should remain constant regardless of the changing polymer lengths, on the other hand if it is the mesh spacing it will vary with changing temperature, as shown by the data presented in this research. This knowledge will aid in understanding and characterizing how bacteria cause infections.

In natural habitats bacteria predominantly grow and survive as biofilms, which are densely populated assemblages of cells encased in self-produced matrices. Biofilms face the challenge of resource limitation due to poor substrate diffusion and consumption by cells closer to the periphery. When terminal electron acceptors for metabolism, such as oxygen, are limiting, reducing equivalents accumulate in the cell, leading to an imbalanced redox state and disruption of metabolic processes. The opportunistic pathogen Pseudomonas aeruginosa possesses various redox-balancing strategies that facilitate disposal of excess reducing power, including (i) production of phenazines, redox-active compounds that mediate extracellular electron shuttling; (ii) use of nitrate as an electron acceptor via the denitrification pathway, and (iii) fermentation of pyruvate. However, if the biofilm grows to a point where these metabolic strategies become insufficient, the community adopts a “structural” strategy: the cells collectively produce extracellular matrix to form wrinkle features, which increase surface area and oxygen availability, ultimately oxidizing (i.e., rebalancing) the cellular redox state. Though the broad physiological effects of these metabolic and structural strategies are known, details of their regulation and coordination in biofilm communities have remained elusive. The work presented in this thesis was aimed at elucidating the (cross-)regulation and coordination of different redox-balancing strategies in biofilms of P. aeruginosa strain PA14. Studies described in Chapter 2 demonstrate novel regulatory links between phenazines and microaerobic denitrification, including a redox-mediated mechanism for control of the global transcription factor Anr, which is traditionally thought to be regulated solely by oxygen. This chapter also presents observations of the spatial segregation of denitrification enzymes in a colony biofilm, which is suggestive of metabolic specialization and substrate crossfeeding between different groups of cells. Chapters 3 and 4 describe work examining the physiological functions and regulation of pyruvate and lactate metabolism in P. aeruginosa. These studies were motivated by pyruvate’s role as a “hub” for central metabolism, the unique structural biochemistry of the P. aeruginosa pyruvate carboxylase, and the intriguing complement of “lactate dehydrogenase” genes in P. aeruginosa. These genes include two that encode canonical and non-canonical respiration-linked L-lactate dehydrogenases. My results in Chapter 3 show that the non-canonical L-lactate dehydrogenase gene can substitute for the canonical one to support aerobic L-lactate utilization and that it is induced specifically by the L- enantiomer of lactate. This enzymatic redundancy for L-lactate utilization could be an adaptation that enhances virulence, given that host organisms (e.g. humans and plants) produce L-lactate but not D-lactate. In addition, Chapter 3 includes studies of pyruvate-lactate metabolism in the context of biofilm communities, where aerobic and anaerobic zones coexist in proximity. Evidence is provided that cells in biofilms have the potential to engage in crossfeeding of anaerobically generated D-lactate, which would constitute a new instance of bacterial multicellular metabolism. Finally, Chapter 4 shows that mutants of pyruvate carboxylase, which converts pyruvate to oxaloacetate, have a matrix-overproducing, hyperwrinkling biofilm phenotype indicative of an imbalanced cellular redox state. This result suggests that disruption of pyruvate carboxylase shunts metabolic flow through pyruvate dehydrogenase, converting pyruvate to acetyl-CoA and generating an excess of reducing power. Together, the findings presented in Chapter 3 and 4 underscore the importance of pyruvate metabolism in the contexts of redox homeostasis and community behavior. When metabolic strategies are insufficient to balance the redox state, biofilms can ameliorate the problem of electron acceptor limitation by forming wrinkle structures, which increase the community’s surface area-to-volume ratio. Wrinkle formation depends on the production of extracellular matrix. Matrix production is also required for the formation of pellicles, biofilms that reside at air-liquid interfaces. Experiments described in Chapter 5 investigate properties of the P. aeruginosa matrix from a socio-evolutionary perspective. My results show that matrix production confers a competitive advantage in pellicle biofilms but not in colony biofilms. The evolutionary landscape of matrix production in biofilms is complex and context-specific; i.e., each microenvironment selects for a subset of phenotypes that confers fitness only in that specific microenvironment. Chapter 6 describes the dynamic processes of pellicle formation in the gram-positive bacterium Bacillus subtilis as well as the gram-negative P. aeruginosa in a time-resolved manner. In these two distantly related species, we observed a conserved mechanism for pellicle formation that involves motility, chemotaxis and aerotaxis. These findings indicate that motility is more than just a unicellular behavior: cells collectively migrate to a microniche and initiate biofilm formation. Finally, Appendix A describes efforts to characterize proteinaceous components of the matrix isolated from P. aeruginosa PA14. In conclusion, this work has elucidated mechanistic details of various redox-balancing strategies in P. aeruginosa, particularly from the perspective of multicellular community development.

Introdução. Pseudomonas aeruginosa é um patógeno clinicamente importante. Existem diversos mecanismos de resistência aos antimicrobianos em P. aeruginosa, dentre eles a produção de enzimas (β-lactamases) e sistemas de efluxo se destacam, uma vez que são capazes de conferir resistência aos carbapenêmicos. Objetivo. Avaliar os sistemas de efluxo MexAB-OprM e MexXY em isolados clínicos de P.aeruginosa de pacientes atendidos no Hospital de Clínicas de Porto Alegre-RS e relacionar a expressão destes sistemas com a CIM de meropenem em isolados produtores e não produtores de metalo-beta-lactamases (MBL). Metodologia. Um total de 86 isolados de P. aeruginosa com suscetibilidade reduzida aos carbapenêmicos foram avaliados. A hiperexpressão dos sistemas MexAB e MexXY foi determinada fenotipicamente utilizando inibidor seletivo da bomba (PAβN). MBLs foi determinada por PCR utilizando primers específicos. Resultados. O fenótipo de hiperexpressão dos sistemas de efluxo estudados foi observado em 34 (47,8%) dos 71 isolados negativos para a produção de MBL e em 14 (93.3%) dos 15 isolados MBL positivos. Na presença de PaβN, todos os isolados não produtores de MBL apresentaram uma redução da CIM para meropenem para valores na faixa de suscetibilidade. Entretanto, das 13 P. aeruginosa produtoras de MBL que diminuíram a CIM, essa redução não foi para valores dentro da faixa de sensibilidade. Conclusão. Os isolados de P. aeruginosa não produtores de MBL apresentam resistência ao meropenem devido a hiperexpressão de MexAB-OprM. Na presença de PaβN, independente de apresentarem produção de MBL, o CIM de meropenem reduziu para valores ≤ 8 mg/L. Contudo, quando isolados não apresentavam MBL, a CIM de meropenem reduziu para níveis de sensibilidade. / Introduction. Bacterial efflux pump systems are resistance mechanisms which may lead to therapeutic failure of antibiotic treatment since many antimicrobial agents are substrate for these mechanisms. The aim of the study was to evaluate the expression of the MexAB, MexXY pump efflux and to determine its influence on meropenem MIC in carbapenemase producing and non-producing P. aeruginosa. Methods. A total of 86 non-repetitive clinical isolates of P. aeruginosa with reduced susceptibility to carbapenems were evaluated. Overexperession of MexAB and MexXY efflux systems were evaluated phenotypically using the PAβN selective inhibitor. Metallo-β-lactamases (MBL) were detected by PCR using specific primers. Results. The efflux pump-overexpressed phenotype was observed in 34 (47.8%) MBL-negative and in 14 (93.3%) MBL-positive isolates. In the presence of the PaβN, all non-producers of MBL presented a reduction of meropenem MICs to the range of susceptibility. In contrast, the 13 P. aeruginosa MBL-producing isolates decreased meropenem MICs at least 16-fold but this reduction did not reach the range of susceptibility. Conclusion. P. aeruginosa non-MBL-producing showed resistance to meropenem due to overexpression of MexAB-OprM. In the presence of PaβN, the isolates harboring or not MBL genes, the meropenem MICs were reduced to values ≤8 mg/L. However, when the isolates harbor MBL genes, the meropenem MIC values were reduced to the susceptibility.

Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.

No description available.

Determina la frecuencia de los genes blaIMP, blaVIM y blaNDM productores de Metalo-ß-lactamasas en aislamientos de Pseudomonas aeruginosa no sensibles a carbapenemes. Recolecta 149 aislamientos de Pseudomonas aeruginosa procedentes de muestras clínicas de los siguientes nosocomios: Hospital Nacional Daniel A. Carrión, Hospital Nacional Edgardo Rebagliati Martins – ESSALUD, Hospital Alberto Sabogal Sologuren – ESSALUD, Hospital General Fuerza Aérea Peruana (FAP) y el Hospital Nacional Docente Madre Niño San Bartolomé. Los aislamientos se almacenaron en el cepario del NAMRU-6 desde julio del 2010 hasta julio del 2012. Realiza el perfil de susceptibilidad a antibióticos mediante el método de “Kirby – Bauer” de acuerdo a los lineamientos del CLSI (Clinical Laboratory Standars Institute). La detección fenotípica de MBL se realizó mediante el método de aproximación de discos de EDTA (Ácido etilendiaminotetraacetico) y la detección de los genes blaIMP, blaVIM y blaNDM mediante un PCR multiplex. Encuentra que en 28 aislamientos de Pseudomonas aeruginosa se detecta la presencia del gen blaIMP mediante PCR, siendo la frecuencia de este gen 18.8%. No se detectaron los genes blaVIM y blaNDM. Concluye que la detección de los genes productores de MBL por PCR permitió detectar la frecuencia real de los aislamientos de Pseudomonas aeruginosa productores de MBL. / Tesis