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

Bacterial biofilm infection is a common (~ 2 to 4%) complication for recipients of surgically implanted medical devices. Due to the tremendous increase in antibiotic resistance when these bacteria enter the biofilm phenotype, present treatment requires explantation and replacement of the device, often with multiple surgeries and always with much longer patient recovery time. The specific objective of this study was to quantify the degree of biofilm deactivation from exposure to thermal shock for varying temperature and time durations. While extreme temperature (>150˚C) is routinely used to sterilize (e.g. autoclaves), such temperatures have a severe cost within the body. Despite extensive studies on thermal deactivation of bacteria in the planktonic phenotype over a wide range of temperatures (e.g., pasteurization protocols), surprisingly little is known about the thermal deactivation of biofilms except under extreme conditions. Here, the deactivation of Pseudomonas aeruginosa biofilms is reported. These biofilms were cultured at 37°C for 24 hours in a drip-flow reactor and subjected to heat shocks on the range of 50°C to 80°C for durations of 1 to 30 minutes. Heat shocks were delivered by immersion in thermostatted media for the prescribed time and the resulting concentration of colony forming units (CFU/mL) were quantified using direct enumeration. Up to 6.6 orders of magnitude reduction in CFU concentration was observed, indicating that thermal deactivation is a reasonable approach to biofilm mitigation. Integrating this approach with a magnetic nanoparticle implant coating will result in an innovative treatment for implant infections in situ without explantation or device replacement.

Upon forming a biofilm, bacteria undergo several changes that prevent them from being eradicated with antimicrobials alone. These biofilms manifest as persistent infections and biofouling in the medical and industrial world, respectively, constituting an ongoing medical crisis and creating a huge financial burden. Biofilms on implanted medical devices cause thousands of patients each year to undergo multiple surgeries to explant and replace the implant, driving billions of dollars in increased health care costs due to the lack of viable treatment options for in situ biofilm eradication. Heat has been used to reliably eliminate biofilms for many years, but the temperatures employed are infeasible for many applications, particularly in vivo medical treatment. Remotely activated localized heat can be applied through a superparamagnetic iron oxide nanoparticle polymer coating when paired with an alternating magnetic field. However, there is very little known about the temperatures required to kill the biofilms and the effects of the heat in conjunction with antibiotics. To better understand the required parameters to effectively kill off bacteria in biofilms a variety of heat treatments were investigated for a variety of Pseudomonas aeruginosa biofilms grown in different conditions. Additionally, these heat treatments were combined with antibiotics to better understand any combined effects of the two orthogonal treatment plans. It was found that heat is an effective method for killing the bacteria in biofilms. Temperatures ranging from body temperature, 37 °C, to 80 °C were used to heat shock the biofilms for 1 to 30 minutes. Higher temperatures for short exposure times yielded similar results to lower temperatures for longer exposure time. Biofilms grown in different conditions did vary in their susceptibility to the heat shocks; however, at the higher temperatures the differences became negligible. Therefore, the more effective treatments were the higher temperature heat shocks with shorter exposure times to maximize bacterial cell death and minimize the potential heat transfer to the surrounding tissue. Regrowth studies indicate a critical post-shock bacterial loading (~103 CFU/cm2) below which the biofilms were no longer viable, while films above that loading slowly regrew to their previous population density. Combined treatments with antibiotics had synergistic effects for all antibiotics across a window of heat shock conditions. Erythromycin in particular, which showed no effect on the biofilm alone, decreased biofilm population by six orders of magnitude at temperatures which had no effect in the absence of antibiotics. These studies will evolve the understanding of biofilms and how to efficiently eradicate them on implant surfaces. The introduction of such a novel coating in conjunction with antibiotics could obviate thousands of surgeries and save billions of dollars spent on explantation, recovery, and re-implantation.

Pseudomonas aeruginosa bacterial biofilms are the leading cause of mortality among cystic fibrosis (CF) patients. Biofilms contain bacteria attached to a surface and encased in a protective matrix. Since bacteria within a biofilm are less susceptible to antibiotics, a new approach is to use dispersion compounds that cause the biofilms to release free-swimming bacteria. Our approach has focused on combining nutrient dispersion compounds with antibiotics to increase eradication of bacteria within biofilms. This approach takes advantage of the enhanced susceptibility of free-swimming bacteria to antibiotics, compared to bacteria within biofilms. Ultimately, this research will guide the development of an aerosol therapy containing both antibiotic and dispersion compounds to treat bacterial biofilm infections. To study the effect of antibiotic and dispersion compound treatments on biofilm eradication, a high-throughput screening assay was used to assess the effect on young Pseudomonas aeruginosa biofilms. In addition, a Lab-Tek chambered coverglass system imaged via confocal microscopy was used to assess the effect on mature Pseudomonas aeruginosa biofilms. Seven antibiotics (amikacin disulfate, tobramycin sulfate, colistin sulfate, colistin methanesulfonate (CMS), polymyxinB sulfate, erythromycin, and ciprofloxacin hydrochloride) were tested alone or in combination with four nutrient dispersion compounds (sodium citrate, succinic acid, xylitol, and glutamic acid) to assess the level of eradication of bacteria within biofilms. For young biofilms, 15 of 24 combinations significantly eliminated more live bacteria within the biofilms (measured in colony forming units per milliliter) compared to antibiotics alone. In the more mature biofilm system, only 3 out of 26 combinations resulted in a higher percentage of live biofilm bacteria being eliminated compared to antibiotics alone, showing the importance of biofilm age in the effectiveness of these potential combination therapies. To aid in confocal microscopic analysis of biofilms, an automated quantification program called STAINIFICATION was developed. This new program can be used to simultaneously investigate connected-biofilm bacteria, unconnected bacteria (dispersed bacteria), the biofilm protective matrix, and a growth surface upon which bacteria are grown in confocal images. The program contains novel algorithms for the assessment of bacterial viability and for the quantification of bacteria grown on uneven surfaces, such as tissue. The utility of the viability assessments were demonstrated with confocal images of Pseudomonas aeruginosa biofilms. The utility of the uneven surface algorithms were demonstrated with confocal images of Staphylococcus aureus biofilms grown on cultured human airway epithelial cells and Neisseria gonorrhoeae biofilms grown on transformed cervical epithelial cells. Finally, a proof-of-concept study demonstrated that dry powder aerosols containing both antibiotic and nutrient dispersion compounds could be developed with properties optimized for efficient deposition in the lungs. A design of experiments study showed that solution concentration was the most significant parameter affecting aerosol yield, particle size, and in vitro deposition profiles. Collectively this work demonstrated that bacterial dispersion from biofilms can enhance antibiotic susceptibility and can be better quantified using the new STAINIFICATION software. Formulation of dispersion compounds and antibiotics into a dry powder aerosol could enable more effective treatment of biofilm infections in the lungs.

Pseudomonas aeruginosa (P. aeruginosa) is a frequently isolated pathogen that causes septicaemia and chronic respiratory infection. It exhibits a higher mortality rate than other gram-negative bacteria and the need for effective immunotherapy is emphasised by the frequency of antibiotic resistance associated with this organism. Mucosal immunisation with a whole killed cell P. aeruginosa vaccine has previously demonstrated a significant immune response in both rodent studies and human trials. This study is a continuation of that research, with the major goal being the identification of a purified protein antigen that could form the basis of a mucosal vaccine against P. aeruginosa. Specifically, the aims of this study were the development of purification protocols for the isolation of previously untested protein antigens, assessment of the efficacy of these antigens to enhance bacterial clearance in an animal model of acute respiratory infection, determination of the immune parameters that are associated with the resolution of P. aeruginosa respiratory infection and finally, cloning of an identified antigen which demonstrated vaccine efficacy. Protocols were established to isolate proteins for use as antigens in immune response studies. The proteins purified in this study were Pa 13, Azurin, acyl carrier protein (ACP), Amidase, Aminopeptidase, KatA and Pa70. These proteins were used to immunise rats by intestinal intra-Peyer's patch (IPP) inoculation and intratracheal (IT) boost. The immunisation protocol employed was designed to target mucosal antigen-specific immune responses where the route of immunisation, Peyer's patch (PP) intestinal inoculation, is akin to the oral delivery of antigens to the gut-associated lymphoid tissue (96). Investigations of a previously uncharacterised antigen, Pa60, later identified this protein as the P. aeruginosa catalase, KatA. This study demonstrated enhanced bacterial clearance of both homologous and heterologous challenge following immunisation with KatA. The level of clearance demonstrated by KatA was promising when compared to that of killed whole cell immunisation. KatA was cloned and studies with the recombinant protein showed enhanced bacterial clearance commensurate with that of the native protein. Immunisations with other proteins identified four additional antigens which enhanced bacterial clearance; Pa13, Pa40, Pa45 and Pa70. Amino acid sequence analysis indicated that Pa13 may be a novel protein, whereas Pa40 was determined to be amidase and Pa45, aminopeptidase. Pa70 was not successfully sequenced. These proteins were effective in significantly enhancing bacterial clearance of homologous P. aeruginosa challenge. For KatA, Pa13 and Pa70, clearance was associated with a marked phagocytic cell recruitment. In contrast, amidase and aminopeptidase demonstrated clearance with a minimal cellular response. Proteins; azurin and ACP were non-protective, failing to clear a live P aeruginosa challenge. Analysis of the antigen-specific responses of these nonprotective proteins and comparison with those antigens which enhanced bacterial clearance were used to determine factors that may contribute to the resolution of an acute pulmonary infection. The study has demonstrated that mucosal immunisation using purified protein antigens can enhance the clearance of pulmonary infection with P. aeruginosa. It has also contributed to the understanding of immune responses to newfound antigens of P. aeruginosa and identified antigen-specific responses which confirm their potential as vaccine candidates.