Tesi sul tema "Pyocyanin"

Sepsis is a leading cause of death among critically ill patients that results in metabolic alterations including hypercatabolism, lipoatrophy, and muscle wasting, contributing to the development of cachexia. Septic cachexia is associated with loss of body weight, fat mass, and lean mass and dysregulated immune function. There are currently no efficacious treatment strategies for septic cachexia, and nutritional interventions have limited success in preventing hypercatabolic wasting. Pyocyanin is a virulence factor produced by sepsis-causing Pseudomonas aeruginosa that has been shown to activate the aryl hydrocarbon receptor (AhR), increase inflammation, and produce reactive oxygen species. Thus, pyocyanin represents a novel mechanistic target in the development of septic cachexia. In Aim 1, we hypothesized that pyocyanin reduces adipocyte differentiation and activates AhR in vitro and in vivo. In vitro, pyocyanin reduced differentiation of 3T3-L1 cells to adipocytes and promoted expression of proinflammatory cytokines. These effects were associated with activation of AhR. We established an in vivo model of pyocyanin-induced cachexia using repeat intraperitoneal exposure to pyocyanin in male and female C57BL/6J mice. Acutely, pyocyanin reduced differentiation of stem cells isolated from adipose stromal vascular tissue and augmented expression of proinflammatory cytokines. Chronically, pyocyanin reduced body weight and fat mass, which was associated with adipose-specific AhR activation. Pyocyanin had sexually dimorphic effects on lipolysis and adipocyte inflammation. These data suggest a role of pyocyanin in adipose cachexia associated with sepsis. In Aim 2, we hypothesized that pyocyanin activates adipocyte AhR to promote adipose tissue wasting and cachexia. To test this hypothesis, we used a mouse model of adipocyte-specific deficiency of AhR and chronically administered pyocyanin to male and female mice. In male mice with adipocyte AhR deficiency, effects of pyocyanin to promote adipose wasting and cachexia were attenuated. In contrast, female adipocyte AhR deficient mice had an augmented response to pyocyanin to decrease body weight. Results suggest divergent mechanisms of pyocyanin to regulate adiposity and body weight through adipocyte AhR between male and female mice. These data support a role for pyocyanin in the development of adipose cachexia associated with Pseudomonas aeruginosa sepsis that is partially regulated by adipocyte AhR. Targeting pyocyanin’s effects on adipocytes represents a potentially novel therapeutic approach for septic cachexia that could mitigate septic cachexia, a condition associated with increased risk of mortality in this population.

Arginine utilization in P. aeruginosa as the source of carbon, nitrogen, and energy is controlled by a complicated regulatory mechanism. While ArgR in the presence of exogenous arginine is required for auto-induction of the aotJQMOP-argR operon for arginine uptake and regulation, this operon is subjected to carbon catabolite repression via an unknown mechanism. This study demonstrated that succinate exerted a stronger repression effect than glucose or pyruvate on arginine induction of an aotJ::lacZ fusion in wild type PAO1. Expression of the aotJ::lacZ fusion was analyzed against three different backgrounds, cbrAB, crc, and vfr, that have been suggested to play a role in carbon catabolite repression. These mutations exerted a negative effect on the arginine-responsive induction to different extents, with the order of cbrAB > vfr > crc. A series of aotJ::lacZ fusions with deletions in the aotJ regulatory region were constructed and the effect of exogenous arginine examined in the argR mutant and its parent strain. The results indicated that a 250-bp sequence upstream of the ArgR operator is required for the optimal induction of the operon by exogenous arginine, and revealed the presence of a cryptic promoter (P0) in this region. Electromobility shift assays with crude cell-free extracts of PAO1 revealed that a DNA-binding protein other than ArgR binds to the aforementioned 250-bp region. Through reverse genetics, two regulatory proteins MvaT and MvaU were identified and specifically interacted with the aotJ-argR regulatory region. The MvaT/MvaU are involved in the regulation of the P0 promoter. The importance of MvaT and MvaU for bacterial growth was supported by the notion that no true mvaT mvaU double knockout mutant can be constructed. This is the first case to characterize the growth phenotypes of quasi-mvaT mvaU double mutants complemented with fusions for arginine-inducible expression of mvaT or mvaU. Further analysis of the physiological significance of MvaT and MvaU revealed their involvement in swarming response and pyocyanin synthesis. The defect in pyocyanin synthesis was correlated to the diminished level of PQS, an important chemical signal compound in the quorum sensing network of P. aeruginosa.

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CNPq
Pseudomonas aeruginosa é uma bactéria Gram negativa, oportunista e ubíqua, frequentemente associada a infecções graves em pacientes imunocomprometidos. Em razão do aumento de resistência dessa bactéria aos múltiplos antimicrobianos, surgem à preocupação e a procura por novas alternativas terapêuticas, com as substâncias bioativas de origem natural representando uma importante fonte para obtenção desses medicamentos. O objetivo do presente estudo foi determinar a atividade sinérgica do timol e agentes antimicrobianos frente a cepas de Pseudomonas aeruginosa multirresistentes e avaliar os efeitos dessa interação sobre a biossíntese de biofilme e de piocianina. Para isso, numa primeira etapa foi determinada a concentração inibitória e bactericida mínima do timol e de antimicrobianos (Polimixina B, ceftazidima, piperacilina/tazobactam, cefepima, ciprofloxacino e meropenem) frente a dez cepas de Pseudomonas aeruginosa. O estudo da interação entre o timol e os agentes antimicrobianos foi realizado pelo método do tabuleiro de xadrez. Os critérios utilizados para avaliar a atividade sinérgica foram definidos pelo Índice da Concentração Inibitória Fracionada (FIC índex). A partir dos melhores valores do FIC índex das associações timol/antimicrobiano foram avaliadas a atividade sobre a produção de biofilme e piocianina. Três cepas (LFBM 01, LFBM 02, LFBM 16) apresentaram um perfil de resistência ao meropenem e cefepima e um efeito sinérgico foi observado entre o timol e meropenem ou cefepima sobre essas cepas. A associação timol/cefepima inibiu a biossíntese do biofilme em até 99,76%, e a associação timol/meropenem mostrou ser mais eficaz na inibição da piocianina cujos valores foram de até 84,33%. O timol associado ao meropenem ou cefepima, age sinergicamente, inibindo cepas de Pseudomonas aeruginosa multirresistentes e interferindo na biossíntese de biofilme e piocianina.
Pseudomonas aeruginosa is a Gram negative bacteria, opportunistic and ubiquitous, often associated with severe infections in immunocompromised patients. Due to the increased resistance of the bacteria to multiple antibiotics, there are the concerns and the search for new therapeutic alternatives, with the bioactive substances of natural origin represents an important source for obtaining these drugs. The aim of this study was to determine the synergistic activity of thymol and antimicrobials agents multiresistant Pseudomonas aeruginosa strains and evaluate the effects of this interaction on the biofilm biosynthesis and pyocyanin. For this, a first step was determined and the minimum inhibitory concentration of thymol and bactericidal antibiotics (polymyxin B, ceftazidime, piperacillin / tazobactam, cefepime, ciprofloxacin and meropenem) compared to ten strains of Pseudomonas aeruginosa. The study of the interaction between the thymol and antimicrobial agents was carried out by the checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Index of Fractional Inhibitory Concentration (FIC index). From the best FIC index values of associations thymol / antimicrobial activity were evaluated on the production of biofilm and pyocyanin. Three strains (LFBM 01, LFBM 02, LFBM 16) showed an meropenem resistance profile and cefepime and a synergistic effect was observed between the thymol and meropenem or cefepime on these strains. The thymol / cefepime combination inhibited biofilm biosynthesis up to 99.76%, and thymol association / meropenem was more effective in inhibiting pyocyanin with values of up to 84.33%. The thymol associated with meropenem or cefepime, acts synergistically by inhibiting multidrug-resistant Pseudomonas aeruginosa strains and interfering in the biosynthesis of biofilm and pyocyanin.

La pyocyanine (PYO) est une phénazine de couleur bleu-vert, produite spécifiquement par la bactérie pathogène opportuniste Pseudomonas aeruginosa (Pa). La toxicité aérobie de la PYO envers les cellules de mammifères, les levures et les bactéries a été décrite de longue date, mais la compréhension des mécanismes d'action est encore lacunaire, en particulier en conditions de limitation en O2 (conditions rencontrées dans le contexte infectieux). De plus, il a récemment été montré que la PYO peut apporter des effets bénéfiques pour la souche productrice en hypoxie. Au cours de ce travail, nous avons réexaminé les effets de la PYO sur un large panel de bactéries dont son propre producteur (Pa) ainsi que sur un modèle cellulaire eucaryote Saccharomyces cerevisiae exposées à différentes tensions en O2. Nos données suggèrent que la toxicité aérobie de la PYO envers S. cerevisiae est multifactorielle, impliquant à la fois une interaction avec le complexe III de la chaîne respiratoire et l'induction d'un stress oxydatif. Pour la première fois, nous avons mis en évidence une toxicité de la PYO exacerbée en anaérobiose chez un eucaryote (S. cerevisiae). Le mécanisme d'action impliquerait le PYO radical. Nous avons également montré que la PYO peut inhiber la croissance aérobie et anaérobie des microorganismes concurrents, plus particulièrement S. aureus en bloquant le complexe III de la chaîne respiratoire. A l'inverse, la PYO peut stimuler la respiration de Pa surtout dans les conditions mimant le contexte infectieux (hypoxie, vie ralentie). Le complexe III et/ou les oxydases terminales cbb3 serait impliqué favorablement. En conclusion, la PYO jouerait à la fois un rôle de poison hypoxique mais aussi un rôle de navette redox bénéfique pour la survie et la virulence de Pa en hypoxie.

Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

Doctor of Philosophy(PhD)
Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

Thesis (Ph.D. in Cancer Biology) -- University of Colorado at Denver and Health Sciences Center, 2006.
Typescript. Includes bibliographical references (leaves 112-124). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las.
Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent.

The opportunistic pathogen Pseudomonas aeruginosa excretes redox-active small molecules called phenazines. This thesis addresses the possibility that the phenazine pyocyanin acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined by comparing the physiological status of the wild type to a mutant defective in phenazine production. Quantification of intracellular NADH and NAD+ pools revealed a more reduced intracellular redox state in the phenazine-null mutant compared to the wild type, consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of culture metabolites showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways.

We set out to identify mechanisms allowing P. aeruginosa to catalyze pyocyanin redox cycling. Through a genetic screen, we found two loci required for full pyocyanin-dependent ferric citrate reduction activity in P. aeruginosa PA14: (1) the gene gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) the operon fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA had oxidized cytoplasms and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, mutants lacking a functional cytochrome bc1 complex produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction.

Pyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form wrinkled (rugose) colonies, while the wild type forms smooth colonies. Using this colony biofilm assay, we showed that the ΔgpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in stimulating smooth colony formation. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results imply that rugosity is an adaptation to electron acceptor limitation.

The work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa, mechanisms controlling intracellular redox state in bacteria, and mechanisms that may contribute to P. aeruginosa virulence.

碩士
輔英科技大學
醫學檢驗生物技術系碩士班
100
Pseudomonas aeruginosa is an aerobic Gram-negative bacterial species and widely present in terrestrial, freshwater and marine environments that causes several opportunistic human infections. One important virulence factor, pyocyanin (PYO), is a blue-green pigment produced by Pseudomonas aeruginosa. Recent study demonstrated that production of pyocyanin by Pseudomonas aeruginosa was inhibited in saline stress condition but the mechanisms remain unknown. The major difference between freshwater and sea water is the constituents of salts. This study investigated the effects of salinity on bacterial growth, production of pyocyanin, mRNA expression of relevant genes, and protein-profile in Pseudomonas aeruginosa. The results indicated that Pseudomonas aeruginosa grew well in all conditions including media either with or without supplemented salts. However, the growth curves showed a log-phase-delay in media prepared with various concentrations of saline water. Furthermore, the pyocyanin was extracted and quantified. The results showed that the pyocyanin production of Pseudomonas aeruginosa cultured in medium with supplemented artificial sea water were 17% to 21% (24 h to 72 h) of those without supplemented salts. The decreases of pyocyanin were proportional to the concentrations of NaCl. Additionally, this study assessed the mRNA expression of phzM, phzS, and phz operon which are related to pyocyanin biosynthesis. The mRNA was measured using qRT-PCR. The results showed that decrease of pyocyanin was due to low mRNA expression of phzM, phzS, phzE, phzD, phzF, phzA1/ phzB1 and phzA2/phzB2 in artificial sea water. Finally, the effects of saline water on protein expression were analyzed by 2D gel electrophoresis and MALDI-TOF-TOF. The protein profiles for Pseudomonas aeruginosa cultured in media either with or without supplemented artificial sea water were compared. In conclusion, these results allow us to understand the molecular mechanisms for seawater affecting the production of pyocyanin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous bacterium that is commonly isolated from soil and water. Additionally, this bacterium can cause infections in individuals with compromised immune systems and in those with underlying debilitating conditions. Individuals with cystic fibrosis, burn wounds, AIDS and diabetes are more likely to being infected by P. aeruginosa than healthy individuals. In individuals with CF, there is a marked increase in the accumulation of lung mucus that serves as a source of nutrition for P. aeruginosa and other bacterial species resulting in chronic and often fatal infections. While CF lung infections are initially caused by more than one species of bacteria, over time P. aeruginosa emerges as the dominant species. P. aeruginosa also causes chronic infections in association with other bacteria in wounds. Microbes within these infections are engaged in complex interactions with each other. Often, these interactions are synergistic resulting in infections that are recalcitrant to antimicrobial therapy. While many studies have documented the occurrence of synergistic polymicrobial infections, little is known about the molecular mechanisms prevailing in these infections. Interestingly, production of virulence factors by P. aeruginosa has been shown to correlate with the presence of specific nutrients in their growth environment. Expanding on the idea of available nutrients affecting virulence, I demonstrate the ability of N-Acetylglucosamine (GlcNAc) and GlcNAc-containing polymers such as peptidoglycan to induce production of virulence factors in P. aeruginosa. Peptidoglycan shed by Gram-positive bacteria acts as a cue for P. aeruginosa in polymicrobial environments, to enhance production of virulence factors. In the context of a polymicrobial infection, this results in enhanced pathogenesis. Here, I provide insights into mechanisms influencing such interspecies interactions between the opportunistic pathogen Pseudomonas aeruginosa and S.aureus.
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Biofilms occur when microorganisms attach to surfaces, replicate and secrete dense polymers to the extent where the organisms become physically encapsulated. As biofilms often arise where there is a high level of diversity of microbial species, there is a high-likelihood that biofilms will contain more than one species of microorganism. These mixed-species biofilms have been shown to be advantageous to either one or multiple species within these biofilms. Pseudomonas aeruginosa and Escherichia coli are two bacteria of interest that are commonly involved in infections of humans and are known biofilm-producers that have generated dual-species biofilms in vitro. In recent years it has been shown that the formation of mixed-species biofilms of these two bacterial species is beneficial to either one or both bacteria in terms of adherence to surfaces, formation of the biofilm matrix, viable cell concentrations within mixed-species biofilms and resistance to external threats in the form of synthetic antimicrobial agents. The interactions between these two organisms in large part had been attributed to secretion of quorum-sensing signals and others molecules produced as a by-product of metabolic processes. This study investigated how the cell-signalling molecules, indole produced by E. coli and n-acyl homoserine lactones, n-butyryl homoserine lactone (C4-HSL) and n-3-oxo-dodecanoyl (3-oxo-C12-HSL) produced by P. aeruginosa would affect interactions between these organisms within mixed-species biofilms. Previous work had suggested that cell signally molecules would result in cross species effects. The work conducted within this study had found that n-acyl homoserine lactones had no discernible effect on E. coli biofilm formation and that indole did not have a strong influence over biofilm and production of the metabolic by-product and virulence factor pyocyanin, produced by P. aeruginosa PaO1. Pyocyanin however produced within mixed-species biofilms was shown to reduce viable wild-type E. coli K-12 to similar concentrations to that of an indole-deficient strain. The results within this thesis also found that reducing the incubation temperature increased biofilm formation of E. coli. For instance, viable cell retention increased from 2.20x 105 cfu at 37ºC to 8.73x107 cfu at 25ºC within 72 hours whereas total biofilm production increased from an absorbance reading of 0.22 at 37ºC to an absorbance reading of 0.98 at 30ºC within 24 hours. Reductions in pyocyanin in P. aeruginosa PaO1 cultures also coincided with reductions in temperature thus reducing the temperature from 37ºC to 25ºC reduced pyocyanin concentrations of P. aeruginosa PaO1 cultures, grown for 72 hours from 149.34 μM to 23.10 μM. A result of reducing the incubation temperature of mixed-species biofilm cultures, other than noticeable reductions in concentrations of pyocyanin, was an increase in E. coli cell recovery. For instance, within 72 hours E. coli viable cell recovery increased from 1.9x102 cfu to 2.0 x105 cfu when the temperature was reduced from 37ºC to 25ºC. This indicated that E. coli within mixed-species biofilms alongside P. aeruginosa in an enclosed system was more likely to survive at lower temperatures than higher temperatures ergo are more likely to survive within mixed-species biofilms outside rather than inside of the human body with a core temperature of approximately 37ºC. This highlights a potential issue where the bacteria contained within these biofilms that have been shown in some instances to be more resistant to antimicrobials than single-species biofilms, have a higher likelihood of remaining viable for an extended period of time outside of the host reservoir. Increasing the duration of viability of E. coli and P. aeruginosa in mixed-species biofilms in areas that are prone to containing biofilms and are commonly associated with the presence of human beings can potentially increase the chances of contracting these infectious agents.