The pulmonary infection with Pseudomonas aeruginosa is considered as one of the main causes of health deterioration in cystic fibrosis patients (CF). Efficient management of P. aeruginosa in CF remains difficult mainly with the emergence of multidrug-resistant strains leading ultimately to death. There is a pressing need for new approaches to control these Pseudomonal infections. Current studies on the antimicrobial efficacy of liposomal antibiotics have shown conflicting results. We sought to assess whether the incorporation of clarithromycin into liposomes could improve its antibacterial activity against clinical isolate of Pseudomonas aeruginosa from CF patients. Different formulations of liposomal clarithromycin were prepared, characterized and their antibacterial activities against resistant strains of P. aeruginosa were investigated. These formulations reduced the biofilm formation, the virulence factors production and the bacterial motilities compared to free drug. The therapeutic importance of liposome containing macrolides in the management of experimental pseudomonal lung infection in animals is warranted.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.

Pseudomonas aeruginosa is an opportunistic human pathogen which, like many other Gram negative pathogens, employs quorum sensing - regulated virulence factors to establish an infection in its host. Quorum sensing in P. aeruginosa populations is controlled by low molecular weight (hapten) signalling molecules known as homoserine lactones (HSLs). Blocking bacterial communication using antibodies is an attractive strategy for infection control as QS takes a central role in P. aeruginosa infections, and antibodies can recognise their targets with exquisite specificity. There are two well-studied QS circuits in P. aeruginosa- the Las system and the Rhl system, controlled by two autoinducers compounds, 3-oxo-C12-HSL and C4-HSL respectively. Antibodies raised against HSL compounds can reduce the expression of virulence factors controlled by QS circuit and the immunomodulatory effects of 3- oxo-C12-HSL. In order to generate antibodies with high sensitivities against the autoinducer compounds of P. aeruginosa, a panel of HSL compounds was synthesised, conjugated to the carrier protein and used for sheep immunisation. High specificity anti-HSL antibodies were isolated from an immunised sheep antibody repertoire using phage display technology. These phage antibody hits were converted into single chain antibody (scAb) format, which possessed a HuCκ gene for detection and 6x histidine tag for purification. Soluble scAbs expressed in E. coli were purified and characterised using ELISA. Unique clones showing high sensitivity for free HSL compounds were reformatted into sheep-mouse chimeric IgGs, expressed transiently in COS 7 cells and characterised using biochemical assays. These cross-reactive monoclonal antibodies were shown to recognise HSL compounds in low nanomolar concentrations and have the potential to reduce virulence gene expression in P. aeruginosa.

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.

Thesis (Ph.D.)--McMaster University (Canada), 1992. / Source: Dissertation Abstracts International, Volume: 54-08, Section: B, page: 4119. Adviser: D. B. MacLean.

Diss. Umeå : Umeå universitet, 2005.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 6 uppsatser.