Publicação
Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs
| Resumo: | Cystic Fibrosis (CF) is a genetic disorder in which a defect in the cystic fibrosis conductance regulator (CTFR) occurs. This disorder affects many organs, the airways being one of the most affected. Patients with CF lung disease are very susceptible to chronic infections by various pathogens, one of them being Pseudomonas aeruginosa. This pathogen has a high ability of adaptation to the harsh environment found in CF lungs, characterized by inflammatory defences, antibiotic treatments, oxygen restriction, and poor availability of nutrients, among others. It is well established that once P. aeruginosa infection is installed in the lungs, it is almost impossible to eradicate. P. aeruginosa adaptation is achieved by some sophisticated mechanisms, as resistance to antibiotics, hypermutability, biofilm mode of growth, differential expression of virulence factors, and according to the stage of infection in the lungs and other genotypic and phenotypic changes. With the onset of chronic CF, P. aeruginosa diversifies into different morphotypes, with the emergence of mucoid phenotype and small colony variants (SCV) that are known to persist after continuous antibiotic treatment. P. aeruginosa adaptation under microaerophilic and anaerobic conditions was assessed in terms of growth kinetics, colony morphology, mutants frequency, pyocyanin production, antibiotic susceptibility, sub-population characterization and expression of mexA and 16S genes. Results showed, in both microaerophilic (10% CO2 and 5 % O2) and anaerobic environment, similar growth profiles and pyocyanin production. The emergence of intermediate susceptibility profiles by different strains for Ticarcilin/clavulanic acid, ciprofloxacin and imipenem was observed, as well as hypermutability, with anaerobic conditions. Phenotypic diversification was also observed, under all conditions, particularly in microaerophilic conditions with 5 % O2, with the appearance of morphotypes not identified before. Sub-populations characterization shown similar characteristics between morphotypes, with one morphotype standing out in terms of biofilm formation and antibiotic susceptibility. Results obtained with the regard of the effects of the decrease in oxygen tension on gene expression showed high levels of contamination of the RNA samples collected through the study to assess differential expression of mexA gene at different oxygen concentration. Also, it was found that both pair of primers used (16S and mexA) were not adequate for the tested strain, which translate in a lack of specificity and efficiency. Given the results of characterization of growth and phenotypic characteristics, is possible to conclude that oxygen depletion has no significant effect on P. aeruginosa growth, affecting, however, the phenotypic characteristics and antibiotic susceptibility profiles. Oxygen depletion increases the presence of intermediate resistant profiles, and strong mutator phenotypes, as observed in chronic infections. Microaerophilic environment seems to be a turning point of the stage of infection, as some distinctive characteristics were observed, as higher colony diversification, appearance of new sub-populations and slight decrease in antibiotic resistance profiles. In future studies of expression of mexAB-OprM efflux pumps, a more suited, or more than one, reference genes should be selected, as well as specific design of mexA primers for the strains used will be required. It is also recommended the treatment of RNA samples with acid phenol:chlorophorm to reduce possible DNA contaminations. |
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| Autores principais: | Vaz, Raquel Alexandra Palas |
| Assunto: | Engenharia e Tecnologia::Biotecnologia Industrial |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Cystic Fibrosis (CF) is a genetic disorder in which a defect in the cystic fibrosis conductance regulator (CTFR) occurs. This disorder affects many organs, the airways being one of the most affected. Patients with CF lung disease are very susceptible to chronic infections by various pathogens, one of them being Pseudomonas aeruginosa. This pathogen has a high ability of adaptation to the harsh environment found in CF lungs, characterized by inflammatory defences, antibiotic treatments, oxygen restriction, and poor availability of nutrients, among others. It is well established that once P. aeruginosa infection is installed in the lungs, it is almost impossible to eradicate. P. aeruginosa adaptation is achieved by some sophisticated mechanisms, as resistance to antibiotics, hypermutability, biofilm mode of growth, differential expression of virulence factors, and according to the stage of infection in the lungs and other genotypic and phenotypic changes. With the onset of chronic CF, P. aeruginosa diversifies into different morphotypes, with the emergence of mucoid phenotype and small colony variants (SCV) that are known to persist after continuous antibiotic treatment. P. aeruginosa adaptation under microaerophilic and anaerobic conditions was assessed in terms of growth kinetics, colony morphology, mutants frequency, pyocyanin production, antibiotic susceptibility, sub-population characterization and expression of mexA and 16S genes. Results showed, in both microaerophilic (10% CO2 and 5 % O2) and anaerobic environment, similar growth profiles and pyocyanin production. The emergence of intermediate susceptibility profiles by different strains for Ticarcilin/clavulanic acid, ciprofloxacin and imipenem was observed, as well as hypermutability, with anaerobic conditions. Phenotypic diversification was also observed, under all conditions, particularly in microaerophilic conditions with 5 % O2, with the appearance of morphotypes not identified before. Sub-populations characterization shown similar characteristics between morphotypes, with one morphotype standing out in terms of biofilm formation and antibiotic susceptibility. Results obtained with the regard of the effects of the decrease in oxygen tension on gene expression showed high levels of contamination of the RNA samples collected through the study to assess differential expression of mexA gene at different oxygen concentration. Also, it was found that both pair of primers used (16S and mexA) were not adequate for the tested strain, which translate in a lack of specificity and efficiency. Given the results of characterization of growth and phenotypic characteristics, is possible to conclude that oxygen depletion has no significant effect on P. aeruginosa growth, affecting, however, the phenotypic characteristics and antibiotic susceptibility profiles. Oxygen depletion increases the presence of intermediate resistant profiles, and strong mutator phenotypes, as observed in chronic infections. Microaerophilic environment seems to be a turning point of the stage of infection, as some distinctive characteristics were observed, as higher colony diversification, appearance of new sub-populations and slight decrease in antibiotic resistance profiles. In future studies of expression of mexAB-OprM efflux pumps, a more suited, or more than one, reference genes should be selected, as well as specific design of mexA primers for the strains used will be required. It is also recommended the treatment of RNA samples with acid phenol:chlorophorm to reduce possible DNA contaminations. |
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