Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients
| Autor: | Kevin M. Guttman, Lindsay Jackson, Amanda J Morris, Valerie Waters, William H. DePas, Yvonne C. W. Yau |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
medicine.drug_class General Chemical Engineering Antibiotics medicine.disease_cause Cystic fibrosis General Biochemistry Genetics and Molecular Biology Microbiology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine medicine DAPI General Immunology and Microbiology medicine.diagnostic_test Pseudomonas aeruginosa business.industry General Neuroscience Biofilm medicine.disease Staining 030104 developmental biology chemistry Sputum medicine.symptom business 030215 immunology Fluorescence in situ hybridization |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61631 |
| Popis: | Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy. |
| Databáze: | OpenAIRE |
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