Diversification of Pseudomonas aeruginosa After Inhaled Tobramycin Therapy of Cystic Fibrosis Patients: Genotypic and Phenotypic Characteristics of Paired Pre- and Post-Treatment Isolates
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Culture Media
2.2. Random Amplification of Polymorphic DNA (RAPD)–PCR Analysis
2.3. Unweighted Pair–Group Method with Arithmetic Mean (UPGMA) Analysis
2.4. Growth Curve Analysis
2.5. Biofilm-Growth Evaluation
2.6. Invasion of A549 Cells
2.7. Fluorescence Microscopy
2.8. Motility Assays
2.9. Determining the Tobramycin Susceptibility of the Strains
2.10. Phenotypic Tests on the Effects of Sub-MICs of Tobramycin
2.11. Viability of Bacteria Cultivated in the Presence of Quarter or Half MIC of Tobramycin
2.12. Scanning Electron Microscopy (SEM)
2.13. Statistical Analysis
3. Results and Discussion
3.1. Molecular Epidemiological Typing by RAPD-PCR
3.2. Growth Parameters
3.3. Biofilm Growth
3.4. Invasion and Intracellular Reproduction of the Bacteria in Cultured A549 Cells
3.5. Motility
3.6. Tobramycin Susceptibility of the Paired Strains
3.7. Phenotypic Shifts in the Strains in the Presence of Sub-MICs of Tobramycin
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Pair | Strain | Patient Age at the Time of Strain Isolation (Years) | Number of Inhaled Tobramycin Cycles |
---|---|---|---|
Pair 1 | PaT-1 | 15 | 0 |
PaT-2 | 16 | 3 | |
Pair 2 | PaT-3 | 15 | 0 |
PaT-4 | 16 | 3 | |
Pair 3 | PaT-5 | 13 | 0 |
PaT-6 | 16 | 15 | |
Pair 4 | PaT-7 | 25 | 0 |
PaT-8 | 27 | 2 | |
Pair 5 | PaT-9 | 10 | 0 |
PaT-10 | 11 | 2 | |
Pair 6 | PaT-11 | 20 | 0 |
PaT-12 | 21 | 3 |
Strain | Swimming Motility (cm) | Swarming Motility (cm) | Twitching Motility (cm) | ||||||
---|---|---|---|---|---|---|---|---|---|
Control | Quarter MIC | Half MIC | Control | Quarter MIC | Half MIC | Control | Quarter MIC | Half MIC | |
PaT-1 | 2.77 ± 0.08 | - | - | 0.5 ± 0.08 | - | - | - | - | - |
PaT-2 | 2.8 ± 0.29 | - | - | 0.375 ± 0.09 | - | - | 1 ± 0 | 0.15 ± 0.05 | - |
PaT-3 | 2.6 ± 0.33 | - | - | 0.6 ± 0.08 | - | - | 1.075 ± 0.09 | - | - |
PaT-4 | 2.7 ± 0.12 | - | - | 0.6 ± 0.08 | - | - | 1.125 ± 0.05 | 1.125 ± 0.05 | 0.825 ± 0.12 |
PaT-5 | 2.1 ± 0.21 | - | - | 0.65 ± 0.12 | 0.4 ± 0.8 | - | - | - | - |
PaT-6 | 3.5 ± 0.32 | - | - | 0.75 ± 0.23 | - | - | - | - | - |
PaT-7 | 3.75 ± 0.16 | - | - | 0.825 ± 0.05 | 0.6 ± 0.08 | - | - | - | - |
PaT-8 | 2.6 ± 0.12 | - | - | 0.625 ± 0.17 | - | - | - | - | - |
PaT-9 | 2.1 ± 0.27 | - | - | 0.45 ± 0.13 | - | - | - | - | - |
PaT-10 | 1.47 ± 0.08 | - | - | 0.125 ± 0.05 | - | - | - | - | - |
PaT-11 | 2.95 ± 0.1 | - | - | 0.85 ± 0.21 | - | - | - | - | - |
PaT-12 | 1.95 ± 0.20 | - | - | 0.374 ± 0.15 | 0.275 ± 0.05 | 0.2 ± 0.08 | - | - | - |
PA O1 | 2.5 ± 0.43 | - | - | 1.25 ± 0.28 | - | - | 2 ± 0.16 | - | - |
Strain | MIC (µg mL−1) | Susceptibility | |
---|---|---|---|
Pair 1 | PaT-1 | 0.25 | S |
PaT-2 | 0.25 | S | |
Pair 2 | PaT-3 | 0.50 | S |
PaT-4 | 0.75 | S | |
Pair 3 | PaT-5 | 0.50 | S |
PaT-6 | 1.00 | S | |
Pair 4 | PaT-7 | 0.38 | S |
PaT-8 | 0.75 | S | |
Pair 5 | PaT-9 | 1.50 | S |
PaT-10 | 1.50 | S | |
Pair 6 | PaT-11 | 0.75 | S |
PaT-12 | 1.50 | S | |
Reference | PAO1 | 1.5 | S |
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Borisova, D.; Strateva, T.; Dimov, S.G.; Atanassova, B.; Paunova-Krasteva, T.; Topouzova-Hristova, T.; Danova, S.T.; Tropcheva, R.; Stoitsova, S. Diversification of Pseudomonas aeruginosa After Inhaled Tobramycin Therapy of Cystic Fibrosis Patients: Genotypic and Phenotypic Characteristics of Paired Pre- and Post-Treatment Isolates. Microorganisms 2025, 13, 730. https://doi.org/10.3390/microorganisms13040730
Borisova D, Strateva T, Dimov SG, Atanassova B, Paunova-Krasteva T, Topouzova-Hristova T, Danova ST, Tropcheva R, Stoitsova S. Diversification of Pseudomonas aeruginosa After Inhaled Tobramycin Therapy of Cystic Fibrosis Patients: Genotypic and Phenotypic Characteristics of Paired Pre- and Post-Treatment Isolates. Microorganisms. 2025; 13(4):730. https://doi.org/10.3390/microorganisms13040730
Chicago/Turabian StyleBorisova, Dayana, Tanya Strateva, Svetoslav G. Dimov, Borjana Atanassova, Tsvetelina Paunova-Krasteva, Tanya Topouzova-Hristova, Svetla T. Danova, Rositsa Tropcheva, and Stoyanka Stoitsova. 2025. "Diversification of Pseudomonas aeruginosa After Inhaled Tobramycin Therapy of Cystic Fibrosis Patients: Genotypic and Phenotypic Characteristics of Paired Pre- and Post-Treatment Isolates" Microorganisms 13, no. 4: 730. https://doi.org/10.3390/microorganisms13040730
APA StyleBorisova, D., Strateva, T., Dimov, S. G., Atanassova, B., Paunova-Krasteva, T., Topouzova-Hristova, T., Danova, S. T., Tropcheva, R., & Stoitsova, S. (2025). Diversification of Pseudomonas aeruginosa After Inhaled Tobramycin Therapy of Cystic Fibrosis Patients: Genotypic and Phenotypic Characteristics of Paired Pre- and Post-Treatment Isolates. Microorganisms, 13(4), 730. https://doi.org/10.3390/microorganisms13040730