Title Page
Contents
1. Abstract 6
2. Introduction 8
3. Materials and Methods 10
3.1. Bacterial strains 10
3.2. Determination of minimum inhibitory concentrations 10
3.3. Vesicles Isolation 11
3.4. Visualization of Vesicles 12
3.5. Particle size distribution and measurement of zeta potential 12
3.6. Preparation of whole cell lysates 12
3.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 13
3.8. In-gel Digestion 13
3.9. Transformation 14
3.10. Effect of OMVs on the growth of bacteria in the presence of β-Lactam antibiotics 15
3.11. Quantification of β-Lactamase activity 15
3.12. Measurement of antibiotic concentrations 16
3.13. LC-MS/MS 17
3.14. Quantitative protein profiling, statistics and database searching 17
3.15. In silico analysis of functional associations 18
3.16. Statistical analysis 18
3.17. Data availability 18
4. Results 19
4.1. Physical characterization of MRSA ST541 EVs 19
4.2. Antibiotic-resistant characterization of MRSA ST541 EVs 20
4.3. EVs-mediated transformation and Characterization of the transformants 21
4.4. Alteration in MIC level by transformation 24
4.5. Physical characterization of OMVs secreted from RC85-T and RC85 cells during in vitro growth 25
4.6. Production and β-lactamase activity of OMVs 26
4.7. OMVs from RC85-T can protect RC85 from β-lactam antibiotic 28
4.8. Total proteins obtained from RC85-T and RC85 OMVs 30
5. Discussion 32
6. Conclusion 36
References 37
Table 1. Physical characterization of EVs ST541 cells. 20
Table 2. The MIC of several antibiotics against the β-lactam-susceptible Staphylococcus aureus ATCC29213 and β-lactam-resistant S. aureus ST541 21
Table 3. Identification of transformants by using MALDI-Biotyper. 23
Table 4. Minimum inhibitory concentrations (MICs) of 16 antibiotics against the tested bacteria. 24
Table 5. Physical characterization of OMVs from RC85-T cells and RC85 cells. 26
Table 6. Quantitative protein profiling related to antibiotic resistance by comparing the OMVs secreted from RC85-T and RC85 using LC-ESI-MS/MS. 31
Fig. 1. Physical characterizations of EVs derived from methicillin-resistant Staphylococcus aureus (MRSA) ST541 cells. 19
Fig. 2. EVs from MRSA ST541 can defend β-lactam-susceptible S. aureus and fully protect them from β-lactam antibiotic-induced growth inhibition. 21
Fig. 3. Transformation frequency according to treatment DNase. 22
Fig. 4. Representative growth profiles of RC85 and EVs mediated transformed bacteria RC85-T. 23
Fig. 5. Physical characterizations of OMVs from RC85-T cells and RC85 cells. 26
Fig. 6. Production of OMVs isolated from RC85-T cells and RC85 cells. 27
Fig. 7. Investigation of the differences in β-lactamase activity between whole-cell lysates (WCL), and OMVs from RC85-T cells and RC85 cells. 27
Fig. 8. OMVs from OMVs from RC85-T cells and RC85 cells can defend β-lactam-susceptible RC85 and fully protect them from β-lactam antibiotic-induced growth inhibition. 29
Fig. 9. LC-QQQ-based assessment of the concentration of antibiotics following incubation with RC85-T or RC85 OMVs in a cell-free system. 30