Title Page
Contents
ABSTRACT 14
Background 17
PART 1. Antibiotic resistance and virulence of Klebsiella pneumoniae 20
1.1. ABSTRACT 21
1.2. INTRODUCTION 23
1.3. MATERIALS AND METHODS 25
1.3.1. Bacterial strains, plasmids, and culture conditions 25
1.3.2. Antimicrobial susceptibility testing 25
1.3.3. String test 27
1.3.4. Mucoviscosity assay and CPS quantification 27
1.3.5. Biofilm formation and serum resistance assays 28
1.3.6. Drosophila melanogaster (fruit fly) infection 29
1.3.7. Macrophage infection assay 29
1.3.8. Sequencing for assessing gene mutations 30
1.3.9. Quantitative RT-PCR 30
1.3.10. Transcriptomic analysis by mRNA sequencing 31
1.3.11. Construction of ompR deletion mutants 34
1.3.12. Cloning of ompR and complementation to mutants 34
1.3.13. Galleria mellonella infection assays 34
1.3.14. Statistical analysis 35
1.4. RESULTS 36
1.4.1. Decreased hypermucoviscosity in TGC-IR mutants 36
1.4.2. Antimicrobial susceptibility profiles 36
1.4.3. Biofilm formation and serum resistance 41
1.4.4. Fruit fly and macrophage infection 41
1.4.5. Alterations of rpsJ, ramR, soxR, acrR, and marR genes in TGC-IR mutants 45
1.4.6. Expression of genes related to tigecycline resistance 45
1.4.7. Differentially expressed genes in the chromosome of Klebisella pneumoniae 45
1.4.8. mRNA expression of ompK35 and ompR 48
1.4.9. Tigecycline susceptibility 52
1.4.10. Phenotypic changes 52
1.4.11. Serum resistance 56
1.4.12. Survival of G.mellonella larva 56
1.4.13. Statistical analysis 56
1.5. DISCUSSION 60
PART 2. Conversion to colistin susceptibility by tigecycline exposure in colistin-resistant Klebsiella pneumoniae 63
2.1. ABSTRACT 64
2.2. INTRODUCTION 65
2.3. MATERIAL AND METHODS 67
2.3.1. Bacterial strains and culture conditions 67
2.3.2. Antimicrobial susceptibility testing 67
2.3.3. Identification of mutations in genes associated with colistin and tigecycline resistance 69
2.3.4. RNA extraction and quantitative real-time PCR 69
2.3.5. Lipid A isolation from whole cells and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry 71
2.3.6. LPS extraction and silver staining for visualization 72
2.3.7. Time-killing assay 73
2.3.8. Statistical analyses 73
2.4. RESULTS 74
2.4.1. Change of colistin MICs upon tigecycline exposure in colistin-resistant K. pneumoniae strains 74
2.4.2. Genetic alterations and mRNA expression in TIR mutants 74
2.4.3. LPS modification in TIR mutants 78
2.4.4. Comparison of lipid A structure 78
2.4.5. Inhibition of growth of colistin-resistant K. pneumoniae strains by tigecycline With low concentration of colistin 83
2.5. Discussion 85
REFERENCES 87
APPENDIX 97
〈Appendix 1〉 Improvement of transformation efficiency in hypermucoviscous Klebsiella pneumoniae using citric acid 98
Appendix 1.1. ABSTRACT 99
Appendix 1.2. INTRODUCTION 100
Appendix 1.3. MATERIAL AND METHODS 101
Appendix 1.4. RESULTS 104
Appendix 1.5. DISCUSSION 108
Appendix 1.6. REFERENCES 109
논문요약 111
PART 1. Antibiotic resistance and virulence of Klebsiella pneumoniae 10
Table 1.1. Bacterial strains and plasmids used in this study 26
Table 1.2. Primers used in this study 32
Table 1.3. Tigecycline MICs and the results of the string test in Klebsiella pneumoniae strains used in this study 37
Table 1.4. Antimicrobial susceptibility profiles of Klebsiella pneumoniae strains used in this study 40
Table 1.5. Mutations in the rpsJ gene and transcription regulator genes in TGC-IR mutants compared with that of their parental TGC-S strains 46
Table 1.6. The top 10 genes with differentially expressed levels in wild-type, tigecycline-resistant strains and tigecycline-resistant mutants 50
Table 1.7. Minimum inhibitory concentrations of tigecycline for wild-types and mutants 53
PART 2. Conversion to colistin susceptibility by tigecycline exposure in colistin-resistant Klebsiella pneumoniae 10
Table 2.1. K. pneumoniae strains used in this study and their minimum inhibitory concentrations (MICs) 68
Table 2.2. Primers used in this study 70
Table 2.3. Mutations of ramR 76
Appendix 1. Improvement of transformation efficiency in hypermucoviscous Klebsiella pneumoniae using citric acid 11
Appendix table 1.1. Values of time constant and transformation efficiency in the K. pneumoniae strains used in this study 106
PART 1. Antibiotic resistance and virulence of Klebsiella pneumoniae 12
Figure 1.1. Results of string test. 38
Figure 1.2. Results of mucoviscosity assay and capsular polysaccharide. 39
Figure 1.3. Results of biofilm formation and serum resistance assays. 42
Figure 1.4. Results of fruit fly infections. 43
Figure 1.5. Result of macrophage infection assay. 44
Figure 1.6. Expression levels of genes related to tigecycline resistance. 47
Figure 1.7. Results of transcriptome analysis. 49
Figure 1.8. mRNA expression of ompK35 and ompR. 51
Figure 1.9. Phenotypic changes. 54
Figure 1.10. Results of serum resistance assay. 58
Figure 1.11. Results of G. mellonella larvae infection assay. 59
PART 2. Conversion to colistin susceptibility by tigecycline exposure in colistin-resistant Klebsiella pneumoniae 12
Figure 2.1. Colistin MIC values of each isolates. 75
Figure 2.2. Expression levels of tigecycline and colistin resistant associated genes. 77
Figure 2.3. LPS profile of the WTs and colistin-susceptible mutants. 79
Figure 2.4. MALDI-TOF mass spectrometry of lipid and modified structure. 80
Figure 2.5. Time-killing assay of colistin-resistant K. pneumoniae using tigecycline and colistin combination. 84
Appendix 1. Improvement of transformation efficiency in hypermucoviscous Klebsiella pneumoniae using citric acid 13
Appendix figure 1.1. Production of capsular polysaccharide (CPS) in K. pneumoniae strains cultured with and without citric acid. 105
Appendix figure 1.2. The results of transformation in K. pneumoniae strains cultured with and without citric acid. 107