Title Page
Contents
Abbreviation 16
Abstract 18
CHAPTER 1. Introduction 20
1.1. Global Crisis of Antibiotic Resistance 21
1.2. Acinetobacter baumannii: An Increasing Threat in Hospital 23
1.2.1. Clinical relevance of the ESKAPE pathogens 23
1.2.2. Acinetobacter baumannii infection 23
1.3. Last-Resort Antibiotics in the Struggle against Multidrug-Resistant Infections 25
1.3.1. Purpose, importance, and implication of last-resort antibiotics 25
1.3.2. Mode of action and resistance mechanisms of colistin 25
1.3.3. Mode of action and resistance mechanisms of tigecycline 27
1.4. Atypical Antibiotic Resistance Phenotypes 28
1.4.1. Colistin dependence 28
1.4.2. Antibiotic heteroresistance 29
CHAPTER 2. Eradication of Colistin Dependence-Developing Acinetobacter baumannii with a Combination of Colistin and Other Antibiotics at Sub-inhibitory Concentration 32
2.1. Introduction 33
2.2. Materials and Methods 35
2.2.1. Bacterial strains 35
2.2.2. Antibiotic susceptibility testing 35
2.2.3. Disk diffusion assay 35
2.2.4. Checkerboard assay 36
2.2.5. Monitoring the development of colistin-dependent mutants 36
2.2.6. In vitro time-killing assay 37
2.2.7. Galleria mellonella killing assay 37
2.2.8. Antimicrobial treatment assay 38
2.3. Results 40
2.3.1. Decrease in antibiotic resistance levels following the development of colistin dependence 40
2.3.2. In vitro efficacy of colistin-based combinations with other antibiotics 40
2.3.3. In vivo efficacy of colistin-based combinations with other antibiotics 41
2.4. Discussion 54
CHAPTER 3. The Mechanism of Resistance Development Following Tigecycline Treatment in Tigecycline-Heteroresistant Acinetobacter baumannii Strain 57
3.1. Introduction 58
3.2. Materials and Methods 60
3.2.1. Bacterial strains and genotyping 60
3.2.2. Plasmid construction 60
3.2.3. Generation of a mimicked tigecycline-heteroresistant strain 60
3.2.4. Determination of minimum inhibitory concentrations 61
3.2.5. Population analysis profiling 61
3.2.6. Comparison of the bacterial fitness 62
3.2.7. Flow cytometry experiments 62
3.3. Results 65
3.3.1. Selection of bacterial strains for mimicking the heteroresistance 65
3.3.2. Verification of the mimicked tigecycline-heteroresistant strain 65
3.3.3. Population dynamics in the mimicked tigecycline-heteroresistant strain in response to tigecycline treatment 65
3.4. Discussion 70
CHAPTER 4. Investigation of the Characteristics and Molecular Mechanisms of Tigecycline Heteroresistance in Acinetobacter baumannii Clinical Isolates 72
4.1. Introduction 73
4.2. Materials and Methods 75
4.2.1. Bacterial isolates and multilocus sequence typing 75
4.2.2. Antimicrobial susceptibility testing 75
4.2.3. Disk diffusion assay 76
4.2.4. Population analysis profiling 76
4.2.5. Efflux pump inhibitor assay 77
4.2.6. Genetic evaluations 77
4.2.7. Gene expression analysis 78
4.2.8. Bacterial killing assay 78
4.2.9. Measurement of survival rate following pre-exposure to tigecycline 79
4.2.10. Experimental evolution 79
4.2.11. Growth curve assay 80
4.2.12. In vitro competition assay 80
4.2.13. Whole genome sequencing 80
4.2.14. Construction of gene overexpression and deletion mutants 81
4.2.15. Statistical analysis 82
4.3. RESULTS 87
4.3.1. Detection of tigecycline heteroresistance in Acinetobacter baumannii clinical isolates 87
4.3.2. Molecular mechanisms of tigecycline resistance in tigecycline-resistant subpopulations 87
4.3.3. Identification of multiple heteroresistance to tigecycline and colistin 92
4.3.4. In vitro efficacy of tigecycline and colistin against heteroresistant strains 93
4.3.5. Killing effect of simultaneous combination treatment using tigecycline and colistin 94
4.3.6. Tigecycline resistance as an instable phenotype in tigecycline-resistant subpopulations 101
4.3.7. Resilience of tigecycline heteroresistance in the absence of antibiotic pressure 101
4.3.8. Fitness of tigecycline-resistant subpopulations and heteroresistance restoration 102
4.3.9. Alterations in nucleotide sequences and transcription levels of tigecycline resistance genes caused by the experimental evolution 103
4.3.10. Effects of target gene overexpression and deletion on tigecycline susceptibility 104
4.3.11. Effects of other two-component regulatory systems on heteroresistance restoration 105
4.4. DISCUSSION 122
CHAPTER 5. Conclusion 128
References 131
논문요약 149
CHAPTER 2. Eradication of Colistin Dependence-Developing Acinetobacter baumannii with a Combination of Colistin and Other Antibiotics at Sub-inhibitory Concentrations 11
Table 2.1. Minimum inhibitory concentrations (MICs) of selected nine antibiotics against Acinetobacter baumannii isolates and their derivatives in this study. WT, wild-type isolate; D, colistin-dependent mutant; R, colistin-resistant mutant; S, susceptible; R, resistant. 39
Table 2.2. The interactions between colistin and other antibiotics indicated by the Fractional Inhibitory Concentration (FIC) index in the wild- type Acinetobacter baumannii strains. MIC, minimum inhibitory concentration; FICI, fractional inhibitory concentration index. 46
CHAPTER 3. The Mechanism of Resistance Development Following Tigecycline Treatment in Tigecycline-Heteroresistant Acinetobacter baumannii Strain 11
Table 3.1. List of plasmids and primers used to generate the green fluorescent protein (GFP) expression system. 64
Table 3.2. Genotypes and minimum inhibitory concentrations (MICs) of tigecycline for TSAB, TRAB, and m-THRAB strains before and after tigecycline treatment. ST, sequence type; S,... 68
CHAPTER 4. Investigation of the Characteristics and Molecular Mechanisms of Tigecycline Heteroresistance in Acinetobacter baumannii Clinical Isolates 11
Table 4.1. List of primers used for DNA sequencing, qRT-PCR, and plasmid construction in this study. Primers for identification of tet(X) gene could detect tet(X2) and tet(X3). Under bar... 83
Table 4.2. List of plasmids used in this study. AMP, ampicillin; TCS, triclosan; GEN, gentamicin. 86
Table 4.3. Characterization of randomly selected Acinetobacter baumannii clinical isolates and tigecycline resistance mechanism of tigecycline-resistant subpopulations. HR₀, parental heteroresistant strain; TGC-RP₀, tigecycline-resistant subpopulation of HR₀ strain; MICs, minimum... 90
Table 4.4. Genotypes and minimum inhibitory concentrations (MICs) of tigecycline and colistin for eight selected tigecycline-heteroresistant Acinetobacter baumannii isolates and their... 95
Table 4.5. Amino acid substitutions on PmrAB two-component regulatory system responsible for colistin resistance in colistin-resistant subpopulations (CST-RP₀ strains). Mutations were identified based on the DNA sequence of their respective parental strains (HR₀ strains). ND,... 97
Table 4.6. Alterations of tigecycline minimum inhibitory concentrations (MICs) in wild-type heteroresistant strains (HR₀ strains) and tigecycline-resistant subpopulations (TGC-RP₀ and TGC-RP₁ strains) during 30 days of laboratory evolution. Four bacterial lineages were... 107
Table 4.7. Summary of the tigecycline minimum inhibitory concentrations (MICs) and genotypes in all evolved mutants. HR, heteroresistant strain; TGC-RP, tigecycline-resistant subpopulation; p30, 30 days of passage; S, susceptible; I, intermediate-resistant; R, resistant; ST, sequence type. 109
Table 4.8. Summary of genetic alterations during the evolution process. Mutations were identified based on the DNA sequence of their respective wild-type heteroresistant strains (HR₀ strains). Genomic variations that determined by whole genome sequencing are bold. 116
Table 4.9. Minimum inhibitory concentrations (MICs) of tigecycline in gene overexpression and deletion mutants. The overexpression mutants were incubated with 0.1 to 1 mM of IPTG to induce expression of plasmids encoded target genes. TGC-HR, tigecycline-heteroresistant strain;... 118
Table 4.10. Conservation of the two-component system genes in Acinetobacter baumannii strain ATCC 17853 and F-1757-HR₀. A1S_2937 and A1S_2938 were not detected in all isolates used in... 119
CHAPTER 2. Eradication of Colistin Dependence-Developing Acinetobacter baumannii with a Combination of Colistin and Other Antibiotics at Sub-inhibitory Concentrations 13
Figure 2.1. Emergence of colistin dependence from the wild-type Acinetobacter baumannii strains on the agar plate supplemented with colistin alone or in combinations. The concentration... 43
Figure 2.2. In vitro efficacy of colistin-based combination treatments against the wild-type Acinetobacter baumannii strains. The concentration of colistin was fixed at 4 mg/L, and the other... 49
Figure 2.3. Colistin susceptibility phenotypes of bacteria surviving treatment with colistin alone or in combinations shown in Figure 2.2. Colistin disk diffusion assay was conducted on only surviving colonies from H08-391 after 24 h of single treatment or simultaneous combination... 52
Figure 2.4. Survival analysis using Acinetobacter baumannii H08-391 and Galleria mellonella larvae (A) G. mellonella killing assay to establish the appropriate inoculum dose of H08-391 for... 53
CHAPTER 3. The Mechanism of Resistance Development Following Tigecycline Treatment in Tigecycline-Heteroresistant Acinetobacter baumannii Strain 13
Figure 3.1. Characterization of tigecycline-susceptible and -resistant Acinetobacter baumannii isolates (TSAB and TRAB, respectively). (A) Determination of tigecycline susceptibility... 67
Figure 3.2. Monitoring the bacterial population using a green fluorescent protein (GFP) expression system and flow cytometry. Green fluorescence versus forward scatter dot plots for TSAB strain (A), TRAB strain (B), and m-TRHAB strain before and after tigecycline treatment... 69
CHAPTER 4. Investigation of the Characteristics and Molecular Mechanisms of Tigecycline Heteroresistance in Acinetobacter baumannii Clinical Isolates 13
Figure 4.1. Determination of tigecycline heteroresistance among Acinetobacter baumannii clinical isolates. (A) The proportions of tigecycline susceptibility and heteroresistance in 323... 89
Figure 4.2. Relative mRNA expression levels of adeB and adeS in wild-type heteroresistant isolates (HR₀ strains) and their respective tigecycline-resistant subpopulations (TGC-RP₀ strains).... 91
Figure 4.3. Detection of multiple heteroresistance to tigecycline and colistin and their colistin resistance mechanism. (A) The result of population analysis profiling (PAP) for colistin in seven... 96
Figure 4.4. The results of an in vitro time-killing assay of both monotherapy and combination treatment of tigecycline and colistin against multiple heteroresistant Acinetobacter baumannii... 98
Figure 4.5. The effect of tigecycline pre-treatments on survival for high doses of subsequent tigecycline treatment. Three tigecycline-heteroresistant strains and two homogenously susceptible isolates were included in this assay. All isolates were pre-treated with varying... 99
Figure 4.6. Survival of resistant subpopulations to tigecycline and colistin on the agar plates supplemented with both antibiotics. Population analysis profiling (PAP) using tigecycline and... 100
Figure 4.7. The schematic of laboratory evolution. Evolution proceeded for 30 days in the absence of tigecycline using tigecycline-resistant subpopulations or wild-type heteroresistant strains. Tigecycline-resistant subpopulations were obtained by exposing the heteroresistant... 106
Figure 4.8. Restoration of a heteroresistance phenotype from tigecycline resistance during the evolution of tigecycline-resistant subpopulations (TGC-RP₀ strains). Alterations in the resistant cell fraction within the entire bacterial population were indicated by the results of population... 108
Figure 4.9. The results of population analysis profiling (PAP) for the novel tigecycline-hetero- resistant strains (TGC-HR₁ strains) and their tigecycline-resistant subpopulations (TGC-RP₁)... 110
Figure 4.10. The results of population analysis profiling (PAP) for the wild-type heteroresistant strains (HR₀ strains) before and after evolution. Mutants obtained after 15 and 30 days of passage... 111
Figure 4.11. Analysis of growth differences between tigecycline-heteroresistant populations (HR₀, HR₀-p30, and TGC-HR₁ strains) and tigecycline-resistant subpopulations (TGC-RP₀ strains).... 112
Figure 4.12. In vitro competitive fitness of tigecycline-heteroresistant strains and tigecycline-resistant subpopulations versus Escherichia coli MG1655. For each lineage, three tigecycline-heteroresistant strains (HR₀, HR₀-p30, and TGC-HR₁ strains) and one tigecycline-resistant strain... 113
Figure 4.13. Survival advantage of tigecycline heteroresistance to tigecycline treatment. The results of an in vitro time-killing assay represent bacterial survival upon tigecycline treatment at... 114
Figure 4.14. Changes in the relative mRNA expression levels of adeB and adeS during experimental evolution. Gene expression was evaluated as a relative quantity by the quantitative... 115
Figure 4.15. The putative model of evolution based on the appearance of genetic mutations. HR, heteroresistant strain; TGC-RP, tigecycline-resistant subpopulations. Red and orange squares... 117
Figure 4.16. The transcript levels of 12 under-represented two-component system genes in the evolved mutants. mRNA expression was assessed as a relative quantity using the quantitative... 120