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Functional Genomic and Transcriptomic Profiling of Pathogenic Multidrug Resistant and Highly Virulent Escherichia Coli of Poultry Origin

摘要第9-12页
Abstract第12-14页
List of Abbreviations第15-16页
1 Introduction第16-29页
    1.1 Poultry Escherichia coli第16页
    1.2 Antibiotic Resistance in Poultry E. coli第16-19页
    1.3 Resistant Factors第19-20页
    1.4 β-lactamases第20页
    1.5 Biofilm Formation第20-21页
    1.6 Adhesion, Invasion and Intracellular Survivability第21-22页
    1.7 Virulent Factors第22-23页
    1.8 Whole Genome Resequencing第23-24页
    1.9 ARDB and VFDB第24-25页
    1.10 RNA-Seq Based Transcriptome Sequencing第25-26页
    1.11 Zoonotic importance of Poultry E. coli第26-27页
    1.12 Aims and Objectives of Study第27-29页
2 Materials and Methods第29-59页
    2.1 Bacterial Strains第29页
    2.2 Species Confirmation and Antimicrobial Susceptibility Test第29页
    2.3 Identification of β-Lactamase Producing Isolates第29-31页
        2.3.1 Phenotypic Confirmation by Double-disk Synergy Test第30页
        2.3.2 Genotypic Confirmation PCR第30-31页
    2.4 Antibiotic Resistant Genes Identification第31-35页
    2.5 Adhesion, Invasion and Intracellular Survivability Assays第35-37页
        2.5.1 Growth Conditions第35-36页
        2.5.2 Adhesion and Invasion Assays第36页
        2.5.3 Intracellular Survivability Assay第36-37页
    2.6 Biofilm Formation Assay第37页
    2.7 Determination of Lethal Dose 50 (LD50)第37-38页
    2.8 Virulent Genes Identification第38-41页
    2.9 Whole Genome Resequencing第41-48页
        2.9.1 Sample Preparation第41页
        2.9.2 Illumina Platform第41-42页
        2.9.3 Bioinformatics Analysis第42-43页
        2.9.4 Genome Sequencing and Assembly第43页
        2.9.5 Data Filtering第43-44页
        2.9.6 Genome Assembly第44页
        2.9.7 Comparative Genomics Analysis第44-45页
        2.9.8 Single Nucleotide Polymorphisms (SNPs) Detection第45-46页
        2.9.9 Species Evolutionary Relationship第46页
        2.9.10 Identification of Acquired Antibiotic Resistance and Virulence Genes第46-48页
    2.10 RNA-Seq Based transcriptome analysis第48-58页
        2.10.1 RNA extraction and purification第48-51页
        2.10.2 RNA-Seq protocol第51-55页
        2.10.3 Validation of library第55页
        2.10.4 Differential gene expression analysis第55-56页
        2.10.5 Bioinformatics analysis第56页
        2.10.6 Validation by RT-qPCR第56-58页
    2.11 Statistical Analysis第58-59页
3 Results第59-104页
    3.1 Bacterial Isolation and Antimicrobial Susceptibility Test第59-63页
    3.2 β-Lactamase Identification第63-66页
        3.2.1 Phenotypic Confirmation by Double-disk Synergy Test第63-65页
        3.2.2 Genotypic Confirmation by PCR第65-66页
    3.3 Antibiotic Resistant Genes Identification by PCR第66-68页
    3.4 Adhesion, Invasion and Survivability Assay第68-72页
        3.4.1 Caco-2 Cells第68-69页
        3.4.2 RAW 264.7 Cells第69-72页
    3.5 Biofilm Formation第72页
    3.6 Determination of Lethal Dose (LD50)第72-73页
    3.7 Virulent Genes Identification by PCR第73-76页
    3.8 Whole Genome Resequencing Analysis第76-90页
        3.8.1 Overview of Illumina Data第76页
        3.8.2 Genome Profile第76页
        3.8.3 Genome Assembly第76-78页
        3.8.4 Comparative genomics第78页
        3.8.5 Reference Genome Homology第78-79页
        3.8.6 SNP Detection and Annotation第79-82页
        3.8.7 Ka/Ks Mutation Ratio第82-83页
        3.8.8 InDel Detection and Annotation第83-85页
        3.8.9 Species Evolution第85-86页
        3.8.10 Identification of Acquired Resistance and Virulence Genes第86-90页
    3.9 RNA-Seq Based Transcriptome Analysis第90-104页
        3.9.1 RNA Extraction and Quality第90-91页
        3.9.2 Library Construction and Quality第91-92页
        3.9.3 Quality of Raw Sequencing Reads第92-93页
        3.9.4 Differential Gene Analysis第93页
        3.9.5 Bioinformatics analysis第93-96页
        3.9.6 Up-regulated Genes第96-100页
        3.9.7 Down-regulated genes第100-101页
        3.9.8 Validation of RNA-Seq Results by RT-qPCR第101-104页
4 Discussion第104-124页
    4.1 Antibiotic Resistance and Multidrug Resistance第104-105页
    4.2 Detection of β-lactamase Producing Isolates第105-106页
    4.3 Detection of Resistant Genes第106页
    4.4 Biofilm Formation第106-107页
    4.5 Adhesion, Invasion and Intracellular Survivability第107-108页
    4.6 Identification of Virulent Genes第108-109页
    4.7 Whole Genome Resequencing第109-111页
    4.8 Resistance Determinants and Their Regulatory Mechanism第111-114页
    4.9 Virulence Determinants and Their Regulatory Mechanism第114-117页
    4.10 Metal Homeostasis第117-119页
    4.11 Stress Tolerance第119-121页
    4.12 Mobile Genetic Elements第121-122页
    4.13 Conclusion第122-124页
5 Summary第124-127页
6 References第127-147页
7 Appendix第147-262页
    7.1 Supplementary Methods第147-149页
    7.2 Supplementary Materials第149-262页
8 Acknowledgement第262-263页
9 Publications第263-265页
10 CURRICULUM VITAE第265-268页

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