青藏高原牦牛大肠埃希氏菌的耐药性，致病性和基因特性研究

发布时间：2022-01-05 16:32

　　大肠埃希氏菌（Escherichia coli,E.coli）是一种常见的食源性、条件性致病菌,一般人畜呈无症状感染,但在一定的条件下能够产生威胁人畜生命安全的并发症。反刍动物是致病性E.coli最重要的宿主之一。牦牛（Bos grunniens或Yak）是生活在青藏高原上特有的反刍动物,也是当地牧民主要肉食品来源。目前,由于药物的滥用,导致大肠杆菌耐药性不断产生,耐药基因不断被发现。因此,越来越多的专家指出研究和评估食用性动物（如牦牛）的抗菌耐药性（Antimicrobial resistance,AMR）和致病因子（Virulence factors）对于公共卫生安全非常重要。现在临床上关于细菌耐药性的监测还不够全面,对动物耐药研究还没形成完整的理论系统,并且关于青藏高原地区牦牛大肠杆菌耐药性报道也较少。因此,本研究开展对青藏高原地区牦牛大肠杆菌中致病基因、遗传谱系、血清型和抗生素抗性模式的研究,以期为青藏高原地区牦牛肉食品安全性评估提供理论依据。研究内容如下:1.青藏高原地区散养牦牛大肠埃希氏菌耐药性研究在我国散养牦牛中关于抗生素耐药性、血清型、遗传谱系和相关毒力特征的研究还未见... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：153 页

【学位级别】：博士

【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
CHAPTER 1 Introduction
    1.1 Antimicrobial resistance (AMR)
    1.2 Susceptibility testing
        1.2.1 Phenotype susceptibility testing methods
        1.2.2 Molecular characteristics of antimicrobial resistance
    1.3 How bacteria acquire resistance
        1.3.1 Mutation
        1.3.2 Horizontal and vertical transfer of resistance
        1.3.3 Mobile genetic elements
    1.4 How antimicrobials exert their effect
    1.5 How bacteria fight back against antimicrobials
    1.6 Main mechanisms of antibiotic resistance development to commonly used antibiotics in livestock (yak) herds
        1.6.1 Beta-lactams
        1.6.2 Tetracyclines
        1.6.3 Fluorquinolones and Quinolones
        1.6.4 Aminoglycosides and Aminocyclitols
        1.6.5 Chloramphenicol and Florfenicol
        1.6.6 Trimethoprim and Sulphonamides
        1.6.7 Co-resistance and cross-resistance
    1.7 Escherichia coli (E. coli) as an indicator organism
    1.8 Pathogenicity of E. coli and AMR
    1.9 Antibiotics usage: General considerations
        1.9.1 Reason for antimicrobial use in livestock
        1.9.2 Antimicrobial use and antimicrobial resistance
        1.9.3 Major multi-drug efflux pump and traditional Chinese medicine
        1.9.4 Challenges of antibiotic usage data collection
    1.10 Antimicrobial resistance in domestic yaks
        1.10.1 How this thesis will fill in the gaps demonstrated in this review
    1.11 Concluding statements
    1.12 Aims &Objectives
        1.12.1 Problem statement
        1.12.2 Aims
        1.12.3 Achieved goals
        1.12.4 Benefits from the project
CHAPTER 2 Antibiotic resistance of Escherichia coli in free-ranging healthy and diarrheic yaks (Bos grunniens) of Qinghai-Tibetan Plateau,China
    2.1 Objective of this study
    2.2 Materials and methods
        2.2.1 Sample collection, isolation, and identification
        2.2.2 Antibiotic sensitivity testing and screening of ESBL-producing strains
        2.2.3 DNA extraction
        2.2.4 Screening for resistance genes and virulence-associated genes
        2.2.5 Phylo groups of E. coli strains
        2.2.6 Sero-grouping
        2.2.7 Codon-based positive selection analysis
        2.2.8 Positive selection on amino acid positions
        2.2.9 Evolutionary analysis of diversifying selection
        2.2.10 Evolutionary finger printing of bla CTX-M gene
        2.2.11 Statistical analysis
    2.3 Results
        2.3.1 Isolation rate of E. coli
        2.3.2 Antibiotic resistance patterns of E. coli isolates
        2.3.3 Detection of resistance and virulence genes
        2.3.4 Occurrence of E. coli pathotypes
        2.3.5 Occurrence of phylogroups
        2.3.6 Association between antimicrobial resistance and virulence genes
        2.3.7 Occurrence of Serogroups
        2.3.8 Detection of ESBL genes
        2.3.9 Codon-based positive selection analysis in ESBL producing CTX-M gene
        2.3.10 Positive selection on amino acid positions
        2.3.11 Evolutionary analysis of diversifying selection
        2.3.12 Evolutionary finger printing of bla CTX-M gene
    2.4 Discussion
CHAPTER 3 Characteristics of integrons and associated gene cassettes in antibiotic resistant Escherichia coli isolated from free-ranging food animals in China
    3.1 Objective of this study
    3.2 Materials and Methods
        3.2.1 Ethics statement
        3.2.2 Study population and bacterial strains
        3.2.3 Characterization of the integrons
        3.2.4 Characterization of class 1 and 2 integron cassette arrays
        3.2.5 Restriction analysis of PCR products and DNA sequencing
        3.2.6 Sequence analysis and nucleotide accession number
        3.2.7 Conjugation studies and plasmid analysis
        3.2.8 Multilocus sequence typing of integrons positive E. coli
    3.3 Results
        3.3.1 Occurrence of class 1 and 2 integrons
        3.3.2 Characterization of integrons and transfer of class 1 integrons
        3.3.3 Multi-locus sequence analysis (MLSA)
        3.3.4 Conjugal transfer and plasmid profiling
    3.4 Discussion
CHAPTER 4 Experimental mouse lethality of Escherichia coli strains isolated from free ranging Tibetan Yaks
    4.1 Objective of this study
    4.2 Materials and Methods
        4.2.1 Ethics Statement
        4.2.2 Strains
        4.2.3 Serogrouping
        4.2.4 Mouse lethality assay
        4.2.5 Statistical analysis
    4.3 Results
        4.3.1 Lethality rate of E. coli strains
        4.3.2 Occurrence of Serogroups
        4.3.3 Mouse lethality assay
    4.4 Discussion
CHAPTER 5 Antibacterial effect and resistant regulation of Houttuynia cordata water extract against multi-drug resistant Escherichia coli
    5.1 Objective of this study
    5.2 Materials and Methods
        5.2.1 Materials and Reagents
        5.2.2 Water Extraction
        5.2.3 Bacterial Strains
        5.2.4 Antibacterial assay of H. cordata water extract
        5.2.5 Determination of minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
        5.2.6 Reverse Transcription Quantitative-Real Time Polymerase Chain Reaction (RT-qPCR)
        5.2.7 Statistical analyses of the results
    5.3 Results
        5.3.1 Antibacterial effects of Houttuynia cordata water extract
        5.3.2 Transcriptional level of AcrA gene by RT-qPCR
    5.4 Discussion
CHAPTER 6 Conclusions and Future directions
    6.1 Study Ⅰ
    6.2 Study Ⅱ
    6.3 Study Ⅲ
    6.4 Study Ⅳ
    6.5 Future directions
References
Acknowledgements
List of Publications
Appendices
    Appendix 1
    Appendix 2
    Appendix 3


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