嗜水氣單胞菌(Aeromonas hydrophila)是人類和水產(chǎn)養(yǎng)殖中常見的病原體,存在于多種水環(huán)境、食品以及食品加工和儲存系統(tǒng)中。該菌能夠在不斷變化的環(huán)境中生存和傳播,在合適的表面可形成生物被膜,從而表現(xiàn)出對不利環(huán)境條件的適應(yīng)性。有研究表明,外膜蛋白(OMPs)可影響細(xì)菌的毒力、耐藥性和生物被膜的形成能力。已有報道某些OMPs與嗜水氣單胞菌的毒力和抗生素耐藥性有關(guān),然而其與生物被膜的相關(guān)性尚不清楚。1.利用比較基因組學(xué)研究嗜水氣單胞菌分類與毒力相關(guān)因子嗜水氣單胞菌隸屬氣單胞菌屬,菌株數(shù)量較多,有時會出現(xiàn)錯誤標(biāo)記或分類。此外,不同來源的嗜水氣單胞菌中可能的毒力因子尚未被系統(tǒng)研究。為了鑒定嗜水氣單胞菌菌株的可靠性及其毒力因素,本試驗進行了泛基因組學(xué)研究。通過平均核苷酸同源性(ANI)、數(shù)字DNA-DNA雜交(dDDH)和多位點序列分型(MLST)鑒定,共確定13株錯標(biāo)菌株和49株有效菌株。多數(shù)菌株基因組中存在前噬菌體多個,且均屬常見的氣單胞菌屬噬菌體phi018。不同菌株間III型分泌系統(tǒng)(T3SS)具有多樣性,而Ⅱ型和Ⅵ型分泌系統(tǒng)(T2SS和T6SS)在菌株間具有一定的保守性。最常...

【文章頁數(shù)】：130 頁

【學(xué)位級別】：博士

【文章目錄】：
List of abbreviations
摘要
ABSTRACT
Chapter 1: Introduction and literature review
    1.1. Aeromonas hydrophila
        1.1.1 Introduction
            1.1.2. Diseases caused by A. hydrophila
            1.1.3. Epidemiology of A.hydrophila infection
            1.1.4 Virulence factors in A.hydrophila
            1.1.5. Antibiotic resistance in A. hydrophila
            1.1.6. Biofilm formation in A. hydrophila
                1.1.6.1. Bacterial attachment-the first phase of biofilm formation
                1.1.6.2. Colony formation and biofilm maturation
                1.1.6.3 Biofilm dispersal
    1.2. Pangenome studies
        1.2.1. Global genomic epidemiology
        1.2.2. In—silico methods
    1.3. Beta barrels and relation to other characters of bacteria
        1.3.1 Role of beta barrels in other characters of bacteria
    1.4 Objectives of the study
    Reference
Chapter 2: Comparative genome analysis provides deep insights into A. hydrophila taxonomy andvirulence-related factors
    2.1. Introduction
    2.2. Materials and Methods
        2.2.1. Genomes, genome properties and gene predictions
        2.2.2. In silico typing, average nucleotide identity and genome-to-genome distance calculation
        2.2.3. Comparative genomic analysis
        2.2.4. Functional annotations
        2.2.5. Comparison of the genome sequences of A. hydrophila with different sequence types(STs)
    2.3. Results
        2.3.1. Core and pangenome analysis of 62 strains
        2.3.2. Characteristics of the valid 49 A.hydrophila genomes
        2.3.3. Functional annotation
        2.3.4. Comparison of A. hydrophila strains with ST-251 and other STs
        2.3.5. The comparative analysis of genome elements
    2.4. Discussion
    2.5. Conclusion
    Reference
Chapter 3: In silico analyses of biofilm related beta barrels and their experimental validation throughgene knockout of selected beta barrel proteins
    3.1. Introduction
    3.2. Materials and methods
        3.2.1. In silico methods
        3.2.2. Strains and plasmids
        3.2.3. Main reagents and instruments
        3.2.4. Primer design
        3.2.5. Construction of gene knockout mutant strains
        3.2.6. Construction of complementary genes
        3.2.7. Real-time quantitative PCR verification
        3.2.8. Determination of growth curve
        3.2.9. Biofilm formation at different intervals
        3.2.10. Conditions for biofilm and planktonic growth
        3.2.11. RNA extraction and qRT-PCR
    3.3. Results
        3.3.1. In silico results
        3.3.2. OMPs distribution among different parameters
        3.3.3. In-house database and OMPs
        3.3.4. Construction and identification of ompAII gene deletion strain
        3.3.5. Construction and identification of omp38 gene deletion strain
        3.3.6. Construction and identification of complementary strains
        3.3.7. Quantitative real time PCR detection
        3.3.8. Determination of growth curve
        3.3.9. Biofilm formation at different intervals
        3.3.10. Expression of ompAII gene under planktonic and biofilm growth conditions
        3.3.11. Expression of omp38 gene under planktonic and biofilm growth conditions
    3.4. Discussion
    3.5. Conclusion
    Reference
Chapter 4: Effect of omp38 and ompAII deletion in A. hydrophila NJ-35 on virulence and drugsensitivity against various antibiotics
    4.1. Introduction
    4.2. Materials and methods
        4.2.1. Bacterial strains and antimicrobial compounds
        4.2.2. Swimming motility test
        4.2.3. Interspecies competition assay
        4.2.4. Relative adhesion test with Hep2 cells
        4.2.5. LD₅₀ in fish
        4.2.6. Minimum inhibitory concentrations (MIC) testing
        4.2.7. Biofilm susceptibly testing
        4.2.8. Effect of Sub-MICs on biofilm inhibition
        4.2.9. Statistical analyses
    4.3. Results
        4.3.1. Swimming motility test
        4.3.2. Inter-strain competition
        4.3.3. Relative adhesion assay
        4.3.4. LD₅₀ in fish
        4.3.5. Bacterial susceptibility to various antibiotics
        4.3.6. Biofllm eradication test
        4.3.7. Effect of Sub-MICs on biofilm
    4.4. Discussion
    4.5. Conclusion
    Reference
Chapter 5: Epi-gene:An R- Language based Package to analyse the pangenome study
    5.1. Introduction
    5.2. Implementation
        5.2.1. R-statistical Language
        5.2.2. External binaries
        5.2.3. FASTA format related functions
        5.2.4. Pan matrix computation
        5.2.5. Analysis of core, accessory and unique genes
        5.2.6. Phylogenetic analyses
        5.2.7. Graphical representation
    5.3. Results
    5.4. Discussion
    5.5. Conclusion
    References
GENERAL CONCLUSIONS
INNOVATIONS
PUBLICATIONS
ACKNOWLEDGEMENT



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