利用泛基因组学方法结合试验研究鉴定嗜水气单胞菌生物被膜形成相关的外膜蛋白
发布时间：2023-02-07 14:40
嗜水气单胞菌(Aeromonas hydrophila)是人类和水产养殖中常见的病原体,存在于多种水环境、食品以及食品加工和储存系统中。该菌能够在不断变化的环境中生存和传播,在合适的表面可形成生物被膜,从而表现出对不利环境条件的适应性。有研究表明,外膜蛋白(OMPs)可影响细菌的毒力、耐药性和生物被膜的形成能力。已有报道某些OMPs与嗜水气单胞菌的毒力和抗生素耐药性有关,然而其与生物被膜的相关性尚不清楚。1.利用比较基因组学研究嗜水气单胞菌分类与毒力相关因子嗜水气单胞菌隶属气单胞菌属,菌株数量较多,有时会出现错误标记或分类。此外,不同来源的嗜水气单胞菌中可能的毒力因子尚未被系统研究。为了鉴定嗜水气单胞菌菌株的可靠性及其毒力因素,本试验进行了泛基因组学研究。通过平均核苷酸同源性(ANI)、数字DNA-DNA杂交(dDDH)和多位点序列分型(MLST)鉴定,共确定13株错标菌株和49株有效菌株。多数菌株基因组中存在前噬菌体多个,且均属常见的气单胞菌属噬菌体phi018。不同菌株间III型分泌系统(T3SS)具有多样性,而Ⅱ型和Ⅵ型分泌系统(T2SS和T6SS)在菌株间具有一定的保守性。最常...
【文章页数】：130 页
【学位级别】：博士
【文章目录】：
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
本文编号：3737009
【文章页数】：130 页
【学位级别】：博士
【文章目录】：
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
本文编号：3737009
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