Genomic, transcriptomic, and metagenomic analyses of Vibrio species and microbial community composition of crabs harvested at different seasons and locations = 패혈증 비브리오균의 유전체 분석을 통한 꽃게 내 전사체 특성 규명 및 계절과 지역에 따른 미생물 균총 조성 분석

다국어 초록 (Multilingual Abstract)

Vibrio vulnificus and Vibrio parahaemolyticus are gram-negative, motile, nonspore-forming opportunistic pathogens that causes foodborne illness associated with the consumption of contaminated seafood. Although many cases of foodborne outbreaks caused by V. vulnificus and V. parahaemolyticus have been reported, the genomes of only few of them have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. vulinificus and V. parahaemolyticus associated with foodborne outbreak in South Korea, new strains V. vulnificus FORC_016 and V. parahaemolyticus FORC_008 were isolated from blood of food-poisoning patient or flounder fish and their genome was completely sequenced. The genomic analysis of FORC_016 revealed that the genome consists of two circular DNA chromosomes, and contains 4,461 predicted open reading frames (ORFs), 129 tRNAs, and 34 rRNA genes. V. parahaemolyticus FORC_008 have two circular DNA chromosomes containing 4,494 predicted ORFs, 129 tRNAs, and 31 rRNA genes. The genomic analysis revealed that the V. vulnificus FORC_016 has major virulence genes such as RTX, cytolysin, and metalloprotases. Furthermore, comparative genome analysis identified unique virulence genes of FORC_016 strain, suggesting that this pathogen have unique pathogenesis mechanism which different from other V. vulnificus. While the strain FORC_008 does not have genes encoding thermo-stable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems, and iron acquisition systems, suggesting that it may be a potential pathogen. Subsequent cytotoxicity test of the strain FORC_008 revealed its high cytotoxicity activity, substantiating this. This report provides an extended understanding on V. vulnificus and V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation, and prevention of foodborne outbreak in South Korea.
Because the foodborne illness occurs via consumption of contaminated food, it is important not only understanding of the virulence factors but also the transcriptome alteration of pathogens caused by contacting with foods. To identify differentially expressed genes of pathogen under contact with foods, V. vulnificus FORC_016, an opportunistic marine pathogen, was selected for the transcriptome analysis. Swimming crab, a common niche of V. vulnificus, was selected for the model foods. The transcriptomic profiles of V. vulnificus exposed or unexporsed to crab in 1 or 4h were analyzed using a strand-specific RNA-sequencing. By analyzing RPKM (reads per kilobase of transcript per million reads) fold changes of each gene, I identified that 922 and 648 genes were differentially expressed under exposure to crab for 1h and 4h (P value < 0.05, 2 fold threshold). Regardless of incubation time, the genes related with energy production, cell growth, oligopeptide transport, and glucose metabolism were up-regulated, while genes associated with amino acid biosynthesis, nitrogen metabolism, and other sugar metabolism were down-regulated. These result suggested that V. vulnificus could metabolize the component of crab. Also, the genes encoding thermolabile hemolysin was up-regulated, suggesting this virulence gene might be have crucial role for pathogenesis of V. vulnificus FORC_016 when consumed the V. vulnificus FORC_016 contaminated crab.
The swimming crab, Portunus trituberculatus, is the most consumed edible crab in South Korea, and their production and consumption have been increased. Although the foodborne illness caused by consuming of swimming crab have been reported each year, the bacterial community in swimming crab has not been fully understood yet. In order to identify the bacterial members in swimming crab depending on seasons and locations, the microbiota in 65 crabs which were collected from different locations in spring and autumn was analyzed by pyrosequencing. The bacterial communities in autumn crab were more diverse in than those in spring. Psychrobacter, Vagococcus, and Carnobacterium were the most abundant genera in spring, whereas Roseovarius was predominant in autumn, but their proportions were influenced by the pathogenic bacterial proportion. These results indicated that the microbiota in swimming crab significantly influenced by seasonal temperature change. The proportion analysis on Vibrio species indicated that intake of crab could cause the foodborne illness. This study provides the extended understanding on composition of bacterial community in swimming crab and the factors influencing crab microbiome.

목차 (Table of Contents)

• Chapter I. Background 1
• I-1. The genus Vibrio 2
• I-2. Vibrio vulnificus 4
• I-3. Vibrio parahaemolyticus 11
• I-4. The objective of this study 15
• Chapter II. Genomic Analysis of Newly Isolated Vibrio vulnificus and Vibrio parahaemolyticus Related with Foodborne Outbreaks 17
• II-1. Introduction 18
• II-2. Materials and Methods 20
• II-3. Results and Discussion 27
• II-3-1. Information of candidates for whole genome sequencing (WGS) 27
• II-3-2. Genome sequencing and assembly results 27
• II-3-3. General genome properties of V. vulnificus FORC_016 and V. parahaemolyticus FORC_008 29
• II-3-4. Comparative genome analysis . 36
• II-3-5. Pathogenesis and virulence factors of V. vulnificus FORC_016 39
• II-3-6. Pathogenesis and virulence factors of V. parahaemolyticus FORC_008 45
• Chapter III. Transcriptome Analysis of Vibrio vulnificus in Response to Crab Exposure 48
• III-1. Introduction 49
• III-2. Materials and Methods 51
• III-3. Results and Discussion 56
• III-3-1. Identification of differentially expressed genes 56
• III-3-2. Identification of differentially expressed genes under exposure to crab 63
• III-3-3. Growth of V. vulnificus in VFMG and VFMG containing crabs 94
• Chapter IV. Seasonal and Local Variation of Microbial Composition in Swimming Crab 96
• IV-1. Introduction 97
• IV-2. Materials and Methods 100
• IV-3. Results 106
• IV-3-1. The comparison of microbiota diversity in swimming crabs 106
• IV-3-2. Composition analysis of seasonal bacterial communities in swimming crab 109
• IV-3-3. Vibrio in swimming crab 117
• IV-4. Discussion 119
• IV-4-1. Seasonal and local effect on bacterial community in swimming crab 119
• IV-4-2. Seasonal variation in bacterial community composition in swimming crab 121
• Chapter V. Conclusion 129
• References 133
• 국문 초록 153

참고문헌 (Reference)

1 Seong Chan Park(박성찬), Chi Nam Seong(성치남), Keun Sik Baik(백근식), Joo Won Kang(강주원), Ji Hee Lee(이지희), "Seasonal Variations in the Bacterial Community of Gwangyang Bay Seawater", 한국생명과학회, 생명과학회지 24: 522-531, 2014