코끼리조개 (Panopea japonica)에서 분리되는 비브리오 속 세균의 다양성 연구 = A Study on the Diversity of Genus Vibrio Bacteria Isolated from Geoduck Clam (Panopea japonica)

다국어 초록 (Multilingual Abstract)

The Genus Vibrio are gram-negative motile curved-rod shape bacteria. More than 100 species have been reported so far, and they have a symbiotic relationship with the host as aquatic resident flora. But, they are also known to show pathogenicity not only in fish but also in bivalves as facultative pathogen that causes disease when the host's immune ability or aquatic environment deteriorates. In case of bivalve shellfish, it is difficult to identify the causative pathogen when disease by bacterial infection occurs because they have various symbiotic microorganisms inside their body due to the filtration feeding, and it is difficult to observe the symptoms because the fleshy part is surrounded by shells, making early diagnosis and treatment difficult.
In this study, we attempted to isolate and identify bacteria that can be a problem in Geoduck clam (Panopea japonica) aquaculture. In particular, we applied MLSA (Multilocus sequence analysis) method to the Genus Vibrio to identify them at the species level. In addition, the phenotype features of the identified isolates and antibiotic susceptibility tests were conducted. Bacteria isolated from Geoduck clam adult and larvae were identified using 16S rDNA gene for general identification, and housekeeping genes (gapA, gyrB, recA, rpoA, pyrH genes) for identification of Family Vibrionaceae bacteria. Among the isolates of V. splendidus, V. tapetis, and V. atlanticus, which are known to be pathogenic to invertebrates, 10 isolates were randomly selected (This study 1-10) and MLSA was performed for clear identification. As a result, it was confirmed that This study 1 and 3 were most close to V. atlanticus C 14.7, This study 2 was V. atlanticus C 2.4, This study 4 was V. tapetis CECT 4600T, and This study 5-10 were most close to V. splendidus LMG 19031T.
In this study, the characteristics of phenotype and identification by 16S rDNA sequencing were highly homologous among isolates, making difficult to identify them to the species level, but the MLSA method can be used to identify them to the species level. The most frequently isolated V. splendidus have been reported to show pathogenicity to various bivalve shellfish such as oysters and scallops, but no symptoms related to V. splendidus infection in Geoduck clam were found in this study. Therefore, it is necessary to conduct continuous monitoring and confirm the pathogenicity of the isolated V. splendidus against Geoduck clam.

목차 (Table of Contents)

• Ⅰ. 서론 ······················································································································· 01
• Ⅱ. 재료 및 방법 ······································································································· 07
• 1. 시료 준비 ··································································································· 07
• 가) 샘플링 ····································································································· 07
• 나) 세균 분리 ······························································································· 07
• 2. 분자생물학적 동정 ··················································································· 11
• 가) 핵산 추출 ······························································································· 11
• 나) Primer 준비 ··························································································· 11
• 다) Polymerase Chain Reaction (PCR) 조건 ········································· 12
• 라) 전기영동 (Electrophoresis) ································································· 12
• 마) DNA sequencing ··················································································· 12
• 바) Multilocus Sequence Analysis (MLSA) ·············································· 13
• 사) 계통수 ····································································································· 13
• 3. 생리학적, 생화학적 특징 조사 ······························································ 16
• 가) 형태학적 특징 ······················································································· 16
• 나) API Kit를 이용한 생화학적 특징 ······················································ 16
• 다) 온도조건별, 염분조건별 배양 ··························································· 16
• 4. 항생제 감수성 검사 ················································································· 18
• Ⅲ. 결과 ······················································································································· 20
• 1. 분자생물학적 동정 ··················································································· 20
• 가) Polymerase Chain Reaction (PCR) 결과 ········································· 16
• 나) 계통수 작성 ··························································································· 21
• 다) Multilocus Sequence Analysis (MLSA) ·············································· 21
• 2. 생리학적, 생화학적 특징 조사 ······························································ 33
• 가) 형태학적 특징 ······················································································· 33
• 나) API Kit를 이용한 생화학적 특징 ······················································ 33
• 다) 온도조건별, 염분조건별 배양 ··························································· 33
• 3. 항생제 감수성 검사 ················································································· 38
• Ⅳ. 고찰 ······················································································································· 40
• Ⅴ. 결론 ······················································································································· 45
• Ⅵ. 참고문헌 ··············································································································· 46
• Ⅶ. 감사의 글 ············································································································· 57