초록

Abstract ▼

□ 연구개요
The frequent use of antibiotics in aquaculture has resulted the development of resistant strains, which have contributed to the inefficacy of antibiotics. To reduce the risk of the development and spreading of microbial resistance, and to control the fish disease in aquaculture, alternativ

□ 연구개요
The frequent use of antibiotics in aquaculture has resulted the development of resistant strains, which have contributed to the inefficacy of antibiotics. To reduce the risk of the development and spreading of microbial resistance, and to control the fish disease in aquaculture, alternative strategies needs to be developed.
• The main goal of this proposal is multidisciplinary approach of developing phage therapy, as an alternative method against antibiotic treatments, to biocontrol antibiotic resistant pathogenic bacteria in fish, and give sound foundation to apply it against bacterial diseases in aquaculture.
• Specific aims were 1) construction of phage library against fish pathogenic and antibacterial resistant bacteria, 2) In vivo studies on investigating protective efficacy of phages against fish disease using zebrafish model, 3) genome sequencing and functional characterization of important phage protein genome.

□ 연구 목표대비 연구결과
Under this project we successfully, isolated and characterized 5 bacteriophages, namely 1) A. salmonicida phage ASP1 from diseased goldfish; 2) A. hydrophila phage AHP1 from zebrafish culture system; 3) E. tarda phage ETP1 from fish farm water; 4) V. cyclitrophicus phage VCP1 from fish farm water); 5) Klebsiella spp phage KP1 from freshwater stream. Moreover, morphological characterization was done based on diameter of head and tail length by TEM analysis. Bacteria growth inhibition and single step growth curve analysis were performed for bacteria and phages, respectively. Results conformed the MOI dependent growth inhibition by all phages. ASP-1, AHP-1, ETP-1 and KPP-1 showed latent period of 40, 50, 50, 20 min and burst size of 109, 80, 4, 2 PFU/infected cell. All the phages exhibited similar trend of infection capabilities after incubation at pH range from 2-14, however, pH 2 and >12 showed complete inactivation. We successfully developed 4 bacteria-zebrafish disease models showing more than 75% cumulative mortality, except for V. cyclitropicus. Phage bath exposure, intra peritoneal injection (i.p.) and oral feeding were effective for phage dispersion and persistancy. Phage dispersion was higher at early time point in i.p. injection than other 2, but exposre and feeding showed higher persistance results. We successfully developed phage enriched Artemia diet as a live tool for efficient phage administration to the adult fish. Protective effect (more than 75% fish survival) was remained in ETP-1 exposed, AHP-1 enriched Artemia oral fed and i.p. injected KPP-1 phage followed by challenged with E tarda, A. hydrophila and K. pneumoniae, respectively. Two combinations of cock tails were suggested for in vitro studies before being tested for in vivo. Antibiotic–phage combination was more effective than either of two added alone. Histopathology examination of phage exposure and fed diet did not show any adverse effect on tissues. Moreover, though few cytokine gene were induced at certain time points, apparent adverse immune stimulation or responses were not detected due to phage administration. Moreover, we sequenced ETP-1, KPP-1 and AHP-1 full genome and was found KPP-1 and AHP-1 were novel. Interestingly, ETP-1 and AHP-1 did not contain the holin or lysin antibacterial genes, where as KPP-1 contained endolysin gene. The recombinant KPP-1 endlolysin showed antibacterial activity against the Klebsiella pneumoniae in vitro. And showed the antibacterial activity by cell penetration and reactive oxygen species generation. However, the in vivo studies did not show the promising results with purified endolysisn against K. pneumoniae or E. tarda infection in zebrafish, though it gave slight protection until 15 h post infection.

□ 연구개발결과의 중요성
• We isolated 5 bacteriophages which 4 had potent important to aquaculture and also for other therapeutics.
• The morphology and genome analysis revealed the relatedness of these phages to the existing phages and their novelty.
• Protective effect (more than 75%) in ETP-1 exposed, AHP-1 enriched Artemia oral fed and i.p. injected KPP-1 phage followed by challenged with E tarda, A. hydrophila and K. pneumoniae, respectively. suggest the possibility of use for phage therapy in aquaculture.
• Phage and antibiotic combination had more efficient inhibition of bacteria than individuals, thus can minimize the dose of antibiotics.
• Important findings that we found was phage enriched Artemia could be a useful live tool for transfering phage into the cultured fish (larvae and adults) as a feed, and this mode of phage administration enhances efficient phage therapy in aquatic animals.
• Genome characterization identified the novel phages (2) with new gene information to the phage database.
• It improves the existing information of the genes that have not been annotated and phage genome sequence information will be useful to develop antimicrobial agents (e.g. endolysin, lysozyme), vaccine adjuvants or vaccines, bacterial detection kits etc.

(출처 : 연구결과 요약문 2p)

목차 Contents

• COVER ... 1
• 연구결과 요약문 ... 2
• 목차 ... 3
• 1. 연구개발과제의 개요 ... 4
• 2. 연구수행내용 및 연구결과 ... 4
• 2.1. Phage source sample collection ... 4
• 2.2. Selection of bacterial strains with antibiotic resistance: ... 4
• 2.3 Optimizing bacteria culture conditions: ... 4
• 2.4. Isolation of 5 phages against antibiotic resistant bacteria: ... 5
• 2.5. Classification of phages by transmission electron micrograph (TEM analysis): ... 5
• 2.6. Bacteria growth inhibition and single step growth curve (SSGC) analysis of phages. ... 5
• 2.7. Host range determination of phages with antibiotic resistant bacteria strains ... 6
• 2.8 Stability analysis of SPI, AHP-1, ETP-1, VCP-1 and KPP-1. under different temperatures, PH,salinity and organic solvents ... 6
• 2.9. Develop bacteria disease model by experimentally challenged zebrafish ... 7
• 2.10. Test the 3 modes of phage therapy administration methods ... 7
• 2.11 Conduct experimental trials to study the protective effects of selected 5 phages against 5 fish pathogenic and MDR bacteria using isolated phages and phage cocktails (based on survival rate) ... 8
• 2.12 Study the antibiotic-phage combination effects to control fish pathogenic bacteria ... 8
• 2.13. Study the effects of phage therapy on host by ... 8
• 2.14 Isolation of phage genomic DNA and estimation of phage genome size by restriction mapping ... 9
• 2.15 Sequencing of phage genomes and genome assembly ... 10
• 2.16 Genome mapping and genome comparison to relative phages ... 11
• 2.17 Screening the phage ORFs that probable for specific importance of phage therapy ... 13
• 2.18 Cloning of phage ORFs and overexpression of recombinant proteins to find probable function of antibacterial activity ... 14
• 2.19 Functional characterization of recombinant endolysin to understand the inhibitory bacteria ... 16
• 2.20 In vivo phage therapy using recombinant endolysin to find phage theraputic different pathogenic bacteria ... 17
• 3. 연구개발결과의 중요성 ... 18
• 4. 참고문헌 ... 18
• 5. 연구성과 (Research achievement) ... 19
• 대표적 연구실적 ... 21
• End of Page ... 33