Effects of bacteriophage and choline as feed additives on physiology and productivity in broilers and pigs

김광호 서울대학교 대학원 2014 국내박사

Effects of Bacteriophage and Choline as Feed Additives on Physiology and Productivity in Broilers and Pigs These experiments were performed to investigate 1) effects of bacteriophage on prevention of Salmonella enteritidis in broilers, 2) effects of bacteriophage on growth performance, fecal properties, blood profiles, and immune response in weaning pigs, and 3) effects of bacteriophage and choline supplementation on physiological responses, growth performance, microbial population, and blood profiles of lactating sows and piglets. Summarized results from each experiment are described as followings: Experiment I. Effects of Bacteriophage on Prevention of Salmonella enteritidis in Broilers The experiment 1 was conducted to investigate the effects of bacteriophage on prevention of Salmonella enteritidis in broilers. A total of 320 one day old male broilers (Ross 308) were allotted by randomized complete block (RCB) design in 8 replicates with 10 chicks per pen. The experimental diets were formulated for 2 phase feeding trial (phase I; 0-2nd wk, phase II; 3rd-5th wk), and 4 different levels (0%, 0.05%; 5×108pfu/g, 0.1%; 1×109pfu/g, and 0.2%; 2×109pfu/g, respectively) of Salmonella enteritidis targeted bacteriophage were supplemented in the basal diet. There were no differences in body weight (BW) gain, feed intake and feed conversion ratio (FCR) during the whole experimental period (P>0.05). Relative weights of liver, spleen, abdominal fat, and tissue muscle of breast obtained from each bacteriophage treatment were similar to control and those values tended to increase when 0.2% (2×109pfu/g) bacteriophage was supplemented. In addition, a numerical difference of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and low density lipoprotein (LDL) cholesterol level were observed when 0.2% (2×109pfu) of bacteriophage were provided even though blood profiles were not affected by supplemented levels of bacteriophage (P>0.05). In the result of a 14 d feeding after Salmonella enteritidis challenge to 160 birds from 4 previous treatments, mortality and Salmonella enteritidis concentration in the cecum were decreased with increasing bacteriophage level (P<0.05). This result demonstrated that supplementation of 0.2% (2×109pfu) Salmonella enteritidis targeted bacteriophage might not cause negative effect on growth, meat production, and it reduced mortality from Salmonella enteritidis challenge. Consequently, bacteriophage could be used as an alternative feed additive to antibiotics in broiler diets. Experiment II. Effects of Bacteriophage on Growth Performance, Fecal Properties, Blood Profiles and Immune Response in Weaning Pigs The experiment 2 was performed to determine the effects of bacteriophage on growth performance, fecal properties, blood profiles and immune response in weaning pigs. A total of 160 pigs [(Yorkshire × Landrace) × Duroc] (BW = 6.78 ± 1.72 kg; weaned at day 24 ± 3) were allotted to 4 groups in a randomized complete block (RCB) design with 5 replication for 5 week growth trial. The experimental diets were formulated for 2 phase feeding trial (phase I; 0-2nd wk, phase II; 3rd-5th wk), and 4 different levels (0%, 0.05%; 5×108pfu/g, 0.1%; 1×109pfu/g, and 0.2%; 2×109pfu/g, respectively) of 16 types of pathogen targeted bacteriophage were supplemented in the basal diet. During the whole experimental period, average daily gain, average daily feed intake, and gain:feed ratio were not affected by bacteriophage levels, resulting in similar BW among all treatments (P>0.05). With increasing bacteriophage level in the diets, fecal microbial population of pathogenic Salmonella spp. (linear, P<0.01; 2wk) and Escherichia coli (linear, P=0.053; 5wk) were decreased. However, the concentration of Lactobacilli was increased in feces when pigs were fed 0.2% of bacteriophage, showing linear response to bacteriophage levels (linear, P<0.05, 2wk; linear, P<0.01, 5wk). The inclusion of bacteriophage in weaning pig diets resulted in decreasing the incidence of diarrhea (linear, P<0.01). In blood immune response, there was a linear decrease in IgA concentration as bacteriophage increased (linear, P<0.05). Although GOT and GPT levels were not affected by bacteriophage levels, total cholesterol (linear, P<0.01, quadratic, P<0.05, 2wk; linear, P<0.01, 5wk) and LDL cholesterol (linear, P<0.01, 2wk) levels were decreased by dietary bacteriophage. This experiment suggested that 16 types of pathogen targeted bacteriophage supplementation did not influence on growth performance. However, 0.2 % (2×109pfu/g) bacteriophage supplementation might have beneficial influences on microbial population, fecal status, immune response, and blood profiles in weaning pigs. Experiment III. Evaluation of Bacteriophage and Choline Supplementation on Physiological Responses, Growth Performance, Microbial Population and Blood Profiles of Lactating Sows and Piglets The experiment 3 was conducted to investigate the effects of bacteriophage and choline supplementation on physiological responses, growth performance, microbial population and blood profiles of lactating sows and piglets. A total of 50 mixed-parity (average= 4.64) crossbred sows (F1, Yorkshire × Landrace; Darby, Korea) with an initial BW of 228.71 ± 15.81 kg were used in a 3 week lactation period and sows were allotted to one of five treatments based on BW and backfat thickness with 10 replicates by 1+2×2 factorial arrangement. The experimental treatments were divided by two levels of bacteriophage (0.05%; 0.5× 108 pfu/g, or 0.1%; 1× 109 pfu/g) and choline chloride (0.05%; 250ppm or 0.1%; 500ppm) and NRC (1998) requirement is regarded as control treatment. The experimental diets were formulated based on corn-soybean meal diets, which contained 3,265 kcal of ME/kg, 16.8% crude protein, 1.08% lysine, respectively. There were no significant differences in BW, backfat thickness and feed intake of lactating sows by bacteriophage and choline supplementation. The BW changes were quadratically decreased in lactation (day 0 to 21) as dietary choline increased (P<0.05). Supplementation of bacteriophage and choline to lactating diets did not influence on mortality, litter weight and piglet weight. However, numerically higher litter weight and piglets weight gain were observed in bacteriophage and choline treatment groups compared to control. No differences were found in estimation of milk production, dry matter, and energy content of milk in lactating sows during the whole lactational period. Bacteriophage and choline supplementation in diets did not alter the population of Escherichia coli and Salmonella in feces of sows as well as piglets. However, the use of bacteriophage to lactation diets altered the concentrations of fecal Lactobacilli (P<0.001). In blood profiles, GOT, GPT, and non-esterified fatty acid (NEFA) levels of lactating sows and piglets were not affected by dietary treatment, while increasing bacteriophage levels tended to decrease GOT levels of lactating sows (linear, P=0.074). Inclusion of bacteriophage and choline did not influence on immunoglobulin concentration of sows at day 21 postpartum. This experiment suggested that choline supplementation in lactating diet showed an improvement of body reserves of lactating sows and increasing of fat contents in sow milks during lactation. But, bacteriophage had no effects on reproductive performance and physiological responses except of sow’s fecal Lactobacilli population. Three experiments demonstrated that positive responses were observed by bacteriophage supplementation in broilers and weaning pigs. However, sows did not show positive performance by dietary bacteriophage but body condition of sows was improved by choline supplementation.

Bacteriophage를 활용한 새싹채소에서의 Bacillus cereus 저감화

박언호 경원대학교 일반대학원 2012 국내석사

Bacillus cereus에 숙주특이성 있는 Lytic Bacteriophage를 분리하여 새싹채소에서의 Bacillus cereus를 제어하고자 하였다. 여러 지역의 토양에서 Soft agar method를 이용하여 분리된 plaque를 순수 분리하여 각 bacteriophage의 host spectrum을 분석하여 우수한 4개의 bacteriophage를 선별하였다. 선별된 bacteriophage는 TEM을 이용한 형태학적 특성과 온도, pH, Ethanol, Isopropanol 등에서의 안정성을 확인하였고, one step growth을 통한 생육특성, bacteriophage에 의한 Bacillus cereus의 생육억제를 확인하였다. 마지막으로 선별된 bacteriophage를 섞어 만든 cocktail을 활용하여 Bacillus cereus로 오염시킨 무순 종자와 무순에 접종시켜 Bacillus cereus의 저감화를 확인하였다. Bacteriophage는 서울 인근 15개의 지역 토양에서 총 18종을 분리하였고 host strain으로 B. cereus KCTC 1094를 분양받아 이용하였다. Host spectrum 분석에는 7종의 B. cereus 표준균주, 15종의 B. thuringiensis 표준균주와 116균주의 B.cereus 분리균과 90균주의 B. thuringiensis를 이용하였다. B. cereus 분리균에서의 bacteriophage들의 host spectrum은 매우 다양하였고, 적게는 5.17%에서 많게는 87.07%까지의 host spectrum을 확인하였다. 이중 host spectrum이 60% 이상인 4개의 bacteriophage를 선별한 결과 7균주의 B.cereus 표준균주와 116균주의 분리균을 99%이상 lysis 시킬 수 있었다. TEM을 이용하여 phage의 형태를 확인한 결과 BCP11은 Myobidae family에 속하였다. 선변된 phage의 One-step growth curve를 분석결과 BCP3은 133의 burst size를 보였으며 BCP9는 약 145의 burst size, BCP11은약 161의 burst size를 가졌다. 4개의 선별된 bacteriophage는 50‘C 이하에서는 대체적으로 안정하였고, 60’C 이상부터는 생육특성이 떨어졌다. 이들은 낮은 pH에도 매우 안정한 결과를 확인할 수 있었고, 100%의 ethanol 첨가하였을 때 모든 virulent phage는 20분 이내에 급격히 사멸하였고 70% ethanol 첨가하였을 때 BCP18을 제외한 virulent phage는 10분 이내에 사멸하였다. 40% ethanol 첨가하였을 때 모든 virulent phage는 101~102pfu/mL의 phage 개채수가 감소하였다. 100% Ethanol 첨가와 마찬가지로 100% isopropanol 첨가하였을 때 20분 이내에 모든 virulent phage가 사멸하였고, 50% isopropanol을 첨가하였을 때 40분 동안 BCP18은 약 102pfu/mL, BCP11은 약 104pfu/mL 감소하였고 나머지 phage들은 20분 이내에 사멸하였다. 선별된 phage를 각각 B. cereus의 대수기 진입 시점인 2hour period에 접종한 결과 B. cereus의 생육이 억제되었다. 또한 선별된 phage를 cocktail 형태로 제조하여 B. cereus가 오염된 무순 종자와 무순에 접종하였을 때 B. cereus의 개체수가 감소하였다.

포유동물의 소화기관내에 박테리오페이지가 미치는 영향

박광서 韓國外國語大學校 大學院 2015 국내석사

포유동물의 소화기관내에 박테리오페이지가 미치는 영향 Bacteriophage는 bacteria에 infection하는 virus로써, 지구상에 가장 많은 비율을 차지하는 life form이다. 오랜 기간 항생제 사용으로 Antibiotics resistant bacteria가 출현하면서 phage therapy가 다시 기존의 conventional antibiotics 대체제로서 주목을 받게 되었다. 그러나 animal 또는 human에 therapy로써 사용되었을 때 안전성은 주요한 문제이다. 또 한 우리 몸에는 bacteriophage가 항상 존재하고 있으며, gastrointestinal tract은 phage에 빈번히 노출되어 있다. Bacteriophage는 홀로 mammalian cells과 interaction할 수 있으며, 간접적으로도 bacteria와 함께 immune system에 영향을 미칠 수 있다. Pathogenic bacteria의 경우 gastrointestinal tract 내로 invasion하여 병원성을 나타낸다. Therapy 과정뿐만 아니라 natural한 상황에서도, bacteriophage는 bacteria가 cells내로 invasion 과정에서 함께 들어갈 수 있다. 그러나 gastrointestinal tract에서 bacteriophages에 의해 발생하는 inflammation에 대한 연구는 미비하다. 본 연구에서는 Mice에 murine norovirus (MNV)또는 bacteriophage T7을 10일간 경구 투여하여 inflammatory responses와 clinical sign을 관찰하였다. 어떠한 clinical sign도 유의 할 만한 차이는 관찰 되지 않았다. 12개의 Inflammatory cytokine의 변화를 보았을 때, IL17A만이 10%정도 증가한 것을 확인 하였다. 또한 gentamycin protection assay와 confocal laser fluorescence microscopy를 이용하여 pathogenic Escherichia coli의 colonic cells invasion을 확인 하였으며, pathogenic E. coli에 infection하는 bacteriophage PBEC 132를 characterization 하였다. Polymerase chain reaction (PCR)을 통하여 Invasion 과정에서 bacteria를 매개한 bacteriophage의 internalization이 일어남을 관찰 하였다.

Electrochemical Behavior of Bacteriophage-Templated Nanowires Combined with Osmium Complex and Its Application in Biofuel Cells

김지태 서울대학교 대학원 2023 국내박사

Bacteriophage is a biomaterial with unique structural and biological properties which is attracting attention as a template material for electrochemistry. As a template material, bacteriophage has several advantages, such as: (1) mass production of bacteriophage by bacterial infection; (2) high aspect ratio ensembles with an average length and width of 1 μm and 6 nm; (3) a highly-ordered protein structure; (4) tunability of coat protein through genetic engineering. Thanks to these characteristics, bacteriophage has been adopted as template materials for energy harvesting, piezoelectric sensing, biomedical applications, and other applications. This dissertation is a study on the electrochemical behavior of bacteriophage-templated redox nanowires and their application characteristics in enzyme electrode. In chapter 1, we provide a brief background on the characteristics of a bacteriophage, osmium redox species, and enzymatic electrode. In chapter 2, we investigate the bacteriophage-templated redox nanowire through an electrochemical approach. A redox nanowire network was formed by covalently binding redox species to filamentous bacteriophages. The combination of highly ordered coat protein structures and redox species exhibited characteristic electron transfer behavior. The apparent electron diffusion of the redox nanowire network is analyzed by electrochemical measurement and Dapp calculation and compared with that of redox polymer. Genetically altered coat proteins have led to an understanding of the correlation between increased mobility of redox species and apparent electron diffusion. The pH dependence of the redox hydrogel was also observed. In chapter 3, bacteriophages function as enzyme stabilizers was investigated. The Michaelis-Menten constant at various glucose concentrations elucidates the substrate affinity of enzymes surrounded by redox nanowires. The activity of the enzyme surrounded by bacteriophage was evaluated by measuring the catalytic oxidation current every week for 2 months. By constructing a biofuel cell, it was confirmed that the bacteriophage-templated enzyme electrode operates spontaneously to generate energy. This study provides electrochemical understanding and insight into the potential use of bacteriophage-templated redox nanowires for electrochemical analysis and applications. 박테리오파지는 독특한 구조적 생물학적 특성 덕분에 전기화학 실험의 재료로써도 주목을 끌고 있다. 박테리오파지는 템플릿 재료로서 다음과 같은 강점을 가지고 있다: (1) 배양을 통해 대량 생산 가능; (2) 길이 1μm, 지름 6 nm의 높은 종횡비; (3) 규칙적인 단백질 구조; (4) 유전자 조작이 가능한 표면 단백질. 이런 특성들 덕분에 박테리오파지는 에너지수집, 압전 센서, 진단 등 다양한 분야에 응용되었다. 본 논문은 박테리오파지 기반의 리독스 나노와이어와 이로 구성된 효소전극에 대한 전기화학적인 분석을 다룬다. 1 장에서는 박테리오파지, 오스뮴 리독스종, 효소전극에 대한 배경지식을 다룬다. 2 장에서는 박테리오파지 기반의 리독스 나노와이어를 전기화학적으로 분석한다. 리독스 나노와이어 네트워크는 리독스종을 박테리오파지에 공유 결합하여 형성시킨다. 규칙적인 표면 단백질 구조와 리독스종의 조합은 특징적인 전자전달 경향을 보여주었다. 리독스 나노와이어의 겉보기 전자확산경향은 전기화학적인 측정과 Dapp 계산을 통해 얻었고, 이를 리독스 폴리머와 비교 분석한다. 표면단백질을 유전자 변형한 실험을 통해서는 리독스종의 이동도와 겉보기 전자확산의 상관관계를 알아냈다. 리독스 하이드로젤 성능에 pH가 미치는 영향도 알아보았다. 3 장에서는 효소의 안정제로써 박테리오파지의 기능을 다룬다. 다양한 글루코스 농도에서의 미카엘리스-멘텐 상수를 통해 리독스 나노와이어에 둘러 쌓인 효소의 기질친화도를 확인할 수 있었다. 박테리오파지를 기반으로 한 효소의 활성을 확인하기 위해서는 두 달 동안 안정성 테스트를 진행했다. 마지막으로 박테리오파지 기반의 효소전극으로 생물연료전지를 구성하여 에너지 생산이 자발적으로 일어나는 것을 확인하였다. 본 연구는 전기화학적 분석과 응용분야에서의 박테리오파지 기반의 리독스 나노와이어의 전기화학적인 특성과 잠재력을 제공한다.

Bacillus cereus bacteriophage의 분리·동정과 살균소독제로의 응용

유혜림 가천대학교 대학원 2013 국내석사

Bacillus cereus group and many bacteria have formed biofilm or extracellular polysaccharide marix(EPM) on natural or artificial surface. Biofilm that resists to external physical and chemical stimulation is not easy to remove by heat or sanitizer treatment at manufacturing process so that it may aid food poisoning when ingested with the pathogens. Therefore, the aims of this study was to isolate the bacteriophages of food-poisoning B. cereus and to analyze the effect on the biofilm reduction. The bacteriophages were isolated from various environments and characterized by restriction enzyme, structure proteins their host spectrum, one step growth curve, stability, and morphological characteristics by TEM. They were applied to the fresh produces with hydroperchloride, organic acid, ethanol, and NaCl for the reduction of B.cereus in the biofilm environments. The twenty-five bacteriophages were isolated and five phages among them identified as BCP1, BCP18, and BCP22 for the broad host spectrum and again BCP3 and BCP16 for the strong halo-forming activities. The higher MOI, the more effective biocontrol of B. cereus. Bacteriophage treatment for 3 hours showed reduction of B. cereus by 84-99%. Through treating sprouts by the five bacteriophage, the effects were confirmed by 58-78% and 58-99% reduction after an hour and 5 hours except BCP3,respectively. Especially, BCP18 showed the best inhibition effect to B. cereus. B.cereus in the biofilm of the glass wool were reduced 60% after 3 hours by BCP22 and BCP cocktail mix, which was highest inhibition effect. By treating organic acids as sanitizer, citric acid(1%) was effective than acetic acid(1%) and lactic acid(1%). The treatment of ethanol(20%), NaCl(10%) and the mix for 15 minutes reduced B. cereus in the biofilm by 18%, 38%, and 58%, respectively. Mixed treatment of ethanol and NaCl was more effective than each ethanol and NaCl treatment. Treatment of 100ppm NaClO and 100ppm QACs for 15 minutes reduced B. cereus in the biofilm by 50% and 58%. Therefore, 100ppm QACs was more effective than 100ppm NaClO. Sequential treatment like 100ppm NaClO for 5 minutes and then bacteriophage cocktail for 30, 60, 180 minutes to B. cereus in the biofilm on the glass wool, bio-control effects were most effectively confirmed by 79, 93, and 93% reduction. Again, the treatment like 100ppm NaClO for 5 minutes and then 1% citric acid+20% ethanol+10% NaCl for 15 minutes was also effective to the B.cereus removal. When applied to B.cereus on the cabbage, the same bio-control effect was confirmed. Consequently, treatment of chemical sanitizer and bacteriophage treatment at the same time showed better inhibition of B. cereus in biofilm. The cells under the biofilm and in planktonic cell were compared for the differences of total cell proteins by two-dimensional gel elctrophoresis. Five particular spots and two spots were up-regulated in planktonic cell and in biofilm, respectively. By MALDI-TOF assay, the seven spots assay were proteins that were involved in a variety of metabolic as bfmbC, carboxylate-dehydrogenase, butanediol dehydrogenase, atpA, and phosphate dehydrogenase. Therefore, the food-poisoning B. cereus on the mininally processed produce might be controlled by the bacteriophage. Bacillus cereus group 등 많은 세균들은 자연적이거나 인위적인 표면에 부착하기 위해 biofilm이라는 extracellular polysaccharide marix(EPM)을 형성한다. 이러한 EPM은 외부 물리화학적 자극에 대한 저항성이 있어서 제조공정에서 열처리나 살균제 처리에도 쉽게 제거되지 않아 섭취 시 식중독을 유발하기도 한다. 따라서 본 연구에서는 식중독 세균인 Bacillus cereus의 bacteriophage를 분리하였고 이를 활용하여 biofilm 저감화 효과에 대하여 연구하고자 한다. 본 연구는 자연으로부터 B. cereus bacteriophage를 분리하였고, 분자생물학적 특성으로 제한효소처리, 단백질구조를 분석하였다. 또한 생물학적 특성으로 안정성, one-step growth curve, 숙주저해특성, TEM을 통해 특성을 분석하였다. Bacteriophage, 차아염소산, 유기산, 에탄올, NaCl을 이용하여 신선편의식품에서 B. cereus의 생육저해와 B. cereus biofilm의 저감화하고자 하였다. B. cereus bacteriophage는 토양과 소 분변 시료로 부터 24개를 분리하여 동정 및 특성분석을 통해 bacteriophage 중에서 숙주저해능력이 좋은 BCP3, BCP16과 숙주저해범위가 넓은 BCP1, BCP18, BCP22을 선정하였다. 선정된 bacteriophage를 접종하여 spectrophotometer로 확인한 결과, MOI가 클수록 B. cereus의 생육저해가 높았다. 또한, viable count로 확인한 결과, bacteriophage 접종 3시간부터 84-99%의 감소율을 나타내어 B. cereus의 생육저해에 대한 효과가 매우 높은 것으로 확인하였다. B. cereus가 감염된 새싹채소에서 1시간과 5시간 후 BCP3을 제외하고 각각 58-78%, 58-99%의 감소율을 확인하였다. 특히, BCP18은 생육저해능력이 가장 좋았다. Two-dimensional gel electrophoresis를 실행하여 B. cereus의 biofilm과 planktonic cell의 총세포단백질 분포를 자세히 분석하였다. 이것으로 planktonic cell에서 더 발현되는 5개의 spot, biofilm에서 더 발현되는 2개의 spot을 확인하였다. 선별한 7개의 spot은 MALDI-TOF을 이용해서 동정하였다. 동정한 결과는 다양한 대사에 관여하는 단백질들로 bfmbC, carboxylate-dehydrogenase, butanediol dehydrogenase, atpA, phosphate dehydrogenas 등이 up-regulated 또는 down-regulated 되는 것으로 나타났다. Glass wool에 부착된 biofilm에 bacteriophage을 접종한 결과, 30분부터 20-26%의 감소율을 나타났고, 3시간일 때 BCP22과 BCP mix(cocktail)의 감소율이 60%으로 biofilm 제어능력이 가장 좋았다. 한편, biofilm을 sanitizer인 유기산을 이용하였을 때, 1% acetic acid 보다 1% citric acid와 1% lactic acid의 효과가 더 높은 것으로 보였다. 또한 20% ethanol과 10% NaCl 처리한 결과, 처리시간 15분 후에 10% NaCl은 38%, 20% ethanol은 18%, 두 개를 혼합 시 58%의 감소율을 보였다. Ethanol과 NaCl 단독처리보다 혼합하여 처리하는 것이 더 효과적이다. 그리고 100ppm 차아염소산과 100ppm QACs을 15분 처리한 결과, 50% 와 58%의 감소율을 나타났고, 100ppm 차아염소산보다 100ppm QACs가 효과가 더 좋았다. Glass wool에 부착된 biofilm에 sanitizer와 bacteriophage 혼합처리 하였을 때, 100ppm 차아염소산 5분 처리 후 bacteriophage로 30분, 60분, 180분 처리하였을 때 79%, 93%, 93%의 감소율을 보였으며 biofilm의 제어에 가장 효과적이였다. 또한 100ppm 차아염소산와 citric acid 복합처리 역시 biofilm의 감소율이 높았다. 양배추에 부착된 biofilm에서는 100ppm 차아염소산 5분 처리 후 citric acid 복합 15분 처리 다음에 bacteriophage로 30분, 60분, 180분 처리하였을 때 68%, 78%, 82%의 감소율로 다른 처리구보다 가장 높았다. 따라서 bacteriophage cocktail 단독 처리할 때보다 sanitizer와 bacteriophage의 혼합 처리할 때 biofilm의 감소에 상승효과를 확인하였다. 그러므로 즉석섭취가 가능한 신선편이식품에 오염된 식중독 세균인 B. cereus는 그의 phage에 의하여 생균 감소 및 biofilm 제어까지 가능한 것으로 사료된다.

Listeria monocytogenes 및 Salmonella spp. 특이성을 가진 bacteriophage의 분리 및 특성과 살균제로서의 응용

서준형 목포대학교 대학원 2022 국내석사

Listeria monocytogenes and Salmonella enterica serovar Typhimurium are one of the most important pathogenic bacteria that can cause diseases such as fever, vomiting, and diarrhea. The causes of listeriosis include milk, dairy products, ice cream, meat products, fruits, vegetables, and cold-preserved foods, and the cause of Salmonellosis include less cooked meat, eggs, suspense, and livestock farms. The outbreak of Listeria and Salmonella food poisoning in the food industry is becoming a global problem. Therefore, in this study, bacteriophages with host specificity for L. monocytogenes and S. enterica ser. Typhimurium were isolated, and host specificity, morphological characteristics, growth characteristics, and stability under various conditions were confirmed to investigate the characteristics of the four isolated bacteriophages. In addition, bacteriophage was utilized to confirm the control effects of L. monocytogenes and S. enterica ser. Typhimurium on the stainless steel surface. Four type of bacteriophages (LmBP-1, LmBP-2, STBP-1 and STBP-2) were isolated from the sewage treatment plants in Jeollanamdo Muan and the host strain was used with Listeria monocytogenes 4244 and Salmonella enterica serovar Typhimurium КСТС 2515. To confirm their host specificity, one type of E. coli, three type of Listeria spp. group, and eleven type of Salmonella spp. group were used. LmBP-1 was confirmed to have host specificity in one type of L. monocytogenes 4244, six types of Salmonella spp. groups, and LmBP-2 was confirmed to have host specificity in one type of L. monocytogenes 4244, three types of Salmonella spp. groups. STBP-1 was confirmed to have host specificity in one type of L. monocytogenes 4244, two types of Salmonella spp. groups, and STBP-2 was confirmed to have host specificity in one type of E. coli ATCC 25922, and ten types of Salmonella spp. groups. Morphological characteristics were confirmed using TEM, and four species of bacteriophages belonged to the Myoviridae family of Caudovirales. One step growth curve growth characteristics confirmed the results, LmBP-1 had a latent time of about 20 minutes and a burst size of about 1.8 log PFU/mL for 60 minutes, and LmBP-2 had a latent time of about 15 minutes and a wide range burst size of about 1.1 log PFU/mL for about 60 minutes. STBP-1 had a latent time of about 10 minutes and a burst size of about 2.3 log PFU/mL for 60 minutes, and STBP-2 had a latent time of about 15 minutes and a burst size of about 2.8 log PFU/mL for 60 minutes. The results of confirming the stability of the pH, ethanol, thermal and the conditions of the conditions, the LmBP-1 and LmBP-2 were relatively stable in the pH 4.0~10.0 range. It was confirmed that STBP-1 and STBP-2 were relatively stable in the pH range of 4.0~8.0 and 6.0~10.0. various range treatments of ethanol was processed, the number of individuals decreased proportionally to the concentration. The result of confirming the stability to themal, it was confirmed that the bacteriophage was maintained in a stable state at 45 °C and 60 °C, and it was confirmed that the bacteriophage decreased proportionally to the treatment time at the high temperature of 75 °C and 90 °C. Currently, stainless steel is widely used as a material for instruments and machines used in the food industry, and chlorine-based disinfectants, which are chemical disinfectants, are often used as disinfectants. Therefore, the treatment effect of bacteriophage was confirmed as a biological disinfectant instead of a chemical disinfectant. After artificially contaminating pathogens on the stainless steel surface, it was treated for 10 minutes, 30 minutes, 1 hour, and 2 hours. The two-hour treatment group of LmBP-1 and LmBP-2 showed a reduction of about 1 log CFU/mL and about 0.7 log CFU/mL, respectively, and the LmBP cocktail showed a reduction of about 2 log CFU/mL or more. In the case of the two-hour treatment group of STBP-1 and STBP-2, they showed reduction of about 1.6 CFU/mL and about 1 log CFU/mL, respectively, and STBP cocktail showed a reduction of about 2 log CFU/mL. Therefore, in this study, it was confirmed that bacteriophage and bacteriophage cocktail treatment were effective against L. monocytogenes and S. enterica ser. Typhimurium on the stainless steel surface. Listeria bacteriophage confirmed that the reduction effect was greater when treated with cocktail than when single treatment was performed, and Salmonella bacteriophage confirmed that the reduction effect was greater when single treatment with STBP-1 than when treated with cocktail. Listeria monocytogenes와 Salmonella enterica serovar Typhimurium은 사람에게 발열, 구토, 설사 등의 질병을 발생시킬 수 있는 중요한 병원성 세균 중의 하나다. Listeriosis의 원인으로는 우유나 유제품, 아이스크림, 식육가공품, 과일, 야채, 저온 보존 식품, 균이 오염된 식기구, 식품 가공 공장 등 있으며, Salmonellosis의 원인으로는 덜 익힌 육류나 달걀, 가금류, 가축 농장 식품 가공 공장 등이 있다. 식품 산업에서 Listeria와 Salmonella 식중독의 발생은 전 세계적으로 문제가 되고 있다. 따라서 본 논문은 L. monocytogenes와 S. enterica ser. Typhimurium에 대한 숙주특이성을 갖는 bacteriophage를 분리하였으며, 분리된 4종의 bacteriophage의 특성을 알아보기 위해 숙주특이성, 형태학적 특성, 생육 특성 및 여러 조건에서의 안정성을 확인하였다. 또한 bacteriophage를 활용하여 식품 공장에서 많이 사용하는 stainless steel 표면에서 L. moncytogens와 S. enterica ser. Typhimurium의 제어 효과를 확인하였다. 전라남도 무안군 하수처리장에서 하수처리장 물을 채취하여 4종의 bacteriophage(LmBP-1, LmBP-2, STBP-1, STBP-2)를 분리하였으며, host strain으로 L. monocytogens 4244와 S. Typhimurium KCTC 2515를 사용하였다. 이들의 숙주 특이성을 확인하기 위해 1종의 E. coli, 3종의 Listeria spp. group, 11종의 Salmonella spp. group를 사용하였으며, LmBP-1은 1종의 L. monocytogenes 4244, 6종의 Salmonella spp. groups에 숙주 특이성을 갖는 것을 확인하였으며, LmBP-2는 1종의 L. monocytogenes 4244, 3종의 Salmonella spp. groups에 숙주 특이성을 갖는 것을 확인하였다. STBP-1은 1종의 L. monocytogenes 4244, 2종의 Salmonella spp. groups에 숙주 특이성을 갖는 것을 확인하였으며, STBP-2는 1종의 E. coli ATCC 25922, 10종의 Salmonella spp. groups에 숙주 특이성을 갖는 것을 확인하였다. TEM을 이용해 형태학적 특성을 확인한 결과, 4종의 bacteriophage(LmBP-1, LmBP-2, STBP-1, STBP-2)는 Caudovirales 목 Myoviridae 과에 속하였다. One step growth curve를 통해 생육 특성을 확인한 결과, LmBP-1은 약 20분의 latent time과 60분간 약 1.8 log PFU/mL의 burst size를 가졌고, LmBP-2은 약 15분의 latent time과 60분간 약 1.1 log PFU/mL의 burst size를 가지를 가졌다. STBP-1은 약 10분의 latent time과 60분간 약 2.3 log PFU/mL의 burst size를 가졌고, STBP-2는 약 15분의 latent time과 60분간 약 2.8 log PFU/mL의 burst size를 가지를 가졌다. 여러 조건의 pH, ethanol, thermal에 대한 안정성을 확인한 결과, LmBP-1과 LmBP-2는 pH 4.0~10.0 범위에서는 비교적 안정적인 것을 확인하였다. STBP-1은 pH 4.0~8.0, STBP-2는 pH 6.0~10.0 범위에서 비교적 안정적인 것을 확인하였다. 다양한 범위의 ethanol을 처리했을 때는 농도에 비례하여 개체 수가 감소하는 것을 확인하였다. Thermal에 대한 안정성을 확인한 결과, 45℃, 60℃에서는 bacteriophage가 안정한 상태를 유지하는 것을 확인하였으며, 75℃, 90℃의 고온에서는 처리 시간에 따라 감소하는 것을 확인하였다. 현재 식품 산업에서 사용되는 기구 및 기계 재질로는 stainless steel을 많이 사용하고 있으며 살균제로는 화학적 살균제인 염소계 살균제를 많이 사용하고 있다. 이에 따른 화학적 살균제 대신 생물학적 살균제로써 bacteriophage의 처리 및 저감화 효과를 확인하였으며, stainless steel 표면을 인위적으로 오염 시켜 초기 농도에서 10분, 30분, 1시간, 2시간으로 처리하였다. 2시간 처리하였을 때 가장 큰 감소를 확인하였으며, LmBP-1과 LmBP-2를 2시간 처리 시 최대 약 1 log CFU/mL, 약 0.7 log CFU/mL 감소하였으며, LmBP cocktail은 최대 약 2 log CFU/mL 이상 감소하였다. STBP-1과 STBP-2를 2시간 처리 시 최대 약 1.6 CFU/mL, 약 1 log CFU/mL 감소하였으며, STBP cocktail 경우 최대 약 1 log CFU/mL 이상 감소하였다. 따라서 본 연구에서 bacteriophage와 bacteriophage cocktail을 제조하여 처리했을 때 stainless steel 표면에 L.monocytogens와 S. enterica ser. Typhimurium의 제어 효과를 확인할 수 있었다. Listeria bacteriophage는 단일 처리했을 때 보다 cocktail로 처리 시 감소 효과가 큰 것을 확인하였으며, Salmonella bacteriophage는 cocktail로 처리했을 때 보다 STBP-1으로 단일 처리 시 감소 효과가 큰 것을 확인하였다.

Characterization and Potential of Bacteriophage for Lactobacillus plantarum Control in Kimchi Fermentation

오지영 가천대학교 일반대학원 2020 국내석사

Kimchi is the Korean traditional fermented food which is made of the vegetables and various seasonings. Since kimchi is not processed with sterilization, there are diverse microorganisms and the number of lactic acid bacteria (LAB) are dominant. The representative LAB in kimchi are the genera of Weissella, Leuconostoc, and Lactobacillus. It was reported that Weissella and Leuconostoc are dominant at the early and mid-stage of fermentation and Lactobacillus is predominant at the late stage of fermentation. Especially, Lb. plantarum is known to be related to the kimchi acidification. The recent studies suggested that many environmental factors affect the kimchi fermentation and the bacteriophages may be a significant determinant of kimchi microbial dynamics. In this study, three different bacteriophages, ΦLCK27, ΦLMB6, and ΦLP12116, were isolated from kimchi and their stability in the diverse conditions like kimchi environment were examined. All of three phages were stable at high salinity, high organic acid concentration, and high acidic conditions and then, it indicated that three phages can survive in kimchi environment. Among the three bacteriophages, Lb. plantarum infecting phage, ΦLP12116, is selected and evaluated the control ability of Lb. plantarum. In kimchi environment, Lb. plantarum showed about 2.2 log CFU/ml of reduction by treating ΦLP12116 compared with non-phage treated Lb. plantarum. Therefore, this study suggested that the growth of Lb. plantarum can be controlled by treating bacteriophage in kimchi environment and it showed the possibility of delay in kimchi acidification by treating bacteriophage.

Characterization, Genomic Analysis, and Application of Salmonella Typhimurium-targeting Bacteriophages

배재우 서울대학교 대학원 2017 국내박사

Salmonella is a Gram-negative, rod-shaped, and flagellated bacteria which can be found in all warm-blooded animals including human and in the environment. Up to date, more than 2,500 serotypes of Salmonella have been discovered and it is noticed to be one of the most important pathogens associated with various foodborne illnesses all over the world. Salmonella infections can cause gastroenteritis with symptoms including nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. In most cases, symptoms of salmonellosis are relatively mild to healthy people and recovered in a few days without specific treatments. However, in some cases of young and elderly patients, Salmonella infection can become severe and even cause death. Recently, infections by antibiotics resistant non-typhoid Salmonella have emerged as one of the important problems by pathogenic bacteria. Due to the emergence of multidrug-resistant Salmonella, Salmonella-infecting bacteriophages have been considered as promising alternative biocontrol agents to antibiotics. To develop a novel biocontrol agent against S. Typhimurium, 26 new bacteriophages targeting S. Typhimurium were newly isolated and characterized. Host receptor analysis identified five different cell wall receptors including flagella, O-antigen, BtuB, LPS core oligosaccharide (OS) region, and OmpC which are utilized by S. Typhimurium phages. For further understanding of the host-phage interactions, whole genomes of selected phages were sequenced and analyzed. Comparative genomic analysis among the phages showed that phage tail and tail fiber structures are important to determine the host ranges as well as the host receptor. Based on the receptor study, three phages (BSPM4, BSP101 and BSP22A) target different receptors including flagella, O-antigen, and BtuB, respectively were selected. Genome sequence analysis results revealed that all three phages neither have lysogen module nor toxin genes, supporting that they are strictly virulent and safe to be developed as biocontrol agents. A phage cocktail comprised of three phages was designed and its antimicrobial efficiency was evaluated. In-vitro treatment of the phage cocktail showed a significant reduction in the development of bacterial resistance to phage infection. Since a significant number of foodborne outbreaks and sporadic infections of Salmonella are mediated by contaminated fresh produces, the antimicrobial efficiency of the phage cocktail was evaluated using two fresh produces, lettuce and cucumber, as food models. The phage cocktail significantly inhibited S. Typhimurium growth in fresh produces for 12 h. These results suggest that the phage cocktail composed of phages targeting three different host receptors would be a useful material for developing a novel biocontrol agent against S. Typhimurium to ensure the safety of fresh produces. Bacteriophage endolysin, a peptidoglycan hydrolase encoded by phage genomes, are synthesized at the end of the phage life cycle and play important roles in the host cell lysis after phage replication and propagation. Since the endolysins show specific activities only to the peptidoglycan layer generally found in bacteria, they have been considered as safe to humans. Therefore, they have also been suggested as a novel biocontrol agent as well as a natural food preservative to control food-borne pathogens in foods. However, the use of endolysins are still limited to control of Gram-positive bacteria because of the presence of the outer membrane in Gram-negative bacteria which prevent endolysin assessment to the peptidoglycan substrate. On this account, studies of endolysins targeting Gram-negative bacteria are still in the beginning stage. Therefore, further studies of endolysins from Gram-negative bacteria targeting phages are required to develop endolysin-based novel antimicrobial agents against Gram-negative bacteria. For this purpose, a novel endolysin designated M4LysA was newly identified from the phage BSPM4 genome and characterized. Bioinformatics analysis revealed that M4LysA was not homologous to the previously known endolysins. However, when M4LysA was induced in E. coli cell, rapid cell lysis was observed, suggesting that M4LysA is a host cell lysis protein. Indeed, colorimetric assay revealed that M4LysA have endopeptidase activity. Domain analysis results showed that M4LysA is a membrane protein having an apparent transmembrane domain (TMD). By deletion of the C-terminal TMD from M4LysA, solubility was increased while the peptidoglycan lysis activity still remained, suggesting that M4LysA cause cell lysis by degrading the peptidoglycan. Since M4LysA contains unusual membrane domain in C-terminal region, it was revealed to be secreted Sec-translocase pathway independently. Instead, TMD of C-terminal region seemed to be important for its translocation to the periplasm. In addition, the host ranges of M4LysA were broader than those of the parental phage BSPM4, supporting its potential use as a novel antimicrobial agent against Gram-negative bacteria. Despite the advantages of endolysins as biocontrol agents, their applications to the Gram-negative bacteria still have limitations because of the outer membrane barrier. In order to overcome this problem, newly purified endolysin BSP16Lys which is revealed to have N-acetylmuramonyl-L-alanine amidase activity was encapsulated into the lipid vesicles and its antimicrobial activity was evaluated. Without outer membrane permeabilizers addition, the amount of S. Typhimurium was successfully reduced (3-log CFU/mL) within 1 h at room temperature (25ºC) by treating BSP16Lys endolysin-encapsulated liposome. In addition, a liposome containing commercial lysozyme also showed antimicrobial activity without any other membrane permeabilizers. The results suggested the promising use of peptidoglycan hydrolases-encapsulated liposomes as antimicrobial agents against Gram-negative bacteria. In this study, I suggested novel approaches to control S. Typhimurium by utilizing and maximizing the advantage of bacteriophages and endolysins as biocontrol agents. These results will provide not only deep insight into the phage biology but also advanced application strategies of the phages and endolysins as novel antimicrobial agents.

Studies of bacteriophage receptor and domains of endolysin for control and detection of Clostridium perfringens

하은수 서울대학교 대학원 2020 국내박사

클로스트리디움 퍼프리겐스 (Clostridium perfringens)는 그람 양성 균으로 혐기 조건에서 잘 자라고 포자를 형성할 수 있는 세균이다. 이 세균은 자연 환경에서 흔히 존재하고 식중독을 일으킬 수 있는 주요 병원 균 중 하나이다. 또한, 클로스트리디움 퍼프리겐스가 형성한 포자는 높은 열에도 살아남을 수 있어 포자가 오염된 식품을 충분한 열 처리 없이 조리하여 섭취 시 사람의 장 안에서 균으로 발생하여 식중독을 일으킬 수 있다. 전 세계적으로 항생제 다재 내성균이 증가함에 따라 균을 죽이는데 있어 항생제를 대체할 새로운 물질을 개발하려는 노력이 이루어지고 있다. 박테리오파지는 항생제 대체할 수 있는 물질로 각광받으며 많은 연구가 이루어지고 있다. 본 연구자는 본 연구에서 클로스트리디움 퍼프리겐스에 특이적으로 감염하는 용균성 파지 CPS1을 분리하였다. 유전체 분석 결과 CPS1은 19 kbps의 작은 유전체를 가지고 있었고 작은 비수축성 꼬리를 지니고 있어 Podoviridae의 하위 구성인 Picovirinae로 분류할 수 있었다. CPS1의 숙주 수용체를 알아보기 위하여, 무작위로 유전자를 돌연변이 시킬 수 있는 EZ-Tn5를 사용하여 클로스트리디움 퍼프리겐스 ATCC 13124 돌연변이 라이브러리를 구축하고 CPS1을 감염시켜 CPS1에 살아남는 균주를 분리하고자 하였다. 살아남은 균주 중 유전자 CPF_0486이 망가진 균주를 선별하였다. 이 유전자는 이전에 UDP-글루코오스 4-에피머레이스 (GalE)를 발현하는 galE 유전자로 알려져 있었다. 그러나 CPF_0486에서 발현된 단백질을 생화학적 분석을 통해 알아본 결과, CPF_0486 단백질은 GalE 활성뿐만 아니라 UDP-N-아세틸글루코사민 4-에피머레이스 (Gne) 활성 또한 지니고 있었다. 본 연구자는 CPF_0486 유전자가 돌연변이 된 균주가 야생주에 비해 캡슐 다당질 (capsular polysaccharides)이 감소했음을 알 수 있었다. 본 연구자는 또한 CPS1이 숙주에 흡착 시 글루코사민과 갈락토사민이 각각 경쟁적으로 이를 방해함을 발견했다. 이 결과들은 캡슐 다당질이 CPS1의 숙주 수용체라는 것을 보여준다. 클로스트리디움 퍼프리겐스를 저해하기위한 항생제 대체제와 생물학적 검출 제재를 찾기 위해 본 연구자는 클로스트리디움 퍼프리겐스를 특이적으로 감염하는 박테리오파지 CPS2를 닭 똥으로부터 분리하고 특성을 분석했다. CPS2 파지의 유전체는 18 kbps 정도의 이중가닥 DNA이고 25 개의 해독틀 (open reading frame) 정보를 담고 있다. CPS2 또한 CPS1과 마찬가지로 Podoviridae의 하위 구성인 Picovirinae로 분류할 수 있었다. 생물정보학적 분석을 통해 CPS1과 CPS2 파지의 유전 정보 안에 엔도라이신 (endolysin)으로 추정되는 단백질 LysCPS1과 LysCPS2를 찾아냈다. LysCPS1은 pH 7에서 9 사이에 용균 활성을 보이고 pH 7.5에서 0.5 M 염 농도 안에서도 활성을 가지고 있었다. LysCPS2는 pH 7.5에서 pH 10, 25℃에서 65℃, 그리고 넓은 범위의 염 농도 안에서 강한 용균 활성을 보였다. 흥미롭게도 LysCPS2는 95℃에서 10분 간 두어도 용균 활성의 30%가 보존되는 뛰어난 열 안정성을 보였다. 추가적으로 LysCPS2의 C-말단에 존재하는 세포벽 결합 도메인 (cell wall binding domain)은 클로스트리디움 퍼프리겐스에 특이적으로 붙은 것을 알 수 있었다. LysCPS2는 클로스트리디움 퍼프리겐스 용균성 파지로부터 분리한 내열성 엔도라이신에 관한 첫번째 연구로 그 의의가 있다. 클로스트리디움 퍼프리겐스 용균성 파지 CPD7으로부터 유래한 엔도라이신 LysCPD7을 연구하는 과정 중에 본 연구자는 LysCPD7의 C-말단 부위가 클로스트리디움 퍼프리겐스 포자에 붙는다는 것을 발견하였다. 엠체리 (mCherry) 형광 단백질과 결합한 C-말단 부위는 클로스트리디움 퍼프리겐스 영양 세포 (vegetative cell)에는 붙지 않지만 포자에 붙는다는 것을 알 수 있었고, 이를 포자 결합 도메인 (spore binding domain)으로 명명하였다. 면역 금 전자 현미경 기법 (immunogold electron microscopy)과 부착 정도를 비교하는 기법 (binding assay)을 통해 포자 결합 도메인이 포자피질 (spore cortex)과 포자핵 주변에 붙는 것을 확인하였다. 생물정보학적 분석을 활용하여 LysCPD7 C-말단의 다섯 잔기들 (E187K, R192E, Q196A, K201E, E208K)을 포자 결합에 영향을 미치는 주요 잔기로 추정하였다. 점 돌연변이 기법 (point mutation analysis)에서 추정한 다섯 개의 잔기 중 두 개의 잔기가 포자 결합에 영향을 끼치는 것을 알 수 있었다. 점 돌연변이 LysCPD7 유도체들 중 네 개는 야생형 LysCPD7과 비교해서 활성이 떨어졌다. 이들 중 포자 결합에 영향을 주지만 엔도라이신 활성에는 영향을 주지 않는 E187K 돌연변이를 본 연구에 활용하였다. 추후 연구를 위해 파지 유전체 편집 기술을 사용하여 포자 결합 도메인에 점 돌연변이된 LysCPD7 E187K 돌연변이가 포함된 재조합 박테리오파지를 구축하였다. 야생형 파지와 재조합 파지를 포자 형성 조건에서 키운 클로스트리디움 퍼프리겐스에 접종하여 감염된 숙주의 생장 정도를 분석하는 실험을 진행하였다. 본 실험에서 야생형 파지가 감염된 숙주는 재조합 파지가 감염된 숙주보다 포자를 덜 형성함을 확인하였고, 이를 통해 포자 결합 도메인이 클로스트리디움 퍼프리겐스의 포자 형성을 방해한다는 것을 제안할 수 있었다. 본 연구자는 박테리오파지의 수용체를 규명하였고 엔도라이신의 도메인들에 대해 연구하였다. 본 연구 결과를 통해 파지와 엔도라이신을 활용하여 클로스트리디움 퍼프리겐스를 효과적으로 저해할 수 있는 방법과 더불어 엔도라이신의 도메인들이 세균 검출 기술 개발에 활용될 수 있는 가능성을 제시하였다. Clostridium perfringens is a Gram-positive, anaerobic, and spore forming bacterium that is widely distributed in the environment and is one of the most common causes of foodborne illnesses. Besides, C. perfringens spores are important food contaminants because they can survive high temperature in a dormant state and germinate in a food or in a human body. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Bacteriophages are regarded as one of the most promising alternatives to antibiotics in controlling antibiotic-resistant pathogenic bacteria. In this study, virulent C. perfringens phages CPS1 was isolated. Analysis of CPS1 genome and morphology revealed that CPS1 has a small genome (19 kbps) and a short noncontractile tail, suggesting that CPS1 can be classified as a member of Picovirinae, a subfamily of Podoviridae. To determine its host receptor, the EZ-Tn5 random transposon mutant library of C. perfringens ATCC 13124 was constructed and used for screening to select CPS1 resistant mutant. Analysis of the CPS1-resistant mutants revealed that a gene named CPF_0486 was disrupted by Tn5. The CPF_0486 was annotated as galE, a gene encoding UDP-glucose 4-epimerase (GalE). However, biochemical analyses demonstrated that the encoded protein possessed dual activities of GalE and UDP-N-acetylglucosamine 4-epimerase (Gne). I found that the CPF_0486::Tn5 mutant produced a reduced amount of capsular polysaccharides (CPS) compared with the wild type. I also discovered that glucosamine and galactosamine could competitively inhibit host adsorption of CPS1. These results suggest that CPS acts as a receptor for this phage. To find an alternative antibiotic to control and a biological detection agent against C. perfringens, I isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken, additionally. The CPS2 phage contains a 17,961 bp double-stranded DNA genome with 25 putative ORFs, and belongs to the Picovirinae, subfamily of Podoviridae like CPS1 phage. Bioinformatic analysis of the CPS1 and CPS2 genome revealed that both have putative endolysins, LysCPS1 and LysCPS2, respectively. LysCPS1 has a lytic activity at pH 7 to 9 and retains the activity in 0.5 M salt condition at pH 7.5. LysCPS2 showed strong lytic activity against C. perfringens with optimum conditions at pH 7.5–10, 25–65℃, and over a broad range of NaCl concentrations. Interestingly, LysCPS2 was found to be highly thermostable, with up to 30% of its lytic activity remaining after 10 min of incubation at 95℃. The cell wall binding domain (CBD) in the C-terminal region of LysCPS2 showed a binding spectrum specific to C. perfringens strains. The study of LysCPS2 is the first report to characterize highly thermostable endolysin isolated from virulent C. perfringens bacteriophage. In the process of identifying an endolysin LysCPD7 from C. perfringens virulent bacteriophage CPD7, I discovered the spore binding activity of C-terminal region of LysCPD7. The mCherry-fused C-terminal region cannot bind to C. perfringens cells, however, the fusion protein can bind to C. perfringens spores, suggesting that the C-terminal region of LysCPD7 contains a spore binding domain (SBD). Both immunogold electron microscopy and binding assay indicated that the SBD binds to the spore cortex. Bioinformatic analysis of the C-terminal region of LysCPD7 revealed five residues (E187, R192, Q196, K201, E208) supposed to be important for spore binding. Point mutation analysis revealed that two (E187K, R192E) of these five residues are essential for spore binding activity. The LysCPD7 derivatives containing the single point mutation at the four residues (R192E, Q196A, K201E, E208K) showed lower lytic activity compared to the wild-type LysCPD7. I selected the E187K mutant that retains lytic activity as wild type LysCPD7 but impairs spore binding activity. Further, a recombinant phage including E187K of LysCPD7 derivatives was constructed by phage genome editing. The bacterial challenging assay with the recombinant phage or wild type CPD7 phage was performed in C. perfringens sporulation condition. The assay revealed that host cells infected with the recombinant phage showed higher sporulation efficiency than those infected with wild type phage, suggesting that the role of SBD hinders sporulation of the C. perfringens. In this study, I identified a receptor of a C. perfringens-infecting phage CPS1. I characterized endolysins from phage CPS1 and CPS2 as C. perfringens biocontrol agents. CBD and SBD, domains of endolysins, are characterized to detect C. perfringens cells and spores. Further, the biological role of SBD is identified as inhibitor of host sporulation. These results suggest as tools for controlling C. perfringens by application of phage and endolysin. In addition, the results reveal as C. perfringens vegetative cells and spores detection methods by using CBD and SBD of endolysin.

Application of Bacteriophages for Control and Detection of Foodborne Pathogens

정래승 강원대학교 대학원 2024 국내박사

The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. The first part of study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium. The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic- resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009. P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Shiphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60oC) and pHs (5.0 to 11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (>5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction). The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutic for the control of multidrug-resistant pathogens. The second part of study was designed to investigate the possibility of using bacteriophage for detecting viable Shigella boydii in food products. The Shigella bacteriophage belonging to a member of the Siphoviridae family was isolated from swine fecal sample. The free bacteriophages were highly stable against pH 4.0 to 9.0 and temperature change (z-value=17.1oC). The bacteriophage amplification assay could selectively detect S. boydii in the bacterial mixture of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium. The number of S. boydii bacteriophages enumerated by the bacteriophage amplification assay was highly correspond to the number of viable S. boydii in single (r = 0.987) and mixed (r = 0.969) cultures. The bacteriophage-based detection of S. boydii was highly reproducible in lettuce (6.3 log CFU/ml and 4.9 log PFU/ml) and cooked chicken breasts (6.1 log CFU/ml and 6.0 log PFU/ml). These results suggest that the bacteriophage amplification assay can be used as an alternative for rapid, selective, and cost-effective detection of S. boydii in food. This provides useful information for designing a quick and simple detection kit.