3 research outputs found
Isolation and characterization of bacteriophages and endolysins targeting Clostridium perfringens
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κ³Όνλν μν곡νκ³Ό, 2019. 2. μ μμ΄.Clostridium perfringens is responsible for a variety of diseases in humans and animals. Since incidence of C. perfringens in the poultry industry has been increasing and the prevalence of antibiotic-resistant bacteria has also increased, it has been required to develop alternatives to typical antimicrobial treatments. Bacteriophages are bacterial viruses and endolysins are phage-encoded peptidoglycan hydrolases, and they both have received considerable attention as promising antibacterial agents. In this study, I newly isolated and characterized seven bacteriophages showing lytic activity against C. perfringens. Among these phages, CPD2 had the remarkable thermal stability and CPD7 had inhibition activity against C. perfringens FORC 25, that carries chromosomal cpe gene considered to be the virulence factor responsible for causing the several common gastrointestinal diseases. Thus, they were selected for further study. Bioinformatic analysis of CPD2 and CPD7 genome revealed a putative endolysins, LysCPD2 and LysCPD7, which had homology with N-acetylmuramoyl-L-alanine amidase. The antimicrobial spectrum was relatively broad since LysCPD2 and LysCPD7 could infect not only C. perfringens strains but also other Gram-positive bacteria such as B. cereus and B. subtilis strains. Also, as expected, LysCPD2 retained about 80% of the lytic activity up to 95Β°C, indicating remarkable thermal stability and LysCPD7 exhibited considerable antimicrobial activity against C. perfringens FORC 25 than LysCPD2. Hence, LysCPD2 was assumed to maintain its activity at the heat-shock condition (75Β°C for 20 min) for C. perfringens spore germination and it showed significant antibacterial ability against heat- activated germinating spores. Moreover, the bactericidal activity of LysCPD7 against C. perfringens FORC 25 was determined in food such as milk and beef broth. The data presented here suggest that these phages and endolysins can be used as an alternative biocontrol agent for C. perfringens.ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€(Clostridium perfringens)λ μμ€λ
μ λ°κ· μΌλ‘μ μΈκ°κ³Ό λλ¬Όμ κ°μΌνμ¬ λ€μν μ§λ³μ μ λ°ν μ μλ€. νΉν, νμμ λ΄μ±μ νμ°μ λ§κΈ°μν΄ μ±μ₯μ΄μ§μ© νμμ μ μ¬μ©μ κΈμ§ν μ¬λ¬ μ λ½ κ΅κ°μμ κ°κΈλ₯μ ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€λ‘ μΈν λ°λ³ μ¦κ°λ‘ μΈνμ¬ κΈ°μ‘΄μ νμμ λ₯Ό λ체ν μ μλ μλ‘μ΄ νμΈκ· μ μ μ κ°λ°μ΄ νμνλ€. λ°ν
리μ€νμ§ λ° νμ§μμ λΆλ¦¬λ μΈν¬λ²½ λΆν΄ ν¨μμΈ μλλΌμ΄μ μ λ³μκ· μ λν΄ νΉμ΄μ μΌλ‘ μμ©νκ³ λ΄μ±κ· μμ± νλ₯ μ΄ μ μ΄ μλ‘μ΄ νμλ¬Όμ§λ‘μ κ°κ΄λ°κ³ μλ€. λ³Έ μ°κ΅¬μμλ ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€λ₯Ό κ°μΌν μ μλ 7κ°μ νμ§λ₯Ό λΆλ¦¬νκ³ κ·Έ νΉμ±μ λΆμνμλ€. κ·Έ μ€μμ λ΄μ΄μ±μ΄ λ°μ΄λ CPD2μ μ£Όμ λ³μμ± μ μ μμ μνλcpe μ μ μλ₯Ό κ°μ§ κ· μ£Όλ₯Ό μ μ΄ν μ μλ CPD7μ μ μ ν΄ μλλΌμ΄μ μ€νμ μ§ννμλ€. CPD2μ CPD7μ μ μ μλ₯Ό λΆμν κ²°κ³Ό μλλΌμ΄μ μΌλ‘ μΆμ λλ μ μ μλ₯Ό λ°νλκ³ , μ΄λ€μ κΈ°μ‘΄μ N-acetylmuramoyl-L-alanine amidaseμ μλμ±μ 보μλ€. κ°κ°μ μλλΌμ΄μ LysCPD2μ LysCPD7μ λͺ¨λ ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€ λΏλ§ μλλΌ λ°μ€λ¬μ€ μΈλ μ°μ€(Bacillus cereus)μ λ°μ€λ¬μ€ μλΈνΈλ¦¬μ€(Bacillus subtilis)μλ μ μ΄ν¨κ³Όλ₯Ό 보μλ€. λν μμν λ°μ κ°μ΄ LysCPD2λ 95Β°Cμμ 80%μ νμ±μ μ μ§νμ¬ λ°μ΄λ λ΄μ΄μ±μ λνλλ€. LysCPD2μ λ΄μ΄μ±μ λ°νμΌλ‘ ν¬μλ₯Ό λ°μ μ‘°κ±΄μΈ 75Β°Cμμ 20λΆκ° LysCPD2μ ν¨κ» λ°°μνμκ³ , μ΄μ μν΄ νμ±νλ ν¬μμ λν΄ νκ· λ₯λ ₯μ νμΈνμλ€. λ§λΆμ¬ LysCPD7μ LysCPD2λ³΄λ€ ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€ FORC 25 κ· μ£Όμ λν΄ λμ μ©ν΄λ₯μ 보μκ³ , μ°μ μ κ³ κΈ°μ‘μμμλ νκ· νμ±μ νμΈνμλ€. μ΄λ¬ν κ²°κ³Όλ‘ λ―Έλ£¨μ΄ μ€νμ μ¬μ©λ νμ§μ μλλΌμ΄μ μ΄ ν΄λ‘μ€νΈλ¦¬λμ νΌνλ¦°μ μ€λ₯Ό μ μ΄ν μ μλ μλ‘μ΄ λ―Έμλ¬Ό μ μ΄ λ°©μμΌλ‘μ κ°λ₯μ±μ κ°μ§ λ¬Όμ§μμ μ μ μμλ€.ABSTRACT.....................................................................................................i
CONTENTS..................................................................................................iii
List of Figure..................................................................................................vi
List of Table..................................................................................................vii
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. INTRODUCTION..................................................................................1
β
‘. MATERIALS AND METHODS...........................................................5
1. Bacterial strains and growth condition........................................5
2. Isolation and bacteriophages........................................................5
3. High-titer stock preparation of bacteriophages..........................6
4. Transmission Electron Microscopy (TEM) Analysis..................7
5. Bacterial-challenge test in liquid culture.....................................7
6. DNA purification and whole genome sequencing of
bacteriophages................................................................................8
7. Endolysin production....................................................................9
8. Lytic activity assay.......................................................................10
9. Antimicrobial activity of LysCPD7 in food samples.................12
10. Amidase assay............................................................................12
11. Spore preparation and purification.........................................13
12. Nucleotide sequence accession number....................................14
13. Statistical analysis.....................................................................14
β
’. RESULTS..........................................................................................15
1. Isolation and host ranges of bacteriophages.................................15
2. Morphology of the phages and challenge assay...........................17
3. Thermal stability of the phages..................................................20
4. Genome analysis of the phages...................................................21
5. Identification and expression of endolysins...............................23
6. Antimicrobial spectrum of the endolysins.................................29
7. Effects of NaCl, pH, and temperature on the endolysins..........31
8. Determination of thermal stability.............................................33
9. Antibacterial ability of LysCPD7 against C. perfringens in milk and beef broth..................................................................................35
10. Antibacterial ability of endolysins against heat-activated spores...............................................................................................38
β
£. DISCUSSION.......................................................................................40
β
€. REFFERENCES..................................................................................43
κ΅λ¬Έμ΄λ‘.......................................................................................................49Maste
FormalinμΌλ‘ μ λλ ν΅μ¦ λͺ¨λΈμμ capsaicin μ½μΉ¨μ ν΅μ¦ μ΅μ ν¨κ³Ό
Dept. of Medical Science/μμ¬Acupuncture combined with pharmaceutical agents has been used to treat diseases in humans and animals. Capsaicin, the active element in hot chili peppers, has selective actions on unmyelinated C-fibres and thinly myelinated A primary sensory neurons. Capsaicin induces depolarization of nerve cell by intracellular accumulation of cation. However, high concentration of intracellular calcium ion is known as voltage sensitive cation channelβs blocker for long time. As a result, continuous intracellular accumulation of cations induce desensitization of nociceptive neuron. This implies capsaicin may be used to relieve pain as one of pharmaceutical agents which can be combined with acupuncture. Manganese-enhanced magnetic resonance imaging (MEMRI) is based upon neuronal activity-dependent manganese (Mn) uptake which can provide useful information about functions of the nervous system. However, no systematic studies regarding processing of pain using MEMRI have not been conducted. The present study was conducted to determine whether capsaicin acupuncture as one of pharmaceutical acupuncture can relieve pain effectively, using formalin test, MEMRI, and c-Fos immunohistochemistry in rats. Male Sprague-Dawley rats (230 ~ 250 g) were used in this study. For MEMRI investigation, MnCl2 solution (50 γ) was injected into subarachnoid space. After then, capsaicin was injected by concentration (0, 0.1, 1, and 2%) at the Zusanli (ST36) acupoint of the left leg. Formalin solution (5 %, 50 γ) was injected into the plantar side of left hind paw and pain-related behavior (flinching) was immediately observed for 1 hr. After behavioral test, MEMRI was performed in a Biospec 4.7 T MRI system. For analyzing MnCl2 distribution, a set of noncontiguous T1-weighted (T1W) images were acquired. Lastly, immunohistochemistry was performed to observe c-fos expression.
Capsaicin injected at ST36 acupoint inhibited formalin-induced pain behaviors. In formalin test, flinching behavior of high concentration capsaicin-injected rats was significantly reduced compared to 0% capsaicin-injected group. MEMRI results showed difference of intensity induced by manganese ion between formalin only treated group and capsaicin treated group. In addition, c-Fos expression level was effectively decreased in capsaicin-treated group compared to formalin only treated group. In conclusion, this experiment using formalin test, MEMRI and c-Fos immunohistochemistry support that capsaicin can reduce formalin-induced pain. It suggests that capsaicin acupuncture as one of pharmaceutical acupunctures may be effective in relieving pain.restrictio
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Background: Hypercalcemia is an important metabolic emergency condition in cancer patients. Bisphosphonate is the treatment of choice for hypercalcemia, whereas calcitonin and hydration with furosemide are recommended for acute supportive therapy