• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1916-1924
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• 2019

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Hg²⁺, and Zn²⁺) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.

• 대한한방부인과학회지
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• 제19권2호
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• pp.92-106
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• 2006

Purpose : To address the ability of Olibanum to induce cell death, we investigated the effect of olibanum on cell apoptosis. Twenty-four hours later, apoptosis occurred following olibanum exposure in a dose-dependent manner. Methods : We culture HeLa cell which is human metrocarcinoma cell in D-MEM included 10% fetal bovine serum(Hyclone Laboratories) below $37^{\circ}C$, 5% CO2. Then we observed apoptosis of log phage cell which is changed cultivation liquid 24 Hours periodically. Results : The treatment of BAPTA-AM regulated olibanum-induced apoptosis in HeLa human cervical carcinoma cells. The 24 hr-earlier -thapsigargin-pretreated cell showed the resistance against olibanum-induced apoptosis and the Ru360-mitochondrial uniporter-inhibited olibanum-induced apoptosis, too. It means that olibanum leads to the accumulation of calcium and the resultant apoptosis in HeLa cells. Immunoblotting data also shows that the expression of GRP78, ER stress marker protein, was induced by the olibanum. Bcl-2, anti-apototic protein, was decreased and that the expression of Bax, pro-apoptotic protein, was increased by the addition of olibanum. Interestingly, the olibanum increased the activity of caspase-8 as well as calpain cysteine pretense in HeLa cervical carcinoma cells. Calpain inhibitor-calpastatin as well as caspase-8C/A expression abrogated olibanum-induced apoptosis in the carcinoma cells. The inhibition of caspase-8 regulated olibanum-induced calpain activation but the inhibition of calpain did not have any effect on the caspase-8 activation in HeLa human cervical carcinoma cells. Conclusion : We conclude that olibanum induces the accumulation of calcium and the resultant apoptosis in which caspase-8 and calpain are involved.

• 미생물학회지
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• 제55권2호
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• pp.87-102
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• 2019

대부분의 만성창상(chronic wounds)은 생물막으로 인해 상처 치유시 염증단계를 지속시킨다. 생물막은 항생제(antibiotics)에 대한 저항성을 가지며 침투력을 저하시키고 살균제(biocides)에 대한 내성을 지니며 국소면역반응을 약화시킨다. 또한 생물막은 주변의 조직에 단단히 붙어 있어 제거하는 작업이 매우 어렵다. 그러므로 주변 조직을 손상시키지 않으면서 단단한 생물막을 제거하는 전략은 매우 중요하다. 그 중에 하나가 분산기작을 이용한 생물막의 해체이며 지금까지 많은 연구가 수행되어 왔다. 본 고찰논문에서는 특별히 화학주성, 파지요법, 다당류, 다양한 효소(당분해효소, 단백질분해효소, DNA 분해효소), 계면활성제, 분산신호, 자기유도인자, 조절인자, 억제제 등이 소개되었으며 더 나아가 항생제 치료 및 다른 치료와의 병행을 통한 병합요법도 소개되었다. 앞으로 본 논문에서 제시된 생물막의 분산기작의 지식을 이용하여 만성 창상 감염치료의 가능성이 더 높아지길 기대한다.

• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.816-823
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• 2022

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 µM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 µM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

• 미생물학회지
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• 제34권4호
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• pp.236-242
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• 1998

박테리오파지 P2-P4 시스템은 바이러스 조립과정 기작 연구를 위한 뛰어난 실험 소재로 연구되어 왔으나, 이 시스템에 유전자 조작상 필수적인 유용한 플라스미드가 없는 관계로 그의 필요성이 대두되고 있다. 본연구에서는 도움파지가 없을 때 플라스미드 상태로 존재하는 P4 ash8 (sid71)을 시작물질로, 항생제 내성 유전자를 도입하여 선택성을 줄 목적으로 P4 파지 증식에 불필요한 P4 DNA 단편을 pUC4-K 플라스미드의 kmr(kanamycin resistance) 유전자로 치환하여 P4 ash8(sid71) kmr을 생성하였다. 이 플라스미드는 박테리오파지 P2로 induction하여 박테리오파지 P4 상태로 증식시킬 수 있게 그 genome 크기를 조정하였으며, 파지 상태로 전환된 것을 burst size 결정 및 CsCl 부양 균등밀도 편차실험을 통해 입증하였다. 생성된 P4 플라스미드 유도체를 유전자 조작하여 P4의 integrase 기능을 잃은 변이체를 쉽게 만들 수 있었으며, 역시 박테리오파지 P4 상태로 증식시킬 수 있었다. 이러한 변이체 플라스미드의 안정성 실험을 수행하여, P4의 integrase 기능이 지속적인 플라스미드 상태 유지를 위해 필요하다는 것을 보여주었다.

• Journal of Dairy Science and Biotechnology
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• 제32권2호
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• pp.93-100
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• 2014

치즈시장이 심각한 국제경쟁을 직면하게 되면서 최적화된 생산과정은 유제품회사가 성공하기 위해 더 중요해졌다. 치즈 배치로부터 생성되는 유청(whey)은 최종 생산품의 산출량 증가, 질감 개선, 영양가 증진을 하기 위한 목적으로 유제품 생산과정 중에 종종 재사용된다. 유청크림(whey cream)과 미립자 유청 단백질(particulated whey protein)은 흔하게 재활용된다. 그러나 이 물질들 내에 저온살균 처리 후에도 남아있는 대부분의 박테리오파지(bacteriophage)가 치즈 제조 과정 중에 들어가 높은 수치로 증식할 위험이 있다. 유청분리는 종종 aerosol-borne 파지를 생성하여 공장 생산과정을 오염시키기 때문에, 박테리오파지에 의한 유청오염은 치즈 생산과정에 문제를 일으킬 수 있으며, 또한 치즈주형으로 재사용되는 유청과 유청 단백질 내에는 내열성 파지(thermo-resistant phage)가 존재할 수 있다. 수분이 제거된 치즈로부터 생성된 유청의 경우, 파지에 의해 $10^9$파지/mL까지 감염될 수 있다. 유청배치를 농축시켰을 경우에는 파지 역가(phage titer)가 10배까지 증가하게 되는데, 이 수치까지 도달하게 되면 파지를 완전히 제거하는 것은 불가능해진다. 유청 재활용 과정 중에 발생하는 발효실패를 방지하기 위해서는 유청 내 존재하는 파지의 수를 감소시키는 처리법이 필요하다. 따라서 열처리, 자외선(UV), 막여과 등의 기술을 사용하여 유청 내의 파지를 불활성화시키는 처리법에 대해 중점적으로 연구자 진행되고 있는 분야이다. 열로 파지를 불활성화시키는 방법이 가장 흔히 사용되는 처리법이지만 내열성 파지를 열로 불활성화 시킬 경우에 자연산 유청 단백질이 받게 되는 부정적인 영향으로 인해 이 처리법의 사용에는 제한이 있다. 따라서 온도를 높이고 시간을 늘리는 방법보다는 다른 결합된 처리법을 사용하여 유청 내의 파지를 제거해야 것이 요구되어진다. 열처리를 막여과 또는 UV와 함께 사용할 경우, 유청 내의 파지 수를 효과적으로 감소시킬 수 있을 것으로 사료되어진다.

• 미생물학회지
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• 제17권4호
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• pp.165-178
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• 1979

Phages of Lactobaciilus casei (PLC) isolated from plant drainage were classified and characterized. The results are as follows : 1. On the basis of host range pattern, phages could be divided into 2 groups (PLC-B and PLC-C). PLC-B group phages could be further divided into 5 sub-groups $(B_1, \;B_2, \;B_3, \;B_4, \;and\;B_5)$. Although PLC-C group phages had the same host range, they could be also divided into 2 sub-groups $(C_1\;and\;C_2)$ by morphlogical type. 2. It was $B_3$ group phages that represented a major proportion (44.4%) of phages tested. However, $B_1$ group phages were shown to have the widest host range. 3. Electron micrographs revealed that the phages fell into three different morphological types. $(B_1, \;B_2, \;and\;B_3)$ group phages hd a hexagonal head (52nm in diameter) and a sheathless noncontractile (245 nm in length). $B_4\;and\;C_2$ group phages had a hexagonal head (56 nm) and a short flexible tail (169nm) having no sheath. $B_5\;and\;C_1$ group phages were shown to have a hexagonal head (81 nm) and a contractile tail (140 nm) having a sheath, a base plate and tail fibers. 4. The inactivation of the phages by antisera indicated that serological relationships correlated completely with morphological types. 5. $B_1, \;C_1\;and\;C_2$ group phages produced a large (1, 2 mm in diameter) plaque with a clear ring. The morphology of plaques of $B_3\;and\;B_5$ group phages was the same as those produced by the above, but the average plaque sizes for $B_3\;and\;B_5$ were 0.8 mm abd 0.5 mm, respectively. $B_2\;and\;B_4$ group phages produced a small (0.5 mm) turbid plaque with an irregular edge. 6. The latent period and the average burst size of $B_1\;and\;B_3$ group phages were 90 min and 100, respectively. These phages reuqired calcium ions for their miltiplication. 7. $B_3$ group phages could not be absrobed to R-variant $KC_1$. 8. The order of resistance of phages to heat was $B_2\;>\;B_1, B_4\;and\;B_5\;>\;B_3\;and\;C_2, \;B_5$ group phages were more stable than $B_3$ in various pH values. $C_2$ group phages were more sensitive to UV irradiation than $B_1\;and\;B_3$ group phages. 9. Strains YIT9018 and IAM 1043 were induced by mitomycin C treatment. Phage particles detected in the lysates had a hexagonal head (38 and 49 nm, respectively), but no tail. Any sensitive indicator strain could not be isolated in spite of repaeated trials.

• 대한미생물학회지
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• 제21권1호
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• pp.73-95
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• 1986

A total of 5,462 isolates suspicious of Salmonella, Shigella and E. coli which were isolated during 1983 to 1985 by 12 City Hygine Laboratories and General Hospital Laboratories were received and identified at the National Salmonella Center, Seoul, Korea. The result of identification of these strains were summarized as follows: 1. It was confirmed that the total organisms broke down into 2,014 strains of Salmonella 1,294 of which were S. typhi, 887 strains of Shigella and 2,561 strains of E. coli. 2. For seasonal distribution of enteric pathogens, July was the month with the highest out breaks of salmonellosis, May was the month of Shigellosis, and April was of the highest month it in the case of E. coli. 3. Salmonella typhi with the highest incidence of isolation was shown to belong to various phage types, especially with the strains detected in Seoul. M1 type was widely distributed all over the country, but the majority was E1 type in 1983. 4. For age distribution of patients, the 20-29 age group had the highest incidence of salmonellosis whileas the 1 to 9 age group had the highest incidence of Shigellosis. 5. For sexuly distribution of Salmonella and Shigella infections seemed to be relatively higher in the female than in the male. However, E. coli. had no relationship to both sex. 6. The antibiotic sensitivity patterns of S. typhi cultures showed a tendancy to be resistant to colistin, gentamycin, neomycin, tetracycline and streptomycin. 7. The isolates of S. paratyphi-A, S. typhimurium and S. enteritidis seemed to have a tendency of multiple drug resistance. 8. 93.9 percent of 1,568 E. coli strains showed negative reactions to the antisera of enteropathogenic E. coli and 15.6 percent of them produced a heat-labile enterotoxin, but positive reaction to the antisera was 6.1 percent and 11.6 percent of them producled the enterotoxin.