항 Methicillin Resistant Staphylococcus aureus 활성을 가지는 해양미생물 Pseudomonas sp. YJ-1의 분리와 특성

우예주,정성윤 한국환경생물학회 2017 환경생물 : 환경생물학회지 Vol.35 No.4

The aim of this study was to isolate and identify marine bacterium with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and to purify the anti-MRSA compound, as well as to determine its activity and synergistic effects. Among the marine bacteria isolated in this study, the YJ-1 isolate had the strongest anti-MRSA activity. The YJ-1 isolate was identified on the basis of its biochemical characteristics and an analysis of 16S rRNA gene sequences. The YJ-1 isolate showed over 99.2% homology with Pseudomonas stutzeri, and was designated as a Pseudomonas sp. YJ-1. The optimal culture conditions were 25℃ and initial pH 7.0. For the purification of the anti-MRSA compounds, the YJ-1 was cultured in Pa PES-II medium, and the culture filtrates were extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by a C18 ODS column, silica gel chromatography and a reverse phase HPLC, to yield three anti-MRSA agents, the MR1, MR2, and MR3 compounds. When the MR1 compound of 250 μg mL-1 concentration was applied to the MRSA cells, over 95% of bacterial cells was killed within 48 hr. Compared with vancomycin and ampicillin, the MR1 compound showed significant anti-MRSA activity. In addition, the anti-MRSA activity was increased by dose and time dependent manners. Furthermore, the combination of an MR1 compound with vancomycin produced a more rapid decrease in the MRSA cells than did the MR1 compound alone. Taken together, our results suggest that the Pseudomonas sp. YJ-1 and its anti-MRSA compounds could be employed as a natural antibacterial agent in MRSA infections. 본 연구의 목적은 항 methicillin-resistant Staphylococcus aureus (MRSA) 활성을 가지는 해양미생물을 분리, 동정하 고 항MRSA 물질을 분리 정제하여 그 물질의 활성과 시너 지 효과를 밝히는 것이다. 본 연구에서 분리한 해양미생물 중에서, YJ-1 분리 균주가 가장 강한 항MRSA 활성을 나타 내었다. YJ-1 균주는 생화학적 특성과 16S rRNA 유전자 염 기서열에 기초하여 분류 동정되었다. YJ-1 균주의 염기서 열은 Pseudomonas stutzeri와 99.2%의 상동성을 나타내어, Pseudomonas sp. YJ-1이라 명명하였다. 이 균주의 최적 성 장조건은 25℃와 초기 pH 농도 7.0이었다. 항MRSA 물질들 을 분리정제하기 위하여 YJ-1 균주를 PPES-II 배지에 배양하 였으며, 배양 상등액을 ethyl acetate, hexane과 80% MeOH 로 순차적으로 추출하였다. 활성을 보인 80% MeOH 분획을 C18 ODS 칼럼 크로마토그래피, silica gel 크로마토그래피와 역상 HPLC법으로 순차적으로 정제하여 항MRSA 활성을 가지는 3개의 순수물질 MR1, MR2 및 MR3를 얻었다. 250 μg mL-1 농도의 MR1 물질을 MRSA 세포에 접종했을 때, MRSA 세포의 95%가 48시간 이내에 사멸하였다. 활성을 vancomycin 및 ampicillin과 비교해 보아도, MR1 물질은 보 다 우수한 항MRSA 활성을 나타내었다. 또한 항MRSA 활성 은 투여량과 시간에 비례하여 증가하였다. 더욱이 MR1 물질 과 vancomycin을 조합하여 시너지 효과를 보았을 때, MR1 물질을 단독으로 투여했을 때보다 신속한 MRSA 세포의 감 소를 관찰할 수 있었다. 이상의 결과를 종합해보면, MRSA 감염에 있어서 Pseudomonas sp. YJ-1과 이것이 생산하는 항MRSA 물질은 천연 항균제로서 기여할 수 있으리라 판단 된다.

임상분리 Methicillin Resistant Staphylococcus aureus에 대한 항균활성 시너지 효과

정성윤 ( Seong-yun Jeong ) 대구가톨릭대학교 자연과학연구소 2017 자연과학연구논문집 Vol.15 No.1

최근 병원 감염의 주 원인균이며 기존 항생제에 다제내성을 보이는 MRSA(Methicillin Resistant Staphylococcus aureus)가 문제가 되고 있다. 다제내성균에 대한 치료제로 vancomycin이나 teicoplanin이 사용되고 있으나 그 사용이 아주 제한적이며, 최근에는 이들 항생제에 대한 내성균주가 많이 출현하여 새로운 항생제 개발이 절실히 요구되고 있다. 따라서 본 연구에서는 해양 미생물에서 분리한 항MRSA 활성을 나타내는 YJ-1 균주의 최적배양 조건을 확립하고, 이 균주가 생산하는 항균활성물질을 분리, 정제하여 신규 항생제제로서의 가능성을 제시하고자 하였다. 울진에서 해수를 sampling한 후 가장 강력한 anti-MRSA 활성을 나타내는 YJ-1 균주를 선택하여 분류, 동정하여 Pseudomonas sp. YJ-1으로명명하였다. 본 균주의 최적 성장 온도는 25℃였으며, 20℃와 30℃에서 성장에 큰 차이는 없었으며, 10℃와 4℃에서는 거의 성장하지 못 하였다. 최적 pH는 7.0으로 나타났으며, pH 6.0-9.0 사이에서 비교적 양호한 성장을 보였고, pH 4.0과 10.0에서는 거의 성장하지 못 하였다. 최적 NaCl 농도는 1%에서 가장 높게 나타났지만 NaCl 0% 농도에서와 큰 차이는 없었고, 3.0%까지 양호한 성장률을 보였으나 염농도가 높을수록 점점 성장률이 낮아지다가 염농도 7.0 이상에서는 거의 성장하지 못 하였다. 각종 유기용매 추출, C18 ODS open column, silica gel chromatography, reverse-phase HPLC 등을 이용하여 anti-MRSA 활성을 나타내는 순수 물질을 얻었으며, 이 중 MR1 물질은 임상분리 MRSA 균주인 DA 12와 DA 16 균주에 대해서도 비교적 우수한 활성을 나타내었다. MR1 물질을 농도별, 시간별로 DA 12와 DA 16 균주에 접종하여 항균활성을 비교한 결과, MR1은 vancomycin과 비슷하거나 오히려 높은 항균활성을 나타내어 MRSA에 대한 새로운 항생제 개발의 가능성을 제시 할 것으로 판단된다. 또한 MR1과 vancomycin과 함께 처리했을 때의 항MRSA의 시너지 효과를 조사한 결과, 임상분리 균주인 DA 12와 DA 16에 대해서 모두 우수한 시너지 효과를 나타내었다. 이상의 결과 향후 YJ-1으로부터 정제한 최종 물질의 화학적 특성과 구조를 규명하고, 작용기작을 밝힌다면 차세대 항생제로서의 개발 가능성이 높을 것으로 판단된다. It was isolated marine bacterium, isolate YJ-1 which produced a bactericidal antibiotics against MRSA. This isolate YJ-1 was identified to the genus Pseudomonas and was designated Pseudomonas sp. YJ-1. Pseudomonas sp. YJ-1 relatively grows well in the 25℃, pH 7.0, NaCl 1.0 %. For the purification of the bioactive compounds, YJ-1 was fermented in 37 L PPES-Ⅱ medium, and culture filtrates of YJ-1 was extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by C18 ODS open column, silica gel chromatography and reverse phase HPLC to yield three anti-MRSA agent, MR1 compound. Compared with vancomycin and ampicillin, the MR1 has the potent activity against MRSA. In addition, the combination of MR1 compound with vancomycin produced a more rapid decrease in the clinical isolates, DA 12 and DA 16 MRSA cells than MR1 compound alone. Taken together, our results suggest that Pseudomonas sp. YJ-1 and its anti-MRSA compound could be employed as a natural antibacterial agent in MRSA infection.

Anti-MRSA Properties of Prodigiosin from Serratia sp. PDGS 120915

Keunho Ji(지근호),Tae Hyug Jeong(정태혁),Young Tae Kim(김영태) 한국생명과학회 2015 생명과학회지 Vol.25 No.1

천연의 붉은 색소인 prodigiosin은 Serratia marcescens에 의해서 생산되며 이는 pyrrolylpyrromethane 골격으로 구성되어 있다. 이 색소는 그 분자가 가진 특성 때문에 넓은 범위에서 활용되고 있다. 또한 항암제, 면역억제제, 항진균제, 살조제 등 다양한 분야의 효과가 보고되소 있다. Methicillin-resistant Staphylococcus aureus (MRSA)는 세계적으로 가장 주요한 원내 감염균으로써, 미국에서 해마다 HIV로 사망하는 수보다 더 많은 사망률을 보이고 있다. 그러므로 MRSA에 대한 새로운 치료제의 개발이 매우 중요하며 급박한 문제이다. 본 연구에서는 중증오염도를 가진 하천수로부터 Serratia sp. PDGS 120915를 분리하였으며 항 MRSA 활성을 가진 prodigiosin에 대하여 연구하였다. 본 연구를 위하여 HPLC를 이용하여 물질을 정제하였으며, 분리된 물질의 항 MRSA 활성을 확인하였다. Prodigiosin의 MRSA에 대한 최소억제농도(Minimal inhibitory concentration; MIC)는 32 μg/ml 이었으며, 분할저해농도(Fractional inhibitory concentration; FIC)는 ampicillin과 penicillin에서 상승작용이 있는 것으로 나타났다. Prodigiosin, a member of natural red pigment family, is produced by Serratia marcescens, and characterized by a common pyrrolylpyrromethane skeleton. This pigment has been reported with the effects of anticancer, immunosuppressant, antifungal, and algicidal activities. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital infections. In this study, anti-MRSA properties of prodigiosin isolated from Serratia sp. PDGS 120915 were investigated. We identified and purified prodigiosin using high performance liquid chromatography (HPLC) and evaluated anti-MRSA activity. Purified prodigiosin inhibited the growth of MRSA. The minimum inhibitory concentrations (MICs) of prodigiosin were determined to 32 μg/ml against the MRSA strains. Fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were indicated synergistic effects of prodigiosin on MRSA.

해양 방선균 유래 항 Methicillin Resistant Staphylococcus aureus 물질의 활성 및 상승 효과

정성윤 (사)한국해양바이오학회 2022 한국해양바이오학회지 Vol.14 No.2

We isolated marine actinomycetes, strain D-5 which produces anti-methicillin resistant Staphylococcus aureus (anti-MRSA) compound. Streptomyces sp. D-5 relatively grew well in the 20∼25℃, pH 8.0, and NaCl 3.0%. The ethyl acetate extract of D-5 culture was separated by C ODS open column and reverse phase HPLC to yield anti-MRSA compound. The molecular weight of this compound was determined to be 898 by a Liquid chromatograph-mass spectrometer (LC-MS). Compared with penicillin G, this compound showed significant anti-MRSA activity. It also exhibited an inhibition zone of 26 mm at a concentration of 64 µg/disk and an inhibition zone of 16 mm at a concentration of 16 µg/disk against the MRSA KCCM 40511. Furthermore, the co-treatment of HPLC peak 5 compound and vancomycin caused a more rapid decrease in MRSA cells than each compound alone. It showed 86.8% growth inhibition activity within 12 hours at a low concentration of 50 µg/mL during co-treatment, and 97.1% growth inhibition activity within 48 hours against MRSA KCCM 40511. Taken together, our results suggest that Streptomyces sp. D-5 and its anti-MRSA compound could be employed as a potent agent in MRSA infection.

길경 추출물의 항균활성

이보미 ( Bo Mi Lee ),이보은 ( Bo Eun Lee ),정한규 ( Han Gyu Jeong ),강은현 ( Eun Hyun Kang ),최민정 ( Min Jung Choi ),정성윤 ( Seong Yun Jeong ) 대구가톨릭대학교 자연과학연구소 2012 자연과학연구논문집 Vol.10 No.1

전통 한의학에서 많이 사용되는 길경은 도라지 (Platycodon grandiflorum A. DC)의 뿌리 껍질을 벗기거나 또는 그대로 말린 것을 말하며, 염증성 호흡기 질환에 많이 사용되어져 오고 있다. 본 연구에서는 길경의 열수 추출물과 각종 유기용매 추출물의 항균활성을 밝히고자 MRSA를 비롯한 병원성 세균에 대한 영향을 조사하였다. 그 결과 길경의 핵산추출물과 열수추출물은 우수한 항균활성을 나타내었으며, 에틸아세테이트 추출물과 부탄올 추출물도 비교적 우수한 항균활성을 나타내었다. 또한 길경의 모든 추출물은 MRSA KCCM 40511, DA16 (MRSA), Mycobacterium smegmatis KCTC 2002, Aeromonas hydrophilia KCTC 2358에 항균 활성을 나타내었다. 길경의 핵산추출물은 임상분리한 포도상구균인 DA16과 MRSA KCCM 40511에 대해, 각각 15와 13 mm의 억제환을 나타내었다. 또한 200 μg/disk의 농도에서Mycobacterium smegmatis와 Shigella flexneri에 대해 각각 14와 10 mm의 억제환을 형성하였다. 그러나 고초균 Bacillus subtilis에 대해서는 항균활성을 나타내지 않았다. 길경의 열수추출물은 DA16 (MRSA) 균주에 대해 가장 높은 20 mm의 항균활성을 나타내었으며, 식중독균인 Aeromonas hydrophilia에 대해 17 mm의 높은 생육저지력을 나타내었다. 또한 황생포도상구균인 MRSA KCCM 40511 균주에 대해 13 mm의 억제환을 형성하였다. 그러나 냉방기에 주로 서식하는 Legionella birminghamensis에 대해서는 7 mm의 약한 활성을 나타내었다. 또한 열 수추출물을 EA와 DW로 이층 분배하여, 항균활성을 측정한 결과, HWE-DW층은 MRSA KCCM 40511, Mycobacterium smegmatis KCTC 2002, Legionella birminghamensis KCTC 2007에 대해 각각 10 mm, 11 mm, 10 mm의 매우 우수한 항균 활성을 나타내었다. 더우기 625 ng/disk의 매우 낮은 농도를 감안한다면, 이상의 결과들은 길경의 HWE-DW층을 이용한 새로운 항균제 개발의 가능성이 있음을 시사하여 준다. Platycodi Radix, the root of Platycodon grandiflorum, commonly known as Doraji, is used as a traditional oriental medicine. Extracts from the roots of P. grandiflorum have been reported to have wide ranging health benefits. In the present study, we investigated the antibacterial activities of hot-water extract and various organic solvent extract from the roots of P. grandiflorum. Hot-water extract showed the significant anti-MRSA (methicillin-resistant Staphylococcus aureus) activity. For the purification of the bioactive extract, hot-water extract was partitioned with ethyl acetate and deionized water. Deionized water extract of hot-water extract (HWE-DW) showed the significant anti-MRSA and antibacterial activity in the concentration of 625 ng/disk. Taken together, these findings suggest that P. grandiflorum has strong potential for development as an antibacterial agent against MRSA and various pathogenic bacteria.

Synergistic Anti-bacterial Effects of Phellinus baumii Ethyl Acetate Extracts and β-Lactam Antimicrobial Agents Against Methicillin-Resistant Staphylococcus aureus

홍승복,이만휘,윤봉식,임영훈,송형근,신경섭 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.2

Background: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with β-lactams against MRSA. Methods: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and β-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. Results: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 μg/mL, respectively. The PBEAE significantly reduced MICs of all β-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-β-lactams. Time-killing assays showed that the synergistic effects of two β-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Δlog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the β-lactams and the PBEAE. Conclusions: PBEAE can enhance the efficacy of β-lactams for combined therapy in patients infected with MRSA.

Original Articles : Antimicrobial Activity of Brown Alga Eisenia bicyclis against Methicillin-resistant Staphylococcus aureus

( Sung Hwan Eom ),( Jae Hong Park ),( Dae Ung Yu ),( Ji Il Choi ),( Jong Duck Choi ),( Myung Suk Lee ),( Young Mog Kim ) 한국수산과학회(구 한국수산학회) 2011 Fisheries and Aquatic Sciences Vol.14 No.4

We screened for antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). Methanolic extract of Eisenia bicyclis exhibited anti-MRSA activity according to a disk diffusion assay. To identify the active compound(s), the methanolic extract was further fractionated using hexane, dichloromethane, ethyl acetate, and n-butanol. The ethyl acetate-soluble fraction showed both the greatest anti-MRSA activity and the highest polyphenol content. The minimum inhibitory concentrations of the ethyl acetate fraction ranged from 32 to 64 μg per mL against methicillin-susceptible S. aureus and MRSA strains. High-performance liquid chromatography analysis revealed that both the methanolic extract and the ethyl acetate soluble fraction contained sizeable quantities of dieckol, which is a known anti-MRSA compound. Thus, these data strongly suggest that the anti-MRSA activity of E. bicyclis may be mediated by phlorotannins such as dieckol.

Production and Biological Activity of Laidlomycin, Anti-MRSA/VRE Antibiotic from Streptomyces sp. CS684

Yoo, Jin-Cheol,Kim, Jun-Ho,Ha, Jung-Wan,Park, Nae-Soo,Sohng, Jae-Kyung,Lee, June-Woo,Park, Seong-Chan,Kim, Mi-Sun,Seong, Chi-Nam The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.1

Culture broth of a streptomycete isolate, Streptomyces sp. CS684 showed antibacterial activity on methicilin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Among purified substances from the organism, CSU-1, which is active against MRSA and VRE, is a $C_{37}H_{62}O_{12}Na\;(M^+,721.3875)$, and identified as laidlomycin. The anti-MRSA and anti-VRE activity of CSU-1 was stronger than oxacillin and vancomycin. Phylogenetic analysis showed that strain CS684 is very similar to Streptomyces ardus NRRL $2817^T$, whereas the ability of Streptomyces sp. CS684 to produce laidlomycin was shown to be unique.

Antimicrobial Activity of Brown Alga Eisenia bicyclis against Methicillin-resistant Staphylococcus aureus

Eom, Sung-Hwan,Park, Jae-Hong,Yu, Dae-Ung,Choi, Ji-Il,Choi, Jong-Duck,Lee, Myung-Suk,Kim, Young-Mog The Korean Society of Fisheries and Aquatic Scienc 2011 Fisheries and Aquatic Sciences Vol.14 No.4

We screened for antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). Methanolic extract of Eisenia bicyclis exhibited anti-MRSA activity according to a disk diffusion assay. To identify the active compound(s), the methanolic extract was further fractionated using hexane, dichloromethane, ethyl acetate, and n-butanol. The ethyl acetate-soluble fraction showed both the greatest anti-MRSA activity and the highest polyphenol content. The minimum inhibitory concentrations of the ethyl acetate fraction ranged from 32 to 64 ${\mu}g$ per mL against methicillin-susceptible S. aureus and MRSA strains. High-performance liquid chromatography analysis revealed that both the methanolic extract and the ethyl acetate soluble fraction contained sizeable quantities of dieckol, which is a known anti-MRSA compound. Thus, these data strongly suggest that the anti-MRSA activity of E. bicyclis may be mediated by phlorotannins such as dieckol.

Antimicrobial Activity of Brown Alga Eisenia bicyclis against Methicillin-resistant Staphylococcus aureus

엄성환,박재홍,유대웅,최지일,최종덕,이명숙,김영목 한국수산과학회 2011 Fisheries and Aquatic Sciences Vol.14 No.4

We screened for antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). Methanolic extract of Eisenia bicyclis exhibited anti-MRSA activity according to a disk diffusion assay. To identify the active compound(s), the methanolic extract was further fractionated using hexane, dichloromethane, ethyl acetate, and n-butanol. The ethyl acetate-soluble fraction showed both the greatest anti-MRSA activity and the highest polyphenol content. The minimum inhibitory concentrations of the ethyl acetate fraction ranged from 32 to 64 μg per mL against methicillin-susceptible S. aureus and MRSA strains. High-performance liquid chromatography analysis revealed that both the methanolic extract and the ethyl acetate soluble fraction contained sizeable quantities of dieckol, which is a known anti-MRSA compound. Thus, these data strongly suggest that the anti-MRSA activity of E. bicyclis may be mediated by phlorotannins such as dieckol.