MRSA(Methicillin Resistant Staphylococcus aureus)에 유효한 항균물질을 생산하는 해양세균의 분리

Abstract

MRSA에 항균활성을 나타내는 해양세균을 분리하여 동정하였으며 분리된 균이 생산하는 항균물질을 통하여 MRSA에 감염된 환자의 치료에 사용되고 있는 vancomycin의 대체 치료제의 개발뿐만 아니라 vancomycin과 병용하여 투여함으로서 vancomycin의 사용량을 줄일 수 있는 연구의 기초자료를 제공하고자 연구한 결과는 다음과 같다. 1. 울진 근해에서 1,000여종의 해양세균을 순수 분리하였으며, 이들 세균들의 MRSA에 대한 항균물질 생성능을 disk diffusion method (NCCLS, 2002)에 따른 확인 시험을 통하여 최종적으로 1종을 선별하였다. 2. 항균물질 생산균주의 동정은 형태적, 생화학적 특성과 분자생물학적 방법인 16S rRNA gene sequencing을 통하여 이루어졌고 동정한 결과 Pseudomonas 속이었으며, MRSA에 항균물질을 생산하는 균주를 Pseudomonas sp. UJ-6로 명명하였다. 3. Pseudomonas sp. UJ-6의 생육 및 항균물질 생산에 대한 최적조건은 25℃, pH 7, 1% NaCl이었다. 4. Pseudomonas sp. UJ-6가 생산하는 crude antibiotics의 MRSA (KCTC 40510)에 대한 MIC (Minimal Inhibitory Concentration)는 160 ㎕/㎖였다. 5. 6종의 그람 양성균과 4종의 그람 음성균을 대상으로 Pseudomonas sp. UJ-6가 생산하는 crude antibiotics의 항균활성을 조사한 결과, 그람 양성균의 경우 6종 모두에 항균활성을 나타내었으며, 그람 음성균의 경우 4종 중 3종에 항균활성을 나타내었다. 6. Pseudomonas sp. UJ-6가 생산하는 crude antibiotics는 4-121℃까지의 온도범위에서 매우 안정적인 항균활성을 가지며, pH 3-8까지의 범위에서 상당히 안정적이었지만 pH 9과 10에서는 항균활성이 다소 떨어지는 것으로 나타났다.

Staphylococcus aureus is one of the most important pathogens that cause suppuration, abscess formation, a variety of pyogenic infection, and even fatal septicemia in human beings. S. aureus is proved to be susceptible to the earliest antimicrobial substance. However, as antibiotic use increased, staphylococcal resistance rapidly developed. MRSA (Methicillin resistant Staphylococcus aureus), having resistance of which was due to penicillin-binding protein 2a (PBP2a) production, was isolated in the early 1960s. MRSA is resistant to not only methicillin and other β-lactams but also to many other antibacterial agents. Since MRSA exhibits multidrug resistance, it has been emerging worldwide as one of the most important hospital and community pathogens. Therefore, in effort to discover new agents needed to treat the MRSA, we screened bacteria that originated from the marine environment for anti-MRSA activity. In order to isolate the marine bacteria producing antibiotics against MRSA, seawater was collected from the coastal water in Uljin. One strain of marine bacteria, showing strong antibiotic activity for MRSA, was isolated by testing disk diffusion method (NCCLS, 2002). This microorganism was tested by examining its morphological, biochemical properties and analysis of 16S rRNA gene sequencing for identification. Isolated strain was tentatively identified to be Pseudomonas sp. and designated as Pseudomonas sp. UJ-6. The optimum culture conditions of Pseudomonas sp. UJ-6 for growth and production of antibiotics against MRSA were initial pH 7, 1% NaCl and 200 rpm agitation at 25℃. Also Pseudomonas sp. UJ-6 relatively grew in conditions which were 20-30℃, pH 5-9, NaCl 0-3%. The MIC (Minimum Inhibitory Concentration) value against MRSA (KCTC 40510) of the crude antibiotics produced by Pseudomonas sp. UJ-6 was estimated as 160 ㎕/㎖. Spectrum of antimicrobial activity was evaluated by examining crude antibiotics to standard strains which are gram positive strains - MRSA (KCTC 40510), Staphylococcus aureus (KCTC 1927), Streptococcus pyrogenes (KCTC 3096), Enterococcus faecalis (KCTC 2011), Bacillus subtilis (KCTC 1028) and Mycobacterium smegmatis (KCTC 1057) and gram negative strains - Vibrio parahaemolyticus (KCTC 2729), Salmonella typhimurium (KCTC 1925), Pseudomonas aeruginosa (KCTC 1637) and Escherichia coli (KCTC 1682). MRSA (KCTC 40510) and B. subtilis (KCTC 1028) did not grow on plate including 160 ㎕/㎖ of crude antibiotics and the others of gram positive strains did not grow at 640 ㎕/㎖. Gram negative strains did not grow at 640 ㎕/㎖ except V. parahaemolyticus (KCTC 2729). The crude antibiotics produced by Pseudomonas sp. UJ-6 was stable within the pH range of 3-8 and temperature range of 4℃-121℃. This crude antibiotics appears to have considerable potential by it's stability.