Antibacterial activity of isolated bacteria against Propionibacterium acnes 273 질인 bacteriocin에대한것이주로보고되었다 (Arihara et al., 1996; Kang et al., 2009).

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1 Korean Journal of Microbiology (2018) Vol. 54, No. 3, pp pissn DOI eissn Copyright c 2018, The Microbiological Society of Korea 여드름을유발하는 Propionibacterium acnes 에대한분리세균들의항균활성 이다솔 송홍규 * 강원대학교생명과학과 Antibacterial activity of isolated bacteria against Propionibacterium acnes causing acne vulgaris Da-Sol Lee and Hong-Gyu Song* Department of Biological Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea (Received July 17, 2018; Revised August 28, 2018; Accepted August 29, 2018) This study was carried out to evaluate antimicrobial activity of isolated bacteria from various soils against two strains of Propionibacterium acnes causing acne vulgaris. Among several hundreds of bacterial strains, Paenibacillus elgii DS381, Paenibacillus elgii DS1515, Burkholderia gladioli DS518, and Streptomyces lienomycini DS620 showed high antimicrobial activities against the strains of P. acnes. All isolated bacteria showed 15.5 to 34.3 mm inhibition zone diameter in an agar well diffusion test, and especially DS620 showed the highest inhibition zone diameters (28.3~34.3 mm). Antibacterial substances were expected as lipopeptide (pelgipeptin and paenipeptin) from strains DS381 and DS1515, protease from DS518, and anthracycline antibiotic (daunomycinone) from DS620, and all these showed very low minimum inhibitory concentration [DS381 and DS1515 (0.078 mg/ml), DS518 (0.312 mg/ml), DS620 ( mg/ml)] against P. acnes. These antibacterial substances could completely kill P. acnes within 24 h in a time-kill assay. These results suggest that antibacterial substances produced by these bacteria may be utilized as useful antimicrobial agent against P. acnes and treatment medicine for acne vulgaris. Keywords: Propionibacterium acnes, acne vulgaris, antibacterial activity, antibacterial substances *For correspondence. hgsong@kangwon.ac.kr; Tel.: ; Fax: Propionibacterium acnes는그람양성, 다형성간균으로혐기성조건에서생장하는데사람의피부에존재하면서다핵백혈구, 단핵백혈구및대식세포를통해염증매개인자를생성함으로써여드름유발병원체로서핵심적인역할을한다고알려졌다 (Vowels et al., 1995; Kang et al., 2009). P. acnes 등에의해유발된여드름은피지샘활동이활발한머리와몸통부근에서주로나타나며사람의피부조직침투나심각한감염을일으키지않는다 (Webster, 2002). 대부분의여드름환자는비염증성여드름, 염증성구진, 농포등이발생하지만일부환자에서는심각한염증이나타나며흉터를남기기도한다. 한편이런여드름진행의정확한기작은알려지지않았다 (Vowels et al., 1995). 여드름치료는쉽지않을뿐만아니라치료를위한항생제처방으로근래여드름균의항생제내성이보고되었으며 (Ross et al., 1997; Nord and Oprica, 2006), 이러한문제를극복하기위해새로운항균물질을찾는것이중요해지고있다 (Luangnarumitchai et al., 2007). 최근의약품분야에서는화학기반의항생제사용을줄이고이에대한대안으로미생물자체혹은미생물유래항균제를이용한시장진출전략을강화하고있다 (Kim et al., 2018). 이런연구동향에따라여드름균을제어하는다양한항균물질자원중약용식물에대한연구가많이이루어져있고 (Nostro et al., 2000; Niyomkam et al., 2010; Zu et al., 2010), 미생물을대상으로한연구에서는유산균이생산하는항균단백

2 Antibacterial activity of isolated bacteria against Propionibacterium acnes 273 질인 bacteriocin에대한것이주로보고되었다 (Arihara et al., 1996; Kang et al., 2009). 미생물은다양한항균물질을생산할수있는자원으로서 bacteriocin 이외에도 biosurfactant, antimicrobial peptide (Jiang et al., 2012; Sharma and Saharan, 2014), 항균및항진균물질인다양한효소와 siderophore 등을생산한다고알려져있지만 (Nagarajkumar et al., 2004) 여드름균을제어하는데아직까지미생물소재를활용하는측면이부족한실정이다. 따라서본연구에서는여러항균활성세균을분리하여 P. acnes에대하여항균활성을평가하고그들이생산하는항균물질의효과를조사하였다. 재료및방법 항균활성세균균주분리및동정춘천과평창일대에서다양한토양시료를채취하고 Nutrient Agar (NA) 와 Luria-Bertani Agar (LA) 배지에시료를도말하여호기적으로배양하였다 (30 C, 7일 ). 계대배양을통해균주를분리하였고경상대학교병원체자원은행 [Gyeongsang National University Hospital Branch of the National Culture Collection for Pathgens (GNUH-NCCP)] 으로부터분양받은 Propionibacterium acnes (strain 2875와 2876) 에대하여항균활성을평가하였다. 세균균주를 P. acnes 균주와대치배양하는데 (30 C, 24~72시간 ) P. acnes가통성혐기성미생물인점을고려하여 petri dish 주변을 parafilm으로감아외부산소의영향을차단하였다. 이후분리세균균주집락주변에생긴저해대를토대로균주를 1차선별하였다. 선별한세균균주의 16S rrna 유전자염기서열은 마크로젠과 코스모진텍에의뢰하여분석하여미국 National Center for Biotechnology Information (NCBI) 에등록된세균균주와상동성을비교하였다. 추가적으로형태와최적생장온도를조사하고, catalase 와 oxidase 활성을측정하며 API kit (50CHB, 20E와 20NE, biomérieux) 를이용한생리생화학적검사를수행한후, bio- Mérieux의데이터베이스에등록된종과생리생화학적특성의상동성을비교하여균주를최종적으로동정하였다. 균주의항균활성분리균주의항균활성검사는 agar well diffusion 방법 (Ansari et al., 2012) 을이용하여수행하였다. DS381, DS1515 과 DS518 균주는 Luria-Bertani Broth (LB), DS620 균주는 Nutrient Broth (NB) 배지에호기적으로배양하고 (30 C, 48~120 시간 ) 배양액을원심분리하였다 (2,300 g, 4 C, 20분 ). P. acnes 가도말된 NA 배지에 cork borer로 10 mm 직경의 well을뚫고배양상등액을 100 µl 첨가하여 petri dish를 parafilm으로감고배양 (30 C, 24시간 ) 후저해직경을측정하였다. 항균물질추출및분석 Lipopeptide와 protease : Paenibacillus elgii DS381과 Paenibacillus elgii DS1515, Burkholderia gladioli DS518 균주의단백질성항균물질추출은 Oh 등 (2006) 의방법을이용하여수행하였다. 각각의균주를 1 L의 LB 배지에호기적으로배양하고 (30 C, 120시간, 160 rpm), 원심분리 (2,300 g, 4 C, 20 분 ) 후배양상등액에 ammonium sulfate를 300 g 첨가한뒤정치하였다 (4 C, overnight). 원심분리 (2,300 g, 4 C, 40분 ) 하여가라앉은단백질성물질을회수하고동결건조 (-90 C, 5 torr) 하였다. DS381과 DS1515 균주의단백질성물질은추출후 crude oil을퍼트리는계면활성을나타내는지조사한후 thin layer chromatography (TLC) 를수행하여계면활성물질의성질을판단하였다. 그중 lipopeptide 성질과항균활성을동시에나타내는물질을 silica gel column chromatography를이용하여분획하였고, Huang 등 (2017) 의 lipopeptide 분석법중하나인 high-performance liquid chromatography (HPLC) 를이용하여분석하였다 [Waters Co. Breeze Model with UV detector (220 nm), 이동상 : HPLC grade water, isopropanol, acetonitrile, 유속 1 ml/min, 컬럼 : Gemini 5 µ C18 (250 x 4.6 mm, Phenomenex)]. Protease는효소자체로항균활성을나타낼수있을뿐만아니라, 존재하는단백질을가수분해하여항균활성을지닌 peptide 산물을생성하여항균성을띨수있다. 이러한 protease는 Ghorbel 등 (2003) 의 casein을이용한 protease 정량법에따라분석하였다. 항생물질 : Streptomyces 속이흔히생산하는항생물질중 daunomycinone은항암효과와항균활성을모두지닌물질로잘알려져있으며유기용매에잘녹는특성을지녔다. DS620 균주의항생물질은위와같은특성을고려하여 Singh 등 (2014) 의방법을이용하여추출하였다. NB 배지에서 DS620 균주를배양하고 (30 C, 168시간 ) 배양액을원심분리하였다 (2,300 g, 4 C, 40분 ). 배양상등액에동량의 ethyl acetate를첨가하고추출한후유기용매층을분리하여감압증발시키고건조하여 -20 C에서보관하였다. 추출한항생물질은 methanol 에용해하여 Fomichova 등 (1992) 의 anthracycline 항생제분석법중하나인 TLC 방법으로분석하였다 [TLC plate: Kieselgel Korean Journal of Microbiology, Vol. 54, No. 3

3 274 Lee and Song 60, 2.2 mm, Merck, 이동상 (v:v): (1)-chloroform:benzene:MeOH, 20:2:3, (2)-choroform:MeOH=10:1]. 이와관련된 HPLC 분석은 Chu 등 (2016) 의방법을이용하여분석하고물질체류시간을비교하였다 [ 파장 : 254 nm, 이동상 : HPLC grade water (0.05% trifluroacetic acid), acetonitrile, 유속 : 1 ml/min, 컬럼 : Gemini 5 µ C18 (250 x 4.6 mm, Phenomenex)]. 최소저해농도 : P. acnes에대한 P. elgii DS381과 P. elgii 1515, B. gladioli DS518 균주의단백질성항균물질및 S. lienomycini DS620 균주의 ethyl acetate 추출물의최소저해농도 (minimum inhibitory concentration; MIC) 는 Sopirala 등 (2010) 과 Al-Ani 등 (2015) 의방법을이용하여수행하였다. 대상세균을 10 7 CFU/ml로보정하여이용하였으며균주항균물질 (w/v로표시 ) 의경우 DS381과 DS1515, DS518 균주의단백질성항균물질은멸균증류수, DS620 균주항균물질은 methanol로용해하였다. 96 well microplate에액체배지 160 µl, 항균물질 (400~ mg/ml) 20 µl, 균배양액 20 µl를첨가한후배양하였다 (30 C, 24~48시간 ). 배양후균생장이나타나지않은항균물질처리구의농도를 MIC로결정하였다. Time kill assay : P. elgii DS381과 P. elgii 1515, B. gladioli DS518 균주의단백질성항균물질, S. lienomycini DS620 균주의 ethyl acetate 추출물의항균물질처리시 P. acnes가사멸하는양상은 time kill assay (Jayaraman et al., 2010; Al-Ani et al., 2015) 로조사하였다. P. acnes를 10 5 ~10 6 CFU/ml로보정하였고배양액에 1 MIC로항균물질을처리하고배양하였다 (30 C, 160 rpm). 0, 4, 8, 24시간간격으로채취하여도말하고배양 (30 C, 24시간 ) 후생균수를계수하였다. 결과및고찰 항균활성세균균주분리및동정춘천과평창지역의여러토양시료로부터다양한세균균주를순수분리하여대치배양을통해 P. acnes GNUH-NCCP2875 와 2876에대한항균활성을조사하였을때, DS381과 DS1515, DS518, DS620 네균주모두 P. acnes의생장을크게저해하였다. 이네균주는 16S rrna 유전자서열분석 (Table 1) 후각균주에해당하는 API kit를이용하여생리화학적분석 (Tables 2 and 3) 을수행하였다. DS381과 DS1515 균주는비교균주인 Paenibacillus elgii SD17 균주와 protease, urease에양성과 L-arabinose에음성 (Table 2) 및 catalase 양성과 oxidase 음 Table 1. Identification of strains DS381, DS1515, DS518, and DS620 showing antimicrobial activity by 16S rrna gene sequencing Strain Type strain Pairwise similarity (%) DS381 Paenibacillus elgii SD DS1515 Paenibacillus elgii MER_ DS518 Burkholderia gladioli IHB B DS620 Streptomyces lienomycini C.P 성 ( 결과미제시 ) 을나타내는등의공통적인특성을지녔으며, DS518 균주는 API 대조균주와 potassium nitrate, D-manitol, N-acetylglucosamine, oxidase 양성이라는공통적인특성을지녔다. 이러한결과를토대로 biomérieux의데이터베이스에등록된종과생리생화학적특성의상동성을비교하여균주는최종적으로각각 Paenibacillus elgii DS381, Paenibacillus elgii DS1515, Burkholderia gladioli DS518 및 Streptomyces lienomycini DS620로동정되었다. 균주의항균활성 Agar well diffusion test : P. acnes GNUH-NCCP2875와 2876 균주에대한 DS381과 DS1515, DS518, DS620 균주배양상등액의억제직경조사결과, 네균주모두 P. acnes 두균주의생장을저해하였다, 그중특히, DS620과 DS1515 균주의배양상등액은최대활성범위인 26 mm 이상의저해직경을나타내었고, 네균주중 DS620 균주의활성이가장우수하였다 (Table 4). 이러한결과는 Luangnarumitchai 등 (2007) 연구에서 black pepper oil과 citronella oil을포함한다양한식물유래항균물질의저해대와비교하여, 대부분높은활성을나타내며, 특히 galanga oil과 ginger oil이대체로 10 mm 이하의저해대를보인것과비교하면뛰어난항균활성이었다. 또한 DS620 균주의배양상등액의경우 P. acnes 균주에따라약간의차이를보이지만양성대조군인 clindamycin과 erythromycin 과도유사한활성을보여 ( 결과미제시 ), 균주의항균활성이뛰어난것을알수있었다. 여드름균제어와관련하여유산균의항균활성이다수보고되었는데 Arihara 등 (1996) 의연구에서다양한 lactic acid bacteria의대부분균주들이 P. acnes를억제하지못하였고, 활성을보인 Lactobacillus salivarius subsp. salicinius TI 40의경우에도 P. acnes를 7 mm 수준으로저해하였다. 따라서본연구의네균주의여드름균제어활성이높다고할수있고이러한점은대부분의연구에서보고된유산균과는다른성질을지닌새로운물질들이여드름균을효과적으로제어한다는점을시사한다. 또한 P. acnes에대하여 0.5~6.3 mm의저해대를나타낸해초추출물이나 (Choi et al., 2013) 2 미생물학회지제 54 권제 3 호

4 Antibacterial activity of isolated bacteria against Propionibacterium acnes 275 Table 2. Biochemical characteristics of strain DS381 and DS1515 using API kit (50CHB and 20E) Characteristic DS381 DS1515 Characteristic DS381 DS1515 Gram stain + + Melibiose + + Morphologie rod rod Sucrose + + Optimum temperature ( C) Trehalose + - Control - - Inuline - - Glycerol - - Melezitose + - Erythritol - + D-raffinose + - D-Arabinose - - Starch - + L-Arabinose - - Glycogen - + Ribose - - Xylitol - - D-xylose + - β-gentiobiose + + L-xylose - - D-Turanose + + Adonitol - - D-Lyxose - - β-methyl-xylopyranside + - D-Tagatose - - Galactose + - D-Fucose - - D-Glucose + + L-Fucose - - D-Fructose + + D-Arabitol - - D-Mannose - + L-Arabitol - - L-Sorbose - - Gluconate - - Rhamnose keto-gluconate - - Dulcitol keto-cluconate - + Inositol nitrophenyl-β-D-galactopyranoside + + Mannitol + - L-arginine - - Sorbitol - - L-lysine - - α-methyl-d-mannopyranside + - L-omithine - - α-methyl-d-glucoside + + Trisodium citrate - + N-acethyl-glucosamine + + Sodium thoisulfate - - Amygdaline + + Urea + + Arbutine + + L-tryptophane (TDA) - - Esculine + + Indole (IND) - - Salicine + + Sodium pyruvate + - Cellobiose + + Gelatin (bovine origin) + + Maltose + + D-glucose - - Lactose + + Table 3. Biochemical characteristics of strain DS518 using API kit (20NE) Characteristic DS518 Characteristic DS518 Gram stain + L-arabinase - Morphologie rod D-mannose - Optimum temperature ( C) 30 D-mannitol + Potassium nitrate + N-acetyl-Glucosamine + L-tryptophane - D-maltose - D-glucose - Potassium gluconate - L-arginine - Capric acid - Urea - Adipic acid - Esculin ferric citrate - Matic acid - Gelatine (bovine origin) - Trisodium citrate - 4-nitrophenyl-B-D-galactopyranoside - Phenylacetic acid - D-glucose - Oxidase + Korean Journal of Microbiology, Vol. 54, No. 3

5 276 Lee and Song Table 4. Growth inhibition of P. acnes by culture supernatant of strains DS381, DS1515, DS518 and DS620 in agar well diffusion test Inhibition zone diameter (mm) Target organism DS381 DS1515 DS518 DS620 P. acnes GNUH-NCCP ± ± ± ± 5.1 P. acnes GNUH-NCCP ± ± ± ± 1.5 (A) (B) Fig. 1. HPLC chromatogram of purified compounds isolated from the culture supernatants of P. elgii DS381 (A) and P. elgii DS1515 (B). cm 이하의저해대를보인 3% 누에동충하초추출물 (Gal et al., 2009) 과비교해도본연구의네균주의항균활성이더우수하였다. 항균물질의항균활성 : 항균활성분리균주의주된항균물질을분석하였을때, 먼저단백질특성을나타내고있는 DS381과 DS1515 균주의항균물질은계면활성을나타내는특성을지녔다. Oil spreading test에서 crude oil을최대 5 cm 까지퍼트리는계면활성을나타내었고 TLC를통해서계면활성을나타내는물질은 lipopeptide 성질을지녔다고판단되었다 ( 결과미제시 ). 이러한계면활성은대부분의미생물이공통적으로가지는세포막을표적으로광범위한항균활성을나타낼수있다. Paenibacillus elgii가생산하는 lipopeptide에는 pelgipeptin과 paenipeptin 같은물질이대표적으로알려져있는데, 본연구에서의물질또한위의물질과마찬가지로광범위한항세균, 항진균활성을나타낼뿐만아니라다양한온도와 ph 범위에서도안정하여유사한특징을나타내었다 ( 결과미제시 ). 이러한특성을고려하여 Huang 등 (2017) 의 HPLC 결과와비교했을때 pelgipeptin 및 paenipeptin과유사한 7분대의체류시간과 peak 모양을나타내었다 (Fig. 1). 위와같이계면활성, TLC 및 HPLC 분석을통하여 DS381과 DS1515 균주의항균물질을 lipopeptide성물질로추정할수있었다. DS518 균주의항균물질의경우다른세균주와비교하여 protease 정량시 mg/l로월등히높은 protease 생산량을나타내었으며, 추출물에서도유사한양상이나타났다. 또한추출한단백질성물질은 ph 6~8 범위에서항균활성을나타내었는데 protease가 ph 6~9 범위에서활성을띤다는 Ghorbel 등 (2003) 의결과와일치하므로 protease가이균주의항균활성에대부분기여한다고추측할수있다. DS620 균주의항균물질은항균범위가넓고, 적은양으로도효과적으로미생물을제어하였다. 이물질을 TLC로분석하였을때 Fomichova 등 (1992) 이보고한 anthracycline 항생물질계열중 daunomycinone 의결과와비교하여 chloroform:benzene:meoh (20:2:3) 과 choroform:meoh (10:1) 이동상에서각각 0.81과 0.92로 Rf값이일치하였다 (Fig. 2A). 그중특히 Rf값이 0.81이었던분획물의경우 P. acnes에대하여다른분획물에비하여월등히높은 미생물학회지제 54 권제 3 호

Antibacterial activity of isolated bacteria against Propionibacterium acnes 277 (A) (B) (C) Fig. 2. TLC (A), antimicrobial activity (B), and HPLC chromatogram of anthracyclic antibiotics of S.

6 Antibacterial activity of isolated bacteria against Propionibacterium acnes 277 (A) (B) (C) Fig. 2. TLC (A), antimicrobial activity (B), and HPLC chromatogram of anthracyclic antibiotics of S. lienomycini DS620 (C). 항균활성을나타내었고 (Fig. 2B), 활성대부분에이물질이기여하고있다고판단하였다. 본연구의항생물질과일치하는 TLC 결과를나타내었던 daunomycinone은이전연구에서항균활성이보고된항암제로서주황색또는노란색의물질로친수성용매에잘녹지않고, chloroform, ethyl acetate 등의유기용매에잘녹는특성을지녔다 (Chu et al., 2016). DS620 균주의항생물질의경우에도마찬가지로위와같은특성을공통적으로지니고있고 HPLC 분석에서 Chu 등 (2016) 의결과와비교하여 peak 모양이 daunomycinone과유사한특성을지녔다 (Fig. 2C). 그러나체류시간이 18.3분과본연구의 21분대로약간다른데분석조건이완전히동일하지않기때문인것으로추정되며이에대해서는추가적인조사가필요하다. P. acnes GNUH-NCCP2875와 2876 균주에대한 DS381과 DS1515, DS518 균주의단백질성물질과 DS620 균주의 ethyl acetate 추출물의 MIC를조사하였을때, 네균주의항균물질모두낮은농도로도 10 6 CFU/ml의 P. acnes 생장을저해하였다. DS381과 DS1515 균주의 lipopeptide 항균물질의경우 mg/ml, DS518 균주의 protease는 mg/ml, DS620 균주의 anthracycline 항생물질은가장우수한활성으로 mg/ml의최소저해농도를나타내었다. 이는 lipopeptide 항생물질인 daptomycin이 Propionibacterium spp. 에대하여, MIC 90 이 2 mg/ml로나타난것 (Steenbergen et al., 2005) 과비교하여더높은항균활성이다. Andrographis paniculata와 Azadirachta indica를포함한 19종의약용식물이 P. acnes에대하여최소 mg/ml, 그리고대부분 0.625~5 mg/ml의 MIC를나타낸결과 (Chomnawang et al., 2005) 와비교해서더낮은농도로 P. acnes를제어할수있었다. Luangnarumitchai 등 (2007) 이조사한식물추출물의대부분이 P. acnes에대하여 1~4% 이상의 MIC를보인결과나태국의 ethyl acetate와 methanol 허브추출물의대부분이 P. acnes에대하여활성을보이지못하였고, 활성을보인경우도대체로 156~312 µg/ml의고농도로 P. acnes를제어하였으며 (Niyomkam et al., 2010), 다양한종류의긴사슬다중불포화지방산이 P. acnes에대하여 32~128 mg/ml의 MIC를나타낸결과 (Desbois and Lawlor, 2013) 와비교하여본연구의분리균주의항균물질들이훨씬효율적으로 P. acnes의생장을제어하였다. Time kill assay : P. acnes에대한네가지항균활성균주의항균물질처리시사멸하는양상을조사하였을때, DS381 균주의 lipopeptide는 P. acnes 두균주를 8시간째에완전히사멸시켰으며, DS1515 균주의 lipopeptide 물질은배양 8시간후절반의개체수감소를보이고 24시간째완전히사멸시켰다. DS518 균주의 protease는 P. acnes GNUH-NCCP2875과 2876 균주에대하여배양 4시간후각각 3.51와 4.35 log로개체수를감소시켰고배양 24시간째에는완전히사멸시켰다. 마지막으로 DS620 균주의 anthracycline 항생물질은 P. acnes 2875과 2876를각각배양 8시간과 24시간후완전히제거하였다 (Fig. 3). 결과적으로모든균주의항균물질이배양 24시간이내의빠른시간내에 P. acnes를완전히사멸시키는효과를보였다. 이는더적은개체수의 P. acnes에대해쟈스민 essential oil이배양 2시간후에도개체수감소를크게변화시키지않은결과 (Zu et al., 2010) 와비교하면이 4가지분리균주는 P. acnes에대하여비교적우수한항균활성을보임을알수있다. 본연구의 4가지균주들의항균물질은 P. acnes에대한생장저해대가이전에보고된식물추출물이나미생물유래항균물질과비교하여크기때문에실제치료제로활용시안전성문제가제기될수도있으나, 현재여드름치료제로이용되고있는 erythromycin과 tetracyclin, clindamycin이 P. acnes CCARM 9010에대하여각각 과 , mg/ml의최소저해농도를나타낸다는보고 (Lim et al., 2007) Korean Journal of Microbiology, Vol. 54, No. 3

7 278 Lee and Song (A) (B) (C) (D) Fig. 3. Effects of antimicrobial substances (1 MIC) of P. elgii DS381 (A), P. elgii DS1515 (B), B. gladioli DS518 (C), and S. lienomycini DS620 (D) on survival of P. acnes GNUH-NCCP2875 ( ) and P. acnes GNUH-NCC 2876 ( ). 와비교하여유사하거나약간높은최소저해농도를나타낸다. 따라서본연구의항균물질들의안전성은문제가없을뿐만아니라계면활성물질이나 protease 처럼항균물질에대한여드름균의내성이나타날가능성이낮기때문에기존여드름치료항생제의대안이될수있다. 오히려앞으로이항균물질들의조합을이용하여항균활성의시너지효과를조사할필요가있다. 적요 본연구는여드름을유발하는세균인 Propionibacterium acnes에대해다양한토양에서분리된세균균주들의항균효과를조사하기위해수행되었다. 수백개의분리세균균주중 Paenibacillus elgii DS381과 Paenibacillus elgii DS1515, Burkhoderia gladioli DS518, Streptomyces lienomycini DS620 는 2가지균주의 P. acnes에대해높은항균활성을나타내었다. 이분리균주들은 agar well diffusion test에서 15.5~34.3 mm 직경의저해대를형성하였으며, 특히 DS620는가장큰저해대직경 (28.3~34.3 mm) 을나타내었다. 분리균주가생성하는항균물질은 DS381과 DS1515 균주의경우 lipopeptide (pelgipeptin, paenipeptin), DS518은 protease, 그리고 DS620은 anthracycline 인것으로추정되며, 이들모두 P. acnes에대해매우낮은최소저해농도를나타내었다 [DS381와 DS1515 (0.078 mg/ml), DS518 (0.312 mg/ml), DS620 ( mg/ml)]. P. acnes를대상으로한 time-kill assay에서는네균주의항균물질이모두 24시 간이내에 P. acnes를완전히사멸시켰다. 이결과는네가지항균활성균주들이분비하는항균물질들이여드름을유발하는 P. acnes에대하여효율적인치료소재로사용될수있는가능성을보여준다. 감사의말 본연구는 2016년도강원대학교대학회계학술연구조성비로연구되었음 ( 과제번호 ). References Al-Ani I, Zimmermann S, Reichlinga J, and Wink M Pharmacological synergism of bee venom and melittin with antibiotics and plant secondary metabolites against multi drug resistant microbial pathogens. Phytomedicine 22, Ansari A, Aman A, Siddiqui NN, Iqbal S, and Qader SA Bacteriocin (BAC-IB17): Screening, isolation and production from Bacillus subtilis KIBGE IB-17. Pak. J. Pharma. Sci. 25, Arihara K, Ogihara S, Mukai T, ltoh M, and Kondo Y Salivacin 140, a novel bacteriocin from Lactobacillus salrivarius subsp. salicinius TI 40 active against pathogenic bacteria. Lett. Appl. Microbiol. 22, Choi JS, Bae HJ, Kim SJ, and Choi IS In vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, Propionibacterium acnes. J. Environ. Biol. 미생물학회지제 54 권제 3 호

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Korean Journal of Microbiology (2018) Vol. 54, No. 4, pp pissn DOI eissn Copyright

Korean Journal of Microbiology (2018) Vol. 54, No. 4, pp. 354-361 pissn 0440-2413 DOI https://doi.org/10.7845/kjm.2018.8053 eissn 2383-9902 Copyright c 2018, The Microbiological Society of Korea Bacillus