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pissn 1229-1153 J. Fd Hyg. Safety Vol. 29, No. 1, pp. 26~30 (2014) http://dx.doi.org

1 pissn J. Fd Hyg. Safety Vol. 29, No. 1, pp. 26~30 (2014) Journal of Food Hygiene and Safety Available online at 식품에서분리된리스테리아모노사이토젠스의분포및항생제내성 정효원 박상훈 * 이집호 김수진 류승희 송미옥 박선희 조정윤 박건용 최성민 서울특별시보건환경연구원미생물관리팀 Prevalence and Antibiotic Resistance Patterns in Listeria Monocytogenes Isolated from Food Hyo-won Jeong, Sang-hun Park, Jib-ho Lee, Soo-jin Kim, Seung-hee Ryu, Mi-ok Song, Sun-hee Park, Jeong-yun Jo, Geon-yong Park, and Sung-min Choi Microbiological control Team, Seoul Metropolitan Government Research Institute of Public Health & Environment, 30, Janggunmaeul 3-gil, Gwacheon-si, Gyeonggi-do, Korea (Received June 10, 2013/Revised August 12, 2013/Accepted February 19, 2014) ABSTRACT - Listeria monocytogenes continues to be a important food safety concern. The aims of the present study were to investigate the prevalence and antimicrobial susceptibility of L. monocytogenes. A total of 1,042 samples was collected from 2010 to 2011 in Seoul and twelve L. monocytogenes were isolated. Isolation rate was Gimbap (0.8%), Beef (yukhoe) (2.6%), Pork (cooked) (5.9%), Fish(cooked) (6.3%), and Salmon (11.1%), respectively. In this study, most of the isolates were susceptible to antibiotics. The most common resistance was cefotetan on 11 isolates, followed by cefotaxime on 7 isolates, cepefime on 6 isolates and tetracyclin on 3 isolates. Key words : L. monocytogenes, Antibiotics, Tetracyclin Listeria 속은아포를형성하지않는통성혐기성그람양성간균으로자연계에흔히존재하며 Listeria denitrificans 등 10개의종으로분류되고 L. monocytogenes와 L. ivanovii 만이병원성이고그중 L. monocytogenes가사람에게감염을일으킨다. L. monocytogenes는 1 o C에서 44 o C의광범위한생육온도를가지고냉동과건조에비교적잘견디며, 또한 ph 5 이하의산과높은염농도에서도생육이가능한것으로보고되고있다 1,2,3). L. monocytogenes는기회감염으로리스테리아증을일으키고수막뇌염, 유산, 패혈증같은임상증상이나타내며, 발병률은높지않지만발병하면사망률이 30% 에육박할정도로위험하여지정전염병으로지정되어있다 4,5) 년까지국내식품의 L. monocytogenes가식중독을일으킨경우는없지만 6) 유럽과미국에서는육류와치즈에오염된 L. monocytogenes 때문에식중독이종종발생되며사망자도나오고있다 7). 특히 2011년발생한미국 Rocky Ford Cantaloupes 사태에서 147명감염에 33명이사망하였고임 *Correspondence to: Sang-hun Park, Seoul Metropolitan Government Research Institute of Public Health & Environment 30, Janggunmaeul 3-gil, Gwacheon-si, Gyeonggi-do, Korea Tel: , Fax: sanghun93@seoul.go.kr 산부 1명이유산하였으며감염자의평균나이가 81세에이를정도로 8) 신생아, 임산부, 노약자, 면역이저하되어있거나억제되어있는사람이위험군이다 3). 리스테리아증치료에는 ampicillin이나 penicillin의단독요법혹은 gentamycin과의병용요법이사용된다 9). L. monocytogenes는흔히 penicillins, aminoglycosides, tetracycline 등다양한항생제에대해감수성을가지고있으며 sulfomethoxazole, cephalosporins에는저항성을가지고 fluoroquinolones을제외한 quinolones에도저항성을가진다 10,11). 국내식품에서 L. monocytogenes는다른식중독균에비해많은연구가이루어지지않고있다. 따라서본연구에서는국내식품중 L. monocytogenes에대한기초자료를제공하고자 2010년에서 2011년동안서울소재재래시장과백화점그리고대형마트에서수거된식품을대상으로 L. monocytogenes의오염도와분리주에대한항생제감수성실험을실시하였다. 재료및방법 시료채취본실험에사용된시료는총 1,042 건으로 2010 년에서 2011 년까지서울지역 25 개구청에서무작위로수거하였고 26

2 Prevalence and Antibiotic Resistance Patterns in Listeria Monocytogenes Isolated from Food 27 Table 1. The kinds and number of samples Ready-to-eat food (RTE) Meat product Fish product kinds of samples Number of samples (%) Gimbap 498 (47.8) Sandwich & Burger 96 (9.2) Dosirak 49 (4.7) Beef (yukhoe) 152 (14.6) Duck & chicken (cooked) 118 (11.3) Pork (cooked) 17 (1.6) Sliced raw fish 41 (3.9) Sushi 37 (3.6) Salmon 18 (1.7) Fish(cooked) 16 (1.5) Total 1042 (100) 모든시료는식품의약품안전청 (KFDA) 식품공전규격 12,13) 기준에따른미생물시험을실시하였다. 그중 643건은즉석섭취식품, 287건은육류함유식품, 112건은어류함유식품이었다 (Table 1). Listeria monocytogenes의분리및동정시료를무균적으로채취하여 UVM-modified Listeria broth (Becton Dickinson) 에서 30 o C, 24시간배양하고배양액 0.1 ml를취하여 Fraser Listeria broth (Becton Dickinson) 에 37 o C, 24시간 2차배양하였다. 선택배지인 Oxford agar (Oxoid) 에 2차배양액을획선도말하여 30 o C, 24~48시간배양하고집락모양이진한갈색또는검은색환으로둘러싸인집락을선발하여 Brain Heart Infusion agar (Becton Dickinson) 에배양해서 API Listeria 키트 (BioMerieux) 를이용하여동정하였다. 항생제감수성시험본연구에사용한항생제디스크는 β-lactam 계중 Penicillin 계 penicillin (PEN: 10 IU), ampicillin (AMP: 10 µg), amoxicillin/clavulanic acid (AMC: 30 µg), ampicillin/sulbactam (SAM: 10/10 µg), Cepalosporin계 cephalothin (CEF: 30 µg), cefotetan (CTT: 30 µg), cefotaxime (CTX: 30 µg), cefepime (FEP: 30 µg) 이고 aminoglycoside계 gentamicin (GEN: 10 µg), streptomycin (STR: 10 µg), neomyicn (NEO: 30 µg), quinolone 계 norfloxacin (NOR: 10 µg), ciproploxacin (CIP: 5 µg), glycopeptide계 Vancomycin (VAN: 30 µg), Tetracycline계 tetracycline (TET: 30 µg), 그외 trimethoprim/sulfamethoxazole (SXT: 1.35/23.75 µg), erythromycin (ERY: 15 µg), chloramphenicol (CHL: 30 µg) 의총 18종을사용하였으며모두 Becton-Dickinson 사의 BBL 제품을사용하였다. 항생제감수성시험의방법과기준은 Clinical and Laboratory Standard Institute (CLSI) 의디스크확산법 14) 를준용하였다. 결과 Listeria monocytogenes 검출률 2010년에서 2011년까지다양한식품으로부터분리한 L. monocytogenes 검출률은 0% 부터 11.1% 까지다양한분포를보였다 (Table 2). 총 1,042건의시료중 12건에서 L. monocytogenes가검출되어 1.2% 의검출률을나타냈다. 검체의종류별로는김밥과육회에서각 4건씩, 연어제품에서 2건, 돈육식품과조리된생선에서각 1건씩검출되었으나검출률에서는연어제품이 18건의시료에서 2건이검출되어 11.1% 로가장높은검출률을나타냈다. 샌드위치와햄버거, 도시락, 오리고기와계육식품, 생선회, 초밥에서는검출되지않았다 (Table 2). 항생제감수성테스트항생제감수성테스트결과는 Table 3과같다. Penicillin 계항생제에는대부분내성이없었지만같은 β-lactam계인 cephalosporin계는 1세대항생제인 cephalothin을제외한 2,3,4세대인 cefotetan (11 균주 ), cefotaxime (7 균주 ), Table 2. Prevalence of L. monocytogenes isolated from samples Samples Number of samples Antimicrobial Number (%) of isolates Gimbap 498 CTT, CTX, FEP, TET 4(0.8) Sandwich & Burger 96 0(0.0) Dosirak 49 0(0.0) Beef (yukhoe) 152 CTT, CTX, FEP 4(2.6) Duck & chicken (cooked) 118 0(0.0) Pork (cooked) 17 FEP 1(5.9) Sliced raw fish 41 0(0.0) Sushi 37 0(0.0) Salmon 18 CTT, CTX, FEP, TET 2(11.1) Fish (cooked) 16 CTT 1(6.3) Total (1.2)

3 28 Hyo-won Jeong et al. Table 3. Antimicrobial susceptibility rates of L. monocytogenes isolated from samples Antimicrobial Penicillin Cepalosporins Aminoglycosides Number of isolates Resistant Intermediate Susceptible PEN AMP AMC SAM CEF CTT CTX FEP GEN STR NEO Quinolone NOR CIP Glycopeptide VAN Tetracyclines TET SXT Others ERY CHL PEN: penicillin, AMP: ampicillin, AMC: amoxicillin/clavulanic acid, SAM: ampicillin/sulbactam, CEF: cephalothin, CTT: cefotetan, CTX: cefotaxime, FEP: cepefime, GEN: gentamicin, STR: streptomycin, NEO: neomycin, NOR: norfloxacin, CIP: ciprofloxacin, VAN: vancomycin, TET: tetracycline, SXT: trimethoprim/sulfamethoxazole, ERY: erythromycin, CHL: chloramphenicol Table 4. Multiresistance patterns of Listeria monocytogenes isolated from samples Number of resistance Number (%) of isolates Antimicrobial resistance patterns (No. of isolates) Samples (Number) (42) CTT (4) Beef (yukhoe) (3) Fish (cooked) (1) FEP (1) Pork (cooked) (1) 2 1 (8) CTT, CTX (1) Salmon (1) 3 4 (33) CTT, CTX, FEP (3) Gimbap (2) Beef (yukhoe) (1) CTT, CTX, TET (1) Gimbap (1) 4 2 (17) CTT, CTX, FEP, TET (2) Gimbap (1) Salmon (1) cefepime (6 균주 ) 은내성이높게나타났다. Tetracycline계의 tetracycline도 3균주에서내성이발견이되었다. 다른항생제에서는내성을나타내지않았다. 12개의분리주모두하나이상의항생제에내성을보였고 2개이상의항생제에내성을보인균주는 7주로 58% 의비율을보였다. 단일항생제에내성을보인분리주가 5주, 3개의항생제에내성을보인분리주 4주, 4개의항생제에내성을보인분리주는 2주, 2개의항생제에내성을보인분리주는 1주가있었으며항생제내성에대한검출률은각각 42%, 33%, 17%, 8% 순이었다 (Table 4). 고찰 L. monocytogenes는리스테리아증을일으키는원인균으로다양한환경에서널리퍼져있으며극한조건에서도생존이가능하기때문에식품제조과정중이나냉장에도살아남아증식할수있다. 냉장유통으로원유나치즈, 육류등여러식품의장거리이동이자유로워지고다시가열하지않아도섭취가가능한식품이늘어나고있으며, 현대인들의스트레스증가등으로인해면역력이약해지고 15) 암환자의증가 16) 와고령화로위험군이늘어나므로 L. monocytogenes 식중독발생의위험성은날로커지고있다. 따라서본연구에서는 2010부터 2011년까지 1,042 개의시료에서 L. monocytogenes 균분리를시도하였으며 12건이검출되어 1.2% 의검출률을보였다. 국내연구결과를살펴보면즉석섭취 편의식품에서 L. monocytogenes가검출되지않았고 17) L. monocytogenes는몇가지감염증예 19,20,21) 가있지만식중독원인균으로밝혀진예는없어국내에선아직다른균종보다위험에대한인식이부족하다. 그러나시료의범위와지역이다르지만외국의경우는 He Yan 등이 Hebei 지방의식품을실험한결과 4.13% 의검출률을보였고 22) E.A. Martins 등은 So Paulo에서햄을조사하여 3.5% 의결과를얻었으며 23) Seza Arslan 등은수제치즈에서 9.2% 를검출되었으며 2) 본연구와같이다양한식품에서분리된검출률이 2.1% 정도로비교적낮게검출되고있다 18). 또한국내와는달리해외에서는 L. monocytogenes가다수발견이되며가끔씩대형식중독사고도일으키는것으로보고되고있다 8). 특히김밥과육회에서많이검출된원인은리스테리아는전세계적으로원료육에오염도가높으며가공중바이오필름을형성해서교차오염이발생되며 26) 김밥은제조과정중미생물의오염요인이크며, 수분활성이높고미생물의증식이용이한원료로구성되어있어보존이어렵고, 식품위생상의식성병해를일으키기쉬운문제점이있다 27). 육회는원료육을그대로썰어조미를가해먹는형태이므로원료육이오염되어있을가능성이높다 18). 원료육에서 L. monocytogenes 검출률이적게는 3.32% 많게는 30% 이상검출된다고보고하고있다 3). 본연구의결과를보면전반적으로항생제에대한내성이높지는않았지만 12균주모두 cephalosporins에내성을나타내었다. β-lactam계항생제중 Penicillins보다 cephalosporins에대한내성을나타냈는데이는다른연구자 10,11) 들의보고에의해서도확인되고있다. 일부 cephalosporins를

4 Prevalence and Antibiotic Resistance Patterns in Listeria Monocytogenes Isolated from Food 29 제외하면 Conter, M. 등 24) 의결과와같이본연구에서도 L. monocytogenes의항생제내성은비교적낮게나타났다. 하지만임상치료에많이사용되는 penicillin, ampicillin, tetracyline과 gentamicin에내성을나타내는것은사망률이높은리스테리아증의치료에서는위험하다. 본연구에서 tetracycline이 3균주 (25%) 에서높은내성 25) 을보였는데다른연구 3,4,22) 에서도다른항생제보다높은내성을가지고있는것으로보고되고있다. 향후 tetracycline의내성유전자에대한연구가이어져야할것으로생각된다. 본연구는조리과정을거치지않고사람이바로섭취하는다양한식품의 L. monocytogenes 오염도조사와항생제내성의패턴변화에대한연구로서지속적으로감시해간다면공중보건학적연구의기초자료로서사용될수있을것으로기대된다. 요약 본연구에서는식품에서의 L. monocytogenes에대한기초자료를제공하고자 2010년부터 2011년까지서울지역에서수거된식품총 1042건에서 L. monocytogenes를분리하여항생제감수성검사를하였다. 김밥 4건 (0.8%), 육회 4건 (2.6%), 연어제품 2건 (11.1%), 돈육식품과조리된생선에서각 1건씩 ( 각5.9%, 6.3%) 총 12균주가분리동정되었다. 분리균주를 20종의항생제에검사한결과대다수항생제 (16종) 에감수성을나타내었지만 cefotetan (11균주), cefotaxime (7균주), cefepime (6균주) 에서내성을나타냈으며 3균주에서임상치료에자주사용되는 tetracycline에대해서내성을나타내었다. 참고문헌 1. Vázquez-boland, J., Kuhn, M., Berche, P., Chakraborty, T., Domínguez-bernal, G., Goebel, W., González-zorn, B., Wehland, J. and Kreft, J.: Listeria Pathogenesis and Molecular Virulence Determinants, Clin. Microbiol. Rev., 14, (2001). 2. Arslan, S. and Özdemir, F.: Prevalence and antimicrobial resistance of Listeria spp. in homemade white cheese, Food Control, 19, (2008). 3. 이지윤 : 식육에서분리한 Listeria monocytogenes 의분자생물학적특성및항생제내성에관한연구, 박사학위논문, 건국대학교대학원 (2010). 4. Jeon, S.B., Lee, S.B., and Hong, C.H.: Antimicrobial resistance of Listeria monocytogenes isolated form pork cutting plants and meat shops. J. Prev. Vet. Med., 34, (2010). 5. Vázquez-boland, J., Domínguez-bernal, G., González-zorn, B., Kreft, J. and Goebel, W.: Pathogenicity islands and virulence evolution in Listeria. Microbes Infect., 3, (2001) Todd, E.C.D. and Notermans, S.: Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food Control, 22, (2011) /index.html. 9. Kalstone, C.: Successful antepartum treatment of listeriosis. Gynecol Obstet., 164, (1991). 10. Swaminathan, B. and Gerner-Smidt, P.: The epidemiology of human listeriosis. Microbes Infect. 9, (2007). 11. Charpentier, E. and Courvalin, P.: Antibiotic Resistance in Listeria spp. Antimicrob Agents Chemother., 43, (1999) jsp? idx= ber &mid=95&searchword= &division=&pageno= 1&seq =2642&cmd=v. 14. CLSI, Performance Standards for Antimicrobial Susceptibility Testing. CLSI Document M100-S20. Clinical and Laboratory Standards Institute., Wayne, PA. (2011). 15. Chang, S.l., Koh, S.B., Park, J.K. and Cha, B.S.: The Effect of social Support on Chronic Stress and Immune System in Male Manufacturing Workers. Korean J. Prev. Med., 35, (2002) IdxMain.jsp?idx_cd= Kim, H.Y., Oh, S.W., Chung, S.Y., Choi, S.H., Lee, J.W., Yang, J.Y., Seo, E.C., Kim, Y.H., Park, H.O., Yang, C.Y., Ha, S.C. and Shin, I.S.: An Investigation of Microbial Contamination of Ready-to-Eat Products in Seoul, Korea. Korean J. Food Sci. Thechnol., 43, (2011). 18. Latorre, L., Parisi, A., Fraccalvieri, R., Normanno, G., La Porta, M.C., Goffredo, E., Palazzo, L., Ciccarese, G., Addante, N. and Santagada, G.: Low prevalence of Listeria monocytogenes in foods from Italy. J. Food Prot., 70, (2007). 19. 신재준, 최지영, 김선민, 이승미, 오경준, 박찬욱, 박중신, 전종관, 박성혜, 신희철 : 임신중기의리스테리아감염으로인한자궁내태아사망 1 예, 대한산부인과학회지, 53, (2010). 20. 정언석, 이은성, 이경욱, 민주영, 이경주 : 임신제 3 삼분기초기리스테리아감염증 1 예, 대한산부인과학회지, 48, (2005). 21. 이승현, 손동우, 심소연, 서일혜, 김석영 : 혈액및양수에서확인된신생아산모리스테리아증 1 예, 대한주산의학회, 19, (2008). 22. Yan, H., Neogi, S.B., Mo, Z., Guan, W., Shen, Z., Zhang, S., Li, L., Yamasaki, S., Shi, L. and Zhong, N.: Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, Int. J. Food Microbiol., 15, (2010). 23. Martins, E. A. Leal Germano, P. M.: Listeria monocytogenes in ready-to-eat, sliced, cooked ham and salami products, marketed in the city of So Paulo, Brazil: Occurrence, quanti-

5 30 Hyo-won Jeong et al. fication, and serotyping, Food Control 22, (2011). 24. Conter, M., Paludi, D., Zanardi, E., Ghidini, S., Vergara, A. and Ianieri, A.: Characterization of antimicrobial resistance of foodborne Listeria monocytogenes, Int. J. Food Microbiol., 128, (2009). 25. Sakaridis, I., Soultos, N., Iossifidou, E., Papa, A., Ambrosiadis, I., and Koidis, P.: Prevalence and antimicrobial resistance of Listeria monocytogenes isolated in chicken slaughterhouses in Northern Greece. J. Food Prot. 74, (2011). 26. Jemmi, T., and Stephan, R.: Listeria monocytogenes: foodborne pathogen and hygiene indicator. Rev. Sci. Tech. 25, (2006). 27. Kim, D.H.. Song, H.P., Kim, J.K., Kim, J.O., Lee, H.J., and Byun, M.W.: Determination of microbial contamination in the process of rice rolled in dried laver and improvement of shelf-life by gamma irradiation. J. Korean Soc. Food Sci. Nutr. 32, (2003).

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???춍??숏 Suseong gu Council Daegu Metropolitan City www.suseongcouncil.daegu.kr Contents SUSEONG GU COUNCIL DAEGU METROPOLITAN CITY 10 www.suseongcouncil.daegu.kr 11 SUSEONG GU COUNCIL DAEGU METROPOLITAN CITY