Table 1. Allelic profiles of 194 C. jejuni analyzed in this study Gene locus Fragment length (bp) No. of alleles No. of polymorphic sites (%) Ave. p d

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1 연구논문, Research article 국내설사환자분리캄필로박터제주니의유전형분석 질병관리본부감염병분석센터세균분석과정수미, 강병학, 홍사현, 김재옥 * * 교신저자 : kimjo70@korea.kr, Genetic analysis of Campylobacter jejuni isolates from diarrheal patients in South Korea (Jan Feb. 2017) Jung Su-Mi, Hong Sahyun, Kang Byung Hak, Kim Jae Ok Division of Bacterial Diseases, Center for Laboratory Control of Infectious Diseases, KCDC Background: Campylobacter jejuni is a major bacterial cause of human gastroenteritis worldwide, being associated with the ingestion of contaminated chicken. The aim of this study is to determine the molecular epidemiology of C. jejuni strains collected from diarrheal patients between January 2012 and February Methodology: To identify the genetic variations in C. jejuni, multilocus sequence typing (MLST) was performed on 194 C. jejuni strains isolated from the cases of sporadic acute gastroenteritis (n = 150), food-poisoned diarrhea outbreak (n=26) and overseas travel-associated diarrhea (n = 18) in South Korea. The phylogenetic tree was constructed using the neighborjoining algorithm with the software MEGA 6. Results: MLST analyses of the 194 clinical isolates generated 58 different sequence types (STs) and 19 clonal complexes (CCs). MLST gene fragments varied in length from 402 to 507 bps, with average p distances of (unca gene fragment) to (pgm gene fragment). Clonal complex 21 (45.9%) including ST-19, ST-21, ST-50, ST-760, ST-1811 and ST-4253 was dominant in this study. In CC 21, ST-21 was the most prevalent ST of C. jejuni in South Korea. A neighbor-joining tree was classified into three clusters, wherein 63.4% of the isolates was found in cluster 3. Conclusions: In this study, domestic isolates and overseas travel-associated isolates can be distinguished by MLST analysis. Our results provide information on MLST type distribution and genetic relatedness of C. jejuni strains in South Korea. Keywords: Gastroenteritis, Campylobacter jejuni, Genotic variation, Multilocus sequence typing, Phylogeny 280

2 Table 1. Allelic profiles of 194 C. jejuni analyzed in this study Gene locus Fragment length (bp) No. of alleles No. of polymorphic sites (%) Ave. p distance aspa (2.1) glna (2.3) glta (1.5) glya (5.3) pgm (5.4) tkt (4.1) unca (1.2) 들어가는말 캄필로박터속균들은그람음성의나선형간균이며 3~5% 의 CO 2 분압하에서성장을하는미호기성세균으로서인체감염시복통, 설사, 권태감, 발열, 구역질및구토등을유발한다 [1]. 질병관리본부감염병분석센터세균분석과에서실시하고있는급성설사질환실험실감시사업 (EnterNet) 은수인성 식품매개성 제주니균 (Campylobacter jejuni) 에대한유전형분포를확인하기위해 Multilocus sequence typing(mlst) 시험법을이용하여분석하였다. MLST 시험법은균주의계통발생학을추적하기위한분자유전학의유용한도구이며, 이시험법을이용하여유전적변화및계통발생학적분포, Sequence types(sts) 간의연관관계를조사하여국내및해외여행자의설사환자로부터분리된캄필로박터제주니균의특성을확인하였다. 감염병의원인병원체에대한상시감시체계로, 전국협력병원에서 설사환자의검체와임상자료를수집하고시 도보건환경연구원에서원인병원체에대한검사를실시하며, 세균성병원체에대해수집한결과를종합분석하여설사질환의유행양상및병원체정보를공개하고있다. 급성설사질환실험실감시사업 (EnterNet) 결과, 캄필로박터속 ( 屬 ) 균 (Campylobacter spp.) 은 2013년에감시대상병원체가확인된전체검체중 158건 (0.8%) 으로확인되었고 2014년은 215건 (1.4%), 2015년은 202건 (1.5%). 2016년 146건 (1.5%) 분리되었다. 식품의약품안전처에서공개한캄필로박터제주니균에의한국내식중독통계자료를통해확인된발생건수 ( 환자수 ) 는 2013년에 6건 (231명), 2014년 18건 (490명), 2015년 22건 (805명), 2016년 15건 (831명 ), 2017년에는 6건 (101 명 ) 이었다 [2]. 이글에서는수집된캄필로박터균중캄필로박터 몸말 급성설사질환실험실감시사업 (EnterNet) 을통해수집된국내및해외여행자의설사환자로부터분리된캄필로박터제주니균 194주 ( 산발적급성설사질환환자분리 150주, 식중독환자분리 26주, 해외여행설사환자분리 18주 ) 를대상으로유전형을분석하였다. 194 균주를대상으로 7개 Housekeeping gene(aspa, glna, glta, glya, pgm, tkt, unca) 의염기서열분석을통해유전자정보를얻었으며 MLST 웹사이트 ( 에서염기서열유형 (Sequence types, STs) 을확인하였다. 7개의 Housekeeping gene을분석한결과유전자는 13~25 가지대립유전자 (allele) 의수를나타냈고, 염기서열의가장큰 281

3 Isolates (%) Clonal complexes (CCs) Figure 1. Distribution of clonal complexes(ccs) of C. jejuni isolates(%) from human stools 변화를보인유전자는 pgm 유전자로 27곳 (5.4%) 에서염기서열이달랐으며두서열이다른뉴클레오티드사이의비율을표시하는평균 p 거리 (Average p distance) 가 이었다. 가장적은변화를보인 unca 유전자는염기서열 6곳 (1.2%) 에서달랐으며평균 p 거리가 0.008을나타내었다 (Table 1). 염기서열유형 (Sequence types, STs) 은 58종류가확인되었으며 ST-21(24.2%, 47주 ), ST-50(11.9%, 23주 ), ST- CC354, CC443, CC460이포함되어있으며, 세개의무리중가장많은해외여행설사환자를포함하고있다. 세번째무리 (Cluster 3) 에는국내에서가장높은분리율을보이는 CC21과 CC49, CC52, CC353, CC446, CC574, CC607이포함되었다. 분포율을비교해보면첫번째무리에는 20.1%(39주 ), 두번째무리에는 16.5%(32주 ) 이며, 세번째무리에는 63.4%(123주 ) 로가장많이분포를보였다 (Table 2). 4253(7.7%, 15 주 ), ST-51(7.2%, 14 주 ), ST-4739(5.7%, 11 주 ) 순으로 가장많이확인되었고, MLST site에서정의된클론복합체 (Clonal complexes, CCs) 는 19가지유형으로 CC21(45.9%, 89주 ), CC443(8.8%, 17주 ), CC22(4.1%, 8주 ), CC464(3.6%, 7주 ) 순으로분포를보였다 (Figure 1). CC21에속한 STs는 ST-19(2주 ), ST- 21(47주 ), ST-50(23 주 ), ST- 760(1주 ), ST-1811(1 주 ), ST-4253(15 주 ) 이었다. STs의분석을위해 MEGA6에서염기서열을분석한 Neighborjoining 방법을이용하여 Tree를그려본결과, 크게 3가지무리 (Cluster) 로나누어졌다 (Figure 2). 첫번째무리 (Cluster 1) 에는 CC22, CC42, CC45, CC48, CC283, CC353, CC464, CC658이포함되어있고, 두번째무리 (Cluster 2) 에는 CC206, CC257, CC353, 맺는말 이연구에서는국내및해외여행자의설사환자로부터분리된캄필로박터제주니균의 MLST 유전자형을분석하여국내유행양상에대한정보를제공하고자하였다. 분리유형에따른계통발생학적인차이는크게나타나지않았지만해외여행설사환자로부터분리된균주가두번째무리 (Cluster 2) 에 61.1% 로집중하는경향을보였다. 또한, 국내인체분리주의대표적인유형으로 CC21을확인하였다. CC21은국내오리에서도분리보고된바있으며 [3], 해외유전형분석보고를통해스위스 [4], 282

4 Cluster 1 Cluster 2 Cluster 3 Sporadic Outbreak Overseas travel-associated Figure 2. A neighbor-joining tree constructed from MLST comparisons of 194 C. jejuni isolates 이탈리아 [5], 뉴질랜드 [6] 에서도대표적인유형으로지목되었다. 이연구에서 CC48-ST918 은국내집단발생에서분리되었지만, 리투아니아에서는아이들과개로부터공통적으로분리된유형으로 보고되었으며 [7], CC21 과 CC353 이리투아니아의임상분리주, 젖소, 구이용닭제품으로부터대표적인유전형이었다고보고되었다 [8]. 또한, CC354-ST354 는이연구에서산발적급성설사질환 283

5 Table 2. Clonal complex profiles from 194 C. jejuni in 3 clusters No. of isolates Clonal complex Sporadic acute gastroenteritis Food-poisoned diarrhea outbreak Overseas travelassociated diarrhea Total of isolates (%) (n=150) (n=26) (n=18) Cluster 1 CC (20.1) CC CC CC353 3 CC45 4 CC CC CC658 1 Not found * 3 1 Cluster 2 CC (16.5) CC353 1 CC257 2 CC CC460 2 CC206 1 Not found * Cluster 3 CC (63.4) CC607 6 CC353 1 CC49 4 CC CC574 2 CC446 1 Not found * *Not found; Non-typeable 환자로부터분리된유형으로중국에서는닭과식품으로부터공통적으로분리되었다 [9]. 이번 MLST 분석자료는캄필로박터제주니균이원인으로유추되는수인성 식품매개질환의역학자료와비교 분석하여원인병원체를규명하는데유용한자원으로활용될것으로기대된다. 참고문헌 1. 질병관리본부. 주간건강과질병. 9권 27호 식품의약품안전처. ( 식품안전나라 > 위해 예방정보 > 식중독정보 > 식중독통계 [Online]. 3. Wei B, et al. Antimicrobial susceptibility profiles and molecular typing of Campylobacter jejuni and Campylobacter coli isolates from ducks in South Korea. Appl Environ Microbiol. 2014;80(24):

6 4. Niederer L. et al. Genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli isolates from domestic and travel-associated human cases. Appl Environ Microbiol. 2012;78(1): Piccirillo A. et al. Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli from humans and chickens in North- Eastern Italy. New Microbiol. 2014;37(4): Friedrich A. et al. Seasonality of Campylobacter jejuni isolates associated with human campylobacteriosis in the Manawatu region, New Zealand. Epidemiol Infect. 2016;144(4): Ramonaite S. et al. Prevalence and genotypes of Campylobacter jejuni from urban environmental sources in comparison with clinical isolates from children. J Med Microbiol. 2014;63(Pt9): Ramonaite S. et al. MLST genotypes of Campylobacter jejuni isolated from broiler products, dairy cattle and human campylobacteriosis cases in Lithuania. BMC Infect Dis. 2017;17(1): Zhang G. et al. Multilocus Sequence Types of Campylobacter jejuni Isolates from Different Sources in Eastern China. Curr Microbiol. 2015;71(3):

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°Ç°�°úÁúº´6-2È£ K O R E A C E N T E R S F O R D I S E A S E C O N T R O L & P R E V E N T I O N PHWR Vol. 6 No. 2 www.cdc.go.kr 2013 1 11 6 2 ISSN:2005-811X Flavivirus surveillance in mosquitoes collected from the quarantine