한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 한국연안산패류중 Vibrio parahaemolyticus 의분포및항생제내성특성비교 유홍식 * 오은경 1 신순범 2 박용수

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1 한수지 47(5), 5-515, 214 Original Article Kor J Fish Aquat Sci 47(5),5-515,214 한국연안산패류중 Vibrio parahaemolyticus 의분포및항생제내성특성비교 유홍식 * 오은경 1 신순범 2 박용수 이희정 3 김지회 송기철 1 국립수산과학원식품안전과, 1 국립수산과학원서해수산연구소, 2 국립수산과학원남서해수산연구소, 3 식품의약품안전평가원미생물과 Distribution and Antimicrobial Resistance of Vibrio parahaemolyticus Isolated from Korean Shellfish Hongsik Yu*, Eun-Gyoung Oh 1, Soon Bum Shin 2, Yong Su Park, Hee-Jung Lee 3, Ji Hoe Kim and Ki Cheol Song 1 Food Safety Division, National Fisheries Research and Development Institute, Busan , Korea 1West Sea Fisheries Institute, National Fisheries Research and Development Institute, Incheon 4-42, Korea 2Southwest Sea Fisheries Institute, National Fisheries Research and Development Institute, Yeosu , Korea 3Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Cheongju 363-7, Korea The contamination status of Vibrio parahaemolyticus in commercially valuable shellfish from the south and west coasts of Korea and the antimicrobial resistance patterns of isolated V. parahaemolyticus were investigated from July through October, 211. The range of V. parahaemolyticus concentrations in oysters Crassostrea gigas and short neck clams Venerupis philippinarum was <3~29 MPN/1 g and <3~46, MPN/1 g, respectively, and greater than 1, MPN/1 g of V. parahaemolyticus was detected from 7 of 4 short neck clams. During the survey period, 436 strains of V. parahaemolyticus were isolated (129 from oysters and 37 from short-neck clams) and the antimicrobial resistance patterns of all of the isolates were examined. Antimicrobial resistance against at least one antibiotic was seen in 79.% of the oyster isolates (13 strains) and 63.% of the short neck clam isolates (196 strains). The antimicrobial resistance patterns were relatively simple because the antimicrobial resistance of the isolates was simply due to resistance to ampicillin. Only one oyster isolate and three short neck clam isolates showed multiple antibiotic resistance, i.e., resistance against more than four antibiotics. Key words: Vibrio parahaemolyticus, Antimicrobial resistance, Multiple antibiotic resistance 서론 % 36 66%, 3.1% (MOF, 214).,, (Hunter et al., 1999; Mallin et al., 21). (Metcalf, 197; Miossec et al., 19; Lipp et al., 21). (Sobsey and Jaykus, 1991). Kor J Fish Aquat Sci 47(5) 5-515, October 214 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 25 August 214; Revised 3 September 214; Accepted 12 September 214 *Corresponding author: Tel: Fax: address: yhspknu@korea.kr Copyright 214 The Korean Society of Fisheries and Aquatic Science 5 pissn: , eissn:227-15

2 패류유래장염비브리오균의항생제내성특성 59 (Vibrio parahaemolyticus) ( Ortigosa et al., 1994; Diggles et al., 2; Urakawa et al., 2). 195,. V. parahaemolyticus 7 11 % (KFDA, 214).,., V. parahaemolyticus., (Rhodes et al., 2)., 7% (Kümmerer, 29). (, ) V. parahaemolyticus V. parahaemolyticus.. 패류시료 재료및방법 V. parahaemolyticus ( ) ( ) (Crassostrea gigas) (Venerupis philippinarum) YSI 556 multiprobe system (Yellow Springs, YSI Life Science, OH, USA). V. parahaemolyticus 의분리및동정 V. parahaemolyticus Bacteriological Analytical Manual (Elliot et al., 25) 3 tube MPN. 12 1, 2 g phosphate buffered saline (PBS: 14 mm NaCl, 5 mm anhydrous Na 2 HPO 4, and 1.5 mm KH 2 PO 4 ph 7.4) 2. ml PBS 2 g 1. 1, 1,. alkaline peptone water (ph.5.2, 2% NaCl ) Thiosulfate Citrate Bile Salt Agar (TCBS agar, Difco), Triple Sugar Iron Agar (Difco) 35 24,., 42, ONPG (O-nitrophenyl- -D-galacto-pyranoside) VITEK system (BioMerieux Vitek, France). 1 g MPN. 독소유전자시험 V. parahaemolyticus tdh, trh PCR. tdh, trh primer VPD-1/VPD-2 VPR-1/VPR-2 (Takara, Japan). Tryptic Soy Broth (Difco, USA) 1 ml 37, heating block (EYELA, MG-12, Japan) 5 L, deoxynucleoside triphosphate mixture (1 mm datp, 1 mm dctp, 1 mm dttp, 1 mm dgtp) 2 L, 1 PCR 5 L, primer (1 pmol/ L) 1 L, Taq polymerase 1.25 U, 5 L Thermal Cycle (Takara, Japan) 94 6, 6 6, PCR 항균제감수성시험 V. parahaemolyticus Acar and Goldstein (1991) NCCLS (National Committee for Clinical Laboratory Standards).

51 오은경ㆍ신순범ㆍ박용수ㆍ이희정ㆍ김지회ㆍ송기철ㆍ유홍식 Muller Hinton Broth (Merck, Germany) 35 1-24 McFarland No..5. 1% NaCl 4 mm Muller Hinton Agar (Merck, Germany) 5 ( mm) 15.

3 51 오은경ㆍ신순범ㆍ박용수ㆍ이희정ㆍ김지회ㆍ송기철ㆍ유홍식 Muller Hinton Broth (Merck, Germany) McFarland No..5. 1% NaCl 4 mm Muller Hinton Agar (Merck, Germany) 5 ( mm) 15. Amikacin (3 g), Ampicillin (1 g), Amoxicillin/clavulanic acid (3 g), Cefepime (3 g), Cefotaxime (3 g), Chloramphenicol (5 g), Ciprofloxacin (5 g), Erythromycin (5 g), Gentamycin (1 g), Nalidixic acid (3 g), Rifampin (5 g), Streptomycin (1 g), Sulfamethoxazole/trimethoprim (25 g), Tetracycline (5 g), Ttrimethoprim (5 g) 15 (BBL, USA). Muller Hinton Agar 35, 16-1 (inhibition zone) caliper, Escherichia coli ATCC 결과및고찰 V. parahaemolyticus 검출현황 V. parahaemolyticus Table , psu (Fig. 1).. 6 V. parahaemolyticus 5%, 7. (Annie and Marta, 212), 7 9 V. parahaemolyticus %. 7, % (3/) % Salinity ( ) Temp ( ) Jul oyster Jul Aug Sep Oct Nov oyster short-neck calm Aug Sep Oct Nov short-neck calm Fig. 1. Monthly variation of salinity and water temperature during survey period. Table 1. Distribution of Vibrio parahaemolyticus from shellfish farm in the coast of Korea Month No. of samples analyzed No. of VP 1 positive samples Oysters Short neck clams Sum (1%) (1%) 16 (1%) 7 (7.5%) (1%) 15 (93.%) 7 (7.5%) (1%) 15 (93.%) 4 (5.%) (1%) 12 (75.%) 3 (37.5%) 7 (7.5%) 1 (62.5%) Total 29 (72.5%) 39 (97.5%) 6 (5%) No. of pathogenic VP positive samples 1 Vibrio parahaemolyticus. (%) (%) (%) (%) (%) (%)

4 패류유래장염비브리오균의항생제내성특성 % (7/). V. parahaemolyticus V. parahaemolyticus. Ju (193),,,, V. parahaemolyticus, V. parahaemolyticus 9.% (36/367) 1.% (59/313). Chao et al. (29) Das et al. (29) V. parahaemolyticus 47.2% 55%. Kirs et al. (211) 5 55 V. parahaemolyticus 94.%. Chesapeake Bay 1% (Parveen et al., 2), 1% (Zimmerman et al., 27). V. parahaemolyticus V. parahaemolyticus. V. parahaemolyticus <3-29 MPN/1 g, 7 11 <3 MPN/1 g (Fig. 2). V. parahaemolyticus 4 7% (2 ) <3-1 MPN/1 g, 11-1, MPN/1 g 3% (12 ). Annie and Marta (212) V.bparahaemolyticus (MPN/1 g) oyster short-neck calm 1 Jul Aug Sep Oct Nov Fig. 2. Monthly variation of Vibrio parahaemolyticus concentration in shellfish. Rhode Island V. parahaemolyticus, 7 V. parahaemolyticus, Alabama V. parahaemolyticus, , CFU/1 g 12 3 <1 CFU/1 g (Angelo et al., 23). V. parahaemolyticus <3-46, MPN/1 g, <3 MPN/1 g. <3-1 MPN/1 g 4 9 (22.5%), 11-1, MPN/1 g 2 (5%), 1,1-1, MPN/1 g 4 (1%), 1, MPN/1 g 7 (17.5%). V. parahaemolyticus 11-1, MPN/1 g, 1, MPN/1 g % (4 ) V. parahaemolyticus. V. parahaemolyticus (Thermostable direct hemolysin, TDH) (TDH-related hemolysin, TRH) V. parahaemolyticus. V. parahaemolyticus V. parahaemolyticus -. V. parahaemolyticus 분리주의항균제내성특성 V. parahaemolyticus 436 ( 129, 37 ) 15 Table 2 Table 3. V. parahaemolyticus (2.2%) 15, 13 (79.%). 1 penicillin ampicillin 79% trimethoprim, streptomysin 3.1% 1.6%. 1% (Table 2). V. parahaemolyticus (36.2%), 196 (63.%). V. parahaemolyticus ampicillin

5 512 오은경ㆍ신순범ㆍ박용수ㆍ이희정ㆍ김지회ㆍ송기철ㆍ유홍식 Table 2. Antimicrobial resistance of Vibrio parahaemolyticus isolated from oysters Class Antimicrobial agents Drug amount (μg)/disk Diffusion zone breakpoint (mm) 1 No. of isolates (%) Penicillins Ampicillin (79.1) Amoxicillin/ clavulanic acid 3 13 (.) Cephalosporins Cefepime 3 14 (.) Cefotaxime (.) Aminoglycosides Streptomycin (1.6) Gentamycin (.) Amikacin 3 14 (.) Fluoroquinolones Ciprofloxacin 5 15 (.) Quinolones Nalidixic acid (.) Sulfonamides Trimethopenem/ sulfamethoxazole 1.25/ (.) Trimethoprim 5 1 4(3.1) Phenicols Chloramphenicol 5 12 (.) Tetracyclines Tetracycline 5 14 (.) Ansamycins Rifampin (.) Macrorides Erythromycin 5 13 (.) 1 US National Committee for Clinical Laboratory Standards. Table 3. Antimicrobial resistance of Vibrio parahaemolyticus isolated from short neck clams Class Antimicrobial agents Drug amount (μg)/disk Diffusion zone breakpoint (mm) 1 No. of isolates (%) Penicillins Ampicillin (61) Amoxicillin/ clavulanic acid 3 13 (.) Cephalosporins Cefepime 3 14 (.) Cefotaxime 3 14 (.) Aminoglycosides Streptomycin (2.9) Gentamycin (1) Amikacin 3 14 (.) Fluoroquinolones Ciprofloxacin 5 15 (.) Quinolones Nalidixic acid (1) Sulfonamides Trimethopenem/ sulfamethoxazole 1.25/ (.) Trimethoprim (5.2) Phenicols Chloramphenicol (.3) Tetracyclines Tetracycline 5 14 (.) Ansamycins Rifampin (2.3) Macrorides Erythromycin 5 13 (.) 1 US National Committee for Clinical Laboratory Standards. 61.2%. trimethoprim, streptomysin, rifampin 5.2%, 2.9% 2.3% (Table 3)., V. parahaemolyticus ampicillin

6 패류유래장염비브리오균의항생제내성특성 513. Oh et al. (211) V. parahaemolyticus, ampicillin 57.%, rifampin 11.9%, trimethoprim 6.4%, streptomysin.7% 4%. V. parahaemolyticus ampicillin. Son et al. (25) V. parahaemolyticus 97.9% ampicillin, (Lee et al., 2) (Lee et al., 27) V. parahaemolyticus ampicillin. Oh et al. (29), V. parahaemolyticus (5.9%) ampicillin. Jakarta Bay V. parahaemolyticus 97% ampicillin (Moritori et al., 195), La Maddmlena, Tavolara Islands Aleghero gulf Vibrio % ampicillin (Zanetti et al., 21). V. parahaemolyticus Vibrio ampicillin ampicillin Vibrio. V. parahaemolyticus Table 4 Table 5. V. parahaemolyticus Table 4. Antimicrobial resistance profiles of Vibrio parahaemolyticus isolates from oysters No. of resisted antimicrobials Most frequent patterns No. of isolated strains Subtotal, no(%) (2.2) 1 AM (73.6) TMP 1 2 AM-TMP 2 6 (4.7) AM-S 2 Others(2 patterns) 2 3 AM-S-CTX 1 1 (.) 6 AM-S-AMC-RA- NA-TMP 1 1 (.) AM, ampicillin; AMC, Amoxillin/clavulanic acid; CTX, cefotaxime; NA, nalidixic acid; RA, Rifampin; S, streptomycin; TMP, trimethoprim. 22 V. parahaemolyticus. 1 ( -95 ; 73.6%, -172 ; 56.%), 4 (Multiple antibiotic resistance) 1 (.%), 3 (1.%). ampicillin (Table 4, Table 5). (Neela et al., 27), V. parahaemolyticus (Oh et al., 29). plasmid, transposon, integron (mobile element) (horizontal transfer). V. parahaemolyticus Table 5. Antimicrobial resistance profiles of Vibrio parahaemolyticus isolates from short neck clams No. of resisted antimicrobials Most frequent patterns No. of isolated strains Subtotal No(%) (36.2) 1 AM (56.) TMP 3 Others (5 patterns) 5 2 AM-TMP 6 13 (4.2) AM-S 4 Others (4 patterns) 3 3 AM-NA-TMP 2 (2.6) AM-S-AN 2 Others (4 patterns) 4 4 AM-GM-S-AMC 1 3(1.) AM-S-RA-TMP 1 AM-GM-RA-TMP 1 AM, ampicillin; AN, amikacin; C, chloramphenicol; CIP, ciprofloxacin; E, erythromycin; NA, nalidixic acid; RA, rifampin; S, streptomycin; SXT, trimethpenem/sulfamethoxazole; TE, tetracycline; TMP, trimethoprim.

7 514 오은경ㆍ신순범ㆍ박용수ㆍ이희정ㆍ김지회ㆍ송기철ㆍ유홍식. 사사 (RP-214- FS-13). References Acar JF and Goldstein FW Disk susceptibility test. In: Antibiotics in laboratory medicine. Lorian V, ed. Williams & Wilkins, Baltimore, USA, Angelo D, Jessica LN, John CB, Joy GW, and David WC. 23. Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters. Appl Environ Microbiol 69, Annie MC and Marta GC Vibrio parahaemolyticus in Rhode Island Coastal Ponds and the Estuarine Environment of Narragansett Bay. Appl Environ Microbiol 7, Chao G, Jiao X, Zhou X, Yang Z, Huan J, Pan Z, Zhou L and Qian X. 29. Serodiversity,319 pandemic O3:K6 clone, molecular typing, and antibiotic susceptibility of foodborne and clinical 32 Vibrio parahaemolyticus isolates in Jiangsu, China. Foodborne Pathog Dis 6, Das B, Manna SK, Sarkar P and Batabyal K. 29. Occurrence of Vibrio parahaemolyticus in different finfish and shellfish species. J Food Safety 29, org/1.1111/j x. Diggles BK, Moss GA, Carson J and Anderson CD. 2. Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harvey. Dis Aquat Org 43, Elliot EL, Kaysner CA, Jackson L and Tamplin ML Vibrio cholerae, V. parahaemolyticus, V. vulnificus and other Vibrio spp. In: Bacteriological Analytical Manual, Association of Official Analytical Chemists, Arlington, U.S.A., Hunter C, Perkins J, Tranter J and Gunn J Agricultural land-use effects on the indicator bacterial quality of an upland stream in the Derbyshire Peak District in the UK. Water Res 33, Ju JW Studies on Vibrio parahaemolyticus on the southern seas of Korea -On the isolation of V. parahaemolyticus from sea water, sea mud and marine products in Juju, Koeje, Namhae, Yockji, Busan and Masan. J Korea Soc Microbiol 1, 1-9. KFDA Food poisoning statistics by pathogen and month. Retrieved from do?nmenucode=32 on August Kirs M, DePaola A, Fyfe R, Jones JL, Krantz J, Van Laanen A, Cotton D and Castle M A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnicus. Int J Food Microbiol 147, Kümmerer K. 29. Antibiotics in the aquatic environment: a review - partⅠ. Chemosphere, org/1.116/j.ijfoodmicro Lee H, Oh YH, Park SG and Choi SM 27. Antibiotic susceptibility and distribution of Vibrio parahaemolyticus isolated from the seafood. Kor J Env Hlth 33, Lee HW, Lee SK and Kim MS. 2. Characteristics of ampicillin-resistant Vibrio spp. isolated from a west coastal area of Korean Peninsula. J Kor Fish Soc 42, Lipp EK, Farrah SA and Rose JB. 21. Assessment and impact of microbial fecal pollution and human enteric pathogens in a coastal community. Mar Pollut Bull 42, Mallin MA, Ensign SH, McIver MR, Shank GC and Fowler PK. 21. Demographic, landscape, and meteorological factors controlling the microbial pollution of coastal waters. Hydrobiologia 46, org/1.123/a: Metcalf TG Indicators of viruses in shellfish. In: Indicators of viruses in water and food. Berg G, ed. Ann Harbor Science, Michigan, Miossec L, Guyader F, Haugarreau L, Comps MA and Pommepuy M. 19. Possible relation between a winter epidemic of acute gastroenteritis in France and viral contamination of shellfish. J Shellfish Res 17, MOF Fisheries production statistics. Retrieved from ss/ss_info.jsp?menudepth=711 on August Morris JG and Tenny J Jr Antibiotic therapy for Vibrio vulnificus infection. J Am Med Assoc 253, Neela FA, Nonaka L and Suzuki S. 27. The diversity of multidrug resistance profiles in tetracycline-resistant Vibrio species isolated from coastal sediments and seawater. J Microbiol 45, Oh EG, Son KT, Ha KS, Yoo HD, Yu HS, Shin SB, Lee HJ and Kim JH. 29. Antimicrobial Resistance of Vibrio strains from Brackish Water on the Coast of Gyeongsangnamdo. J Kor Fish Soc 42, Oh EG, Son KT, Yu HS, Lee TS, Lee HJ, Shin SB, Kwon JY, Park KBW and KIM JH Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from Farmed Fish in Korea during J Food Protec 74, Ortigosa M, Garay E and Pujalte MJ Numerical taxonomy of vibrionaceae isolated from oysters and seawater along an annual cycle. Syst Appl Microbiol 17, Parveen S, Hettiarachchi KA, Bowers JC, Jones JL, Tamplin ML, McKay R, Beatty W, Brohawn K, Dasilva LV, Depaola A. 2. Seasonal distribution of total and pathogenic Vibrio

8 parahaemolyticus in Chesapeake Bay oysters and waters. Int J Food Microbiol 12, ijfoodmicro Rhodes BJ, Smith HL Jr. and Ogg JE Isolation of non- O1 Vibrio cholerae serovars from surface waters in Western Colorado. Appl Environ Microbiol, 51, Sobsey MD and Jaykus LA Human enteric viruses and depuration of bivalve molluscs. In: Molluscan shellfish depravation. Otwell WS, Rodrick GE and Martin RE, eds. CRC Press, Boca Raton, Florida, USA, Son KT, Oh EG, Lee TS, Lee HJ, Kim PH and Kim JH. 25. Antimicrobial susceptibility of Vibrio parahaemomlyticus and Vibrio alginolyticus from fish farms on the southern coast of Korea. J Kor Fish Soc 3, Urakawa H, Yoshida T, Nishimura M and Ohwada K. 2. Characterization of depth related population variation in microbial communities of coastal marine sediment using 16S rdna-based approaches and quinone profiling. Environ Microbiol 2, Zanetti S, Spanu T, Deriu A, Romano L, Sechi LA and Fadda G. 21. In vitro susceptibility of Vibrio spp. isolated from the environment. Int J Antimicrob Agents 17, Zimmerman AM, DePaola A, Bowers JC, Krantz JA, Nordstrom JL, Johnson CN and Grimes DJ. 27. Variability of total and pathogenic Vibrio parahaemolyticus densities in northern Gulf of Mexico water and oysters. Appl Environ Microbiol 73, 패류유래장염비브리오균의항생제내성특성 515

674 권순재ㆍ정연중ㆍ윤현진ㆍ목종수ㆍ권지영,.., (Watts et al., 2017)., 70% (Kummerer, 2009). (Witte, 2000)., (Souli et al., 2008)., % ,113 (NIFS, 2017

674 권순재ㆍ정연중ㆍ윤현진ㆍ목종수ㆍ권지영,.., (Watts et al., 2017)., 70% (Kummerer, 2009). (Witte, 2000)., (Souli et al., 2008)., % ,113 (NIFS, 2017 한수지 51(6), 673-681, 2018 Original Article Korean J Fish Aquat Sci 51(6),673-681,2018 용남광도해역의굴 (Crassostrea gigas) 및육상오염원에서분리한대장균 (Escherichia coli) 의항생제내성 권순재 정연중 윤현진 목종수 1 권지영 * 국립수산과학원남동해수산연구소, 1 국립수산과학원식품위생가공과