한수지 49(5), , 2016 Original Article Korean J Fish Aquat Sci 49(5), ,2016 곰소만해역해수에서분리한장염비브리오 (Vibrio parahaemolyticus) 의항균제내성및최소발육억제농도의구명 김

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1 한수지 49(5), , 206 Original Article Korean J Fish Aquat Sci 49(5), ,206 곰소만해역해수에서분리한장염비브리오 (Vibrio parahaemolyticus) 의항균제내성및최소발육억제농도의구명 김태옥 엄인선 김희대 박권삼 * 군산대학교식품생명공학과, 충북도립대학바이오생명의약과 Antimicrobial Resistance and Minimum Inhibitory Concentrations of Vibrio parahaemolyticus Strains Isolated from Gomso Bay, Korea Tae-Ok Kim, In-Seon Um, Hee-Dai Kim and Kwon-Sam Park* Department of Food Science and Biotechnology, Kunsan National University, Gunsan 5450, Korea Department of Biotechnology and Biomedicine, Chungbuk Provincial College, Cheongju 2860, Korea Seventy-nine Vibrio parahaemolyticus isolates from surface seawater from Gomso Bay, west coast of Korea, were analyzed for the presence of virulence genes and their susceptibility to 30 different antimicrobials. All 79 isolates were examined for the presence of two virulence genes (tdh or trh) using polymerase chain reaction (PCR); however, no isolates possessed either the tdh or trh gene. According to a disk diffusion susceptibility test, all of the strains studied were resistant to oxacillin, penicillin, and vancomycin, followed by ticarcillin (97.5%), ampicillin (96.2%), clindamycin (86.%), erythromycin (0.%), streptomycin (7.6%), cefoxitin (6.3%), amikacin (2.5%), and cephalothin (2.5%). However, all of the strains were susceptible to 9 other antimicrobials including cefepime, cefotaxime, chloramphenicol, gentamycin, nalidixic acid, sulfamethoxazole/trimethoprim, and trimethoprim. All 79 isolates (00%) were resistant to four or more classes of antimicrobials, and two strains exhibited resistance to eight antimicrobial agents. The average minimum inhibitory concentrations (MICs) for V. parahaemolyticus for ampicillin, penicillin, ticarcillin, and vacomycin were 946.5,,305.9,,032.3, and 45.0 µg/ml, respectively. Key words: Vibrio parahaemolyticus, Antimicrobial resistance, Virulence genes, Minimum inhibitory concentration, Gomso Bay 서론 (Vibrio parahaemolyticus),,,,,, (Honda and Iida, 993; Zhang and Orth, 203) % 5.4% (MFDS, 206). (thermostable direct hemolysin, TDH), (TDH-related hemolysin, TRH) type III secretion systems (TTSS 2) effector (Honda and Iida, 993; Park et al., 2004; Letchumanan et al., 204; Wang et al., 205). Korean J Fish Aquat Sci 49(5) , October 206 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 8 August 206; Revised 3 August 206; Accepted September 206 *Corresponding author: Tel: Fax: address: parkks@kunsan.ac.kr Copyright 206 The Korean Society of Fisheries and Aquatic Science 582 pissn:0374-8, eissn:

2 장염비브리오의항균제내성및최소발육억제농도 583 (Sakazaki et al., 968; Honda and Iida, 993)..,. (Son et al., 2005; Lee et al., 2007; Oh et al., 2008; Lee et al., 2009; Ryu et al., 200; Yu et al., 200; Kim et al., 204; Kang et al., 206; Kim et al., 206).,,., plasmid transposon, plasmid (Kuhl et al., 978). integron DNA transposon (Rowe-Magnus and Mazel, 2002) 사용균주및시약 재료및방법 RIMD (Makino et al., 2003) TH3996 (Park et al., 2000). Escherichia coli ATCC Staphylococcus aureus ATCC Takara (Otsu, Japan), Becton Dickinson (BBL Sensi-Disk, Sparks, MD, USA) Sigma (St. Louis, MO, USA). 장염비브리오의분리및동정 (MFDS, 206) ml double strength alkaline peptone water (Merck, Darmstadt, Germany) 0 ml thiosulfate-citrate-bile-salts (TCBS) agar (Difco, Detroit, MI, USA) TCBS 3-5 mm triple sugar iron agar (Difco, Detroit, MI, USA) API 20E system (BioMerieux, Marcy-I'Etoil, France). toxr (Kim et al., 999) hns (No et al., 20) PCR assay.. Luria-Bertani (tryptone %, yeast-extract 0.5%, NaCl 3%) broth 5% cryovial storage box (Simport, Canada) -80. DNA 증폭용 primer set DNA primers DNA Table. Primers Bioneer (Daejon, Korea). TDH, TRH, toxr H-NS DNA PCR , 55 30, DNA. DNA.5% agarose gel ethidium bromide Vilber Lourmat (Bio-Paint ST4, France) Gel-Doc system. 항균제감수성시험 Acar and Goldstein (99) NCCLS (National Committee for Clinical Laboratory Standards, 2002). 3% LB broth 35 2 McFar-

3 584 김태옥ㆍ엄인선ㆍ김희대ㆍ박권삼 Table. Primers used in this study Target gene Oligonucleotide sequence Amplicon size (bp) Reference toxr tdh trh hns 5'-AGCCCGCTTTCTTCAGACTC-3' 5'-AACGAGTCTTCTGCATGGTG-3' 5'-GTAAAGGTCTCTGACTTTTGGAC-3' 5'-TGGAATAGAACCTTCATCTTCACC-3' 5'-TTGGCTTCGATATTTTCAGTATCT-3' 5'-CATAACAAACATATGCCCATTTCCG-3' 5'-AAACACGTTAACCTATTAATAGG-3' 5'-AACGGGAGCCTTTTTAAACAAGA-3' 399 Kim et al., Lee and Park, Lee and Park, No et al., 20 land No mm Muller Hinton agar (Merck, Germany) amikacin (AK; 30 µg), ampicillin (AMP; 0 µg), cefepime (FEP; 30 µg), cefotaxime (CTX; 30 µg), cefotetan (CTT; 30 µg), cefoxitin (FOX; 30 µg), cefuroxime (CXM; 30 µg), ceftriaxone (CRO; 30 µg), cephalothin (KF; 30 µg), cephazolin (KZ; 30 µg), chloramphenicol (C; 30 µg), ciprofloxacin (CIP; 5 µg), clindamycin (CC; 2 µg), erythromycin (E; 5 µg), gentamicin (GN; 0 µg), imipenem (IPM; 0 µg), kanamycin (K; 30 µg), nalidixic acid (NA; 30 µg), nitrofurantoin (F; 00 µg), norfloxacin (NOR; 0 µg), oxacillin (OX; µg), penicillin (P; 0 µg), pipemidic acid (PIP; 20 µg), rifampin (RD; 5 µg), streptomycin (S; 0 µg), sulfamethoxazole/trimethoprim (SXT; 23.75/.25 µg), tetracycline (TE; 30 µg), ticarcillin (TIC; 75 µg), trimethoprim (W; 5 µg), vancomycin (VA; 30 µg) 30. 최소발육억제농도 (Minimum Inhibitory Concentration, MIC) 측정 NCCLS (National Committee for Clinical Laboratory Standards, 2002). Muller Hinton broth (Merck, Germany) 2,048 µg/ml µg/ml 2 ml. 3% LB broth 3 µl 결과및고찰 곰소만표층해수에서분리한장염비브리오의특성 Fig.. Location of sampling stations in Gomso Bay, Korea, from June 204 to October Fig.., toxr (Kim et al., 999) hns (No et al., 20). 79 toxr hns DNA DNA ( ). toxr hns 6.. toxr hns. PCR assay,, 79 TDH

4 장염비브리오의항균제내성및최소발육억제농도 585 TRH ( ). (Shirai et al., 990), (Deepanjali et al., 2005; Jones et al., 202; Ellingsen et al., 203; Gutierrez et al., 203; Kang et al., 206; Kim et al., 206). 79. 장염비브리오의항균제내성양상 amikacin, ampicillin, cephalothin, cefoxitin, gentamicin, kanamycin, rifampin, streptomycin vancomycin (Son et al., 2005; Lee et al., 2009; Ryu et al., 200; Han et al., 202; Kim et al., 204; Kang et al., 206; Kim et al., 206) Table , 9. oxacillin, penicillin, vancomycin, ticarcillin, ampicillin, clindamycin, erythromycin, streptomycin, cefoxitin, amikacin cephalothin. oxacillin, penicillin vancomycin 00%, ticarcillin (97.5%), ampicillin (96.2%), clindamycin (86.%), erythromycin (0.%), streptomycin (7.6%), cefoxitin (6.3%), amikacin (2.5%) cephalothin (2.5%). Cefotaxime 9.,, (Son et al., 2005: Lee et al., 2007; Lee and Park, 200; Ryu et al., 200; Han et al., 202; Kim et al., 204; Kang et al., 206; Kim et al., 206). 79 Table 3. 4 (.3%), 5 9 (.4%), 6 54 (68.4%), 7 3 (6.5%) 8 2 (2.5%). AMP-CC-OX-P-TIC-VA 50 (63.3%), AMP-OX-P-TIC-VA 8 Table 2. Antimicrobial susceptibility and resistance of Vibrio parahaemolyticus strains isolated from surface seawater in Gomso Bay Antimicrobials Disc content (μg) No. of isolates Resistant Intermediate Susceptible Amikacin (AK) Ampicillin (AMP) Cefepime (FEP) Cefotaxime (CTX) Cefotetan (CTT) Cefoxitin (FOX) Cefuroxime (CXM) Ceftriaxone (CRO) Cephalothin (KF) Cephazolin (KZ) Chloramphenicol (C) Ciprofloxacin (CIP) Clindamycin (CC) Erythromycin (E) Gentamicin (GN) Imipenem (IPM) Kanamycin (K) Nalidixic acid (NA) Nitrofurantoin (F) Norfloxacin (NOR) Oxacillin (OX) Penicillin (P) Pipemidic acid (PIP) Rifampin (RD) Streptomycin (S) Sulfamethoxazole /Trimethoprim (SXT) Tetracycline (TE) Ticarcillin (TIC) Trimethoprim (W) Vancomycin (VA) (0.%) AMP-CC-OX-P-S-TIC-VA 6 (7.6%) 4 (Table 3). 4.,

5 586 김태옥ㆍ엄인선ㆍ김희대ㆍ박권삼 Table 3. Antimicrobial resistance patterns of Vibrio parahaemolyticus strains isolated from surface seawater in Gomso Bay Resistance type No. of resistant strains CC-OX-P-VA AMP-OX-P-TIC-VA 8 CC-FOX-OX-P-VA AK-CC-E-OX-TIC-VA AMP-E-OX-P-TIC-VA 2 AMP-CC-OX-P-TIC-VA 50 AMP-KF-OX-P-TIC-VA AMP-CC-OX-P-S-TIC-VA 6 AMP-CC-E-OX-P-TIC-VA 3 AMP-CC-FOX-OX-P-TIC-VA 3 AMP-CC-KF-OX-P-TIC-VA AK-AMP-CC-E-OX-P-TIC-VA AMP-CC-E-FOX-OX-P-TIC-VA Total 79 AK, amikacin; AMP, ampicillin; CC, clindamycin; E, erythromycin; FOX, cefoxitin; KF, cephalothin; OX, oxacillin; P, penicillin; S, streptomycin; TIC, ticarcillin; VA, vancomycin. plasmid bacteriophage. 장염비브리오의최소발육억제농도측정 Table 4. Amikacin 2 MIC 32 6 µg/ml, 77 MIC µg/ml. Ampicillin MIC 2,048 µg/ ml 8 (0.%),,024 µg/ml MIC 4 (5.9%), 52 µg/ml MIC 26 (32.9%) 256 µg/ml MIC (.3%) 3 (3.8%) MIC 2.0 µg/ml. Ampicillin MIC µg/ml. ampicillin (Tanil et al., 2005; Lee et al., 2009; Lee and Park, 200; Kim et al., 204). Clindamycin MIC 6.0 µg/ Table 4. Minimum inhibitory concentration of Vibrio parahaemolyticus strains isolated from surface seawater in Gomso Bay Antimicrobials Amikacin Ampicillin Cindamycin Erythromycin Cefoxitin Cephalothin Oxacillin Penicillin Streptomycin Ticarcillin Vancomycin µg/ml > ,024 2, (97.4%) 7 (89.8%) 74 (93.7%) 73 (92.4%) (3.9%) 3 (3.8%) 6 6 (20.3%) (77.2%) 3 (3.8%) 26 (32.9%) 4 (5.%) 3 (3.8%) 2 (2.5%) (.3%) 29 (36.7%) (.3%) 2 (2.5%) 5 (6.3%) 6 (7.6%) (.3%) 3 (6.5%) (.3%) 43 (54.4%) 30 (38.0%) 3 (39.2%) (.3%) (.3%) 2 (2.5%) 42 (53.2%) 4 (5.%) 26 (32.9%) 6 (7.6%) 6 (7.6%) 23 (29.%) 4 (5.9%) 4 (5.9%) 40 (50.6%) 8 (0.%) 28 (35.4%) 3 (6.5%)

6 장염비브리오의항균제내성및최소발육억제농도 587 ml MIC 2.0 µg/ml. erythromycin, streptomycin cefoxitin MIC. Cephalothin MIC 256 µg/ml, oxacillin MIC µg/ml. Penicillin ticarcillin MIC,305.9 µg/ml,032.3 µg/ml, vancomycin MIC 45.0 µg/ml. ampicillin, penicillin, ticarcillin oxacillin cephalothin MIC amikacin, clindamycin, erythromycin, cefoxitin, streptomycin, vancomycin MIC (Table 4) ampicillin, penicillin, ticarcillin MIC.. DNA. References Acar JF and Goldstein FW. 99. Disk susceptibility test. In: Antibiotics in Laboratory Medicine, Lorian V, ed. Williams & Wilkins, Baltimore, U.S.A., Deepanjali A, Kumar HS, Karunasagar I and Karunasagar I Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India. Appl Environ Microbiol 7, Ellingsen AB, Olsen JS, Granum PE, Rørvik LM and González- Escalona N Genetic characterization of trh positive Vibrio spp. isolated from Norway. Front Cell Infect Microbial 3, Gutierrez West CK, Klein SL and Lovell CR High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary. Appl Environ Microbiol 79, org/0.28/aem Han AR, Yoon YJ and Kim JW Antibiotic resistance and plasmid profile of Vibrio parahaemolyticus strains isolated from Kyunggi-Incheon coastal area. Korean J Microbiol 48, Honda T and Iida T The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev Med Microbiol 4, Jones JL, Ludeke CH, Bowers JC, Garrett N, Fischer M, Parsons MB, Bopp CA and DePaola A Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates. J Clin Microbiol 50, Kang CH, Shin Y, Kim W, Kim Y, Song K, Oh EG, Kim S, Yu H and So JS Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from oysters in Korea. Environ Sci Pollut Res Int 23, org/0.007/s Kim SK, An SR, Park BM, Oh EG, Song KC, Kim JW and Yu HS Virulence factors and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from the oyster Crassostrea gigas. Korean J Fish Aquat Sci 49, Kim TO, Eum IS, Jo SM, Kim HD and Park KS Antimicrobial-resistance profiles and virulence genes of Vibrio parahaemolyticus isolated from seawater in the Wando area. Korean J Fish Aquat Sci 47, org/0.5657/kfas Kim YB, Okuda J, Matsumoto C, Takahashi N, Hashimoto S and Nishibuchi M Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxr gene. J Clin Microbiol 37, Kuhl SA, Pattee PA and Baldwin NJ Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus. J Bacteriol 35, Lee H, Oh YH, Park SG and Choi SM Antibiotic susceptibility and distribution of Vibrio parahaemolyticus isolated from the seafood. Kor J Env Hlth 33, Lee HW, Lim SK and Kim MN Characteristics of ampicillin-resistant Vibrio spp. isolated from a west coastal area of Korea peninsula. J Kor Fish Soc 42, Lee KW and Park KS Antibiotic-resistance profiles and the identification of the ampicillin-resistance gene of Vibrio parahaemolyticus isolated from seawater. Korean J Fish Aquat Sci 43, KFAS Letchumanan V, Chan KG and Lee LH Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front Microbiol 5, Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, Iijima Y, Najima M, Nakano M, Yamashita A, Kubota Y, Kimura S, Yasunaga T, Honda T, Shinagawa H, Hattori M and Iida T Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae. Lancet 36, Ministry of Food and Drug Safety (MFDS) Retrieved from grp=menu_grp02 on August. National Committee for Clinical Laboratory Standards (NC- CLS) Performance standards for antimicrobial sus-

7 588 김태옥ㆍ엄인선ㆍ김희대ㆍ박권삼 ceptibility testing. Twelfth informational supplement M00- S2. Wayne, Pennsylvania, U.S.A., No AR, Okada K, Kogure K and Park KS. 20. Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization. Lett Appl Microbiol 53, dx.doi.org/0./j x x. Oh EG, Yu HS, Shin SB, Son KT, Park KB, Kwon JY, Lee TS and Lee HJ Trimethoprim resistance of Vibrio parahaemolyticus isolated from the fish farm. J Kor Fish Soc 4, Park KS, Iida T, Yamaichi Y, Oyagi T, Yamamoto K and Honda T Genetic characterization of DNA region containing the trh and ure genes of Vibrio parahaemolyticus. Infect Immun 68, Park KS, Ono T, Rokuda M, Jang MH, Okada K, Iida T and Honda T Functional characterization of two type III secretion systems of Vibrio parahaemolyticus. Infect Immun 72, Rowe-Magnus DA and Mazel D The role of integrons in antibiotic resistance gene capture. Int J Med Microbiol 292, Ryu SH, Hwang YO, Park SG and Lee YK Antibiotic susceptibility of Vibrio parahaemolyticus isolated from commercial marine products. Korean J Food Sci Technol 42, Sakazaki R, Tamura K, Kato T, Obara Y and Yamai S Studies on the enteropathogenic, facultatively halophilic bacterium, Vibrio parahaemolyticus. 3. Enteropathogenicity. Jpn J Med Sci Biol 2, Shirai H, Ito H, Hirayama T, Nakamoto Y, Nakabayashi N, Kumagai K, Takeda Y and Nishibuchi M Molecular epidemiological evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis. Infect Immun 58, Son KT, Oh EG, Lee TS, Lee HJ, Kim PH and Kim JH Antimicrobial susceptibility of Vibrio parahaemolyticus and Vibrio alginolyticus from farms on the southern coast of Korea. J Kor Fish Soc 38, Tanil GB, Radu S, Nishibuchi M, Rahim RA, Napis S, Maurice L and Gunsalam JW Characterization of Vibrio parahaemolyticus isolated from coastal seawater in peninsular Malaysia. Southeast Asian J Trop Public Health 36, Wang R, Zhong Y, Gu X, Yuan J, Saeed AF and Wang S The pathogenesis, detection, and prevention of Vibrio parahaemolyticus. Front Microbiol 6, org/0.3389/fmicb Yu HS, Park KB, Oh EG, Lee TS, Shin SB, Kwon JY, Kim JH and Son KT Trimethoprim resistance by class I integron in Vibrio parahaemolyticus from a fish farm. Korean J Fish Aquat Sci 43, Zhang L and Orth K Virulence determinants for Vibrio parahaemolyticus infection. Curr Opin Microbiol 6,

한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 MPN 및 H-NS 유전자를표적으로하는 PCR assay 를병용한장염비브리오 (Vibrio parahaemolyticus)

한수지 47(5), , 2014 Original Article Kor J Fish Aquat Sci 47(5), ,2014 MPN 및 H-NS 유전자를표적으로하는 PCR assay 를병용한장염비브리오 (Vibrio parahaemolyticus) 한수지 47(5), 556-561, 2014 Original Article Kor J Fish Aquat Sci 47(5),556-561,2014 MPN 및 H-NS 유전자를표적으로하는 PCR assay 를병용한장염비브리오 (Vibrio parahaemolyticus) 의정량 김태옥 박권삼 * 군산대학교식품생명공학과 Quantification of Vibrio