한수지 49(5), 582-588, 206 Original Article Korean J Fish Aquat Sci 49(5),582-588,206 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). 2006 205 0 6 0 2.5% 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). http://dx.doi.org/0.5657/kfas.206.0582 Korean J Fish Aquat Sci 49(5) 582-588, October 206 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) 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: +82. 63. 469. 822 Fax: +82. 63. 469. 7448 E-mail address: parkks@kunsan.ac.kr Copyright 206 The Korean Society of Fisheries and Aquatic Science 582 pissn:0374-8, eissn:2287-885
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).. 204 6 205 0 79. 사용균주및시약 재료및방법 204 6 205 0 79 RIMD220633 (Makino et al., 2003) TH3996 (Park et al., 2000). Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923. Takara (Otsu, Japan), Becton Dickinson (BBL Sensi-Disk, Sparks, MD, USA) Sigma (St. Louis, MO, USA). 장염비브리오의분리및동정 (MFDS, 206). 4. 0 ml double strength alkaline peptone water (Merck, Darmstadt, Germany) 0 ml 35 8-24 thiosulfate-citrate-bile-salts (TCBS) agar (Difco, Detroit, MI, USA) 35 8-24. TCBS 3-5 mm triple sugar iron agar (Difco, Detroit, MI, USA) 35 24 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 95 3 95 30, 55 30, 72 30 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-
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., 999 269 Lee and Park, 200 486 Lee and Park, 200 465 No et al., 20 land No. 0.5 0.4 mm Muller Hinton agar (Merck, Germany). 35 6-8. 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 35 8. 결과및고찰 곰소만표층해수에서분리한장염비브리오의특성 Fig.. Location of sampling stations in Gomso Bay, Korea, from June 204 to October 205. 204 6 205 0 0 0 00 79. 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
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). 79 30 Table 2. 30 79, 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) 30 2 4 73 Ampicillin (AMP) 0 76 3 0 Cefepime (FEP) 30 0 2 77 Cefotaxime (CTX) 30 0 0 79 Cefotetan (CTT) 30 0 2 77 Cefoxitin (FOX) 30 5 6 58 Cefuroxime (CXM) 30 0 5 74 Ceftriaxone (CRO) 30 0 0 79 Cephalothin (KF) 30 2 7 70 Cephazolin (KZ) 30 0 68 Chloramphenicol (C) 30 0 78 Ciprofloxacin (CIP) 5 0 8 7 Clindamycin (CC) 2 68 0 Erythromycin (E) 5 8 6 65 Gentamicin (GN) 0 0 78 Imipenem (IPM) 0 0 0 79 Kanamycin (K) 30 0 78 Nalidixic acid (NA) 30 0 78 Nitrofurantoin (F) 00 0 22 57 Norfloxacin (NOR) 0 0 3 76 Oxacillin (OX) 79 0 0 Penicillin (P) 0 79 0 0 Pipemidic acid (PIP) 20 0 2 77 Rifampin (RD) 5 0 4 75 Streptomycin (S) 0 6 2 7 Sulfamethoxazole /Trimethoprim (SXT) 25 0 0 79 Tetracycline (TE) 30 0 78 Ticarcillin (TIC) 75 77 2 0 Trimethoprim (W) 5 0 2 77 Vancomycin (VA) 30 79 0 0 (0.%) AMP-CC-OX-P-S-TIC-VA 6 (7.6%) 4 (Table 3). 4.,
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 946.5 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 > 2 4 8 6 32 64 28 256 52,024 2,048 77 (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%)
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