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 619-75, 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 556-23, 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 211 461 14.3% 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). http://dx.doi.org/1.5657/kfas.214.5 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 (http://creativecommons.org/licenses/by-nc/3./)which 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: +2. 51. 72. 2632 Fax: +2. 51. 72. 2619 E-mail address: yhspknu@korea.kr Copyright 214 The Korean Society of Fisheries and Aquatic Science 5 pissn:374-111, eissn:227-15
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 211 7 11 V. parahaemolyticus.. 패류시료 재료및방법 V. parahaemolyticus ( ) ( ) (Crassostrea gigas) (Venerupis philippinarum) 211 7 11. 1. 4. 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 ) 3 35.5 1-24. Thiosulfate Citrate Bile Salt Agar (TCBS agar, Difco), 35 1-24. 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, 1-24 1 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, 72 6 35 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. 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 25922. 결과및고찰 V. parahaemolyticus 검출현황 211 7 11 4 4 V. parahaemolyticus Table 1. 16.4-26.9, 23.6-33.1 psu (Fig. 1).. 6 V. parahaemolyticus 5%, 7. (Annie and Marta, 212), 7 9 V. parahaemolyticus. 4 29 72.5%. 7, 11 37.5% (3/). 4 39 97.5% Salinity ( ) Temp ( ) 4 3 2 1 4 3 2 1 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 7 9 1 11 No. of samples analyzed 16 16 16 16 16 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. (%) (%) (%) (%) (%) (%)
패류유래장염비브리오균의항생제내성특성 511. 7 1 11 7.5% (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) 1 1 1 1 1 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, 4 11 1-1, CFU/1 g 12 3 <1 CFU/1 g (Angelo et al., 23). V. parahaemolyticus <3-46, MPN/1 g, 7 11 4 11 1 <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 7. 57% (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 129 26 (2.2%) 15, 13 (79.%). 1 penicillin ampicillin 79% trimethoprim, streptomysin 3.1% 1.6%. 1% (Table 2). V. parahaemolyticus 37 111 (36.2%), 196 (63.%). V. parahaemolyticus ampicillin
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 1 13 12(79.1) Amoxicillin/ clavulanic acid 3 13 (.) Cephalosporins Cefepime 3 14 (.) Cefotaxime 3 14 1(.) Aminoglycosides Streptomycin 1 11 2(1.6) Gentamycin 1 12 1(.) Amikacin 3 14 (.) Fluoroquinolones Ciprofloxacin 5 15 (.) Quinolones Nalidixic acid 3 13 1(.) Sulfonamides Trimethopenem/ sulfamethoxazole 1.25/23.75 1 1(.) Trimethoprim 5 1 4(3.1) Phenicols Chloramphenicol 5 12 (.) Tetracyclines Tetracycline 5 14 (.) Ansamycins Rifampin 5 17 1(.) 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 1 13 1(61) Amoxicillin/ clavulanic acid 3 13 (.) Cephalosporins Cefepime 3 14 (.) Cefotaxime 3 14 (.) Aminoglycosides Streptomycin 1 11 9(2.9) Gentamycin 1 12 3(1) Amikacin 3 14 (.) Fluoroquinolones Ciprofloxacin 5 15 (.) Quinolones Nalidixic acid 3 13 3(1) Sulfonamides Trimethopenem/ sulfamethoxazole 1.25/23.75 1 (.) Trimethoprim 5 1 16(5.2) Phenicols Chloramphenicol 5 12 1(.3) Tetracyclines Tetracycline 5 14 (.) Ansamycins Rifampin 5 17 7(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
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 177 152 (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(%) 26 26 (2.2) 1 AM 94 95 (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(%) 111 111 (36.2) 1 AM 164 172 (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.
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