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1 8 6 Vol. 8 No Journal of Food Safety and Quality Jun., 白 瑶 1, 叶淑瑶 2, 闫韶飞 1, 韩海红 1, 余东敏 1, 李志刚 1, 杜春明 1, 李凤琴 1* (1., ; 2., ) 摘要 : 目的 2015 方法, 1046 tlh tdh trh,, / 结果 (92.3%), 92.2%, 10, 3 (85.4%) (59.4%) / (49.3%) 1046 tlh, 19 (1.8%)trh, 4 (0.4%)tdh, 结论 2015, ; 关键词 : ; ; ; Virulence and antimicrobial characteristics of foodborne Vibrio parahaemolyticus strains in China in 2015 BAI Yao 1, YE Shu-Yao 2, YAN Shao-Fei 1, HAN Hai-Hong 1, YU Dong-Min 1, LI Zhi-Gang 1, DU Chun-Ming 1, LI Feng-Qin 1* (1. Key Lab of Food Safety Risk Assessment of National Health and Family Planning Commission, China National Centre for Food Safety Risk Assessment, Beijing , China; 2. College of Food Science and Engineering, Northwest Agriculture and Forestry University, Yangling , China) ABSTRACT: Objective To evaluate virulence distribution and antimicrobial resistance of Vibrio parahaemolyticus isolated from food in China in Methods A polymerase chain reaction method was used to detect virulence genes of tlh, tdh and trh. The susceptibility of 1046 isolates of Vibrio parahaemolyticus to 12 antibiotics were determined by a broth microdilution susceptibility test. The antibiotics of ampicillin, amoxicillin-clavulanic acid, cefazolin, cefotaxime, ceftazidime, imipenem, gentamicin, tetracycline, ciprofloxacin, levofloxacin, chloramphenico 基金项目 : (2016YFD ) Fund: Supported by the National Key Research and Development Program of China, the Ministry of Science and Technology of the People's Republic of China (2016YFD ) * 通讯作者 :,,, lifengqin@cfsa.net.cn *Corresponding author: LI Feng-Qin, Professor, China National Center for Food Safety Risk Assessment, 7# Panjiayuan Nanli, Chaoyang District, Beijing , China. lifengqin@cfsa.net.cn

2 6, : and trimethoprim/sulfamethoxazole were chosen. Results Totally 965 strains (92.2%) presented resistance to single or multiple antibiotics. All strains were sensitive to gentamicin and levofloxacin, and presented resistance to other antibiotics. The highest resistance rates were cefazolin (85.4%), ampicillin (59.4%) and amoxicillin-clavulanic acid (49.3%). All the 1046 isolates were positive to tlh gene, including 19 isolates were positive to trh gene and 4 isolates were positive to tdh gene. All the positive strains were not resistance to multiple antibiotics, and some of them were resistance to penicillins and the first-generation cephalosporin. Conclusion There is a lower positive rate of virulence genes in Vibrio parahaemolyticus isolated from food in China in 2015, though with a higher resistance rate to penicillins and the first-generation cephalosporin antibiotics. There is an urgent need to strengthen the management and standardization of antibiotics in a breeding process in the farming areas. KEY WORDS: Vibrio parahaemolyticus; virulence; antimicrobial; food 1 引言 (Vibrio parahaemolyticus, VP),,,,, [1] [2], [3] Ⅲ [4],, (thermolabile hemolysin, TLH) (thermostable direct hemolysin, TDH) TDH (TDH related hemolysin, TRH), 3 tlh tdh trh [5,6] TLH,, min [4] TDH, min TDH, (Kanagawa phenomenon, KP), KP TRH, TLH, TRH KP, 2,,,,,,,, [7,8], , (polymerase chain reaction, PCR) 3, 2 材料与方法 2.1 材料与仪器 实验菌株 (44 ) (16 ) (13 ) (57 ) (15 ) (232 ) (81 ) (60 ) (66 ) (8 ) (97 ) (6 ) (10 ) (16 ) (16 ) (5 ) (13 ) (8 ) (60 ) (7 ) (7 ) (85 ) (99 ) (24 ) (1 ) GB (Esherichia coli)atcc ATCC PCR ATCC17802 CICC21528 CICC 仪器与试剂 Thermo 1389 ( Thermo ); FORMA 3131 ( Thermo ); SWB25 ( Thermo ); Legend Micro 21R ( Thermo ); C W ( Bio Rad ); PowerPAC ( Bio Rad ); Versodoc MP 4000 ( Bio Rad ); MLS-3780 ( Sanyo ); Vitek 2 Compact 60 ( Bio Mérieux ) (CM1135B, Oxoid ); (CM1136, Oxoid ); Mueller-Hinton (CM0405, Oxoid ); DP DNA ( ); PCR Master Mix(A6001,

3 Promega ) (, ) 2.2 试验方法 抗生素敏感性测定 (Clinical and Laboratory Standards Institute, CLSI) [9] ( ),, / 12 (minimum inhibitory concentrations, MIC), CLSI (1) 3%NaCl BHA, 37,, 3%NaCl BHA,, 3mL,, 0.5 (McFarland), 10 8 CFU/mL (2) 60 μl 12 ml, 96, ( ), 100 μl, 37 18~24 h MIC 1 [9] 毒力基因检测 (1) DNA 3%NaCl BHA, 37, DNA, DNA, -20 (2) PCR PCR, 2 PCR (25 μl): 2 Taq PCR Master Mix 12.5 μl(0.1u/μl Taq polymerase, 500 mmol/l dntp, 20 mmol/l Tris-HCl, 100 mmol/l KCl, 3 mmol/l MgCl 2 ), 1 μl, DNA 1 μl, dd H 2 O 9.5 μl PCR : 94 3 min; s s 72 1 min, 30 ; 72 7 min 5 μl PCR 1.5%, 90 V, 50 min, 表 1 副溶血性弧菌药物敏感性测试用抗生素及判定标准 Table 1 Antibiotic andcriteria in antibiotic susceptibility test of Vibrio parahaemolyticus (μg/ml) (μg/ml) (μg/ml) AMP / AMC 32/16 16/8 8/4 CFZ CTX CAZ IMP GEN TET CIP LEV SXT 4/76 / 2/38 CHL : (R):antibiotic resistance; (I):intermediate resistance; (S):susceptible Jorgensen [9] 表 2 PCR 反应引物 Table 2 Primer pairs used in PCR (bp) tlh tdh trh F:5'-AAAGCGGATTATGCAGAAGCACTG-3' R:5'-GCTACTTTCTAGCATTTTCTCTGC-3' F:5'-GTAAAGGTCTCTGACTTTTGGAC-3' R:5'-TGGAATAGAACCTTCATCTTCACC-3' F:5'-TTGGCTTCGATATTTTCAGTATCT-3' R:5'-CATAACAAACATATGCCCATTTCCG : PCR Nishibuchi [10]

4 6, : 结果与分析 3.1 抗生素敏感性实验 1046, 965 1, 92.2%, 10, (85.4%) (59.4%) / (49.3%) 2.0% 1.9% 1.3% 0.9% 0.8%( 1) ( 3 3 ) 43.5%(455/1046), 4.6%(48/1046) 5 5 : - / - (372/1046, 35.6%) - (102/1046, 9.8%) - / (55/1046, 5.3%), 93.3%, 91.1% %, 43.5%, 98% 95% 80%,, 50% ( 2) Fig Antibiotic resistant rates of 1046Vibrio parahaemolyticusstrains to12 kinds of antibiotics from foods Fig. 2 Resistant spectrumof1046vibrio parahaemolyticusstrainsto12 kinds of antibiotics from foods

5 %(965/1046), 25,, [11-16], β- [17,18], β-,,,,, 98%, 50%,, 3.2 毒力基因检测 PCR,, 1046 tlh, 22 (2.10%) tlh tdh trh, 19 tlh trh, 4 tlh tdh, 3,,, 4,,, tdh trh 2 1, [19,20] 1046, 0.4% tdh, 1.8% trh, 2 2.1%, [20-23], : tdh trh 97.5% [24,25], tdh trh 100% [26], tdh 93.0% [27], tdh trh,, tdh trh, tdh trh [28] 4 结论与讨论, 25, 1046 ( ) ( ),,, 2015, (81.8%, 18/22),,,,,,,,, Table 3 表 3 22 株食源性副溶血性弧菌毒力基因阳性菌株耐药谱 Resistant spectrumof 22Vibrio parahaemolyticusstrains with virulence gene from foods * ( ) 18 tlh(+) tdh(-) trh(+) 1.70% 3 tlh(+) tdh(+) trh(-) 0.30% (4) AMP-CFZ(2) AMP-AMC(2) AMP-AMC-CFZ(4) CFZ(6) CFZ(2) AMC(1),, 1 tlh(+) tdh(+) trh(+) 0.10% CFZ(1) : +, - * 4,

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7 susceptibility and molecular characteristics of clinical Vibrio parahaemolyticus strains circulating in southeastern China from 2009 to 2013 [J]. Clin Microbiol Infect, 2016, 22(3): [28] Shirai H, Ito H, Hirayama T, et al. Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis [J]. Infect Immun, 1990, 58(11): ( 责任编辑 : 姜姗 ) 作者简介 白瑶, 博士, 助理研究员, 主要研究方向为食品微生物 baiyao@cfsa.net.cn 李凤琴, 博士, 研究员, 博士生导师, 主要研究方向为食品微生物 lifengqin@cfsa.net.cn