8 6 Vol. 8 No. 6 2017 6 Journal of Food Safety and Quality Jun., 2017 2015 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., 100021; 2., 712100) 摘要 : 目的 2015 方法, 1046 tlh tdh trh,, / 结果 1046 965 (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 100021, China; 2. College of Food Science and Engineering, Northwest Agriculture and Forestry University, Yangling 712100, China) ABSTRACT: Objective To evaluate virulence distribution and antimicrobial resistance of Vibrio parahaemolyticus isolated from food in China in 2015. 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 : (2016YFD0401102) 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 (2016YFD0401102) * 通讯作者 :,,, E-mail: lifengqin@cfsa.net.cn *Corresponding author: LI Feng-Qin, Professor, China National Center for Food Safety Risk Assessment, 7# Panjiayuan Nanli, Chaoyang District, Beijing 100021, China. E-mail: lifengqin@cfsa.net.cn
6, : 2015 2319 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,, 60 10 min [4] TDH, 100 10 min TDH, (Kanagawa phenomenon, KP), KP TRH, TLH, TRH KP, 2,,,,,,,, [7,8], 2015 25 1046, (polymerase chain reaction, PCR) 3, 2 材料与方法 2.1 材料与仪器 2.1.1 实验菌株 2015 25 1046 (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 4789.7-2013 (Esherichia coli)atcc 25922 ATCC 35218 PCR ATCC17802 CICC21528 CICC21618 2.1.2 仪器与试剂 Thermo 1389 ( Thermo ); FORMA 3131 ( Thermo ); SWB25 ( Thermo ); Legend Micro 21R ( Thermo ); C1000 96W ( 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 ); DP302-02 DNA ( ); PCR Master Mix(A6001,
2320 8 Promega ) (, ) 2.2 试验方法 2.2.1 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] 2.2.2 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; 94 30 s 58 30 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 32 16 8 / AMC 32/16 16/8 8/4 CFZ 4 2 1 CTX 4 2 1 CAZ 16 8 4 IMP 16 8 4 GEN 16 8 4 TET 16 8 4 CIP 4 2 1 LEV 8 4 2 SXT 4/76 / 2/38 CHL 32 16 8 : (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-3 450 269 500 : PCR Nishibuchi [10]
6, : 2015 2321 3 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% 3 45.0%, 43.5%, 98% 95% 80%,, 50% ( 2) Fig. 1 1 1046 12 Antibiotic resistant rates of 1046Vibrio parahaemolyticusstrains to12 kinds of antibiotics from foods 2 1046 12 Fig. 2 Resistant spectrumof1046vibrio parahaemolyticusstrainsto12 kinds of antibiotics from foods
2322 8 92.2%(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,
6, : 2015 2323 参考文献 [1],,. [J]., 2005, 26(4): 75 83. Yang SL, Wang YG, Dong SG. Progress of research on vibriosis in marine cultured fish [J]. Prog Fish Sci, 2005, 26(4): 75 83. [2] Newton A1, Kendall M, Vugia DJ, et al. Increasing rates of vibriosis in the United States, 1996-2010: Review of surveillance data from 2 systems [J]. Clin Infect Dis, 2012, (54): 391 395. [3] Su YC, Liu C. Vibrio parahaemolyticus: a concern of seafood safety [J]. Food Microbiol, 2007, 24(6): 549 558. [4] Theethakaew C, Feil EJ, Castilloramírez S, et al. Genetic relationships of Vibrio parahaemolyticus isolates from clinical, human carrier, and environmental sources in Thailand, determined by multilocus sequence analysis [J]. Appl Environ Microbiol, 2013, 79(7): 2358 2370. [5] Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of thermostable direct hemolysin and related hemolysins [J]. Rev Med Microbiol, 1993, 4(2): 106 113. [6] Ceccarelli D, Hasan NA, Huq A, et al. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors [J]. Front Cell Infect Microbiol, 2013, 3(8): 97. [7] Okoh AI, Igbinosa EO. Antibiotic susceptibility profiles of some Vibrio strains isolated from wastewater final effluents in a rural community of the Eastern Cape Province of South Africa [J]. BMC Microbiol, 2010, 10(1): 143. [8] Jun JW, Kim JH, Jr CC, et al. Isolation, molecular characterization, and antibiotic susceptibility of Vibrio parahaemolyticus in Korean seafood [J]. Foodborne Pathog Dis, 2012, 9(3): 224 231. [9] Jorgensen JH. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria [D]. Wayne: Clinical and Laboratory Standards Institute, 2015. [10] Bej AK, Patterson DP, Brasher CW, et al. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh and trh [J]. J Microbiol Method, 1999, 36(3): 215 225. [11] He Y, Jin L, Sun F, et al. Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China [J]. Environ Sci Pollut Res, 2016, 23(15): 1 8. [12] Alothrubi SM. Antibiotic Resistance of Vibrio parahaemolyticusisolated from cockles and shrimp sea food marketed in Selangor, Malaysia [J]. Clin Microbiol, 2014, 3(3): 148. [13] Melo LM, Almeida D, Hofer E, et al. Antibiotic resistance of Vibrio parahaemolyticus isolated from pond-reared Litopenaeus vannamei marketed in natal, Brazil[J]. Br J Microbiol, 2011, 42(4): 1463 1469. [14] Kang CH, Shin Y, Jang S, et al. Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes [J]. Mar Pollut Bull, 2017, 118(1/2): 261 266. [15] Kang CH, Shin Y, Kim W, et al. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from oysters in Korea [J]. Environ Sci Pollut Res, 2016, 23(1): 1 9. [16] Xu X, Cheng J, Wu Q, et al. Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China [J]. BMC Microbiol, 2016, 16(1): 32. [17],,,. [J]., 2013, 29(5): 516 519. Jiang YH, Yao L, Li FL, et al. Isolation and antimicrobial resistance of Vibro parehaemolyticus in farmed marine shrimps from Qingdao markets [J]. Chin J Zoono, 2013, 29(5): 516 519. [18],, 禤,. 3 [J]., 2016, 37(6): 29 34. Chen SF, Chen J, Xuan JC, et al. Isolation, identification and drug resistance analysis of three vibrio species in aquaculture area of Beihai [J]. Prog Vet Med 2016, 37(6): 29 34 [19] Nishibuchi M, Kaper JB. Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium [J]. Infect Immun, 1995, 63(6): 2093 2099. [20] Gutierrez WCK, Klein SL, Lovell CR. High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary [J]. Appl Environ Microbiol, 2013, 79(7): 2247 2252. [21],,. [J]., 2010, 22(3): 239 243. Li WW, Wang XY, Guo YC. Distribution of virulence genes in Vibrio parahaemolyticusisolated from seafood in China [J]. Chin J Food Hyg, 2010, 22(3): 239 243. [22] Vuddhakul V, Soboon S, Sunghiran W, et al. Distribution of virulent and pandemic strains of Vibrio parahaemolyticus in three molluscan shellfish species (Meretrix meretrix, Perna viridis, and Anadara granosa) and their association with foodborne disease in southern Thailand [J]. J Food Prot, 2006, 69(11): 2615. [23] Bhoopong P, Palittapongarnpim PR, Kiatkittipong A, et al. Variability of properties of Vibrio parahaemolyticus strains isolated from individual patients [J]. J Clin Microbiol, 2007, 45(5): 1544 1550. [24],,,. 2011-2014 [J]., 2015, 42(24): 4468 4470. Yan HQ, Zhang X, Huang Y, et al. Analysis on the characteristics of serotype and virulence gene of Vibrioparahaemolyticus in Beijing during from 2011 to 2014 [J]. Mod Prev Med, 2015, 42(24): 4468 4470. [25],,,. [J]., 2011, 32(12): 1255 1258. Qu M, Zhang X, Liu GR, et al. Analysis on the etiological and molecular epidemiological characteristics of Vibrio parahaemolyticusin Beijing [J]. Chin J Epidemiol, 2011, 32(12): 1255 1258. [26],,,. [J]., 2011, 23(2): 114 118. Chen HY, Chen M, Sheng YY, et al. Distribution of serotype, ribotype and virulence gene of Vibrio parahaemolyticus isolated from foodborne disease outbreaks [J]. Chin J Food Hyg, 2011, 23(2): 114 118. [27] Chen Y, Chen X, Yu F, et al. Serology, virulence, antimicrobial
2324 8 susceptibility and molecular characteristics of clinical Vibrio parahaemolyticus strains circulating in southeastern China from 2009 to 2013 [J]. Clin Microbiol Infect, 2016, 22(3): 258 259. [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): 3568 3573. ( 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 E-mail: 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 E-mail: lifengqin@cfsa.net.cn