微生物学通报 Microbiology China tongbao@im.ac.cn 专论与综述 Oct. 20, 2014, 41(10): 2112 2121 2014 by Institute of Microbiology, CAS DOI: 10.13344/j.microbiol.china.140027 副溶血性弧菌 Vibrio parahaemolyticus O3:K6 大流行克隆的溯源 徐苗苗 1,2 刘静雯 (1. 361021) (2. 361021) 1,2* 摘要 副溶血性弧菌 (Vibrio Parahaemolyticus) 是一种革兰氏阴性嗜盐性海洋细菌 1950 年从日本一次暴发性食物中毒中首次分离发现 作为一种食源性人鱼共患致病菌, 副溶血性弧菌在全球的河口 海洋和沿海广泛传播, 由其引起的食物中毒已跃居其它病原菌之首 副溶血性弧菌在进化过程中通过基因重组和基因水平转移逐渐改善其对环境的适应性, 因而与其它所有致病微生物相比, 副溶血性弧菌的基因型和血清型都具有高度的多样性 本文就副溶血性弧菌, 特别是 1996 年后在世界范围内出现的 O3:K6 新血清型流行株 ( 形成所谓的 O3:K6 大流行克隆 Pandemic clone) 的发现及流行特征 变异分子流行病学特征 在我国的分布及研究进展进行综述, 以期为 O3:K6 大流行克隆的溯源提供更多依据 关键词 副溶血性弧菌,O3:K6 流行株, 发现及分布, 变异分子流行病学特征, 溯源 Traceability of Vibrio parahaemolyticus pandemic O3:K6 serovar clone XU Miao-Miao 1,2 LIU Jing-Wen 1,2* (1. College of Food and Biological Engineering, Jimei University, Xiamen, Fujian 361021, China) (2. Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, Fujian 361021, China) Abstract: Vibrio parahaemolyticus, a gram-negative halophilic bacterium, first identified as a cause of food-borne illness in Japan in 1950, is widely disseminated in estuarine, marine, and coastal environments throughout the world. As one of major pathogens, V. parahaemolyticus can cause bacterial food poisoning and threaten the public health. V. parahaemolyticus isolates are multiple diverse genotype and serotypes faster adapting to the versatile environment. Molecular genetic analysis showed that the genetic diversity may be acquired as a result of gene recombination or horizontal gene transfer. This paper reviewed the finding, distribution, epidemiological characteristics and mutation molecular epidemiological characteristics of V. parahaemolyticus, particularly the global dissemination of pandemic serovar O3:K6 clone, so as to provide evidences 基金项目 : (No. 201305027) (No. M20130910) * 通讯作者 :Tel 86-592-6180470 ljwsbch@163.com 收稿日期 :2014-01-08 接受日期 :2014-05-20 优先数字出版日期 (www.cnki.net) 2014-05-23
: Vibrio parahaemolyticus O3:K6 2113 for the traceability of the isolates. Keywords: Vibrio parahaemolyticus, Pandemic O3:K6 clone, Finding and distribution, Mutation molecular epidemiological characteristics, Traceability (Vibrio parahaemolyticus VP) (TDH) (TRH) tdh trh TDH TRH [1-2] tdh / trh 20 90 tdh O3:K6 O3:K6 1 副溶血性弧菌的发现历程及传播 1.1 副溶血性弧菌的发现及危害 1950 272 20 1951 Pasteurella parahaemolyticus [3] 1963 ( ) Vibrio parahaemolyticus ( 腸 ) 1966 [4] 1960 1970 [5] 1957 [6] 1958 儁 [7] [8-9] 1961 1962 83.8% [10] 68.0% [11] 1.2 我国首株分离株的形态及生理生化特性 1959 1961 7 253 102 22
2114 微生物学通报 Microbiol. China 2014, Vol.41, No.10 0.3 0.5 m ph 7.4 7.6 32 37 C [12] 1.3 传播途径 1950 37 C / / 2007 [13] [14] O3:K6 O3:K6 [15] O3:K6 O3:K6 O3:K6 2 O3:K6 的变异分子流行病学特征 2.1 血清型及其多样性 (O) (K) O K 11 O 71 K [16] O K 1982 1983 1990 1 530 1 429 93.4% [17] O3:K6 O3:K6 1995 1996 2 50% 80% [18] O3:K6 1982 1993 O3:K6 1995 1996 O3:K6 O3:K6
: Vibrio parahaemolyticus O3:K6 2115 [19] O3:K6 toxrs ( ) [20] O3:K6 O3:K6 O K [21] O3:K6 O3:K6 [22] O3:K6 1995 10 [23] 1997 1998 O3:K6 AP-PCR [24] 1983 1988 4 tdh ( ) O3:K6 4 O3:K6 ( >75%) AP-PCR toxrs O3:K6 toxrs Okura O3:K6 tdh O3:K6 O3:K6 [25] O3:K6 ( 1) ph [26] 206 42 O3:K6 O4:K8 O4:K68 O1:K25 O3:K29 O3:K25 O1:K56 14 206 155 51 25 [27] 2.2 分子分型 (Ribotyping) (Pulsed field gel electrophoresis PFGE) (Multilocus sequence typing MLST) (Random amplified polymorphism DNA RAPD) (Arbitrarily primed polymerase chain reaction AP-PCR) [27] PFGE 205 O3:K6 VP 1996 1 [19] MLST / 201 201 63 STs O3:K6 O3:KUT O4:K68 O3:K25 O1:K6 O1:KUT 6 111 ST3 ST331 332 333 433 434 435 444 447 448 452 472 478 ST3 STs [28] O3:K6 7 Vpals 120 MLST O3:K6 (Ancestor strain)
2116 微生物学通报 Microbiol. China 2014, Vol.41, No.10 O3:K6 ST3 O3:K6 ST3 O3:K6 [15] O3:K6 表 1 O3:K6 及衍生血清型在不同国家和地区出现的年代表 Table 1 Chronology of appearance of Vibrio parahaemolyticus O3:K6 and its serovariants in different countries and regions Serotype O1:K25 O1:K33 India (2002) O1:K36 China (2009) Countries, regions and year of isolation India (1998); Thailand (1999); Vietnam (1998 1999); Bangladesh (1999 2000) O1: K41 Thailand (1998 1999); Vietnam (1998 1999) O1:K56 Vietnam (1998 1999) O1:KUT India (1998); Bangladesh (1998, 2000) O2:K3 India (2002) O3:K5 India (2004) O3:K6 O3:K25 China (2010) O3:K68 China (2010) India (1996); Peru (1996); Vietnam (1997); Laos (1997); Indonesia (1997); United States (1997 1998); Korea (1997 1998); Chile (1998, 2004); Taiwan, China (1996 1999); Bangladesh (1998 2000); Japan (1998); Thailand (1999); Ecuador (1999); Angola (1999); Russia (2001); Brazil (2001); France (2004); Mozambique (2004); England (2001, 2012); Mexico (2004); Spain (2004) O3:K75 O3:KUT Vietnam (1998 1999) India (2003 2004) O4:K4 India (2004) O4:K8 Vietnam (1998 1999) O4:K10 India (2004) O4:K12 Thailand (1998 1999); Vietnam (1998 1999); Chile (2004) O4:K48 China (2010) O4:K68 India (1998); Thailand (1999); Bangladesh (1998, 2000); Vietnam (1998); Mozambique (2004) O4:KUT Vietnam (1998 1999); Spain (1999) O5:K17 India (2002) O5:K25 India (2002) O5:KUT Vietnam (1998 1999); India (2004) O6:K18 Taiwan, China (2005) OUT:KUT India (2003 2004)
: Vibrio parahaemolyticus O3:K6 2117 2.3 分子进化 2003 RIMD2210633 (1996 O3:K6 tdh + ) [29] O3:K6 toxrs/new (orf8) orf8 HU-a MTase VP2905 toxrs/new tdh O3:K6 [30] toxrs toxr toxs toxrs 1996 toxrs/old toxrs/new O3:K6 toxrs/old 1995 O3:K6 toxrs/new 1996 [24] Hu-a Hu-a ORF 3 16 kb DNA ORF ORF [31] ORF VP2905 VPal-5 433 bp MTase 233 VPal-1 VP0394 [20] O3:K6 180 93% orf8 96% HU-a MTase 99% VP2905 89% orf8 HU-a Mtase VP2905 [32] 6 4 4 2.4 致病机制 (TDH TRH TLH) (Outer memebrane protein OMP) Ⅲ (Type Ⅲ secretion systems T3SSs) [33-36] (TDH) 100 C 10 min [37-38] 90% TDH TDH TDH C (PKC) Ca 2+ Ca 2+ Cl Cl [38] (TRH) TDH 67% [39] TRH TDH Ca 2+ Cl [40] (TLH) [41]
2118 微生物学通报 Microbiol. China 2014, Vol.41, No.10 Ⅲ T3SS1 T3SS2 T3SSs [42-43] T3SS1 T3SS2 T3SS1 T3SS2 1996 O3:K6 1996 O3:K6 T3SS2 T3SS2 [36] O3:K6 O3:K6 3 O3:K6 大流行克隆在中国的分布 O3:K6 2009 2012 622 27 O3:K6 61.7% [44] 2007 2012 168 168 14 O3:K6 (114 67.9%) O4:K8 (25 14.9%) O3:K29 (6 3.6%) O3:K6 [45] (Enterobacterial repetitive intergenic consensus ERIC) 187 O3:K6 86% (161/187) [46] 2010 63 O3:K6 (26/57) O1:KUT (4/6) [47] 2010 4 12 112 O3:K6 72 63.2% [48] 31 6 4 检测方法 O3:K6 PCR PCR PCR PCR PCR PCR DNA [49] PCR PCR (Cross-priming amplification CPA) PCR [50]
: Vibrio parahaemolyticus O3:K6 2119 5 8 DNA RNA CPA- 60 min Mg 2+ dntps bst DNA ( 1) 10 5.6 10 2 CFU/mL ( 2 ) 图 1 交叉引物恒温扩增结合核酸试纸条检测副溶血性弧菌的特异性 Figure 1 Specificity of CPA-nucleic acid strip assays for detection of Vibrio patahaemolyticus 1 2 3 4 5 6 7 8. Note: 1: Negative control; 2: Vibrio patahaemolyticus; 3: Vibrio cholerae; 4: Vibrio alginnolyficus; 5: Vibrio vulnificus; 6: Staphylococcus aureus; 7: Escherichia coli; 8: Salmonella. 图 2 CPA- 核酸试纸条检测副溶血性弧菌纯培养物的灵敏度 Figure 2 Sensitivity of CPA-nucleic acid strip assays for detection of Vibrio patahaemolyticus in pure cultures M 100 bp DNA 0 1 5.6 10 8 CFU/mL 2 5.6 10 7 CFU/mL 3 5.6 10 6 CFU/mL 4 5.6 10 5 CFU/mL 5 5.6 10 4 CFU/mL 6 5.6 10 3 CFU/mL 7 5.6 10 2 CFU/mL 8 5.6 10 1 CFU/mL. Note: M: 100 bp ladder marker; 0: Negative control; 1: 5.6 10 8 CFU/mL; 2: 5.6 10 7 CFU/mL; 3: 5.6 10 6 CFU/mL; 4: 5.6 10 5 CFU/mL; 5: 5.6 10 4 CFU/mL; 6: 5.6 10 3 CFU/mL; 7: 5.6 10 2 CFU/mL; 8: 5.6 10 1 CFU/mL. 5 结论与展望 O3:K6 tdh O3:K6 1996 O3:K6 O:K O3:K6 O3:K6 ST3 3 (tdh trh tlh) ToxR gyrb flae ( )
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