2007-2009年中国副溶血性弧菌临床分离株分子特征分析

Analysis of molecular features of clinical Vibrio parahaemolyticus strains in China

目的 了解2007-2009年中国副溶血性弧菌临床分离株表型和基因型特征.方法 选取2007-2009年分离自浙江省、江苏省、四川省、广西壮族自治区和辽宁省,经生化鉴定为副溶血性弧菌的临床分离株作为研究对象,共135株.采用PCR方法对其进行毒力基因耐热直接溶血素（tdh）、耐热直接溶血素相关溶血素（trh）,种鉴定基因不耐热溶血素（tlh）、toxR、VPM、gyrB及“大流行菌群”标识基因（GS-PCR、PGS-PCR、orf8、HU-α）检测;采用微量肉汤稀释法分析菌株对8种抗菌素的耐药性;对实验菌株进行血清学检测;采用脉冲场凝胶电泳方法（PFGE）对菌株进行聚类分析.结果 135株临床分离株tlh、toxR、gyrB、VPM均为阳性,85.9％ （116/135）的临床菌株tdh和（或）trh阳性,tdh、trh的携带率分别为85.2％（115/135）、3.0％ （4/135）,其中3株菌同时携带两种毒力基因.GS-PCR、PGS-PCR、orf8、HU-α的携带率分别为66.7％ （90/135）、80.7％ （109/135）、65.2％ （88/135）、66.7％ （90/135）.135株临床株耐受至少1种抗菌素的占8.1％ （11/135）,其中,9株对氨苄青霉素的耐药,2株对复方磺胺甲嗯唑耐药,1株对四环素耐药.所有菌株对头孢噻肟、头孢他啶、庆大霉素、环丙沙星、氯霉素均敏感.135株临床株检出29种血清型,以O3、O4、O1群为主,占89.6％（121/135）,O3∶K6为优势血清型,占56.3％ （76/135）.我国“大流行菌群”包括O3∶K6、O4：K68、O1∶ K36、O1∶K25、O1∶K5、O3∶ K29等血清型.限制性内切酶SfiⅠ和NotⅠ的酶切图谱片段集中在30～700 kb之间,聚类分析各分为6个群和9个群,最低相似度分别为52.6％和58.7％,临床“大流行菌群”菌株分别位于C群和D群.结论 我国大部分副溶血性弧菌临床株携带毒力基因;“大流行菌群”尤其是O3∶K6血清型在我国呈流行趋势,GS-PCR、HU-a可作为判定“大流行菌群”标识基因.PFGE分型可靠且能区分“大流行菌群”和散发菌株.

Objective To explore the phenetic and genetic features of clinical Vibrio parahaemolyticus strains from 2007-2009 in China.Methods A total of 135 clinical Vibrio parahaemolyticus strains,isolated from Zhejiang,Jiangsu,Sichuan,Guangxi,Liaoning Provinces during 2007 to 2009,were selected for the research.The occurrence of virulence genes thermostable direct hemolysin （tdh） and TDH-related hemolysin （trh）,species-specific genes thermoliable hemolsin （tlh）,toxR,VPM and gyrB,the pandemic clone gene markers（GS-PCR,PGS-PCR,orf8 and HU-α） in 135 Vibrio parahaemolyticus strains was detected by PCR.The antimicrobial susceptibilities to eight antimicrobial agents of the experimental strains were determined by the broth microdilution method.All strains were serotyped and underwent the cluster analysis with pulsed-field gel electrophoreses.Results The results of PCR methods claim that all experiment strains carry species-specific genes such as tlh,toxR,gyrB,VPM.Among clinical strains,85.9％ （116/135） carry tdh and/or trh.85.2％ （115/135） were positive for tdh,and 3.0％ （4/ 135） were positive for trh; while 3 strains carried both.66.7％ （90/135）,80.7％ （109/135）,65.2％ （88/135）,66.7％ （90/135）clinical strains carried the genes of GS-PCR,PGS-PCR,orf8,HU-α,respectively.The results of antibiotics susceptibility test showed that 8.1％ （11/135） strains were resistant to at least one agent,including 9 strains were resistant to ampicillin,2 strains were resistant to trimethoprim and sulphame-thoxazole,and 1 strain were resistant to tetracycline.All clinical strains were sensitive to cefotaxme,ceftazidime,gentamicin,ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 29 serotypes were identified for clinical strains,predominantly O3,O4 and O1 groups,accounting for 89.6％ （121/135）.O3∶K6 was dominant serotype,accounting for 56.3％ （76/135）.The pandemic flora in China included O3∶ K6,04∶ K68,O1∶ K36,O1∶ K25,O1∶K5 and O3∶K29 serotypes.Genomic DNAs of 135 clinical strains were digested with Sfi Ⅰ and Not Ⅰ,the molecular size of PFGE restriction fragments used for analysis mainly ranged from 30-700 kb.When subjected to UPGMA clustering,6 and 9 clusters were grouped by Sfi Ⅰ and Not Ⅰ,and the minimal similarity was 52.6％ and 58.7％,and pandemic flora were located in C groups and D group,respectively.Conclusion Most of Vibrio parahaemolyticus strains isolated from clinical sources in China were pathogenic.The pandemic clone,especially O3∶K6 was prevalent.The GS-PCR and HU-α genes were reliable markers to identify the pandemic flora.The serotype by PFGE was reliable to distinguish the pandemic flora and the sporadic strains.