2012-2015年贵州省炭疽芽胞杆菌分离鉴定及MLVA分型分析

Identification and MLVA genotyping of Bacillus anthracis strains isolated in Guizhou Province from 2012 to 2015

目的了解菌株的分子流行病学特征,为贵州省炭疽疫情的预防控制提供科学依据。方法对2012-2015年贵州省间不同地区的409份标本(外环境388份、患者19份和牲畜2份)进行炭疽芽胞杆菌分离培养,采用革兰染色镜检、青霉素抑制试验和噬菌体裂解试验对可疑炭疽菌落进行鉴定,分析菌株检出情况。运用MLVA-15技术对炭疽芽胞杆菌分离株进行基因分型获得各VNTR位点的扩增长度并计算重复单元的重复数目。结合各VNTR位点重复数目,利用NTsys 2.10e软件对不同地区菌株进行聚类分析。结果从409份标本中分离出34株炭疽芽胞杆菌,检出率为8.31%。其中341份土壤检出33株,检出率为9.68%;17份患者皮肤病灶渗出液检出1株,检出率为5.88%。2015年分离出炭疽杆菌的阳性率最高,占48.72%(19/39)。MLVA-15分析显示,34株菌株被分为3个MLVA型;聚类分析显示,34株菌株被分为A和B两簇,其中A簇又被进一步分为A1和A2分支。来自相同地区或年份的多数菌株聚类关系相对较近。结论 2012-2015年贵州省间各种疑似炭疽送检标本中,土壤的检出阳性率最高,贵州省炭疽芽胞杆菌菌株具有MLVA型别多样性,型别分布和聚类关系具有明显的地域性。

In order to provide scientific basis for the control and prevention of anthrax by the epidemiological characteristic of Bacillus anthracis. The 409 samples including 388 from outside environment, 19 from patients and 2 from animals in different regions of Guizhou Province from 2012 to 2015 were used for B. anthracis isolatation and identification by using the traditional Gram staining, penicillin inhibition experiments and bacteriophage lysis test. The B. anthracis genome DNA was amplified using PCR based on 15 variable number of tandem repeats （VNTR）, respectively. The amplification length of each VNTR locus was obtained by capillary electrophoresis. The copy numbers of tandem repeat sequence were subsequently analyzed with the software of NTsys 2.10e. The results showed that 34 B. anthracis strains were isolated from 409 specimens with positive rate of 8.31%. The positive rates of soil and skin lesion exudate samples were 9.68% （33/341） and 5.88%（1/17）, respectively. Among the four years, the year 2015 was with highest positive rate of B. anthracis, with positive rate of 48.72% （19/39）. MLVA-15 genotyping showed that the 34 isolates were typed into 3 MLVA-15 types. Clustering analysis showed that the 34 isolates divided into A and B clusters. Clade A was further divided into A1 and A2 clade. Most isolates come from the same region or year was closely clustered. Results of this study suggested that the soil sample was of the highest positive rate with B. anthracis among all the samples from Guizhou Province during 2012-2015 and B. anthracis isolates in Guizhou Province were of genetic diversity and were clustered geographically.