婴幼儿配方羊乳粉生产环节蜡样芽孢杆菌分离株多位点序列分型分析

Multiple Locus Sequence Typing Analysis of Bacillus cereus Isolates from the Production Line of Goat Milk-Based Infant Formula

为探讨我国婴幼儿配方羊乳粉生产环节阳性分离株的遗传多样性,并将其基因序列与已报道的乳源分离株的基因序列进行比对,确立其特征序列,为婴幼儿配方食品生产体系溯源提供依据,为探究其致病机理和有效防控提供参考依据。实验选取glpF、gmk、ilvD、pta、pur、pycA、tpi 7个管家基因构建多位点序列分析分型方案,鉴定经随机扩增多态性DNA分型得到的42株Bacillus cereus的序列类型。结果表明:42株B.cereus分为7个序列类型(sequence type,ST),分别为ST-770(4.8%,2/42)、ST-1000(71.4%,30/42)、ST-1084(9.5%,4/42)、ST-1348(2.4%,1/42)、ST-1349(2.4%,1/42)、ST-1350(2.4%,1/42)和ST-1351(7.1%,3/42);所有分离株被识别为205、142、23三个不同克隆谱系,2个单态群(ST-770和ST-1351)和2个独株(ST-1348和ST-1349);发现4个新的ST型和4个新的等位基因,已上传至国际数据库得到新的序列号和等位基因号,分别为ST-1348、ST-1349、ST-1350、ST-1351和glp-253、glp-254、ilv-277、pyc-199。

In the present study, we analyzed the genetic diversity of Bacillus cereus isolated from the production line of goat milk-based infant formula, and we compared their gene sequences with published sequences of isolates from milk products, aiming to detect the specific sequence types of the isolates in this study, which will provide useful information for further research on pathogenesis, prevalence, traceability, prevention and control. A total of 350 B. cereus isolates were obtained in this study, which had been subtyped to 12 clusters（42 isolates） by random amplification of polymorphic DNA（RAPD）. The multiple locus sequence typing（MLST） profiles for 42 isolates were conducted based on the house-keeping genes glpF, gmk, ilvD, pta, pur, pycA, and tpi. The clonal complexes for these isolates were analyzed by the online BURST（n-4）. The results showed that 42 isolates were detected and classified into 7 sequence types： ST-770（4.8%, 2/42）, ST-1000（71.4%, 30/42）, ST-1084（9.5%, 4/42）, ST-1348（2.4%, 1/42）, ST-1349（2.4%, 1/42）, ST-1350（2.4%, 1/42） and ST-1351（7.1%, 3/42）. Clonal complexes 205, 142, 23, and two groups（ST-770 and ST-1351） were detected as well as two singletons（3 L33 with ST-1349 and 3 L47 with ST-1350）. Four novel STs（ST-1348, ST-1349, ST-1350, and ST-1351） and four novel alleles（glp-253, glp-254, ilv-277 and pyc-199） were found.