广西市售婴儿食品中克罗诺菌分离株基因特征及进化分类鉴定

目的分析广西市售婴儿食品中的克罗诺菌分离株的种类、表型和基因特性。方法将保存的试验菌株进行复苏,通过API 20E生化条和omp A基因扩增检测进行初步鉴定,扩增16S r RNA基因后进行测序,将获得的全16S r RNA基因序列在Gene Bank数据库上比对,构建进化树,确认是否为克罗诺菌。将试验菌接种于显色平板观察其表型和黄色素的产生情况,通过手工生化进行分型,确定其种属。结果 9株分离菌确定为克罗诺菌,有5种生化型,属于4个克罗诺种。检出Cronobacter sakazakii 6株菌,C.malonaticus、C.muytjensii和C.dublinensis各有1株;7株菌符合API 20E鉴定结果,9株分离菌均可检测到omp A基因。结论广西市售婴儿食品中克罗诺菌的污染以C.sakazakii为主,生化表型、种属及基因型具有多样性;鉴定时仅以单一的生化或其他表型作为判别依据存在很大的局限,需辅以分子生物学手段加以鉴别。

Identification,classification,phenotypic and genetic characterization of Cronobacter spp. isolated from infant food

To investigate the subspecies, phenotypic and genetic characterization of Cronobacter isdates from infant food in 2010.Methods All isolates were reactivated, then suspectable stains were identified preliminarily by two methods, PCR amplifying ompA gene and API 20E. To confirm these stains, full 16S rRNA gene sequences were obtained, and made BLASTN on GeneBank database. The identified strains were streaked onto DFI chromogenic agar and TSA to observe phenotype and yellow pigment production. Biochemical tests were conducted and assigned to biogroups to determine their species.Results Nine strains were identified as Cronobacter spp. These strains were assigned to 5 biogroups, and belonged to 4 species. The majority of strains were Cronobacter sakazakii, and three strains was identified as C.malonaticus, C.muytjensii and C.dublinensis respectively. 7 strains were identified as Cronobacter by API 20E, yet ompA gene amplification of all strains was positive.Conclusion Cronobacter isolated from infant food were diversed in biochemical phenotype and species. There was a risk of Cronobacter in infant food from supermarkets. There was limitation to identify the bacteria only by biochemical phenotype, and supplementary molecular biology methods was necessary.