Differentiation of Cronobacter sakazakii and related taxa using direct sequencing, species-specific PCR, and mini-sequencing assays

Abstract

Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Clearly identifying the specific species of the C. sakazakii group using phenotypic and genotypic techniques is hard. The aim of this study was to use the tuf gene for species discrimination in the C. sakazakii and its phylogenetically closest species, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on 16S rRNA and tuf gene sequence for species identification. The average sequence similarity for the tuf gene among type strains was 96.9 %, and most members of the C. sakazakii group could be distinguished. The species-specific primer was designed according to the 16S rRNA gene sequence, which was employed for PCR with the template DNA of Cronobacter strains. Single 137-bp species-specific band was found only in C. sakazakii and C. malonaticus. A mini-sequencing assay using tuf as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 24 strains of Cronobacter species and was able to unambiguously discriminate strains belonging to the species C. sakazakii. The sequence of the tuf gene is more polymorphic than that of the 16S rRNA gene and can be used to differentiate the C. sakazakii group strains. In addition, a novel method to identify the species of the C. sakazakii and C. malonaticus was also developed by species-specific PCR combined with mini-sequencing.