Multi-locus Genetic Characterization of Cronobacter sakazakii and Related Species

Introduction: Cronobacter sakazakii is an opportunistic Gram negative human pathogenic bacterium known to cause fatal meningitis and necrotizing enterocolitis in infants and immunocompromised individuals.  It can withstand severe dry conditions, and has been linked to powdered infant formula. Nevertheless, it has also been isolated from a wide variety of food and environmental samples. In this study, we have modified the PCR conditions and assessed the robustness of seven published multi-locus PCR protocols, and used for Cronobacter species identification.

Purpose: The major objective of this study was rapid species identification of Cronobacter species isolated from food and other sources by multi-locus sequence characterization.

Methods: In this study, a total of 185 Cronobacter isolates from food and other sources were isolated in our laboratory, initially by performing two-step enrichment followed by streaking on a selective agar. The recovered Cronobacter isolates were subsequently sequenced at seven different loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps) using ABI 3500 XL Genetic Analyzer. The sequencing data was analyzed using BioEdit and GENEIOUS programs.

Results: Multi-locus sequence characterization was done at seven loci including atpD, fusA, glnS, gltB, gyrB, infB, and pps for the 185 isolates of Cronobacter recovered from food environment, surveillance, outbreak, sporadic cases and the ATCC reference cultures. The bi-directional DNA sequencing resulted high quality bases (> 98% HQ - 100% HQ) at all seven loci examined. Multiple alignments of the generated sequences revealed inter- as well as intra-specific genetic polymorphism at all seven loci for all isolates of three Cronobacter species (including C.  dublinensis, C. muytjensii, and C. sakazakii) sequenced.

Significance: The results suggest that multi-locus genetic characterization can be accomplished at seven different loci by modifying PCR conditions for the species-identification of Cronobacter isolated from food, outbreak, sporadic cases, environmental monitoring program, routine surveillance, and consumer complain of public health importance.