Comparative proteomic analysis by iTRAQ-2DLC-MS/MS provides insight into the key proteins involved in Cronobacter sp. biofilm formation

Cronobacter sp., an opportunistic foodborne pathogen, can cause high mortality in neonatal and premature infants because this pathogen is associated with powdered infant formula (PIF) contamination. Biofilm formation is recognized as the most effective means to help Cronobacter survive throughout the long shelf-life of PIF, thus promoting resistance to cleaning agents and disinfectants. Crystal violet (CV) staining and fluorescence microscopy analyses were used to compare the biofilm formation ability among different Cronobacter strains, and then the weak biofilm former Cronobacter sakazakii ATCC 29544 and the strong biofilm former Cronobacter dubliniensis DSM 18707 were selected for further proteomic analysis. Two-dimensional liquid chromatography–tandem mass spectrometry, which was coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling, was employed to quantitatively identify the proteins that were differentially expressed in C. sakazakii ATCC 29544 compared to C. dubliniensis DSM 18707. In total, 1190 proteins were detected. Gene Ontology (GO) analysis indicated that these differentially expressed proteins are related to biological binding, cell structure, signal transduction, cell adhesion, and cellular interaction. Among these differential proteins, the expression levels of 177 non-redundant proteins were altered significantly by more than 5-fold, with 96 up-regulated proteins and 81 down-regulated. This study provides important information regarding the cellular requirements for the underlying mechanism of Cronobacter biofilm formation and its survival in harsh environments.