Prevalence of curli genes among Cronobacter species and their roles in biofilm formation and cell-cell aggregation

Cronobacter species are food-borne opportunistic pathogens that cause sepsis, meningitis, and necrotizing enterocolitis in neonates. Bacterial pathogens such as pathogenic Escherichia coli and Salmonella species express extracellular curli fimbriae that are involved in rugosity, biofilm formation, and host cell adherence. csgBAC operon encodes the major curli structural subunit CsgA and the nucleator protein CsgB. csgDEFG operon encodes the regulatory protein CsgD and putative assembly factors. In this study, the curli operons were analyzed in the sequences of 13 Cronobacter strains and other enteric bacterial pathogens. Isogenic mutants of csgA and csgB were generated in C. turicensis LMG23827 (z3032). csgA and csgB mutants did not express curli fimbriae as indicated by a lack of Congo red binding and absence of curli by electron microscopic evaluation. Compared to the wild type strain, biofilm formation and cell-cell aggregation of csgA and csgB mutants were remarkably decreased. The prevalence of curli operons were investigated in 231 Cronobacter strains isolated from different sources using polymerase chain reaction (PCR) assay. The results of the PCR analysis showed that csgA and csgG were present in 30% clinical isolates, 8% food, and 11% environmental isolates. These genes were present in C. dublinensis, C. malonaticus, C. turicensis, and C. universalis, but not in C. muytjensii and C. sakazakii. Our data indicate that csgBAC and csgDEFG operons were present about three fold higher in clinical isolates than in isolates from other sources. The csgA and csgB genes were shown to be involved in the early stages of biofilm development and cell-cell aggregation in Cronobacter.