PCR-RFLP analysis of the rpoB gene to distinguish the five species of Cronobacter

Members of the genus Cronobacter are opportunistic pathogens associated with life-threatening infections in immuno-compromised individuals. Polyphasic analysis has facilitated the classification of the novel genus Cronobacter containing five species. However, since this recent reclassification there are not many identification methods optimised for differentiation between the five Cronobacter species. This differentiation between the species is of importance as there are indications that the species may be diverse regarding their virulence. The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol to differentiate between the five Cronobacter species. The rpoB gene of 49 Enterobacteriaceae strains, including 33 Cronobacter strains was amplified using conventional PCR, followed by digestion of these PCR products with restriction endonucleases MboI, HinP1I and Csp6I. The PCR-RFLP analysis with single digestions of each of the restriction endonucleases did not distinguish between all five Cronobacter species. This study describes the successful differentiation of the five Cronobacter species based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. This PCR-RFLP assay is an accurate identification method that ensures rapid differentiation between the five species of Cronobacter. (C) 2011 Elsevier Ltd. All rights reserved.