Sequencing of the grxB Gene of Cronobacter spp. and the Development of a PCR Assay for Its Identification

Abstract

Cronobacter spp. (formerly Enterobacter sakazakii), a foodborne pathogen linked to powdered infant formula, is a rare cause of invasive infection with a high mortality rate in neonates. In this study, the Cronobacter sakazakii ATCC 29544 and C. muytjensii ATCC 51329 glutaredoxin 2 (grxB) genes were cloned and sequenced. Based on the unique regions of the Cronobacter grxB genes, two primers were synthesized to develop and optimize a Cronobacter-specific polymerase chain reaction (PCR) method. The PCR assay amplified a 378-bp DNA product from all positive controls, which are composed of 45 strains of Cronobacter spp., but not from any of 45 non-Cronobacter bacterial strains. The detection limits of this method are 10⁴ colony-forming units (CFU)/mL of Cronobacter spp. in infant formula directly and 10⁰ CFU/mL after an 8-h enrichment step. In summary, we have developed a PCR assay based on the grxB sequence. Combined with enrichment culturing, this technique offers a rapid and sensitive method for the detection of Cronobacter spp.