Cloning and Sequencing of the ompA Gene of Enterobacter sakazakii and Development of an ompA -Targeted PCR for Rapid Detection of Enterobacter sakazakii in Infant Formula

Abstract

ABSTRACT Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii . The outer membrane protein A gene ( ompA ), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae . Based on regions of the ompA gene unique to E. sakazakii , two primers were synthesized to develop and optimize an E. sakazakii -specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10 3 CFU/ml of E. sakazakii bacteria in infant formula directly and 10 −1 CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.