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

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.

FIG. 1.

Optimal alignment of the deduced amino acid sequence of E. sakazakii 51329 OmpA with those from other bacteria. E. saka, E. sakazakii 51329; E. aero, E. aerogenes; E. coli, E. coli K-12; S. flex, S. flexneri 2a strain 2457^T; S. Typhi, S. enterica serovar Typhimurium LT2.

FIG. 2.

Detection of E. sakazakii by PCR amplification of the ompA gene. Primer pair ESSF and ESSR and the amplification conditions and gel electrophoresis method described in Materials and Methods were employed. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 17, PCR products from E. sakazakii strains 1 to 17, as listed in Table 1; 18 to 68, PCR products from negative control DNA from bacterial strains 18 to 68, as listed in Table 1; C, control PCR run without any template DNA.

FIG. 3.

Sensitivity of the PCR for detection of E. sakazakii in pure cultures. Primer pair ESSF and ESSR and the template preparation, amplification conditions, and gel electrophoresis method described in Materials and Methods were employed. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 9, PCR products amplified from samples containing 10⁸ to 10⁰ CFU/ml of E. sakazakii (ATCC 51329); 10, control PCR run without any template DNA.

FIG. 4.

Direct detection of E. sakazakii in reconstituted infant formula by PCR. Primer pair ESSF and ESSR and the template preparation, amplification conditions, and gel electrophoresis method described in Materials and Methods were employed. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 9, PCR products amplified from infant formula containing 10⁸ to 10⁰ CFU/ml of E. sakazakii (ATCC 51329); 10, control PCR run with uninoculated, reconstituted infant formula.

FIG. 5.

Detection of E. sakazakii in reconstituted infant formula by PCR after an enrichment step of 8 h. Primer pair ESSF and ESSR and the template preparation, amplification conditions, and gel electrophoresis method described in Materials and Methods were employed. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 6, PCR products amplified from infant formula containing 10³ to 10⁻² CFU/ml of E. sakazakii (ATCC 51329) after 8 h of enrichment; 7, control PCR run with uninoculated, reconstituted infant formula after 8 h of enrichment.

FIG. 6.

Detection of E. sakazakii in reconstituted infant formula by PCR in the presence of S. enterica serovar Typhimurium. Primer pair ESSF and ESSR and the template preparation, amplification conditions, and gel electrophoresis method described in Materials and Methods were employed. (A) PCR amplifications carried out on infant formula samples containing 10⁸ CFU/ml serovar Typhimurium along with 10⁸ to 10¹ CFU/ml of E. sakazakii. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 8, PCR products amplified from infant formula containing 10⁸ to 10¹ CFU/ml of E. sakazakii (corresponding samples on each of the lanes also contained 10⁸ CFU/ml serovar Typhimurium); 9, control PCR run with infant formula containing 10⁸ CFU/ml serovar Typhimurium alone. (B) PCR amplifications carried out on infant formula samples containing 10³ CFU/ml E. sakazakii along with 10⁸ to 10¹ CFU/ml of serovar Typhimurium. Lanes: M, molecular weight marker (GeneRuler DNA Ladder Mix; Fermentas, Hanover, Md.); 1 to 8, PCR products amplified from infant formula containing 10⁸ to 10¹ CFU/ml of serovar Typhimurium (corresponding samples on each of the lanes also contained 10³ CFU/ml E. sakazakii); 9, control PCR run with infant formula containing 10⁸ CFU/ml serovar Typhimurium alone.