Abstract
Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.
Highlights
Cronobacter spp., formerly named as Enterobacter sakazakii, is an emerging food-borne pathogen causing neonatal meningitis, sepsis and necrotizing enterocolitis and with 40–80% fatality rate to neonates, children and even in adults [1,2,3,4]
We developed a real-time PCR assay coupled with high resolution melting (HRM) analysis, which was a more rapid, technically simpler detection and typing method for Cronobacter spp. in powdered infant formula, for the first time, targeting the out membrane protein A (OmpA) gene
Agarose gel electrophoresis indicated that positive amplification products correlated to a size of 70 bp as expected, and nucleotide sequence analyses of the amplicons confirmed 100% identical to C. sakazakii, while a difference of two bases was detected between C. sakazakii and C. muytjensii (BLASTn option, GenBank)
Summary
Cronobacter spp., formerly named as Enterobacter sakazakii, is an emerging food-borne pathogen causing neonatal meningitis, sepsis and necrotizing enterocolitis and with 40–80% fatality rate to neonates, children and even in adults [1,2,3,4]. It has been ranked by The International Commission for Microbiological Specification for Foods as a bacterium having ‘‘severe hazard for restricted populations, life threatening or substantial chronic sequence long duration’’ [5]. Sensitive, simple and accurate techniques for early diagnosis of PIF contaminating pathogens, including Cronobacter spp., are urgently required [10,11]
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