Abstract
BackgroundEnterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Rehydrated powdered infant formulae have been implicated as the source of infection in several outbreaks and sporadic cases. In this work, a real time fluorescence resonance energy transfer PCR assay incorporating an internal amplification control (IAC) was developed for the specific detection of E. sakazakii in foods. Performance of the assay, coupled to an automated DNA extraction system and the E. sakazakii ISO-IDF (TS 22964/RM 210) enrichment procedure, was evaluated on infant formulae and samples from production environment.ResultsThe real-time PCR assay had 100% specificity as assessed using 35 E. sakazakii and 184 non-E. sakazakii strains. According to the E. sakazakii strains tested, the detection limits ranged from 5 to 25 genomic copies. Assays on pure cultures (including real-time PCR and DNA extraction) gave a sensitivity of about 102 to 103 CFU/ml. Out of 41 naturally contaminated infant formulae and environmental samples analysed for the presence of E. sakazakii, 23 were positive by real-time PCR and 22 by the conventional culture method, giving 97.5% concordance with the ISO-IDF reference method.ConclusionThis method, combining specific real-time PCR, automated DNA extraction and ISO-IDF standard enrichments, provides a useful tool for rapid screening of E. sakazakii in food and environmental matrices.
Highlights
Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants
The present study evaluated a real-time PCR-based assay including an internal amplification control (IAC) and an automated nucleic acid extraction method that can be used in combination with the International Standard Organisation (ISO)-International Dairy Federation (IDF) enrichment steps for the routine examination of naturally contaminated powdered infant formulae (PIF)
Specificity of the real-time PCR assay The DNA region located between the tRNA-glu and 23S rRNA genes was selected as target for detecting the E. sakazakii species
Summary
Enterobacter sakazakii is the causative agent of rare but severe food-borne infections associated with meningitis, necrotizing enterocolitis and sepsis in infants. Enterobacter sakazakii, previously known as yellow-pigmented E. cloacae [1], has been identified as the causative agent of rare but often severe invasive infections in infants. Infants in whom meningitis developed were generally < 1 week of age at the onset of infection and had near-term gestational age and birthweight [3]. Compared with infants with meningitis, infants in whom bacteremia alone developed tended to be born very prematurely and have extremely low birthweight (
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