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Cronobacter spp. (formerly Enterobacter sakazakii) is responsible for rare but fatal cases of infection in neonates and immunocompromised infants. The aim of our study was to characterize Cronobacter strains isolated from powdered infant foods in Public Health Authority of the Slovak Republic in 2009–2010. Powdered infant food products have been analysed using currently available standard method ISO/TS 22964: 2006 for the detection of Cronobacter spp. complemented with qPCR confirmation of positive strains. Thirteen Cronobacter strains were isolated from more than 900 powdered infant formulae, milk-based and cereal-based powdered weaning food products. The strains were assigned to five biogroups and ten multilocus sequence typing (MLST) sequence types. In total, twelve strains were identified as Cronobacter sakazakii and one strain as Cronobacter dublinensis. Multiple strains originated from parallel isolation were obtained in three samples and the variability between strains from the same food was observed twice. The results are in agreement with the hypothesis that the Cronobacter contamination detected in infant powdered food is low and originating in various accidental sources.This study characterized Cronobacter strains isolated from powdered infant formulae and weaning foods by biotyping and multilocus sequence typing. The later method was shown to be more discriminative and suitable for both species identification and subtyping. Low level (0·9%) of Cronobacter positivity was observed in 916 samples. Multiple sequence types were observed among strains isolated from the same food product. This highlights that multiple isolates from each single sample should be analysed in epidemiological studies, since more than one genetic subtype may be present.