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Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Identification and characterization of Cronobacter strains isolated from powdered infant foods 1, M. Oriesˇkova 2, L. Oslanecova 2, H. Drahovska 2 and E. Kaclıkova 3 A. Gicova 1 Public Health Authority of the Slovak Republic, Bratislava, Slovakia 2 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia 3 Department of Microbiology, Molecular Biology and Biotechnology, Food Research Institute, Bratislava, Slovakia

Significance and Impact of the study: 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 (09%) 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.

Keywords biotyping, Cronobacter spp., multi-locus sequence typing, powdered infant food. Correspondence Eva Kaclıkov a, Department of Microbiology, Molecular Biology and Biotechnology, Food Research Institute, P.O.Box 25, SK-82475 Bratislava, Slovakia. E-mail: [email protected] 2013/1447: received 18 July 2013, revised 9 October 2013 and accepted 15 October 2013 doi:10.1111/lam.12179

Abstract 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.

Introduction Cronobacter spp. are opportunistic pathogenic bacteria that can cause serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis with low frequency, but high lethality rate (Mullane et al. 2007; Hunter and Bean 2013). Infections in adults also have been reported, in particular among the elderly and immunocompromised patients (FAO/WHO 2008). Cronobacter spp. was originally defined as Enterobacter sakazakii in 1980 (Farmer et al. 1980). Results of several 242

genotyping analyses led to reclassification of E. sakazakii to the new Cronobacter genus with five species of Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter turicensis (Iversen et al. 2008) and with two new species of Cronobacter condimenti and Cronobacter universalis added subsequently (Joseph et al. 2012). Recently, multilocus sequence analysis has been used for taxonomical re-evaluation of Enterobacter genus and three species were transferred from Enterobacter into Cronobacter as Cronobacter zurichensis, Cronobacter helveticus and Cronobacter pulveris (Brady et al. 2013).

Letters in Applied Microbiology 58, 242--247 © 2013 The Society for Applied Microbiology

A. Gicov a et al.

Although Cronobacter is ubiquitous and has been isolated from various foods and environments (Jaradat et al. 2009; Turcovsky et al. 2011), the main vehicle for its transmission in neonatal infections is rehydrated powdered infant formula (PIF) (van Acker et al. 2001; Iversen and Forsythe 2004; Yan et al. 2012). Cronobacter has been found at levels

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