Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

Abstract

SummaryEnterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 108 pfu ml−1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (106 and 102 cfu ml−1). In contrast, broth inoculated with 104 phage and 102 bacteria per ml first showed normal bacterial growth until reaching a cell titre of 105 cfu ml−1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml−1. Phages could be produced with titres of 1010 pfu ml−1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.