Biocontrol of Cronobacter spp. using Bacteriophage in Infant Formula

The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31 (GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models.