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Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a G؉C content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ⌽HAP-1. Vibrio parahaemolyticus is a marine bacterium that has been a major cause of food-borne illness worldwide and is mainly associated with the consumption of contaminated seafood (8). Due to the ubiquitous nature of the bacterium, it is almost impossible to prevent contamination of seafood. Therefore, there is a critical need for more accurate, reliable, sensitive, specific, and fast detection methods. Current methods for the detection of V. parahaemolyticus, such as the most probably number method described by the U.S. FDA (2) are labor-intensive and time-consuming and lack specificity for Vibrio strains (8), and regular molecular methods do not distinguish between live or dead cells, which make them less useful methods for V. parahaemolyticus. An approach to increase the sensitivity of assays for this organism and to decrease the detection time is through the use of bacteriophages, which are viruses that display high host specificity (3). The objectives of this project were to isolate a V. parahaemolyticusspecific phage and determine its sequence in order to develop a detection system. Bacteriophages were isolated from fresh nontreated seawater samples (San Felipe, Baja California, Mexico), applying the method described by Van Twest and Kropinski (9). Phage vB_ VpaM_MAR is a temperate phage that has high specificity to its host, producing infection in 16 out of 21 V. parahaemolyticus strains. Out of a collection of 11 other Vibrio strains, it had weak lytic activity against one strain of Vibrio alginolyticus and one strain of Photobacterium leiognathi. Phage MAR was examined by electron microscopy of negatively stained preparations (2% uranyl acetate) at the University of Guelph. Electron micrographs revealed that MAR belongs to the family Myoviridae (1) and has a contractile tail of 234 by 20 nm and a head of 74 by 69 nm, similar to Vibrio vulnificus phage P147 (7).The DNA of MAR phage was extracted and purified by using the Midi Lambda DNA purification kit (Qiagen), and the genomic sequence was determined using 454 technology at McGill University and the Genome Quebec Innovation Centre (Montreal, QC, Canada). The genome was annotated using MyRAST, with gene calls verified using Kodon (Applied Maths, Austin, TX). For each protein, the number of amino acids, molecular weight, and isoelectric point was calculated using programs at

The genome of Cronobacter sakazakii podovirus vB_CsaP_GAP227 was fully sequenced. The DNA of this lytic phage consists of 41,796 bp and has a G+C content of 55.7%. Forty-nine open reading frames and no tRNAs were identified. This phage is related to Yersinia phages ϕR8-01 and ϕ80-18 and Aeromonas phage phiAS7.