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.

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.

Cronobacter sakazakii is a pathogen that predominantly infects immunocompromised individuals, especially infants, where it causes meningitis. The genome of lytic C. sakazakii myovirus vB_CsaM_GAP31 has been fully sequenced. It consists of 147,940 bp and has a G؉C content of 46.3%. A total of 295 genes, including 269 open reading frames and 26 tRNA genes, were identified. This phage is related to Salmonella phage PVP-SE1 and coliphages vB_EcoM-FV3 and rV5.

c Vibrio parahaemolyticus is recognized as one of the main causes of human gastroenteritis associated with seafood. We have fully sequenced the genome of a newly isolated phage, vB_VpaS_MAR10, which lysed 61.9% of the V. parahaemolyticus strains tested. Phage MAR10 is a temperate siphovirus, and its genome consists of double-stranded DNA (dsDNA) with a size of 78,751 bp, a G؉C content of 49.70%, and 104 open reading frames. Bioinformatic analysis shows that phage MAR10 is closely related to Vibrio phage SSP002. Vibrio parahaemolyticus has been distinguished as the most important cause of human gastroenteritis associated with seafood since its initial identification in 1950 (4, 6). Due to the ubiquitous nature of the bacterium, it is difficult to prevent contamination of seafood (9). Therefore, there is a critical need for more accurate, sensitive, and fast detection methods. Current detection methods of V. parahaemolyticus, such as the most probable number (MPN) method (FDA standard method) (2, 9), have some disadvantages (e.g., that they are labor-intensive, time-consuming, and lacking in specificity among Vibrio strains) (7). Another approach to increase the sensitivity of the detection method of food-borne pathogens is the use of bacteriophages, which are highly host-specific viruses (3). The objectives of the current study were to isolate a novel V. parahaemolyticus-specific phage and determine its sequence in order to develop a detection system. Using the method previously described by Van Twest and Kropinski (8), phages of V. parahaemolyticus were isolated from seawater samples (San Felipe, Baja California, Mexico). Phage vB_VpaS_MAR10 (MAR10) is a temperate phage with high specificity to its host and is able to infect 13 out of 21 (61.9%) V. parahaemolyticus strains tested. The phage preparation was negatively stained (2% uranyl acetate) and examined by electron microscopy at the University of Guelph. Electron micrographs show that phage MAR10 has a noncontractile tail of 160 nm by 10 nm and an elongated head of 94 nm by 50 nm, revealing that this phage belongs to the family Siphoviridae (1).The Midi Lambda DNA purification kit (Qiagen, Mississauga, Ontario, Canada) was used to extract and purify the DNA of phage MAR10, and the genomic sequence was determined using 454 technology (McGill University and Génome Québec Innovation Centre, Montreal, Quebec, Canada). MyRAST was applied to annotate the genome, and gene calls were verified using Kodon (Applied Maths, Austin, TX). For each protein the number of amino acids, molecular weight, and isoelectric point were determined by means of Batch MW and pI Finder (http://greengene.uml.edu/programs/FindMW.html). Homologs were identified using BatchBLAST (http://greengene.uml .edu/programs/NCBI_Blast.html). Protein motifs were predicted using Phobius (http://phobius.sbc.su.se), Pfam (http://pfam.sanger.ac .uk/), and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/).Analysis of the sequence reveals that the genome of phage MAR10 is 78,751 bp double-stranded DNA with ...