Abstract
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.
GENOME ANNOUNCEMENT
Cronobacter sakazakii contaminations in milk-based powdered infant formulae have been the source of infections that cause sepsis, brain abscess, and meningitis in neonates and infants (2, 4), with mortality up to 80% (6). Bacteriophages (phages), due to their high specificity and effectiveness, have been used as alternative agents to control pathogens (3, 5) and could be particularly interesting for the control of Cronobacter because of its intrinsic antibiotic resistance (6). Comprehensive knowledge of a potential therapeutic phage, however, is required to ensure its safety before clinical application. To date, only seven fully sequenced Cronobacter phages have been reported, including three myoviruses (ESSI-2, ES2, and CR3) (10, 12, 14), three siphoviruses (ESP2949-1, phiES15, and ENT39118) (8, 9, 11), and ENT47670 (unclassified; GenBank accession number HQ201308).
Lytic C. sakazakii phages were isolated from sewage samples (Guelph, ON, Canada) by applying the method described by Van Twest and Kropinski (17). Phage vB_CsaM_GAP31 (GAP31) lysed 11 of 14 C. sakazakii strains tested. Phage GAP31 was chosen for further study because of its host range and strong lytic activity. Electron microscopy of negatively stained (2% uranyl acetate) viral preparations was carried out at the University of Guelph, and particle sizes were verified at Laval University. GAP31 is a myovirus (1) with an icosahedral head 83 nm in diameter and a tail of 107 by 19 nm.
Phage DNA was extracted and purified using the Midi Lambda DNA purification kit (Qiagen, Mississauga, ON, Canada). The genomic sequence was determined using 454 technology (McGill University and Génome Québec Innovation Centre, Montreal, QC, Canada). MyRAST was used to annotate the genome, and gene calls were verified in Kodon (Applied Maths, Austin, TX). The number of amino acids, molecular weight, and isoelectric point of each protein were determined using 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 Pfam (http://pfam.sanger.ac.uk/search#tabview=tab1), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and Phobius (http://phobius.sbc.su.se).
The double-stranded DNA genome of phage GAP31 has 147,940 bp with a G+C content of 46.3%. This genome contains 295 genes, including 269 open reading frames (ORFs) and 26 tRNA genes. Comparative proteomic analysis using CoreGenes (18) shows that the genome sequence of GAP31 encodes 202/244 (82.79%), 96/218 (44.04%), and 98/233 (42.06%) proteins that are homologs of those encoded by Salmonella phage PVP-SE1 (13) and coliphages vB_EcoM-FV3 (16) and rV5 (DQ832317), respectively. In addition, this virus is peripherally related to Cronobacter phage CR3, with 35.47% homology (94/265 proteins) (14). Phage GAP31 belongs to the proposed genus “V5likevirus” within the Myoviridae family (7).
The proteome of phage GAP31 was screened against a database of 83 bacterial toxin proteins (including those from Bacillus, Bordetella, Clostridium, Enterobacteriaceae, Listeria, Pseudomonas, Staphylococcus, Streptococcus, and Vibrio spp.) using the BLASTP feature of BioEdit (15). No hits (E value <0.003) were recorded, suggesting that phage GAP31 could be used as a biocontrol agent against Cronobacter sakazakii.
Nucleotide sequence accession number.
The complete genome sequence of phage vB_CsaM_GAP31 is available in GenBank under accession number JN882284.
ACKNOWLEDGMENT
This study was financially supported by the National Sciences and Engineering Research Council of Canada (NSERC).
REFERENCES
- 1. Ackermann H-W. 2005. Bacteriophage classification, p 67–89 In Kutter E, Sulakvelidze A. (ed), Bacteriophages: biology and applications. CRC Press, Boca Raton, FL [Google Scholar]
- 2. Centers for Disease Control and Prevention 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. MMWR Morb. Mortal. Wkly. Rep. 51:297–300 [PubMed] [Google Scholar]
- 3. Goodridge LD, Bisha B. 2011. Phage-based biocontrol strategies to reduce foodborne pathogens in foods. Bacteriophage 1:130–137 doi:10.4161/bact.1.3.17629 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Gurtler JB, Kornacki JL, Beuchat LR. 2005. Enterobacter sakazakii: a coliform of increased concern to infant health. Int. J. Food Microbiol. 104:1–34 [DOI] [PubMed] [Google Scholar]
- 5. Kropinski AM. 2006. Phage therapy—everything old is new again. Can. J. Infect. Dis. Med. Microbiol. 17:297–306 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Lai KK. 2001. Enterobacter sakazakii infections among neonates, infants, children, and adults—case reports and a review of the literature. Medicine 80:113–122 [DOI] [PubMed] [Google Scholar]
- 7. Lavigne R, et al. 2009. Classification of Myoviridae bacteriophages using protein sequence similarity. BMC Microbiol. 9:224 doi:10.1186/1471-2180-9-224 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Lee Y-D, Kim J-Y, Park J-H, Chang H. 2012. Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii. Arch. Virol. 157:199–202 [DOI] [PubMed] [Google Scholar]
- 9. Lee JH, Choi Y, Shin H, Lee J, Ryu S. 2012. Complete genome sequence of Cronobacter sakazakii temperate bacteriophage phiES15. J. Virol. 86:7713–7714 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Lee Y-D, Chang HI, Park J-H. 2011. Complete genomic sequence of virulent Cronobacter sakazakii phage ESSI-2 isolated from swine feces. Arch. Virol. 156:721–724 [DOI] [PubMed] [Google Scholar]
- 11. Lee Y-D, Park J-H. 2012. Complete genome of temperate phage ENT39118 from Cronobacter sakazakii. J. Virol. 86:5400–5401 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Lee Y-D, Park J-H, Chang HI. 2011. Genomic sequence analysis of virulent Cronobacter sakazakii bacteriophage ES2. Arch. Virol. 156:2105–2108 [DOI] [PubMed] [Google Scholar]
- 13. Santos SB, et al. 2011. Genomic and proteomic characterization of the broad-host-range Salmonella phage PVP-SE1: creation of a new phage genus. J. Virol. 85:11265–11273 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Shin H, Lee JH, Kim Y, Ryu S. 2012. Complete genome sequence of Cronobacter sakazakii bacteriophage CR3. J. Virol. 86:6367–6368 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Tippmann HF. 2004. Analysis for free: comparing programs for sequence analysis. Brief. Bioinform. 5:82–87 [DOI] [PubMed] [Google Scholar]
- 16. Truncaite L, et al. Bacteriophage vB_EcoM_FV3: a new member of “rV5-like viruses.”. Arch. Virol. 2012 Aug 21; doi: 10.1007/s00705-012-1449-x. doi: 10.1007/s00705-012-1449-x. [DOI] [PubMed] [Google Scholar]
- 17. Van Twest R, Kropinski AM. 2009. Bacteriophage enrichment from water and soil, p. 15–21 In Clokie MR, Kropinski AM. (ed), Bacteriophages: methods and protocols, vol 1 Humana Press, New York, NY: [DOI] [PubMed] [Google Scholar]
- 18. Zafar N, Mazumder R, Seto D. 2002. CoreGenes: a computational tool for identifying and cataloging “core” genes in a set of small genomes. BMC Bioinformatics 3:12 doi:10.1186/1471-2105-3-12 [DOI] [PMC free article] [PubMed] [Google Scholar]