Abstract
Cronobacter sakazakii is an opportunistic pathogen that causes infant meningitis and is often associated with milk-based infant formula. We have fully sequenced the genome of a newly isolated lytic C. sakazakii myovirus, vB_CsaM_GAP161, briefly named GAP161. It consists of 178,193 bp and has a G+C content of 44.5%. A total of 277 genes, including 275 open reading frames and two tRNA-encoding genes, were identified. This phage is closely related to coliphages RB16 and RB43 and Klebsiella pneumoniae phage KP15.
GENOME ANNOUNCEMENT
Contaminated milk-based powdered infant formulae have been the source of Cronobacter infections that cause sepsis, brain abscess, and meningitis in neonates and infants (2, 5), with mortality up to 80% (7). Because of their high specificity and effectiveness, bacteriophages have been used as alternative agents to control pathogens (4, 6) and may be particularly relevant for the control of Cronobacter because of its intrinsic antibiotic resistance (7). However, complete knowledge of a potential therapeutic phage is required to ensure its safety before clinical application. Currently, there are only seven reported fully sequenced Cronobacter phages, including three members of the Myoviridae (CR3, ESSI-2, and ES2) (11, 13, 15), three members of the Siphoviridae (phiES15, ESP2949-1, and ENT39118) (9, 10, 12), and ENT47670 (unclassified; GenBank accession number HQ201308).
Lytic bacteriophages against Cronobacter sakazakii were isolated from sewage samples (Guelph, ON, Canada) using the method described by Van Twest and Kropinski (17). Phage vB_CsaM_GAP161, briefly named GAP161, lysed 12 of 14 C. sakazakii strains tested.
Based on host range and its strong lytic activity, phage GAP161 was chosen for further study. Electron microscopy of negatively stained (2% uranyl acetate) viral preparations was carried out at the University of Guelph, with sizes verified at Laval University. GAP161 belongs to the Myoviridae family (1), with an elongated cylindrical head that is 110 by 74 nm and a tail that is 113 by 17 nm, and has the characteristic morphology of a T4-like phage.
The DNA was extracted and purified by the Midi Lambda DNA purification kit (Qiagen, Mississauga, ON, Canada), and the genomic sequence was determined using 454 technology (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). Transfer RNAs were predicted using tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/). For each protein, the number of amino acids, molecular weight, and isoelectric point were calculated 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). Transmembrane helices in proteins and transmembrane topology and signal peptides were predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) and Phobius (http://phobius.sbc.su.se), respectively.
Phage GAP161 has a double-stranded DNA genome of 178,193 bp with a G+C content of 44.5%. This genome encodes 277 genes, including 275 open reading frames (ORFs) and two tRNA genes. Bioinformatic analysis using CoreGenes (18) showed that the genome sequence of GAP161 shares 94.07% (254/270), 87.21% (225/258), and 85.27% (249/292) homology with coliphages RB16 and RB43 (14) and phage KP15 of Klebsiella pneumoniae (3), respectively, indicating that GAP161 is a member of the myoviral subfamily Teequatrovirinae in a genus of T4-like viruses (8).
The complete proteome of phage GAP161 was screened against a database of 83 bacterial toxin proteins (including those from Bacillus spp., Bordetella, Clostridium spp., Enterobacteriaceae, Listeria, Pseudomonas, Staphylococcus, Streptococcus, and Vibrio) using the BLASTP feature of BioEdit (16). No hits (E value < 0.003) were recorded, suggesting that phage GAP161 may be applied as a biocontrol agent against Cronobacter sakazakii.
Nucleotide sequence accession number.
The complete genome sequence of phage vB_CsaM_GAP161 is available in GenBank under accession number JN882287.
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. Drulis-Kawa Z, et al. 2011. Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae. Appl. Microbiol. Biotechnol. 90:1333–1345 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Goodridge LD, Bisha B. 2011. Phage-based biocontrol strategies to reduce foodborne pathogens in foods. Bacteriophage 1:130–137 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. 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]
- 6. 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]
- 7. 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]
- 8. Lavigne R, et al. 2009. Classification of Myoviridae bacteriophages using protein sequence similarity. BMC Microbiol. 9:224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. 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]
- 10. 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]
- 11. 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]
- 12. 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]
- 13. 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]
- 14. Petrov VM, et al. 2006. Plasticity of the gene functions for DNA replication in the T4-like phages. J. Mol. Biol. 361:46–68 [DOI] [PubMed] [Google Scholar]
- 15. 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]
- 16. Tippmann HF. 2004. Analysis for free: comparing programs for sequence analysis. Brief Bioinform. 5:82–87 [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 Isolation, characterization, and interactions. 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] [PMC free article] [PubMed] [Google Scholar]