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Research article

Molecular and Cellular Microbiology (MCM)  |  Biochemistry and Physiology

A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii

Hyung Ju Lee 1, Wan Il Kim 1, Young Chan Kwon 1, Kyung Eun Cha 2, 3, Minjin Kim 3 and Heejoon Myung 2, 3*

1Hana High School, Seoul 03305, Republic of Korea, 2The Bacteriophage Bank of Korea, Yongin 17035, Republic of Korea, 3Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 17035, Republic of Korea

Received: May 9, 2016; Accepted: May 27, 2016

J. Microbiol. Biotechnol. 2016; 26(9): 1629-1635

Published September 28, 2016 https://doi.org/10.4014/jmb.1605.05020

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized. It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to Myoviridae as observed under a transmission electron microscope. The major virion protein appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging from 4oC to 55oC for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h. The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula. PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes. Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions, 10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA sequences of genes encoding the major capsid protein revealed a wide geographical distribution of related phages over Asia, Europe, and America.

Keywords

bacteriophage PBES 02, Cronobacter sakazakii, genomic analysis, phage therapy

References

  1. Abbasifar R, Griffiths MW, Sabour PM, Ackermann HW, Vandersteegen K, Lavigne R, et al. 2014. Supersize me:Cronobacter sakazakii phage GAP32. Virology 460-461: 138-146.
    Pubmed CrossRef
  2. Abbasifar R, Kropinski AM, Sabour PM, Chambers JR, MacKinnon J, Malig T, Griffiths MW. 2014. Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae. Arch. Virol. 159: 2 253 2261.
  3. Bragg R, van der Westhuizen W, Lee JY, Coetsee E, Boucher C. 2014. Bacteriophages as potential treatment option for antibiotic resistant bacteria. Adv. Exp. Med. Biol. 807: 97-110.
    Pubmed CrossRef
  4. Das M, Bhowmick TS, Ahern SJ, Young R, Gonzalez CF. 2015. Control of Pierce’s disease by phage. PLoS One 10: e0128902.
    Pubmed PMC CrossRef
  5. Gottesman S, Zipser D. 1978. Deg phenotype of Escherichia coli lon mutants. J. Bacteriol. 133: 844-851.
    Pubmed PMC
  6. Hatfull GF. 2015. Dark matter of the biosphere: the amazing world of bacteriophage diversity. J. Virol. 89: 8107-8110.
    Pubmed PMC CrossRef
  7. Healy B, Cooney S, O’Brien S, Iversen C, Whyte P, Nally J, et al. 2010. Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen. Foodborne Pathog. Dis. 7: 339-350
    Pubmed CrossRef
  8. Hong SS, Jeong J, Lee J, Kim S, Min W, Myung H. 2013. Therapeutic effects of bacteriophages against Salmonella gallinarum infection in chickens. J. Microbiol. Biotechnol. 23:1478-1483.
    Pubmed CrossRef
  9. Hunter CJ, Bean JF. 2013. Cronobacter: an emerging opportunistic pathogen associated with neonatal meningitis, sepsis and necrotizing enterocolitis. J. Perinatol. 33: 581-585.
    Pubmed CrossRef
  10. Jackel C, Hammerl JA, Reetz J, Kropinski AM, Hertwig S. 2015. Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity. BMC Genomics 16: 629.
    Pubmed PMC CrossRef
  11. Kalyantanda G, Shumyak L, Archibald LK. 2015. Cronobacter species contamination of powdered infant formula and the implications for neonatal health. Front. Pediatr. 3: 56.
    Pubmed PMC CrossRef
  12. Kim KP, Klumpp J, Loessner MJ. 2007. Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula. Int. J. Food Microbiol. 115: 195-203.
    Pubmed CrossRef
  13. Kim MS, Myung H. 2012. Complete genome of Staphylococcus aureus phage SA11. J. Virol. 86: 10232.
    Pubmed PMC CrossRef
  14. Kropinski AM, Mazzocco A, Waddell TE, Linghor E, Johnson RP. 2009. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 501: 69-76.
    Pubmed CrossRef
  15. Lee YD, Kim JY, Park JH, Chang H. 2012. Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii. Arch. Virol. 157: 199-202.
    Pubmed CrossRef
  16. Manfioletti G, Schneider C. 1988. A new and fast method for preparing high quality lambda DNA suitable for sequencing. Nucleic Acids Res. 16: 2873-2884.
    Pubmed PMC CrossRef
  17. Nobrega FL, Costa AR, Kluskens LD, Azeredo J. 2015. Revisiting phage therapy: new applications for old resources. Trends Microbiol. 23: 185-191.
    Pubmed CrossRef
  18. Sambrook J, Russell DW. 2006. Purification of bacteriophage λ particles by centrifugation through a glycerol step gradient. CSH Protoc. 2006: 10.1101/pdb.prot3969.
    CrossRef
  19. Shin H, Lee JH, Kim Y, Ryu. S. 2012. Complete genome sequence of Cronobacter sakazakii bacteriophage CR3. J. Virol. 86: 6367-6368.
    Pubmed PMC CrossRef
  20. Singh N, Goel G, Raghav M. 2015. Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii. Virulence 6: 433-440.
    Pubmed PMC CrossRef
  21. Tóthová L, Celec P, Bábí ková J, Gajdošová J, Al-Alami H, Kamodyova N, et al. 2011. Phage therapy of Cronobacterinduced urinary tract infection in mice. Med. Sci. Monit. 17:BR173-BR178.
    Pubmed PMC CrossRef
  22. Viertel TM, Ritter K, Horz HP. 2014. Viruses versus bacteria - novel approaches to phage therapy as a tool against multidrug-resistant pathogens. J. Antimicrob. Chemother. 69:2326-2336.
    Pubmed CrossRef
  23. Yang JA, Kang I, Moon M, Ryu U, Kwon KK, Cho JC, Oh HM. 2016. Complete genome sequence of Celeribacter marinus IMCC12053 (T), t he h ost strain o f marine b acteriophage P12053L. Mar. Genomics 26: 5-7.
    Pubmed CrossRef
  24. Zuber S, Boissin-Delaporte C, Michot L, Iversen C, Diep B, Brüssow H, Breeuwer P. 2008. Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages:prospects and problems. Microb. Biotechnol. 1: 532-543.
    Pubmed PMC CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(9): 1629-1635

Published online September 28, 2016 https://doi.org/10.4014/jmb.1605.05020

Copyright © The Korean Society for Microbiology and Biotechnology.

A Newly Isolated Bacteriophage, PBES 02, Infecting Cronobacter sakazakii

Hyung Ju Lee 1, Wan Il Kim 1, Young Chan Kwon 1, Kyung Eun Cha 2, 3, Minjin Kim 3 and Heejoon Myung 2, 3*

1Hana High School, Seoul 03305, Republic of Korea, 2The Bacteriophage Bank of Korea, Yongin 17035, Republic of Korea, 3Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 17035, Republic of Korea

Received: May 9, 2016; Accepted: May 27, 2016

Abstract

A novel bacteriophage, PBES 02, infecting Cronobacter sakazakii was isolated and characterized.
It has a spherical head of 90 nm in diameter and a tail of 130 nm in length, and belongs to
Myoviridae as observed under a transmission electron microscope. The major virion protein
appears to be 38 kilodaltons (kDa) in size. The latent period of PBES 02 is 30 min and the burst
size is 250. Infectivity of the phage remained intact after exposure to temperatures ranging
from 4oC to 55oC for 1 h. It was also stable after exposure to pHs ranging from 6 to 10 for 1 h.
The phage effectively removed contaminating Cronobacter sakazakii from broth infant formula.
PBES 02 has a double-stranded DNA genome of 149,732 bases. Its GC ratio is 50.7%. Sequence
analysis revealed that PBES 02 has 299 open reading frames (ORFs) and 14 tRNA genes.
Thirty-nine ORFs were annotated, including 24 related to replication and regulation functions,
10 related to structural proteins, and 5 related to DNA packaging. The genome of PBES 02 is
closely related to that of two other C. sakazakii phages, CR3 and CR8. Comparison of DNA
sequences of genes encoding the major capsid protein revealed a wide geographical
distribution of related phages over Asia, Europe, and America.

Keywords: bacteriophage PBES 02, Cronobacter sakazakii, genomic analysis, phage therapy

References

  1. Abbasifar R, Griffiths MW, Sabour PM, Ackermann HW, Vandersteegen K, Lavigne R, et al. 2014. Supersize me:Cronobacter sakazakii phage GAP32. Virology 460-461: 138-146.
    Pubmed CrossRef
  2. Abbasifar R, Kropinski AM, Sabour PM, Chambers JR, MacKinnon J, Malig T, Griffiths MW. 2014. Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae. Arch. Virol. 159: 2 253 2261.
  3. Bragg R, van der Westhuizen W, Lee JY, Coetsee E, Boucher C. 2014. Bacteriophages as potential treatment option for antibiotic resistant bacteria. Adv. Exp. Med. Biol. 807: 97-110.
    Pubmed CrossRef
  4. Das M, Bhowmick TS, Ahern SJ, Young R, Gonzalez CF. 2015. Control of Pierce’s disease by phage. PLoS One 10: e0128902.
    Pubmed KoreaMed CrossRef
  5. Gottesman S, Zipser D. 1978. Deg phenotype of Escherichia coli lon mutants. J. Bacteriol. 133: 844-851.
    Pubmed KoreaMed
  6. Hatfull GF. 2015. Dark matter of the biosphere: the amazing world of bacteriophage diversity. J. Virol. 89: 8107-8110.
    Pubmed KoreaMed CrossRef
  7. Healy B, Cooney S, O’Brien S, Iversen C, Whyte P, Nally J, et al. 2010. Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen. Foodborne Pathog. Dis. 7: 339-350
    Pubmed CrossRef
  8. Hong SS, Jeong J, Lee J, Kim S, Min W, Myung H. 2013. Therapeutic effects of bacteriophages against Salmonella gallinarum infection in chickens. J. Microbiol. Biotechnol. 23:1478-1483.
    Pubmed CrossRef
  9. Hunter CJ, Bean JF. 2013. Cronobacter: an emerging opportunistic pathogen associated with neonatal meningitis, sepsis and necrotizing enterocolitis. J. Perinatol. 33: 581-585.
    Pubmed CrossRef
  10. Jackel C, Hammerl JA, Reetz J, Kropinski AM, Hertwig S. 2015. Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity. BMC Genomics 16: 629.
    Pubmed KoreaMed CrossRef
  11. Kalyantanda G, Shumyak L, Archibald LK. 2015. Cronobacter species contamination of powdered infant formula and the implications for neonatal health. Front. Pediatr. 3: 56.
    Pubmed KoreaMed CrossRef
  12. Kim KP, Klumpp J, Loessner MJ. 2007. Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula. Int. J. Food Microbiol. 115: 195-203.
    Pubmed CrossRef
  13. Kim MS, Myung H. 2012. Complete genome of Staphylococcus aureus phage SA11. J. Virol. 86: 10232.
    Pubmed KoreaMed CrossRef
  14. Kropinski AM, Mazzocco A, Waddell TE, Linghor E, Johnson RP. 2009. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 501: 69-76.
    Pubmed CrossRef
  15. Lee YD, Kim JY, Park JH, Chang H. 2012. Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii. Arch. Virol. 157: 199-202.
    Pubmed CrossRef
  16. Manfioletti G, Schneider C. 1988. A new and fast method for preparing high quality lambda DNA suitable for sequencing. Nucleic Acids Res. 16: 2873-2884.
    Pubmed KoreaMed CrossRef
  17. Nobrega FL, Costa AR, Kluskens LD, Azeredo J. 2015. Revisiting phage therapy: new applications for old resources. Trends Microbiol. 23: 185-191.
    Pubmed CrossRef
  18. Sambrook J, Russell DW. 2006. Purification of bacteriophage λ particles by centrifugation through a glycerol step gradient. CSH Protoc. 2006: 10.1101/pdb.prot3969.
    CrossRef
  19. Shin H, Lee JH, Kim Y, Ryu. S. 2012. Complete genome sequence of Cronobacter sakazakii bacteriophage CR3. J. Virol. 86: 6367-6368.
    Pubmed KoreaMed CrossRef
  20. Singh N, Goel G, Raghav M. 2015. Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii. Virulence 6: 433-440.
    Pubmed KoreaMed CrossRef
  21. Tóthová L, Celec P, Bábí ková J, Gajdošová J, Al-Alami H, Kamodyova N, et al. 2011. Phage therapy of Cronobacterinduced urinary tract infection in mice. Med. Sci. Monit. 17:BR173-BR178.
    Pubmed KoreaMed CrossRef
  22. Viertel TM, Ritter K, Horz HP. 2014. Viruses versus bacteria - novel approaches to phage therapy as a tool against multidrug-resistant pathogens. J. Antimicrob. Chemother. 69:2326-2336.
    Pubmed CrossRef
  23. Yang JA, Kang I, Moon M, Ryu U, Kwon KK, Cho JC, Oh HM. 2016. Complete genome sequence of Celeribacter marinus IMCC12053 (T), t he h ost strain o f marine b acteriophage P12053L. Mar. Genomics 26: 5-7.
    Pubmed CrossRef
  24. Zuber S, Boissin-Delaporte C, Michot L, Iversen C, Diep B, Brüssow H, Breeuwer P. 2008. Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages:prospects and problems. Microb. Biotechnol. 1: 532-543.
    Pubmed KoreaMed CrossRef