Isolation and characterization of broad host-range of bacteriophages infecting Cronobacter sakazakii and its biocontrol potential in dairy products

Main Article Content

Huaxiang Li
Xiao-jun Yang
Xiao-yan Zhu
Lu Gao
Sheng-qi Rao
Lei Yuan
Zhen-quan Yang


Cronobacter sakazakii, bacteriophage, antibacterial effects, genome, biocontrol


Cronobacter sakazakii (C. sakazakii) is an important pathogen contaminating dairy products (e.g., milk pow-der) and causes high mortality in infants. Bacteriophage as a potential biocontrol agent is a good alternative method for the control of this pathogen in dairy production and its environment. Thus, it is important to complete the C. sakazakii phage library by isolating and characterizing the broad host range of bacteriophage against C. sakazakii for control use. In this study, C. sakazakii strains from different sources were used as hosts to isolate and purify phages from human stool and sewage samples by double-layer plates. The biological characteristics, antibacterial properties, and genomes of these phages were then studied. Finally, ten virulent phages (EspYZU01–EspYZU10) infecting C. sakazakii were isolated and identified as belonging to the Myoviridae, Podoviridae, Tectivirus, and Stylovinidae families. Phage EspYZU08 presented the broadest host range and could infect all the five host strains of C. sakazakii. All 10 phages retained their infectivity at 50°C and pH 5–9. Both genomes of EspYZU05 and EspYZU08 were double-stranded DNAs with sizes of 41723 bp and 145582 bp, G+C contents of 55.69% and 46.75%, and open reading frames of 47 and 103, respectively. No toxins and antibiotic resistance genes were detected in both EspYZU05 and EspYZU08. Phage EspYZU08 and phage cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented excellent antibacterial efficacy for C. sakazakii in liquid broth and milk at 4°C, 25°C, and 37°C, suggesting that the phages in this study have great potential for the development of biocontrol agents against C. sakazakii in dairy and its processing environment.

Abstract 106 | PDF Downloads 75 XML Downloads 1 HTML Downloads 27


Abbasifar, R., Kropinski, A.M., Sabour, P.M., Chambers, J.R., MacKinnon, J., Malig, T. and Griffiths, M.W., 2014. Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae. Archives of Virology 159(9): 2253–2261.
Asakura, H., Morita-Ishihara, T., Yamamoto, S. and Igimi, S., 2007. Genetic characterization of thermal tolerance in Enterobacter sakazakii. Microbiology and Immunology 51(7): 671–677.
Bigot, B., Lee, W.-J., McIntyre, L., Wilson, T., Hudson, J., Billington, C. and Heinemann, J. 2011. Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage. Food Microbiology 28(8): 1448–1452.
Brady, C., Cleenwerck, I., Venter, S., Coutinho, T. and De Vos, P., 2013. Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter. Systematic and Applied Microbiology 36(5): 309–319.
Brüssow, H., Canchaya, C. and Hardt, W.D., 2004. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiology and Molecular Biology Reviews 68(3): 560–602.
Carlton, R., Noordman, W., Biswas, B., De Meester, E. and Loessner, M.J., 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application. Regulatory Toxicology and Pharmacology 43(3): 301–312.
Caubilla-Barron J., Hurrell E., Townsend S., Cheetham P., Loc-Carrillo C., Fayet O., Prere M.F. and Forsythe S.J., 2007. Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France. Journal of Clinical Microbiology 45, 3979–3985.
Drudy, D., Mullane, N.R., Quinn, T., Wall, P.G. and Fanning, S., 2006. Enterobacter sakazakii: an emerging pathogen in powdered infant formula. Clinical Infectious Diseases 42(7): 996–1002.
Ellis, D., Whitman, P. and Marshall, R., 1973. Effects of homologous bacteriophage on growth of Pseudomonas fragi WY in milk. Applied Microbiology 25(1): 24–25.
Endersen, L., Buttimer, C., Nevin, E., Coffey, A., Neve, H., Oliveira, H., Lavigne, R., O'Mahony and J., 2017. Investigating the biocontrol and anti-biofilm potential of a three-phage cocktail against Cronobacter sakazakii in different brands of infant formula. International Journal of Food Microbiology 253, 1–11.
Endersen, L., Coffey, A., Neve, H., McAuliffe, O., Ross, R.P. and O'Mahony, J.M., 2013. Isolation and characterisation of six novel mycobacteriophages and investigation of their antimicrobial potential in milk. International Dairy Journal 28(1): 8–14.
Endersen, L., O'Mahony, J., Hill, C., Ross, R.P., McAuliffe, O. and Coffey, A., 2014. Phage therapy in the food industry. Annual Review of Food Science and Technology 5, 327–349.
Farmer, J.J., Asbury, M.A., Hickman, F.W. and Brenner, D.J., 1980. Enterobacter sakazakii: a new species of "Enterobacteriaceae" isolated from clinical specimens. International Journal of Systematic Bacteriology 30(3): 569–584.
Faruque, S.M. and Mekalanos, J.J., 2012. Phage-bacterial interactions in the evolution of toxigenic Vibrio cholerae. Virulence 3(7): 556–565.
Food and Agriculture Organization of the United Nations /World Health Organization (FAO/WHO), 2008. Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae: meeting report. Microbiological Risk Assessment Series No. 15. Rome. 84pp. Available at: _report.pdf.
Forsythe, S., 2005. Enterobacter sakazakii and other bacteria in powdered infant milk formula. Maternal & Child Nutrition 1(1): 44.
Friedemann, M., 2009. Epidemiology of invasive neonatal Cronobacter (Enterobacter sakazakii) infections. European Journal of Clinical Microbiology & Infectious Diseases 28(11): 1297–1304.
Goodridge, L.D. and Bisha, B., 2014. Phage-based biocontrol strategies to reduce food borne pathogens in foods. Bacteriophage 1(3): 130–137.
Hawkins, R., Lissner, C. and Sanford, J.P., 1991. Enterobacter sakazakii bacteremia in an adult. Southern Medical Journal 84(6): 793–795.
Healy, B., Cooney, S., O'Brien, S., Iversen, C., Whyte, P., Nally, J., Callanan, J.J. and Fanning, S., 2010. Cronobacter (Enterobacter sakazakii): an opportunistic food-borne pathogen. Foodborne Pathogens and Disease 7(4): 339–350.
Himelright I., Harris E., Lorch V., Anderson M., Jones T., Craig A., Kuehnert M., Forster T., Arduino M., Jensen B. and Jernigan D., 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula-Tennessee, 2001. Journal of the American Medical Directors Association 287: 2204–2205.
Holý, O. and Forsythe, S., 2014. Cronobacter spp. as emerging causes of healthcare-associated infection. Journal of Hospital Infection 86(3): 169–177.
Iversen, C., Lehner, A., Mullane, N., Bidlas, E., Cleenwerck, I., Marugg, J., Fanning, S., Stephan, R. and Joosten, H., 2007. The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1. BMC Evolutionary Biology 7, 64.
Jepson, C.D. and March, J.B., 2004. Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine 22(19): 2413–2419.
Joseph, S., Cetinkaya, E., Drahovska, H., Levican, A., Figueras, M.J. and Forsythe, S.J., 2012. Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients. International Journal of Systematic and Evolutionary Microbiology 62(Pt 6): 1277–1283.
Kajsík, M., Oslanecová, L., Szemes, T., Hýblová, M., Bilková, A., Drahovská, H. and Turňa, J., 2014. Characterization and genome sequence of Dev2, a new T7-like bacteriophage infecting Cronobacter turicensis. Archives of Virology 159(11): 3013–3019.
Kandhai, M.C., Reij, M.W., Gorris, L.G., Guillaume-Gentil, O. and van Schothorst, M., 2004. Occurrence of Enterobacter sakazakii in food production environments and households. The Lancet 363(9402): 39–40.
Kim, K.P., Klumpp, J. and Loessner, M.J., 2007. Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula. International Journal of Food Microbiology 115(2): 195–203.
Lai, K.K., 2001. Enterobacter sakazakii infections among neonates, infants, children, and adults: case reports and a review of the literature. Medicine 80(2): 113–122.
Lee, J.H., Bai, J., Shin, H., Kim, Y., Park, B., Heu, S., Ryu, S. and Schottel, J.L., 2016a. A novel bacteriophage targeting Cronobacter sakazakii is a potential biocontrol agent in foods. Applied and Environmental Microbiology 82(1): 192–201.
Lee, H.J., Kim, W.I., Kwon, Y.C., Cha, K.E., Kim, M. and Myung, H., 2016b. A newly isolated bacteriophage, PBES 02, infecting Cronobacter sakazakii. Journal of Microbiology and Biotechnology 26(9): 1629–1635.
Lepuschitz, S., Ruppitsch, W., Pekard-Amenitsch, S., Forsythe, S.J., Cormican, M., Mach, R.L., Pierard, D. and Allerberger, F., 2019. Multicenter study of Cronobacter sakazakii infections in humans, Europe, 2017. Emerging Infectious Diseases 25(3): 515–522.
Magnani, C.F., Mezzanotte C., Cappuzzello C., Bardini M., Tettamanti S., Fazio G., Cooper L.J.N., Dastoli G., Cazzaniga G., Biondi A. and Biagi E., 2018. Preclinical efficacy and safety of CD19CAR cytokine-induced killer (CIK) cells transfected with Sleeping Beauty transposon for the treatment of acute lymphoblastic leukemia. Human Gene Therapy 29 (5): 602–613.
Nazarowec-White, M. and Farber, J.M., 1997. Enterobacter sakazakii: a review. International Journal of Food Microbiology 34(2): 103–113.
See, K., Than, H. and Tang, T., 2007. Enterobacter sakazakii bacteraemia with multiple splenic abscesses in a 75-year-old woman: a case report. Age and Ageing 36(5): 595–596.
Stoll B.J., Hansen N., Fanaroff A.A. and Lemons J.A., 2004. Enterobacter sakazakii is a rare cause of neonatal septicemia or meningitis in VLBW infants. JAMA Pediatrics 144: 821–823.
Tey, B.T., Ooi, S.T., Yong, K.C., Ng, M.Y.T., Ling, T.C. and Tan, W.S., 2009. Production of fusion m13 phage bearing the di-sulphide constrained peptide sequence (C-WSFFSNI-C) that interacts with hepatitis B core antigen. African Journal of Biotechnology 8(2): 268–273. Corpus ID: 59467076
Tóthová, Ľ., Celec, P., Bábíčková, J., Gajdošová, J., Al-Alami, H., Kamodyova, N., Drahovska, H., Liptakova, A., Turňa, J. and Hodosy, J., 2011. Phage therapy of Cronobacter-induced urinary tract infection in mice. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research 17(7): BR173.
Yan, Q., Condell, O., Power, K., Butler, F., Tall, B. and Fanning, S., 2012. Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium. Journal of Applied Microbiology 113(1): 1–15.
Zhang, Y., Zhang Z., Ding Y., Fang Y., Wang P., Chu W., Jin Z., Yang X., Wang J., Lou J., and Qian Q. 2021. Phase I clinical trial of EGFR–specific CAR–T cells generated by the piggyBac transposon system in advanced relapsed/refractory non–small cell lung cancer patients. Journal of Cancer Research and Clinical Oncology.
Zuber, S., Boissin-Delaporte, C., Michot, L., Iversen, C., Diep, B., Brüssow, H. and Breeuwer, P. 2008. Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems. Microbial biotechnology, 1(6): 532–543.