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Biocontrol of Cronobacter Spp. Using Bacteriophage in Infant Formula

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Biocontrol of Cronobacter Spp. Using Bacteriophage in Infant Formula i Biocontrol of Cronobacter spp. using Bacteriophage in Infant Formula by Reza Abbasifar A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science Guelph, Ontario, Canada © Reza Abbasifar, May, 2013 ii ABSTRACT Biocontrol of Cronobacter spp. using Bacteriophage in Infant Formula Reza Abbasifar Advisor: Dr. Mansel W. Griffiths University of Guelph, 2013 Co-Advisor: Dr. Parviz M. Sabour The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31 (GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be iii potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models. iv ACKNOWLEDGMENTS I am grateful to my advisor, Dr. Mansel W. Griffiths, for his great advice, support and help through my PhD program. I would also like to express my appreciation to my Co-Advisor, Dr. Parviz M. Sabour for his valuable advice, suggestion, and encoragement that patiently gave me during this long process. I am thankful to my Advisory Committee Member, Dr. Andrew M. Kropinski, who was greatly helpful in my thesis. This thesis would not be completed without my advisors’ help and suggestions. Also, I would like to thank Dr. Hans-W. Ackermann, Dr. Brian Dunphy, Dr. John H.E. Nash, Dr. Jim Chambers, Dr. Rob Lavigne, Dr. Roger Johnson, Dr. Elizabeth (Betty) Kutter, Dr. Keith Warriner, Dr. Kimberly Seed, Joanne MacKinnon, Erika Lingohr, Dr. Haifeng Wang, Dr. Franco Pagotto, Dr. Roger Stephan, Dr. Andrew Chibeu, Dr. Angela M. Tellez, Ann Blake, and William (Bill) Lachowsky for their helpful assistance and suggestions in this project. My sincerest appreciation goes to my family for their unlimited and nonstop support during all the moments of my study. My father and mother have supported me all my life and have continued it while I was doing my thesis although I was far from them. My dearest wife, Argentina Alanis Villa, has supported me continuously with love, caring and courage in school and in other aspects of life. Thanks to my brothers, Dr. Arash and Mehdi for their help and being there for me. I am thankful to The University of Guelph for providing the opportunity to gain my PhD degree and for financial support from NSERC and DFO. v My gratitude goes to all my colleagues in CRIFS and The Department of Food Science. vi TABLE OF CONTENTS Abstract……………………………………………………………………………….….ii Acknowledgements……………………………………………………………………...iv Table of Contents………………………………………………………………………..vi List of Tables……………………………………………………………………………..x List of Figures…………………………………………………………………………..xii List of Table in Appendix……………………………………………………………...xiv Chapter 1. Introduction ...……………………………………………………………….1 1.1. Research Introduction ………………………………………………………..1 1.2. Cronobacter: A food-borne pathogen ………………………………………..2 1.2.1. Epidemiology ……………………………………………………..5 1.2.2. Physiology ………………………………………………………...9 1.2.3. Detection ………………………………………………………...10 1.2.4. Control …………………………………………………………..14 1.3. Overview of Bacteriophage ………………………………………………...18 1.3.1. Discovery of Bacteriophage ……………………………………..18 1.3.2. Biology of Bacteriophage ……………………………………….19 1.3.3. Taxonomy of Bacteriophage …………………………………….21 1.3.4. Lysogenic and Lytic Cycle of Bacteriophage …………………...25 1.4. Biocontrol of Food-borne Pathogens Using Bacteriophage ………………..30 1.4.1. Phage Applications in Food ……………………………………..30 1.4.2. Phage Application as Biocontrol Agent …………………………42 1.4.3. Phage Therapy ...………………………………………………..45 1.5. Research Objectives ………………………………………………………...47 Chapter 2. Isolation and Characterization of Lytic Cronobacter Bacteriophages…49 2.1. Abstract ……………………………………………………………………..49 2.2. Introduction …………………………………………………………………49 vii 2.3. Material and Methods ………………………………………………………51 2.3.1. Bacteria and Bacteriophages ……………………………………...51 2.3.2. Isolation of Bacteriophages ……………………………………….52 2.3.3. Purification of Bacteriophages ……………………………………53 2.3.4. Propagation of Bacteriophages …………………………………...54 2.3.5. Host Range Determination………………………………………...54 2.3.6. Transmission Electron Microscopy (TEM) ………………………55 2.3.7. Cross Infectivity …………………………………………………..56 2.4. Results ………………………………………………………………………57 2.4.1. Isolation of Bacteriophages …………………………...…………..57 2.4.2. Host Range ………………………………………………………..57 2.4.3. Characterization of the Selected Phages ………………………….60 2.4.3.1. Morphology ……………………………………………..60 2.4.3.2. Cross Infectivity ………………………………………...65 2.5. Discussion …………………………………………………………………..67 Chapter 3. Sequencing and Genome Analysis of Cronobacter phage GAP32 …...…72 3.1. Abstract ……………………………………………………………………..72 3.2. Introduction …………………………………………………………………73 3.3. Material and Methods ………………………………………………………74 3.3.1. Bacteria and Bacteriophage ………………………………………74 3.3.2. Phage Purification, DNA Isolation and Sequencing ……………...74 3.3.3. Bioinformatic Analysis …………………………………………...76 3.3.4. Proteomic Analysis ……………………………………………….77 3.3.5. Genome Sequence ………………………………………………...77 3.4. Results ………………………………………………………………………77 3.5. Discussion …………………………………………………………………..80 Chapter 4. Sequencing and Genome Analysis of Cronobacter phages GAP31, GAP52, GAP161 and GAP227…………………………………………………………82 4.1. Abstract ……………………………………………………………………..82 viii 4.2. Introduction …………………………………………………………………83 4.3. Material and Methods ………………………………………………………84 4.3.1. Bacteria and Bacteriophages ……………………………………...84 4.3.2. Phage Purification, DNA Isolation and Sequencing ……………...85 4.3.3. Bioinformatic Analysis …………………………………………...86 4.3.4. Genome Sequences ……………………………………………….87 4.4. Results ………………………………………………………………………87 4.4.1. Features of the Genome of Phage GAP31 ………………………..87 4.4.2. Features of the Genome of Phage GAP52 ………………………..90 4.4.3. Features of the Genome of Phage GAP161 ………………………93 4.4.4. Features of the Genome of Phage GAP227 ………………………95 4.5 Discussion ………………………………………………………………..….97 Chapter 5. Efficiency of Bacteriophage Therapy Against Cronobacter sakazakii in Galleria mellonella (Greater Wax Moth) Larvae...…………………..……………99 5.1. Abstract …………………………………………………………..…………99 5.2. Introduction ………………………………………………………………..100 5.3. Materials and Methods …………………………………………………….103 5.3.1. Bacterial Strains and Culture Conditions ………………………..103 5.3.2. Bacteriophage Isolation and Characterization …………………..104 5.3.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G. mellonella Larvae …...…………….……………………..……...105 5.3.4. Statistical Analysis ………………………………………………108 5.4. Results ……………………………………………………………………..108 5.4.1. Mortality of C. sakazakii in G. mellonella Larvae ………………108 5.4.2. Bacteriophage and Its Persistence in G. mellonella Larvae …………………………………………………………………………..109 5.4.3. Efficiency of Bacteriophage Therapy Against C. sakazakii in G. mellonella Larvae ………………………………………………………110 5.5. Discussion …………………………………………………………………112 ix Chapter 6. Use of Cocktail of Five Phages to Control C. sakazakii in Broth Media and in Infant Formula………………………………………………………………...116 6.1. Abstract ……………………………………………………………………116 6.2. Introduction ………………………………………………………………..117 6.3. Material and Methods ……………………………………………………..122 6.3.1. Bacteria and Bacteriophages …………………………………….122 6.3.2. Host Range Determination ………………………………………123 6.3.3. Effect of the Phage Cocktail on C. sakazakii Strains in Broth and in Reconstituted Infant Formula ………………………………….………124 6.3.4. Statistical Analysis ………………………………………………127 6.4. Results ……………………………………………………………………..128 6.4.1. Host Range ………………………………………………………128 6.4.2. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in Broth ………………………………………………………………...129 6.4.3. Effect of the Phage Cocktail on the Mixture of C. sakazakii Strains in Reconstituted Infant Formula ……………………………………….133 6.4.4. The Correlation Between Luminescence and Plate Count …...…138 6.5. Discussion …………………………………………………………………141 Chapter 7. Conclusions and Future Work ...………………………………………..151 7.1. Thesis Summary and General Conclusion ………………………………...151 7.2. Future Work ……………………………………………………………….158 References……………………………………………………………………………...159 Appendix ………………………………………………………………………………197 x LIST OF TABLES Table 1.1. Global outbreaks and sporadic cases of Cronobacter infections…..………………………………………………………………………………4 Table 1.2. Classification and basic properties of bacteriophages...……………….…………………………………………………………24 Table 2.1. Cronobacter strains used for isolation
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