FACOLTÀ DI SCIENZE AGRARIE E ALIMENTARI Department of Food, Environmental and Nutritional Sciences (DeFENS) Graduate School in Molecular Sciences and Plant, Food and Environmental Biotechnology PhD programme in Food Science, Technology and Biotechnology XXVIII cycle POLYPHASIC APPROACH TO STUDY THE EMERGING PATHOGEN LACTOCOCCUS GARVIEAE Scientific field AGR/16 GIOVANNI ERACLIO Tutor: Prof. Maria Grazia Fortina PhD Coordinator: Prof. Maria Grazia Fortina 2014/2015 LIST OF CONTENTS ABSTRACT ........................................................................................................... 1 RIASSUNTO ......................................................................................................... 3 PREFACE .............................................................................................................. 5 STATE OF THE ART ............................................................................................ 8 Lactococcus garvieae .......................................................................................... 8 Insertion sequences and temperate bacteriophages as promoter of genome plasticity ............................................................................................................. 9 Biocontrol of Lactococcus garvieae .................................................................. 10 Phage therapy ................................................................................................... 11 AIM OF THE STUDY ......................................................................................... 13 REFERENCES ..................................................................................................... 14 1. PHYLOGENETIC RELATIONSHIPS BETWEEN LACTOCOCCUS GARVIEAE AND LACTOCOCCUS LACTIS ......................................................... 19 MATERIALS AND METHODS .......................................................................... 19 Lactococcus garvieae strains ............................................................................. 19 DNA extraction ................................................................................................. 19 Multi Locus Sequence Typing (MLST) ............................................................. 19 Genome analysis and comparison ...................................................................... 20 RESULTS AND DISCUSSION ........................................................................... 21 Multilocus Sequence Typing (MLST) ............................................................... 21 Genome comparison ......................................................................................... 27 CONCLUSIONS .................................................................................................. 30 REFERENCES ..................................................................................................... 31 2. INSERTION SEQUENCE ELEMENTS IN LACTOCOCCUS GARVIEAE ..... 33 MATERIALS AND METHODS .......................................................................... 34 RESULTS AND DISCUSSIONS ......................................................................... 35 CONCLUSIONS .................................................................................................. 42 REFERENCES ..................................................................................................... 43 3. PHENOTYPIC AND GENOTYPIC DIFFERENCES BETWEEN EUROPEAN AND AMERICAN STRAINS OF LACTOCOCCUS GARVIEAE ......................... 45 MATERIALS AND METHODS .......................................................................... 46 I Sampling, selection, culturing and identification of L. garvieae isolates ............. 46 Carbon source utilization ................................................................................... 46 Antibiotic resistance .......................................................................................... 46 Plasmid and total DNA extraction and quantification ......................................... 47 Genomic fingerprinting of L. garvieae............................................................... 47 PCR amplification ............................................................................................. 47 Southern blotting analysis and probes preparation ............................................. 48 Genome analysis ............................................................................................... 48 RNA isolation ................................................................................................... 48 cDNA synthesis and RT-PCR experiments ........................................................ 48 RESULTS AND DISCUSSION ........................................................................... 52 Carbon source utilization ................................................................................... 52 Antibiotic resistance .......................................................................................... 54 Genomic fingerprinting ..................................................................................... 55 Plasmids dissemination and Southern analysis ................................................... 58 Resistance to sulfonamide compounds............................................................... 62 CONCLUSIONS .................................................................................................. 67 REFERENCES ..................................................................................................... 68 4. CHARACTERIZATION OF TEMPERATE BACTERIOPHAGES INFECTING LACTOCOCCUS GARVIEAE REVEALS NEW INSIGHT INTO THE EVOLUTION OF THE SPECIES ................................................................ 71 MATERIAL AND METHODS ............................................................................ 72 Induction assay and transmission electron microscope ....................................... 72 DNA extraction and sequencing ........................................................................ 72 Phage genome analysis and comparison ............................................................ 73 RESULTS AND DISCUSSION ........................................................................... 75 Phage induction ................................................................................................ 75 Ex novo genome sequencing of temperate bacteriophage TB25 ......................... 75 Genome comparison ......................................................................................... 80 CONCLUSIONS .................................................................................................. 91 REFERENCES ..................................................................................................... 92 II 5. ISOLATION AND CHARACTERIZATION OF NEW VIRULENT PHAGES INFECTING LACTOCOCCUS GARVIEAE .......................................................... 95 MATERIALS AND METHODS .......................................................................... 96 Bacterial host and phage isolation ..................................................................... 96 Microbiological assays ...................................................................................... 96 Electron microscopy.......................................................................................... 97 DNA extraction and restriction profile ............................................................... 97 Phage DNA extraction, sequencing, and genome analysis.................................. 97 Structural protein profile ................................................................................... 98 Proteomic tree design ........................................................................................ 98 RESULTS AND DISCUSSION ........................................................................... 98 Phage isolation and lytic spectrum assessment ................................................... 98 Morphological, genomic and structural characterization .................................... 98 Characterization of phage GE1 ........................................................................ 101 Genome of phage GE1 and its cos site ............................................................. 102 Analysis of phage GE1 genes .......................................................................... 104 Proteome of phage GE1 .................................................................................. 104 Comparison of prolate-headed phages ............................................................. 108 Phylogenetic studies of L. garvieae phages ...................................................... 110 CONCLUSIONS ................................................................................................ 111 REFERENCES ................................................................................................... 112 ACKNOWLEDGEMENTS ................................................................................ 116 LIST OF PAPER, ORAL COMMUNICATIONS AND POSTERS ..................... 117 Peer-reviewed publications.............................................................................. 117 Posters and oral communications in scientific congress ................................... 118 III Abstract ABSTRACT This Ph.D. thesis research project aims to increase knowledge on emerging and opportunistic pathogens in food, to improve the current systems of management and control of food safety.