University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2016 The icrM obial Ecology Of Listeria Monocytogenes As Impacted By Three Environments: A Cheese Microbial Community; A Farm Environment; And A Soil Microbial Community Panagiotis Lekkas University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Food Science Commons Recommended Citation Lekkas, Panagiotis, "The icrM obial Ecology Of Listeria Monocytogenes As Impacted By Three Environments: A Cheese Microbial Community; A Farm Environment; And A Soil Microbial Community" (2016). Graduate College Dissertations and Theses. 463. https://scholarworks.uvm.edu/graddis/463 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. THE MICROBIAL ECOLOGY OF LISTERIA MONOCYTOGENES AS IMPACTED BY THREE ENVIRONMENTS: A CHEESE MICROBIAL COMMUNITY; A FARM ENVIRONMENT; AND A SOIL MICROBIAL COMMUNITY A Dissertation Presented by Panagiotis Lekkas to The Faculty of the Graduate College of The University of Vermont In Partial Fulfilment of the Requirements For the Degree of Doctor of Philosophy Specializing in Animal, Nutrition and Food Sciences January, 2016 Defence date: September 18, 2015 Dissertation Examination Committee: Catherine W. Donnelly, Ph.D., Advisor Douglas I. Johnson, Ph.D., Chairperson John W. Barlow, Ph.D. Paul S. Kindstedt, Ph.D. Dean Cynthia J. Forehand, Ph.D., Dean of the Graduate College Abstract The aim of the first study was to investigate the effects of L. monocytogenes on the composition of the surface microflora on washed rind soft cheese. Two trials with washed rind cheeses that were inoculated with 100cfu cm-2 of a L. monocytogenes six strain cocktail were conducted. Surface cheese rind samples were collected from both control and inoculated cheeses every 7 days. Cheese rind samples were analyzed through the standard BAM method for enumeration of L. monocytogenes and through amplification of the V4 region of 16S rRNA and ITS regions for identification of the surface rind bacterial and fungal communities, respectively. Our data showed that Pseudomonas spp. significantly changed the composition of the microorganisms on the surface of the rind while L. monocytogenes had little effect. Although the concentration of L. monocytogenes increased to levels of 106 cfu cm-2 based on the enumeration data, the genetic data was not able to identify it in the flora, which is a limitation of molecular methods used for identification of pathogens in foods. For the second study the presence and incidence of L. monocytogenes on farms that either produce raw milk cheese or supply the milk for raw milk cheese production was investigated. Five farms were visited and in total 266 samples were collected from barn, environmental, and milk sites. L. monocytogenes prevalence was found to be at 6% from all the farms tested. Samples were identified to the genus level through a modified BAM method and speciated though multiplex PCR. Included in the pathogenic isolates was a DUP-1042B L. monocytogenes strain that has been implicated in major outbreaks. Results from this study continue to support the fact that contaminated silage can be an important reservoir of the pathogen in a dairy farm setting. From our data we identified that drinking water sources for the animals are also an important reservoir of L. monocytogenes in farm environments. Lastly manure amended soils in the northeastern U.S. were tested for the presence and survival of rifampicin resistant Escherichia coli (rE. coli), generic E. coli (gE. coli) and Listeria spp.. Both gE.coli and rE.coli samples were processed using either direct enumeration, MPN or bag enrichment methods. Samples were taken from both tilled and surface dairy solid manure-amended plots. Listeria samples were processed using a modified BAM method. Listeria presence was constant throughout the study. In contrast, rE. coli and gE. coli levels declined with time. The main conclusions of this study were that soil type, location and physical characteristics have a significant role in the survival of bacterial populations of rE. coli, gE. coli and Listeria spp. in soil. Tilling of soils results in increased survival of the bacterial population due to the fact that it increases soil pore size and facilitates moisture entry. Data from this research will assist in the creation of preventative measures that lead to the elimination of pathogen reservoirs. It will be further used to verify that a 120 day interval following manure application should be sufficient to ensure food safety of edible crops subsequently planted on these soils. Acknowledgments In my years as a graduate student at the department of Nutrition and Food Science at the University of Vermont, I was lucky to have such a diverse and supportive group of people that helped me compete my studies. I would like to thank all the faculty and staff especially Ann Martin and Lacey Ure for their support all those years and for their understanding through stressful times. I would like to thank the chair of my department, Dr. Harvey, for her support during my program. I want to thank Dr. Douglas Johnson for being my committee chair. He brought a different aspect to my work and was always a source of information throughout my program. I want to thank Dr. John Barlow for being one of the committee members and for guiding me through my projects. In the travels we did he always was willing to talk about my research and provide different insights that were helpful in the completion of my projects. Dr. Paul Kindstedt was instrumental during my first project and was always available to help and provide me with advice on how to properly age cheese in a lab setting. I could have never completed that project without the support I received from him. I would like to thank Dr. Manan Sharma and Dr. Patricia Millner for their support and help during my soil amendments project. Dr. Rachel Dutton and Dr. Julie Button for their help and advice during the molecular analysis of the cheese rinds and for allowing me to use their facilities for part of my work. I would like to thank all my fellow graduate students for their help, jokes, and support through these years. ii I also would like to thank Dr. Donnelly, my advisor, for seeing the potential in me and accepting me into the program. I want to thank her for all the opportunities she provided me with and all the long discussions we had about my projects and future directions. Words cannot fully explain the respect and gratitude I have and will have for her throughout my life. Thank you Dr. Donnelly. Lastly I would like to thank my family and my parents for all the support they provided thought the years. My son Niko especially for being so understanding and not complaining when I had to work late. iii Table of Contents Acknowledgments……………………………………………………………………….ii List of Tables .................................................................................................................. viii List of Figures ................................................................................................................... ix CHAPTER 1: LITERATURE REVIEW ....................................................................... 1 1.1 Introduction ............................................................................................................... 1 1.2 Listeria- Background................................................................................................. 2 1.3 Listeria monocytogenes ............................................................................................. 4 1.4 Pathogenicity and virulence ...................................................................................... 5 1.5 Internalins .................................................................................................................. 7 1.6 Listeria pathogenicity island -1 (LIPI-1) .................................................................. 9 1.7 Host Susceptibility .................................................................................................. 13 1.8 Listeria monocytogenes presence and distribution.................................................. 15 1.9 Methods of detection and identification (speciation) .............................................. 23 1.10 Culture based methods .......................................................................................... 23 1.11 Immuno-based techniques ..................................................................................... 27 1.12 Molecular methods of detection ............................................................................ 28 1.13 Nucleic acid probes ............................................................................................... 28 1.14 Polymerase Chain Reaction Based Methods (Conventional PCR, RT- PCR, Multiplex PCR, Real Time-PCR) ........................................................................ 29 1.15 DNA Microarrays .................................................................................................. 31 1.16 Subtyping .............................................................................................................. 32 1.17 Comparison of cultural vs. culture independent