Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations Spring 2015 Influence and characterization of microbial contaminants associated with the FDA BAM method used to detect Listeria monocytogenes from romaine lettuce Christopher E. Bach Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Part of the Food Microbiology Commons Recommended Citation Bach, Christopher E., "Influence and characterization of microbial contaminants associated with the FDA BAM method used to detect Listeria monocytogenes from romaine lettuce" (2015). Open Access Theses. 560. https://docs.lib.purdue.edu/open_access_theses/560 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School Form 30 Updated 1/15/2015 PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Christopher E. Bach Entitled INFLUENCE AND CHARACTERIZATION OF MICROBIAL CONTAMINANTS ASSOCIATED WITH THE FDA BAM METHOD USED TO DETECT LISTERIA MONOCYTOGENES FROM ROMAINE LETTUCE For the degree of Master of Science Is approved by the final examining committee: Robert E. Pruitt Chair M. Catherine Aime Haley F. Oliver Steve G. Hallett To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy of Integrity in Research” and the use of copyright material. Approved by Major Professor(s): Robert E. Pruitt Approved by: Peter B. Goldsbrough 4/15/2015 Head of the Departmental Graduate Program Date INFLUENCE AND CHARACTERIZATION OF MICROBIAL CONTAMINANTS ASSOCIATED WITH THE FDA BAM METHOD USED TO DETECT LISTERIA MONOCYTOGENES FROM ROMAINE LETTUCE A Thesis Submitted to the Faculty of Purdue University by Christopher E. Bach In Partial Fulfillment of the Requirements for the Degree of Master of Science May 2015 Purdue University West Lafayette, Indiana ii I would like to dedicate this thesis to my grandfather, Robert T. White. iii ACKNOWLEDGEMENTS First and foremost I would like to extend my gratitude to my major advisor Dr. Bob Pruitt. Dr. Pruitt has played an essential role in further cultivating my interests in science by challenging me with projects across different scientific disciplines. Dr. Pruitt is truly a great mentor, scientist and educator. I am also grateful to my committee members, Drs. Cathie Aime, Steve Hallett and Haley Oliver for stimulating good discussion during meetings and any help they have provided during my graduate career. I would also like to extend my appreciation to Dr. Peter Goldsbrough, who is tremendously supportive of graduate students in the department. I would also like to thank the Department of Botany and Plant Pathology and faculty members that provided great lectures for coursework that I took while at Purdue University. I have to give a special thanks to Aime lab members, Rachel Koch, Jorge Diaz and Dr. Merje Toome for letting me bug them with phylogenetics questions and periodically using their gel imager. Lastly, I would like to thank my parents, Gale and Joy Bach and my wonderful sister Emily Bach for all of their continuous support and encouragement. iv TABLE OF CONTENTS LIST OF TABLES............................................................................................................................................ vi LIST OF FIGURES........................................................................................................................................Pageviii ABSTRACT ........................................................................................................................................................ix CHAPTER 1. GENERAL INTRODUCTION ............................................................................................1 1.1 General background on food safety..........................................................................................1 1.2 Trends in food safety of fresh produce ...................................................................................2 1.3 Characterizing relationships between foodborne pathogens and plants ..............4 1.3.1 Mechanisms for contamination of fresh produce by human pathogens .......4 1.3.2 Interactions between human pathogens and plants ...............................................6 1.4 Background on the bacterial foodborne pathogen Listeria monocytogenes .........9 1.4.1 General history of Listeria monocytogenes...................................................................9 1.4.2 Listeria monocytogenes virulence and disease ........................................................ 11 1.4.3 Listeria monocytogenes and fresh produce ............................................................... 12 1.5 Methods for isolation and detection of Listeria monocytogenes from food ....... 16 1.6 References ......................................................................................................................................... 22 CHAPTER 2. EVALUATION OF THE FOOD AND DRUG ADMINISTRATION METHOD USED FOR THE DETECTION OF LISTERIA MONOCYTOGENES FROM ROMAINE LETTUCE ......................................................................................................................................................... 32 2.1 Introduction ...................................................................................................................................... 32 2.2 Materials and Methods ................................................................................................................ 40 2.2.1 Detection of Listeria monocytogenes from Romaine lettuce using the FDA method ................................................................................................................................................... 40 2.2.3 PCR confirmation of Listeria monocytogenes isolates.......................................... 43 2.2.4 Media cross-comparison.................................................................................................... 43 2.3 Results ................................................................................................................................................. 48 2.3.1 Evaluation of traditional FDA media............................................................................ 48 2.3.2 Media cross-comparison.................................................................................................... 52 2.4 Discussion .......................................................................................................................................... 61 2.4.1 Evaluation of traditional FDA media............................................................................ 61 2.4.2 Media cross-comparison.................................................................................................... 67 2.4.3 Phenotypes of presumptive isolates streaked onto Rapid’L.Mono ............... 68 2.4.4 Conclusions .............................................................................................................................. 69 2.5 References ......................................................................................................................................... 71 v CHAPTER 3. CHARACTERIZATION OF FALSE POSITIVE CELLULOMONAS ISOLATES: GENOME ANALYSIS, ANTIBIOTIC RESISTANCE GENES, ANTIBIOTIC TARGET GENESPage AND PHYLOGENETIC ANALYSIS ......................................................................................................... 75 3.1 Introduction ...................................................................................................................................... 75 3.2 Methods .............................................................................................................................................. 78 3.2.1 Selection of Cellulomonas isolates, genome sequencing and assembly....... 78 3.2.2 Genome annotation .............................................................................................................. 78 3.2.3 Multilocus phylogenetic analysis using closely related proteomes .............. 79 3.2.4 Phylogenetic analysis using the 16S rRNA gene..................................................... 80 3.2.5 Identification of antibiotic resistance genes and antibiotic target genes... 81 3.3 Results ................................................................................................................................................. 83 3.3.1 Genome description and comparison.......................................................................... 83 3.3.2 Multilocus phylogenetic analysis ................................................................................... 84 3.3.3 Phylogenetic analysis of the 16S rRNA gene ............................................................ 84 3.3.4 Antibiotic resistance and antibiotic gene targets .................................................. 85 3.4 Discussion .......................................................................................................................................... 95 3.4.1 Genome analysis .................................................................................................................... 95 3.4.2 Phylogenetic analysis .........................................................................................................