The Pennsylvania State University The Graduate School Department of Food Science DETECTING AND UNDERSTANDING THE PERSISTENCE OF A PREDOMINANT LISTERIA MONOCYTOGENES CLONE IN A COMMERCIAL FRESH MUSHROOM SLICING AND PACKAGING FACILITY A Dissertation in Food Science by Latha Murugesan 2016 Latha Murugesan Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2016 The dissertation of Latha Murugesan was reviewed and approved* by the following: Luke F. LaBorde Associate Professor of Food Science Dissertation Co-Advisor Co-Chair of Committee Stephen J. Knabel Professor of Food Science Dissertation Co-Advisor Co-Chair of Committee Mary Ann V. Bruns Associate Professor of Soil Science/Microbial Ecology Edward G. Dudley Associate Professor of Food Science Sara R. Milillo Instructor of Food Science Robert F. Roberts Professor of Food Science Head of the Department *Signatures are on file in the Graduate School ii ABSTRACT A longitudinal survey of non-food-contact surfaces in a commercial mushroom slicing and packaging facility was conducted to determine the prevalence, distribution, and potential routes of transmission of L. monocytogenes. Samples were taken at 3 sampling occasions over a 13- month period. Multi-virulence-locus sequence typing (MVLST) was used to identify persistent and transient L. monocytogenes clones. The longitudinal study demonstrated that L. monocytogenes was present in 18.8% of samples. A trench drain and a wet porous concrete floor were identified as harborage sites for L. monocytogenes. MVLST identified 4 different virulence types (VTs) of L. monocytogenes in the facility: VT11, VT107, VT105, and VT56. Of these clones, VT11 predominated and was persistent at 2 sampling sites during all sampling periods over 13 months. Improvements made in sanitation practices and facilities maintenance between the second and third sampling periods decreased the prevalence of L. monocytogenes; however, complete elimination of the pathogen was not achieved. Therefore, the persistent and transient clones were further evaluated for their ability to tolerate quaternary ammonium compound (QAC) sanitizer, adherence to stainless steel (SS) and concrete coupons, and growth rate and peak cell density in mushroom broth. QAC tolerance among clones was investigated by growing them for 20-h (peak cell density) and 7 days (long-term-survival, (LTS), phase) at 35°C using minimum inhibitory concentration (MIC) and survival (tolerance) assays. The MIC value of QAC for all L. monocytogenes clones was 9.1 ppm. However, VT11 and VT107 grew to a significantly (P ≤ 0.05) higher OD600 at sub-lethal QAC concentrations than VT105 and iii VT56. In addition, LTS phase cells of VT11 were significantly (P ≤ 0.05) more tolerant (2.6 to 220 times) to in-use QAC concentration of 200 ppm compared to other clones. Clones did not differ significantly (P ≤ 0.05) in their ability to firmly adhere to concrete. However, on SS coupons, VT11 populations significantly (P ≤ 0.05) decreased more rapidly than the other clones (after 5 and 7 days) suggesting the former dispersed earlier from the surface. Concrete coupons harbored more cells (2.12 to 8.06 log CFU/cm2) than SS (1.14 to 6.12 log CFU/cm2) and were significantly (P ≤ 0.05) more protective of L. monocytogenes when treated with 200 ppm QAC. Five different fresh mushroom broth (MB) dilution levels, 30%, 10%, 5%, 0.5% and 0.05%, were used to determine growth rate and peak cell density of L. monocytogenes clones at 35°C and 10°C. The results showed that all L. monocytogenes clones were able to grow and/or survive in each of the MB dilutions. The generation time of L. monocytogenes clones in MB ranged between 38.45 and 207.06 min at 35°C and from 9.27 to 28.42 h at 10°C. However, VT11 grew rapidly to a significantly (P ≤ 0.05) higher peak cell density in 0.5% and 0.05% MB dilutions compared to other clones. The results indicate that VT11 may have a growth and survival advantage over other clones in nutrient-limited environments. Overall, the results suggest that the observed predominance and persistence of VT11 in the surveyed mushroom slicing and packaging facility can be at least partially explained by 1) higher peak cell density at low mushroom nutrient levels, 2) higher growth at sub-lethal QAC concentrations, 3) higher tolerance to in-use QAC concentration in the LTS phase, and 4) a more rapid dispersal from surfaces allowing colonization of new areas in the mushroom processing environment. Based on these iv results, a model was proposed to explain how several complex factors provide a competitive advantage for VT11 and thus the observed predominance and persistence within the mushroom processing facility. These results can be used to provide guidance on targeted interventions for reducing or controlling L. monocytogenes contamination in mushroom and other fresh produce processing facilities. v TABLE OF CONTENTS List of Abbreviations ................................................................................................... x List of Figures .............................................................................................................. xii List of Tables ............................................................................................................... xv Acknowledgment ......................................................................................................... xvii Chapter 1 Statement of the problem ............................................................................ 1 Chapter 2 Literature review ......................................................................................... 5 2.1 Foodborne outbreaks and recalls ................................................................... 5 2.1.1 Outbreaks attributed to contaminated fresh produce ............................ 5 2.1.2 Contamination of fresh mushrooms with L. monocytogenes ............... 6 2.2 Listeria ........................................................................................................... 7 2.2.1 Taxonomy of Listeria ........................................................................... 7 2.2.2 Listeria monocytogenes ........................................................................ 7 2.2.3 Growth curve of L. monocytogenes ...................................................... 8 2.2.4 Growth characteristics of L. monocytogenes ........................................ 11 2.2.5 Pathogenesis of L. monocytogenes ....................................................... 12 2.2.6 Epidemiology ....................................................................................... 15 2.2.7 Subtyping methods ............................................................................... 16 2.2.7.1 Serotyping and serogrouping ..................................................... 16 2.2.7.2 DNA fragment-based methods ................................................... 17 2.2.7.3 DNA sequence-based methods ................................................... 18 2.2.8 Lineages of L. monocytogenes ............................................................. 20 2.2.9 Epidemic clones of L. monocytogenes ................................................. 20 vi 2.3 L. monocytogenes in foods and food processing facilities ............................ 21 2.3.1 Current regulations ............................................................................... 21 2.3.2 Prevalence of L. monocytogenes .......................................................... 22 2.3.3 Growth and survival of L. monocytogenes under different environmental conditions in food facilities ............................................ 23 2.3.3.1 Cold ............................................................................................ 23 2.3.3.2 Acid and salt ............................................................................... 24 2.3.3.3 Surface adherence and biofilm formation .................................. 24 2.3.3.4 Antimicrobial compounds .......................................................... 27 2.3.3.5 Quaternary Ammonium Compounds (QACs) ............................ 28 2.3.4 Persistence of L. monocytogenes subtypes in food facilities ............... 29 2.3.4.1 Raw materials ............................................................................. 30 2.3.4.2 Processing environment ............................................................. 30 2.3.5 Factors causing persistence of L. monocytogenes subtypes in food processing facilities ................................................................................ 31 2.3.5.1 Adherence and biofilm formation .............................................. 31 2.3.5.2 Sanitizer tolerance ...................................................................... 32 2.3.5.3 Native microflora ....................................................................... 33 2.3.5.4 Genetic determinants .................................................................. 34 2.3.6 Control of L. monocytogenes in food processing facilities .................. 35 2.4 Mushrooms and mushroom production processes ........................................ 37 2.4.1 Taxonomy ............................................................................................. 37 2.4.2 Commercial Agaricus mushroom production ...................................... 38 2.4.3 Composition of Agaricus mushrooms .................................................. 39 vii 2.4.4 Pre-harvest operations .........................................................................