The role of environmental microbiota in the persistence of Listeria monocytogenes in food processing facilities
Restricted (Penn State Only)
- Author:
- Rolon, Maria Laura
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 16, 2023
- Committee Members:
- Robert Roberts, Co-Chair & Dissertation Advisor
Jasna Kovac, Co-Chair & Dissertation Advisor
Terrence Bell, Outside Unit & Field Member
Luke Laborde, Major Field Member
Robert Roberts, Program Head/Chair
Kerry Kaylegian, Major Field Member - Keywords:
- Listeria monocytogenes
food processing facilities
microbiome
lactic acid bacteria
biological control
cleaning and sanitizing
biofilm
microbiota
amplicon sequencing
environmental microbiota
food safety - Abstract:
- Food safety is of paramount importance for production of wholesome and nutritious food. Throughout my dissertation, I present results of four studies exploring the relationship between Listeria monocytogenes, a deadly foodborne pathogen, and environmental microbiota found in food processing facilities. L. monocytogenes can inhabit a wide range of habitats (e.g., soil, water, animals, insects) and its ability to adapt to different environmental conditions (e.g., low temperatures, high salt concentrations, low pH) allows for its survival in food processing environments, which can result in the potential for recurring contamination of food. To control the presence of L. monocytogenes, food processors rely on cleaning and sanitizing operations coupled with Environmental Monitoring Programs. While L. monocytogenes is typically susceptible to the cleaning and sanitizing protocols applied in food processing facilities, it may survive the action of sanitizers by forming, or residing inside biofilms. However, in food processing environments, L. monocytogenes resides with other environmental microorganisms that are introduced to the facilities with raw products or personnel. The presence of other microorganisms can facilitate formation of robust multi-species biofilms that may enhance the survival and persistence of L. monocytogenes. Currently, limited information is available regarding the potential associations between environmental microbiota and the presence of foodborne pathogens in food processing facilities. My dissertation research aimed to address some of the critical gaps in the understanding of the role of environmental microbiota in the survival and persistence of L. monocytogenes in food processing environments. iv To begin unraveling the role of environmental microbiota in the persistence of L. monocytogenes in food processing environments, I first sought to characterize the microbiota of three tree fruit packing facilities and statistically assess whether certain taxa co-occur with L. monocytogenes (Chapter 2). I observed a recurring presence of L. monocytogenes and a distinct microbiota composition in the monitored facilities throughout two seasons. Importantly, I found that bacterial taxa from taxonomic families Pseudomonadaceae, Xanthomonadaceae, and Microbacteriaceae were present in a significantly higher relative abundance in L. monocytogenes-positive samples, especially in the facility with a persistent L. monocytogenes contamination. While some members of these bacterial families have been studied as food spoilage organisms (e.g., Pseudomonas spp.), emerging human pathogens (e.g., Stenotrophomonas spp.), or plant pathogens (e.g., Xanthomonas spp.), there is limited information available on potential interactions between these species and L. monocytogenes. Furthermore, their role in the survival and persistence of L. monocytogenes in food processing facilities is severely understudied. In Chapter 3, I seek to understand how the detected bacterial taxa interact with L. monocytogenes. Given that the survival of L. monocytogenes under sanitizer pressure may be facilitated by biofilms formed in food processing environment, I aimed to assess the effect of biofilms formed by environmental microbiota on the survival during exposure to a commonly used sanitizer, benzalkonium chloride. I isolated environmental microbiota from families Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae from tree fruit packing environments, and tested their ability to form biofilms in single- and multi-family assemblages with L. monocytogenes. I found that microbial assemblages of v increasing complexity (i.e., increasing number of families) generally formed more biofilm and contained a greater concentration of L. monocytogenes, compared to monoculture biofilms. I further tested the effects of multi-species biofilm formation on the sanitizer tolerance of L. monocytogenes, by exposing multi-species biofilms to sanitizers and quantifying the die-off kinetics of L. monocytogenes throughout a 2-hour period. In Chapter 4, I aimed to assist fruit packers by providing practical tools to enhance the control of L. monocytogenes through traditional chemical-based cleaning and sanitizing. I tested four cleaning and sanitizing cleaning and sanitizing protocols in the fruit packing environments to determine their effectiveness in controlling L. monocytogenes and examined their effect on environmental microbiota. I found that the cleaning and sanitizing protocol that included a disinfectant with a biofilm-degrading ability was most effective in reducing the frequency of detected L. monocytogenes in the sampled areas. While the total microbial load generally decreased after the application of cleaning and sanitizing protocols, the microbiota composition did not appear to be significantly affected by the treatments. In the last research chapter (Chapter 5), I tested the ability of two lactic acid bacteria strains to inhibit L. monocytogenes in a monoculture as well as in the context of environmental microbiota from food processing facilities. Microbial strains with antilisterial activity have been previously assessed as alternative strategy to control L. monocytogenes in food processing environments, however, their performance in the context of environmental microbiota from ice cream processing facilities has not been tested before. Here, I collected environmental microbiota from three small-scale ice cream processing facilities and tested whether two lactic acid vi bacteria could attach and effectively inhibit L. monocytogenes when co-cultured with the collected environmental microbiota. I observed that the microbiota composition of ice cream processing facilities may affect the antilisterial ability of two lactic acid bacteria strains and their attachment to surfaces. In particular, the presence of Pseudomonas significantly reduced the antilisterial ability of the tested strains. The knowledge generated through my studies on the role of food processing facilities’ microbiota will aid in designing tailored cleaning, sanitizing, and/or biological control protocols for control of L. monocytogenes. For example, cleaning and sanitizing could be redesigned to control taxa that facilitate Listeria’s persistence, or new cleaning chemistries can be developed to improve biofilm control in food processing facilities.