Performing Department
(N/A)
Non Technical Summary
The high-mortality pathogen Listeria monocytogenes is endemic and can easily be introduced into meat processing facilities, but the conditions that allow it to persist in these locations are not fully known. Listeria monocytogenes is a bacterial pathogen associated with ready-to-eat meats, soft cheeses, ice cream, and many other food products. It is of particular concern due to a 30% mortality rate and elevated risks of pregnancy loss and long-term disability. Despite monumental efforts by the food industry to prevent contamination, there are regular outbreaks of listeriosis, especially among the young, old, and immunocompromised. In just the last five years it has been the causative agent for at least 15 outbreaks, resulting in 194 illnesses, 176 hospitalizations, and 18 deaths. These outbreaks are not only devastating in terms of public health, but they also lead to large food product recalls, which have a high economic cost and continueto exasperate the global food waste problem. The risk this pathogen poses to our most vulnerable populations makes it worth the continued scientific pursuit of new methods to identify and prevent potential outbreaks before they begin.Understanding why Listeria persists in some meat processing facilities is critical to address this public health issue. These facilities undergo rigorous daily cleaning and sanitation protocols, and this hostile environment has selected for organisms that have survival adaptations; one such adaptation is the ability to form biofilms, a structured network of protein and sugars that protects the organisms living within it. These biofilms can provide a perfect environmental niche for Listeria persistence. However, we still know very little about the role of specific organisms in a biofilm microbiome and their potential impacts on food products. Therefore, in this project, we will determine whether there are meat processing facility-associated biofilm microbial communities that support or inhibit the growth and survival of Listeria monocytogenes.To address this critical research question, we will employ DNA sequencing and 'omics technologies to fully reveal the populations and activities of the microbes involved in facility biofilms. We will work with the meat processing community to collect biofilm samples from small meat facilities located in Alabama and the southeastern United States. Within each facility, we will place stainless steel coupons in select drains throughout the processing spaces and allow biofilms to grow over a period of three months, which will allow us to form a bio-map of biofilm microbes representative of all small processors. Once we have collected the samples, we will divide the coupon into three portions. The first will undergo DNA sequencing, which will allow us to identify the specific microorganisms present in the sample, the relative abundance of each organism in the community, and their overall microbial diversity. The second portion will undergo RNA sequencing, which will allow us to determine the biological functions these organisms are generating (e.g., biofilm formation, competition). The final portion will be cultured with Listeria monocytogenes, and then also undergo RNA sequencing, so we can determine what microbial activities change in response to the addition of the pathogen. We hypothesize that the addition of Listeria to these communities will drive a re-organization of the community, which will be evident by these functional changes. Different communities will likely have different responses, and with this information we can identify biofilm communities more likely to respond with competitive exclusion to Listeria, therefore preventing its colonization.The goal of this research project is to better understand the dynamics of microbes within meat processing facility biofilms. Our methods will allow us to directly address the diversity of microbial communities in these environments across the region and to gain insight into why they form and what roles each member performs. With the introduction of an important pathogen, Listeria monocytogenes, we will also learn how these communities respond to the introduction of new organisms, and whether this increases the amount of competition for nutrients within the limited space. Ultimately, we believe this will lead to the creation of an early-warning system for detecting the growth potential of Listeria in a high-risk space such as meat processing facilities before it is actually colonized by the pathogen. Through this, we will aim to decrease the risk of pathogen contamination for the small processor, decrease the economic burden associated with food recalls and illness, and improve the safety of food products locally, regionally, and, ultimately, nationwide.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Goals / Objectives
Research ObjectivesObjective 1. Identify and describe the resident microbial community composition of biofilms across small meat processing facilities in Alabama.Objective 2. Determine the relationship between meat processing biofilm communities and Listeria monocytogenes survival.Objective 2.1. Identify specific taxa and functional attributes that support or exclude Listeria monocytogenes survival in a biofilm communityObjective 2.2. Identify the physical biofilm community structure during Listeria monocytogenes incorporation.
Project Methods
Objective 1 is intended to address the lack of knowledge regarding the microbial composition of drain biofilms in meat processing facilities, including the environmental variables that influence microbial membership. In order to resolve this definitively, we will address these related questions: What the general microbial communities found in small meat processing facilities consist of, whether there is a core biofilm microbiome common to all facilities, and how specific facility and processing room environmental conditions influence the biofilm microbial community development. We hypothesize that there will be a small core community of microbes that are found across processing facilities; however, there will also be accessory organisms that lead to unique and varied communities driven by location-specific environmental variables. To achieve this objective, we will recruit small and very small meat processors located throughout the state of Alabama and the surroundings. Recruiting will be conducted using introductions and recommendations from members of our research community at Auburn University, through previous work conducted in collaboration with local processors, and through our extension activities (see letters of support)(Rabinowitz et al., 2021). We anticipate no problems with recruitment due to the nature of the project, as we are not directly sampling for pathogens in the processing facility, we are not collecting from food contact surfaces, we will be anonymizing the facilities in reports of our results, and we already have local producers that have committed to enrollment in this study (see letters of support). We will also offer financial rewards for participating in the experiment. Once recruited, we will place stainless steel sampling coupons in drains within each processing facility to collect the biofilms, then sequence the microbial DNA in order to compare the associated microbial communities.The rationale for objective 1 is to provide a survey of microbes present in drain biofilms as a basis for further objectives and future work, as it is important to have a strong foundational knowledge of what is occurring under normal conditions before we can develop modifications.The goal of objective 2.1is to determine the microbial features of a biofilm community that enhances or suppress L. monocytogenes survival. To investigate this, we have designed an experiment that will answer these related questions: which facility-specific biofilm communities can support Listeria introduction and survival, what functional pathways are differentially regulated in response to the introduction of Listeria to either support or competitively inhibit its growth, and what specific traits related to these taxa and functional pathways are most important in regulating Listeria presence in biofilms? We hypothesize that the introduction of L. monocytogenes to a biofilm community will drive the differential regulation of synergistic and competitive microbial functional pathways within the resident biofilm microorganisms. In general, microbial communities assemble in a way that each member fills a specific niche in the ecological space, so the addition of a new organism will require that the current members respond in some way to maintain their position. Often this will occur as a competitive inhibition, but it is also likely that the new organism can be incorporated into the native community by filling another role or by taking advantage of functional redundancy in these ecosystems. To determine how this interplay occurs in processing drain biofilms, we will add L. monocytogenes to the biofilm communities obtained in Objective 1 and monitor how the community dynamics shift. We will do this by sequencing the total RNA produced in the community before and after the addition of L. monocytogenes using standard metatranscriptomics techniques, which will demonstrate which functions are prioritized by the community. The overall rationale for this objective is to connect the environmental variables surveyed in objective 1 with microbial communities that either support or inhibit the presence of the critical pathogen L. monocytogenes. Beyond the immediate applications of this knowledge, these results will also provide information of basic scientific merit surrounding microbial community dynamics in biofilms.Objective 2.2is designed, in combination with Objective 2.1, to improve our understanding of the mechanisms through which L, monocytogenes is incorporated into meat processing facility biofilms and is able to persist in the environment. Objective 2.1 demonstrated the functional traits expressed by the native members of the biofilm microbial communities in response to L. monocytogenes introduction that either supported or competitively excluded the pathogen. This objective will further investigate the biofilms that incorporated L. monocytogenes to determine the spatial and structural mechanisms of L. monocytogenes persistence. The primary questions associated with this objective are: how does the biofilm structure support L. monocytogenes, what is the distribution of L. monocytogenes in the biofilm, and how does L. monocytogenes spatially associate with the native microbes? We hypothesize that L. monocytogenes cells will be homogeneously intermixed with the native microbes and situate within gaps in the biofilm exopolysaccharide filaments. This type of spatial placement has been previously seen in biofilms grown on conveyor belts in meat processing facilities, and given the similarities in sanitation regimes and microbial sources we believe this to be a reasonable expectation for floor drains as well (Fagerlund et al., 2017). More specific knowledge of the structure of these Listeria-containing biofilms will allow the development of more specific interventions and cleaning regimes that can target these spaces in future work, thus directly improving food safety and processing facility management.