Microbiome’s Place in Meat: Contributions of the Meat Processing Built Environment Microbiome to Pathogen Persistence

The Built Environment Microbiome

As the field of microbiome science becomes more well-known and the ease of analysis improves, more researchers are incorporating a survey of the microbial community into their larger experimental designs. This abundance of data has greatly contributed to our ability to understand the vital relationship between hosts, environments, and occupants, whether the environment is as small as a rumen or as large as the soil affected by a wildfire. One specific area of growing interest is the microbiome of the built environment, spaces that are man-made or modified that provide areas for habitation, work, or recreation (U.S. Environmental Protection Agency, 2024). These spaces are generally occupied by humans, but they can also support livestock animals, pets, plants, rodents, and insects (National Academies of Sciences Engineering and Medicine et al., 2017). Recent work has demonstrated that the microbiome of the built environment is greatly impacted by its occupants, but the mechanism by which this occurs depends greatly on the function of the environment itself.

The microbiome of occupied buildings

The majority of research conducted on the built environment thus far has focused on the microbiome of houses and human-occupied buildings, as this has been thought to affect human health most directly. This work has demonstrated that the microbiome of the occupied spaces resembles that of the occupants. In new homes, the microbiome quickly shifts to reflect that of the family that has moved in, which is reflected in the taxonomy present in the house and the similarities in community structure. A large-scale project by Lax et al. (2014) demonstrated that differences in microbiome between houses were larger than different locations within a household, further underscoring the impact of residents. Further, the microbiome of exterior locations in homes is less similar to the human occupants than the interior spaces, showing that the constant occupancy is likely a key reason for this microbial shift (Dunn et al., 2013). It is likely that these microbes are deposited from human sources including the skin, mouth, and feces; these are likely spread via direct contact, aerosols (i.e., those created when flushing a toilet), and skin cells spread via dust (Dunn et al., 2013). This trend follows in other places of consistent human occupation, including the NASA submerged habitat and space station, elementary schools, college campuses, athletic facilities, and hospitals (Lam et al., 2022; Lax et al., 2017; Malli Mohan et al., 2020; Park et al., 2021; Ross and Neufeld, 2015; Singh et al., 2023; Wood et al., 2015). Of particular interest to animal scientists is that these findings extend beyond human housing. A study by Hyde et al. (2016) investigating the habitats of Komodo dragon enclosures showed that the microbiome of their environment was strongly influenced by the dragon saliva, skin, and feces.

An understanding of how the microbiome of the environments that humans inhabit is critical because of the close ties between environmental exposures and animal or human health. An important question still being explored is how the animal microbiome develops; however, environmental exposures from birth through development play an important role in shaping the animal microbiome. If the animal microbiome develops from the environment, but the environmental microbiome is developed from the occupants, it follows that generationally animals are exposed to fewer microbes over time. This connects to the hygiene hypothesis, also called the biodiversity hypothesis, which states that the lack of exposure to a diverse microbial ecology may lead to decreased immune function and more allergenic and autoimmune conditions (Stamper et al., 2016). The microbial communities of the built environment tend to be low diversity and contain similar microbes to those already present in the resident, so time spent in built environments is a key feature of this hypothesis (Bosch et al., 2024; Stamper et al., 2016). Therefore, though we are developing a better idea of the microbial ecology of built environments, it is still unclear whether the trends we have identified are beneficial or detrimental to the health of the occupants.

The microbiome of food processing facilities

In food processing environments, in contrast to the previously discussed houses and other inhabited buildings, the primary occupants that impact the environmental microbiome tend to be the food product and ingredients, while the human employees have limited influence. The food products are interacting directly with the spaces such as conveyors, equipment, tables, and floors via drippings and wash-water, while the human occupants are generally wearing protective equipment that limits their contact. This is especially the case when the ultimate quality of the product depends on the microbial functions, where the product contributes greatly to the environment, such as in fermented food products (Bokulich et al., 2015; Bokulich and Mills, 2013). The environment can impact the health of the employees, such as the rapid spread of SARS-CoV-2 in meat processing facilities during the COVID-19 pandemic (Herstein et al., 2021). However, in these cases the greater concern is generally contaminating the food, with either pathogens that can lead to human illness in the consumers or with spoilage organisms that reduce the shelf-life and eating quality. These have major implications from an economic standpoint in addition to the moral prerogative to produce safe foods.

The microbiome of food processing facilities is directly associated with the product in the facility and is a representation of the cleanliness and healthfulness. In a study of three distinct fruit processing facilities, the microbiomes had a common community composition consisting of Flavobacteriaceae, Moraxellaceae, Weeksellaceae, Xanthomonadaceae, and Burkholderiaceae, though the relative abundances were different by location (Tan et al., 2019). Of these, the facility with the poorest visual cleanliness was associated with the highest occurrence of pathogens including Listeria monocytogenes and a predominance of Pseudomonadaceae and Dipodascaceae organisms in the microbiome (Tan et al., 2019). Further analysis of these data showed that the presence of L. monocytogenes was correlated with the abundance of specific organisms in the facility microbiome, including Pseudomonas, Stenotrophomonas, Microbacterium, and several fungal taxa, which suggests that the facility microbiome could be used as an indicator for important food safety hazards (Rolon et al., 2023). The ability of the facility microbiome to impact the product quality has also been demonstrated. In a cheesemaking plant, where the environmental exposures may be important to product fermentation, the microbiome of the cheese product was similar to the surrounding environment, such that disruptions to the built environment microbiome may actually change the flavor profile of the product (Bokulich and Mills, 2013). In breweries, the built environment microbiome also contributes to the facility, though in brewery product this is considered contamination and can drive product spoilage (Bokulich et al., 2015). Given these important implications of the contributions of the processing facility microbiome to the final product, it is clear why the field of built environment microbiome studies in food is still growing.

The microbiome of meat processing facilities

Meat processing facilities present a unique challenge for microbiome management due to the constant introduction of organisms from sources as varied as live animals, ingredients, and water. As such, these microbiomes should be closely monitored. Currently, federal regulations require monitoring and control plans for several important pathogens or their indicators, but there is not the same level of oversight for the environmental organisms (Food and Drug Administration, 2016). A few studies have sought to characterize the microbiome of these facilities, which is an important initial step towards creating a generalized understanding of the organisms involved. In one of the first studies to evaluate this, Hultman et al. (2015) found that microbes present on contact surfaces resembled the microbiome of the raw ingredients, rather than the final products. Specifically, the organism most prevalent in the final sausage product, Leuconostoc, was only found in very low abundances in the surrounding built environment. In this case, it could be due to oxygen availability in the environment compared with the anaerobic environment of the sausage rather than the sources, but it is demonstrative of the unknown elements in the sources of microbes in the facility microbiome. In another study, evaluating microbial transmission routes through a pork harvest and processing facility, specific organisms were traced through the entire facility to determine their likely origin and impacts on the final product (Zwirzitz et al., 2020). It appeared that Moraxella spp. originated from polishing tunnels, gloves, and railings before contaminating carcasses, while the splitting saw may be the point of origin for Lactococcus spp. Importantly, they also identified points in carcass handling that significantly shifted the microbial community, suggesting these are candidates for applying interventions to alter the microbiome if necessary, which included singeing and the transportation step (Zwirzitz et al., 2020). Additional work has been conducted to determine the core microbial community associated with meat processing. Stellato et al. (2017) collected microbiome samples from twenty distinct processing facilities and were able to identify a core microbiome. This study specifically focused on food contact surfaces and meat products themselves, and within these locations they found 80% of samples to contain Pseudomonas spp., Streptococcus spp., Brochothrix spp., Psychrobacter spp., and Acinetobacter spp., confirming the importance of these organisms identified in other work. These organisms being connected with major spoilage organisms in the product is an important collective finding in this body of work.

We recently conducted a study to determine the origins and establishment of a microbiome in a small meat processing facility (Belk et al., 2022). In this, we tracked the development of the facility-associated microbiome in drains throughout a newly constructed meat processing facility by sampling monthly for the first 18 mo of production. During the experimental period, the microbiome of each drain shifted in beta diversity over time, but these changes became smaller over time, indicating the likely eventual establishment of a stable environmental microbiome. If correct, an understanding of the stable microbiome of a facility could be used as an indicator of environmental changes that may risk food safety. Moreover, our study confirmed patterns seen in other food processing facilities, with microbiomes grouping by different room conditions. Ongoing work is further investigating the relevance of these patterns to food safety and the presence of L. monocytogenes in the environment.

The built environment microbiome is a crucial area of research, for understanding human health, animal health, and food safety and quality. These microbial communities persist in environments with which humans and other animals interact every day, so their influence cannot be understated. The research conducted in food processing built environments specifically is leading to development of indicators of food safety risk and interventions for safety and quality.

Meat processing facility biofilm communities

It is perplexing that stable microbiomes are able to persist in food and meat processing facilities, because these environments are cleaned and sanitized regularly and are designed to limit opportunities for microbial persistence. One known reason for this phenomenon is the presence of biofilms on surfaces within the facilities, which have been widely investigated as sources of pathogens that enter the meat supply chain (Jessen and Lammert, 2003). These microbially secreted exopolysaccharide structures create niches that protect microbes from sanitation and other interventions, allowing them to persist in the environment. The ability of an organism to produce a biofilm can be evaluated by laboratory biochemical tests or by metagenomic sequencing, but non-producing organisms are still able to take advantage of and join biofilms, even if they do not express the ability themselves. This means that the biofilm forming capabilities of the entire community, as opposed to only the pathogens or organisms of interest, is important.

Surveys of meat processing facility microbiomes have found a diverse range of biofilm-forming organisms. These differences have been shown to drive the functionality of the biofilm itself; in one study, biofilms created by Psychrobacter spp. had a lower bacterial load than those formed from Microbacterium spp., and different organism compositions led to different levels of carbohydrate, protein, and environmental DNA production (Wagner et al., 2021). However, biofilms found in meat processing plants tend to be poly-bacterial and identifiable in distinct locations across a facility. In a study of a Russian meat processing facility, biofilms were identified within 10–24 h after sanitation, more quickly than previously described, in distinct locations throughout the facility (Nikolaev et al., 2022). In this study, similar taxa were identified in all collected biofilms, though the relative abundance of these differed, with those collected from walls and floors of the cutting room containing a higher relative abundance of Bacteroidetes compared with equipment from the forming workshop, and a biofilm collected from a joint between the wall and table in the cutting room containing a higher relative abundance of Pseudomonadaceae compared to other locations (Nikolaev et al., 2022). Other work has shown Pseudomonas to dominate most meat processing facility microbiomes in cold rooms, but these results suggest these organisms are free-living in many of these environments rather than members of the biofilms (Belk et al., 2022; Hultman et al., 2015). Interestingly, Nikolaev et al. (2022) also showed meat cells present in the biofilms, suggesting the formation of these structures is influenced by the presence of food residues. This agrees with previous work presented by Iñiguez-Moreno et al. (2019) who grew biofilms in the lab using different substrates and determined that a medium containing meat extract was most effective for the growth of dense, multispecies biofilms. Meat processing facilities are an ideal environment for the formation of these biofilms, and research must continue into the mechanisms of formation and persistence.

Biofilms’ relevance to food safety

The biofilms in meat processing built environments are important to understand for cleaning and maintenance of sanitary conditions in a facility, but also because they can and often do harbor pathogens of importance to food safety. Listeria monocytogenes is of particular interest for this mechanism of persistence because this organism is not brought into the facility from the animal itself so is not as frequently re-introduced. When alone, isolates of L. monocytogenes are able to form biofilms, but the biofilm community is much stronger and more resistant to sanitizers when they are in a multi-species biofilm (Fagerlund et al., 2017). This suggests importance in the community structure to enable L. monocytogenes persistence. This has been especially shown when the L. monocytogenes is co-cultures with Pseudomonas fluorescens, which has been shown to drive increased exopolysaccharide production and increased Listeria survival (Pang and Yuk, 2019). Conversely, other studies of Listeria-containing biofilms have demonstrated that the member organisms may act in competition with the Listeria. In an experiment with simulated biofilms, Heir et al. (2018) showed that complex biofilm microbiomes containing L. inocua and other organisms inhibited L. monocytogenes. Similarly, Ripolles-Avila et al. (2022) showed that Bacillus megaterium and Candida zeylanoides decreased L. monocytogenes adherence in biofilms. Similar work has identified biofilms as a harbor for other pathogens, especially E. coli. Strains of E. coli O157:H7 can form biofilms but are also frequently identified in multispecies biofilms, and highly diverse multi-species biofilms have the greatest ability to protecting the pathogen from sanitizers (Chitlapilly Dass et al., 2020). Given all this, it is likely that the biofilm microbiome is a critical area of continued research to improve food plant sanitation and reduce food safety risk in the future.

Impacts of environmental conditions on meat facility biofilms

Environmental microbiomes are dynamic—influenced by the occupants and materials being introduced as described above, but also by the environmental conditions. Parsing the exact elements of an environment that shift the microbial communities is a challenge, because in real-world scenarios many variables are shifting concurrently. We identified distinct microbial communities forming in meat processing facility spaces that correlate with the room function; however, it is still unclear what drives these microbiomes to form differently, whether it is the room temperature, available substrates, moisture, frequency of use, or something we were unable to measure (Belk et al., 2022). Likely, this is a combination of all these factors, but it will take more research to fully describe the role of each variable. Recently Yang et al. (2023) used a meta-analysis to try to address this issue. They confirmed the correlation between room function and the microbial community, with the extra level of these communities assembling differently by country. Interestingly, the function of the room seemed to have a stronger effect than the species being processed, with the cutting room for beef and pork plants located in Canada containing similar microbes, predominated by Pseudomonas, Janthinobacterium, and Flavobacterium and containing less Enterococcus than rooms of different functions. Authors also generated a network analysis to identify 42 combinations of microbial genera that were associated with each other within a functional room (Yang et al., 2023). Unfortunately, there was insufficient metadata for these authors to fully describe the reason behind these clustering patterns, beyond understanding the room function and biofilm forming abilities of the microbes. The variables can be broken down more easily in simulated laboratory studies, which is a growing area of research. From this it is becoming clear that temperature of work surfaces and environment is very important to the persistence of microbes and the formation of biofilms. In monospecies biofilms, the temperature affects their growth ability differently depending on the microbe involved. Escherichia coli and Bacillus cereus biofilms grown in meat extract appear to be most impacted by temperature, with low temperatures limiting their growth, while Pseudomonas aeruginosa is not impacted by temperature (Iñiguez-Moreno et al., 2019). Multispecies biofilms grown with meat extract are also impacted by temperature, with biofilms containing a higher cell density at higher temperatures, and a notable shift in community composition at low temperatures that aligns with observations of high levels of Pseudomonas spp. in cold rooms from previous studies (Belk et al., 2022; Hultman et al., 2015; Iñiguez-Moreno et al., 2019). While temperature and substrate are clearly important variables for microbiome formation, there is a need for further investigation to identify other environmental features that impact the formation of microbiomes and biofilm community membership.