Impact of intense sanitization on environmental biofilm communities and the survival of Salmonella enterica at a beef processing plant.

Salmonella enterica is a leading cause of foodborne illness in the U.S. In the meat industry, one action taken to address pathogen contamination incidence is an intense sanitization (IS) of the entire processing plant that many large processors perform annually or semiannually. However, this procedure's immediate and long-term impact on environment microbial community and pathogen colonization are unknown. Here we investigated the impact of IS procedure on environmental biofilms and the subsequent S. enterica colonization and stress tolerance. Environmental samples were collected from floor drains at various areas 1 week before, 1 week, and 4 weeks after the IS procedure at a beef plant with sporadic S. enterica prevalence. Biofilm formation by microorganisms in the drain samples without S. enterica presence was tested under processing temperature. The ability of the biofilms to recruit and/or protect a co-inoculated S. enterica strain from quaternary ammonium compound (QAC) treatment was determined. The community structure of each drain sample was elucidated through 16S rRNA amplicon community sequencing. Post-IS samples collected from 8 drains formed significantly stronger biofilms than the respective pre-IS samples. S. enterica colonization was not different between the pre- and post-IS biofilms at all drain locations. S. enterica survival in QAC-treated pre- and post-IS mixed biofilms varied depending upon the drain location but a higher survival was associated with a stronger biofilm matrix. The 16S rRNA amplicon gene community sequencing results exhibited a decrease in community diversity 1 week after IS treatment but followed by a significant increase 4 weeks after the treatment. The IS procedure also significantly altered the community composition and the higher presence of certain species in the post-IS community may be associated with the stronger mixed biofilm formation and Salmonella tolerance. Our study suggested that the IS procedure might disrupt the existing environmental microbial community and alter the natural population composition, which might lead to unintended consequences as a result of a lack of competition within the multispecies mixture. The survival and recruitment of species with high colonizing capability to the post-IS community may play crucial roles in shaping the ensuing ecological dynamics.

Salmonella-induced microbiome profile in response to sanitation by quaternary ammonium chloride.

Salmonella enterica is a prominent cause of foodborne disease in the United States. However, the mechanism and route of pathogen transmission that leads to Salmonella infection in commercial processing plants are poorly understood. This study aimed to investigate the effect of mixed-species biofilms on S. enterica survival and persistence under sanitizer stress [Quaternary ammonium compounds (QACs)] by analyzing 78 floor drain samples from a meat processing facility and three S. enterica strains (serovars Cerro, Montevideo, and Typhimurium) isolated from that facility and an unrelated source. The four test groups were as follows: control, QAC treatment, Salmonella addition, and QAC treatment with Salmonella addition. DNAs were extracted, and 16S rRNA gene based on the variable region V4 amplicon sequencing was performed to analyze the relative abundance, core microbiome, and Alpha and Beta diversity using the qiime2 pipeline. At the genus level, the Brochothrix (45.56%), Pseudomonas (38.94%), Carnobacterium (6.18%), Lactococcus (4.68%), Serratia (3.14%), and Staphylococcus (0.82%) were shown to be the most prevalent in all drain samples. The results demonstrate that the relative abundance of different bacterial genera was affected by both QAC treatment and Salmonella addition, with some genera showing increases or decreases in abundance. Notably, the correlation network was constructed to understand the relationships between the different bacteria. Nitrospira had the greatest number of connections in the floor drain environment network, with two negative and eight positive correlations. The results suggest that Nitrospira in the mixed-species biofilm community may play a role in converting ammonium in the QAC sanitizer into nitrites. Thus, Nitrospira could be a potentially important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms.IMPORTANCESalmonella contamination in meat processing facilities can lead to foodborne illness outbreaks. Our study characterized the microbiome dynamics in beef facility drains and their response to Salmonella addition and common sanitizer (QAC). Nitrospira could be an important genus in providing sanitizer resistance to pathogen-encompassed mixed-species biofilms. The results provide insight into the impact of mixed-species biofilms on Salmonella survival and persistence under sanitizer stress in meat processing facilities. The results highlight the need to consider mixed-species biofilm effects when developing targeted interventions to enhance food safety.

Shotgun-metagenomics reveals a highly diverse and communal microbial network present in the drains of three beef-processing plants.

Background: Multi-species biofilms pose a problem in various environments, especially food-processing environments. The diversity of microorganisms in these biofilms plays a critical role in their integrity and protection against external biotic and abiotic factors. Compared to single-species biofilms, mixed-species biofilms are more resistant to various stresses, including antimicrobials like sanitizers. Therefore, understanding the microbiome composition and diversity in biofilms and their metabolic potential is a priority when developing intervention techniques to combat foodborne pathogens in food processing environments. Methods: This study aimed to describe and compare the microbiome profile of 75 drain biofilm samples obtained from five different locations (Hotscale, Hotbox, Cooler, Processing, & Grind room) of three beef-processing plants (Plant A, B & C) taken over two timepoints 2017-18 (T1) and 2021 (T2) by shotgun sequencing. Results: Core microbiome analysis found Pseudomonas, Psychrobacter, and Acinetobacter to be the top three prevalent genera among the plants and locations. Alpha diversity analysis demonstrated a high diversity of microbiome present in all the plants and locations across the time points. Functional analysis showed the high metabolic potential of the microbial community with abundance of genes in metabolism, cell-adhesion, motility, and quorum sensing. Moreover, Quaternary Ammonium Compound (QAC) resistance genes were also observed, this is significant as QAC sanitizers are commonly used in many food processing facilities. Multi-functional genes such as transposases, polymerases, permeases, flagellar proteins, and Mobile Genetic Elements (MGEs) were found suggesting these are dynamic microbial communities that work together to protect themselves against environmental stresses through multiple defense mechanisms. Conclusion: This study provides a framework for understanding the collective microbial network spanning a beef processing system. The results can be used to develop intervention strategies to best control these highly communicative microbial networks.

A novel method using a differential staining fluorescence microscopy (DSFM) to track the location of enteric pathogens within mixed-species biofilms.

This study developed a new tool, differential staining fluorescence microscopy (DSFM), to measure the biovolume and track the location of enteric pathogens in mixed-species biofilms which can pose a risk to food safety in beef processing facilities. DSFM was employed to examine the impact of pathogenic bacteria, Escherichia coli O157:H7 and three different Salmonella enterica strains on mixed-species biofilms of beef processing facilities. Fourteen floor drain biofilm samples from three beef processing plants were incubated with overnight BacLight stained enteric pathogens at 7 °C for 5 days on stainless steel surface then counter-stained with FM-1-43 biofilm stain and analyzed using fluorescence microscopy. Notable variations in biovolume of biofilms were observed across the fourteen samples. The introduction of E. coli O157:H7 and S. enterica strains resulted in diverse alterations of biofilm biovolume, suggesting distinct impacts on mixed-species biofilms by different enteric pathogens which were revealed to be located in the upper layer of the mixed-species biofilms. Pathogen strain growth curve comparisons and verification of BacLight Red Stain staining effectiveness were validated. The findings of this study show that the DSFM method is a promising approach to studying the location of enteric pathogens within mixed-species biofilms recovered from processing facilities. Understanding how foodborne pathogens interact with biofilms will allow for improved targeted antimicrobial interventions.

Characterization of boar semen microbiome and association with sperm quality parameters.

Elevated levels of bacteria within fresh extended boar semen are associated with decreased sperm longevity, therefore reducing the fertility of a semen dose. The objective of this study was to characterize the bacterial communities using 16S rRNA sequencing in freshly extended boar semen samples and relate the prevalence and diversity of the microbial population to sperm quality parameters 1) between studs, 2) between pooled and single-sire doses, and 3) over a 5-day period. Eight single-sire (n = 4 per stud) and eight pooled (n = 4 per stud) non-frozen extended semen doses were obtained from two boar studs (A and B). Pooled doses were the composite of the boar's ejaculates used in single-sire doses. Doses were subsampled for 5 d post-collection. Ten negative controls of each pooled dose (n = 2) and single-sire dose (n = 8) remained sealed until the last day. Microbiome analysis was achieved by examining the V4 hypervariable region of the 16S rRNA gene of flash-frozen samples. Two evaluators determined the average sperm motility and agglutination (0: no adhesion to 3: >50% adhesion) by averaging their estimates together at 10 random locations per slide. Stud A had greater sperm agglutination (1.6 vs. 1.0 ± 0.1; P < 0.01) than stud B. Sperm motility decreased over the 5-day period (P < 0.01) and tended (P = 0.09) to be greater in stud B than A (67.4% vs. 61.5% ± 0.02%). Compared with stud A, stud B had a greater relative abundance of Proteobacteria (60.0% vs. 47.2% ± 1.5%; P < 0.01) and a lower relative abundance of Firmicutes (22.5% vs. 31.9% ± 1.4%; P < 0.01). Moreover, stud A had a greater relative abundance of Bacteroidetes (6.3% vs. 5.3% ± 0.4%; P < 0.01) and Actinobacteria (11.5% vs. 10.1% ± 0.5%; P = 0.05) than stud B. Differences were found in alpha diversity for both Chao1 (P < 0.01) and Shannon (P < 0.01) diversity indexes among days 2, 3, 4, and 5 post-collection to day 1. For beta diversity, unweighted UniFrac metric on days 2, 3, 4, and 5 post-collection differed from those on day 1 (P < 0.01). There were significant correlations between sperm motility and relative abundance of Prevotella (r = -0.29), Ruminococcus (r = -0.24), and Bacteroides (r = -0.32). Additionally, there were significant correlations between sperm motility and Chao1 (r = -0.50) and Shannon's index (r = -0.36). These results demonstrate that differences in bacterial communities over time and between boar studs can be associated with variation in sperm quality.

The ability to improve production output remains essential to meet the growing global demand for pork. However, the presence of pathogenic bacteria such as Escherichia coli and Clostridium perfringens can hinder production goals by reducing semen quality through increased clumping events and decreased sperm motility. In addition, reduced conception rates and decreased litter size can occur when bacterial-contaminated semen doses are used for artificial insemination. The purpose of this study was to determine the bacterial communities within freshly extended boar semen and associate specific bacterial communities with sperm quality measurements. Current findings suggest that certain bacteria can accumulate within a group of animals or over a short period of time which can impact sperm cell characteristics. Having less diverse bacterial communities also appears to be associated with favorable semen quality. Future research is needed to determine the interactions different bacterial communities have with sperm cells to characterize their nature as pathogenic or commensal.

Understanding how different surfaces and environmental biofilms found in food processing plants affect the spread of COVID-19.

Meat processing plants have been at the center of the SARS-CoV-2 pandemic, with a recent report citing 90% of US facilities having multiple outbreaks during 2020 and 2021. We explored the potential for biofilms to act as a reservoir in protecting, harboring, and dispersing SARS-CoV-2 throughout the meat processing facility environment. To do this, we used Murine Hepatitis Virus (MHV), as a surrogate for SARS-CoV-2, and meat processing facility drain samples to develop mixed-species biofilms on materials found in meat processing facilities (stainless steel (SS), PVC, and ceramic tiles). After exposure to the biofilm organisms for five days post-inoculation at 7°C we conducted quantitative PCR (qPCR) and plaque assays to determine whether MHV could remain both detectable and viable. Our data provides evidence that coronaviruses can remain viable on all the surfaces tested and are also able to integrate within an environmental biofilm. Although a portion of MHV was able to remain infectious after incubation with the environmental biofilm, a large reduction in plaque numbers was identified when compared with the viral inoculum incubated without biofilm on all test surfaces, which ranged from 6.45-9.27-fold higher. Interestingly, we observed a 2-fold increase in the virus-environmental biofilm biovolume when compared to biofilm without virus, indicating that the biofilm bacteria both detected and reacted to the virus. These results indicate a complex virus-environmental biofilm interaction. Although we observed better survival of MHV on a variety of surfaces commonly found in meat processing plants alone than with the biofilm, there is the potential for biofilms to protect virions from disinfecting agents, which has implications for the potential of SARS-CoV-2 prevalence within the meat processing plant environment. Also given the highly infectious nature of SARS-CoV-2, particularly for some of the variant strains such as omicron, having even a residual level of virus present represents a serious health hazard. The increase in biofilm biovolume in response to virus is also a concern for food safety due to the potential of the same being seen with organisms associated with food poisoning and food spoilage.

Murine Hepatitis Virus, a Biosafety Level 2 Model for SARS-CoV-2, Can Remain Viable on Meat and Meat Packaging Materials for at Least 48 Hours.

In 2020 and 2021, many meat processing plants faced temporary closures due to outbreaks of COVID-19 cases among the workers. There are several factors that could potentially contribute to the increased numbers of COVID-19 cases in meat processing plants: the survival of viable SARS-CoV-2 on meat and meat packaging materials, difficulties in maintaining workplace physical distancing, personal hygiene, and crowded living and transportation conditions. In this study, we used murine hepatitis virus (MHV) as a biosafety level 2 (BSL2) surrogate for SARS-CoV-2 to determine viral survival on the surface of meat, namely, stew-cut beef and ground beef, and commonly used meat packaging materials, such as plastic wrap, meat-absorbent material, and Styrofoam. From our studies, we observed the infectivity of MHV inoculated on ground beef and stew-cut beef for 48 h and saw no significant loss in infectivity for MHV from 0 to 6 h postinoculation (hpi) (unpaired t test). However, beginning at 9 hpi, viral infectivity steadily decreased, resulting in a 1.12-log reduction for ground beef and a 0.46-log reduction for stew-cut beef by 48 hpi. We also observed a significant persistence of MHV on meat packaging materials, with Styrofoam supporting the highest viability (3.25 × 103 ± 9.57 × 102 PFU/mL, a 0.91-log reduction after 48 hpi), followed by meat-absorbent material (75 ± 50 PFU/mL, a 1.10-log reduction after 48 hpi), and lastly, plastic wrap (no detectable PFU after 3 hpi, a 3.12-log reduction). Despite a notable reduction in infectivity, the virus was able to survive and remain infectious for up to 48 h at 7°C on four of the five test surfaces. Our results provide evidence that coronaviruses, such as SARS-CoV-2, could potentially survive on meat, meat-absorbent materials. and Styrofoam for up to 2 days, and potentially longer. IMPORTANCE The meat industry has been faced with astronomical challenges with the rampant spread of COVID-19 among meat processing plant workers. This has resulted in meat processing and packaging plant closures, creating bottlenecks everywhere in the chain, from farms to consumers, subsequently leading to much smaller production outputs and higher prices for all parties involved. This study tested the viability of meat and meat packaging materials as potential reservoirs for SARS-CoV-2, allowing the virus to survive and potentially spread among the workers. We used murine hepatitis virus (MHV) as a biosafety level 2 (BSL2) surrogate for SARS-CoV-2. Our results suggest that ground beef, stew-cut beef, meat-absorbent material, and Styrofoam can harbor coronavirus particles, which can remain viable for at least 48 h. Furthermore, our study provides evidence that the environmental and physical conditions within meat processing facilities can facilitate the survival of viable virus.

Biofilm through the Looking Glass: A Microbial Food Safety Perspective.

Food-processing facilities harbor a wide diversity of microorganisms that persist and interact in multispecies biofilms, which could provide an ecological niche for pathogens to better colonize and gain tolerance against sanitization. Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Biofilms are formed in an environment through synergistic interactions within the microbial community through mutual adaptive response to their long-term coexistence. Mixed-species biofilms are more tolerant to sanitizers than single-species biofilms or their planktonic equivalents. Hence, there is a need to explore how multispecies biofilms help in protecting the foodborne pathogen from common sanitizers and disseminate biofilm cells from hotspots and contaminate food products. This knowledge will help in designing microbial interventions to mitigate foodborne pathogens in the processing environment. As the global need for safe, high-quality, and nutritious food increases, it is vital to study foodborne pathogen behavior and engineer new interventions that safeguard food from contamination with pathogens. This review focuses on the potential food safety issues associated with biofilms in the food-processing environment.

Impact of mixed biofilm formation with environmental microorganisms on E. coli O157:H7 survival against sanitization.

Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Meat processing plants may harbor various microorganisms and occasional foodborne pathogens; thus, the environmental microbial community might impact pathogen survival via mixed biofilm formation. We collected floor drain samples from two beef plants with different E. coli O157:H7 prevalence history and investigated the effects of the environmental microorganisms on pathogen sanitizer tolerance. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the plants and drain locations. E. coli O157:H7 cells obtained significantly higher sanitizer tolerance in mixed biofilms by samples from the plant with recurrent E. coli O157:H7 prevalence than those mixed with samples from the other plant. The mixed biofilm that best protected E. coli O157:H7 also had the highest species diversity. The percentages of the species were altered significantly after sanitization, suggesting that the community composition affects the role and tolerance level of each individual species. Therefore, the unique environmental microbial community, their ability to form biofilms on contact surfaces and the interspecies interactions all play roles in E. coli O157:H7 persistence by either enhancing or reducing pathogen survival within the biofilm community.