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

Abstract

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.

Fig 1. CFU counts from biofilm + MHV and biofilm samples on stainless steel, PVC, and tile chips.

(A-C) CFU counts for biofilm + MHV and biofilm samples on (A) stainless steel, (B) PVC, and (C) ceramic tile chips. Each sample was plated in duplicate. Results in this figure are the mean values and standard deviations (error bars) from three independent experiments. Statistical significance was analyzed by unpaired t-test. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Fig 2. qPCR analysis of MHV mixed with biofilm and pre-incubated for 5 days on stainless steel, PVC, and ceramic tile chips.

(A-C) qPCR analysis of MHV mixed with environmental biofilm on stainless steel, PVC and on tile chips. 1.0 x 10³ PFU of MHV were added to a SS, PVC, or ceramic tile chip along with a floor drain biofilm sample collected from the cooler of a single meat processing plant drain. The qPCR samples were analyzed in quadruplicate. Gene copy numbers were calculated from a standard curve of known quantities of MHV RNA in a 25 μL qPCR reaction. Results in this figure are the mean values and standard deviations (error bars) from two independent experiments. Statistical significance was analyzed by unpaired t-test. ns: not significant; *: p < 0.05; ***: p < 0.001, ****: p < 0.0001.

Fig 3. Plaque assay results from biofilm + virus and virus samples on stainless steel, PVC, and ceramic tile chips.

(A-C) Results from plaque assays on samples collected from (A) stainless steel, (B) PVC, and (C) tile chips. Each sample was filtered through a 0.2 μm filter and pipetted onto L2 cells in duplicate. Results in this figure are the mean values and standard deviations (error bars) from two independent experiments. Statistical significance was analyzed by unpaired t-test. *: p < 0.05; **: p < 0.01.

Fig 4. Schematic representation of floor drain biofilm and virus experiment.

(A and B): Experimental set up with Biofilm + Virus, Biofilm, Virus, and Negative Control in duplicate. The experimental set is incubated at 7°C for 5 days. (C). After 5 days, the biofilm was harvested from SS, PVC, or tile chips using a cell lifter and forceps and rinsed with 1 mL of LB-NS. (D) Harvested cells were stored in a screw-cap tube at -80°C until assayed.