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.

Gene Switching and Essentiality Testing.

The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described which can be used to confirm gene essentiality using gene switching, following the construction of a strain carrying its only functional copy on an integrated plasmid (Δ'int). Since deletion mutants cannot be created for essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal gene copy to be deleted. The integrated vector can then be replaced using the gene switching method, where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm functionality of gene homologs and to easily identify essential operon members.

Whole-Genome Sequencing of Mycobacterium tuberculosis Directly from Sputum Samples.

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing pathogens, such as Mycobacterium tuberculosis. However, clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a nonmutagenic approach, we can capture all known variations found within M. tuberculosis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of M. tuberculosis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Whole-Genome Sequencing of Chlamydia trachomatis Directly from Human Samples.

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing, or obligate intracellular pathogens, such as Chlamydia trachomatis. However clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a nonmutagenic approach, we can capture all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

High-Throughput Screening for Novel Inhibitors of Intracellular Pathogens, Including Chlamydia trachomatis.

High-throughput drug screening (HTS) is a powerful tool that can be used rapidly to identify new potential bacterial inhibitors and/or compounds which enhance host cell control of pathogens, which can then go on to be developed as novel therapeutics. Typically screening is commonly done in artificial culture medium; however, obligate intracellular pathogens, such as Chlamydia trachomatis, cannot be tested this way. Intracellular screening methods allow for such pathogens to undergo HTS, while still giving reliable and consistent data. Plus, as well as identifying new potential bacterial inhibitors, it is also able to detect compounds which enhance host cell control of pathogens, to allow for host-directed therapies to be developed.

Whole-Genome Enrichment Using RNA Probes and Sequencing of Chlamydia trachomatis Directly from Clinical Samples.

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow-growing, or obligate intracellular pathogens, such as Chlamydia trachomatis. However clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a non-mutagenic approach, we can capture all known variations found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

LytB1 and LytB2 of Mycobacterium tuberculosis Are Not Genetically Redundant.

Mycobacterium tuberculosis synthesises isoprenoid precursors via the MEP/DOXP pathway and at least five enzymes in the pathway (Dxs1, Dxr/IspC, IspD, IspF, and GcpE/IspG) are required for growth in vitro. We investigated the role of LytB (IspH) in M. tuberculosis; M. tuberculosis is unusual in that it has two homologs-LytB1 and LytB2. We were unable to delete the lytB2 gene unless we provided an additional copy elsewhere, demonstrating that this is the essential homolog. We expressed lytB1 from the lytB2 promoter and confirmed that this could not complement for loss of function of lytB2, despite LytB1 possessing all the previously described conserved critical residues. Interestingly the sole LytB homolog of Mycobacterium smegmatis was able to compensate for loss of LytB2 in M. tuberculosis. We tested translational fusions of LytB1 and LytB2 for functionality in M. tuberculosis, but only a fusion with 90% N-terminal LytB2 and 10% C-terminal LytB1 was functional. In order to identify the key difference between the two proteins, site directed mutagenesis was used to change LytB2 residues into their counterparts in LytB1. None of these amino acid substitutions was essential for function and all lytB2 mutant alleles were functional. In contrast, mutation of the key residues for [Fe4S4] cluster formation, as well as a catalytic residue in LytB1 did not result in functional complementation. Thus, although LytB1 and LytB2 are not genetically redundant, this is not dependent on small amino acid changes, but is likely to be a result of major overall structural differences.

Gene switching and essentiality testing.

The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described that can be used to confirm gene essentiality using gene switching, following the construction of a delinquent strain. Because deletion mutants cannot be created for essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal gene copy to be deleted. The integrated vector can then be replaced using the gene switching method; where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm functionality of gene homologues and to easily identify essential operon members.