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).

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.

Kupyaphores are zinc homeostatic metallophores required for colonization of .

() endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and iron-chelating siderophores, mycobactin, have been known since 1965.

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.

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.

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.

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.

The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

(Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone.

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.

Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains.

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes.

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections.

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.

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis.

The geographic distribution and economic value of climate change-related ozone health impacts in the United States in 2030.

Unlabelled: In this United States-focused analysis we use outputs from two general circulation models (GCMs) driven by different greenhouse gas forcing scenarios as inputs to regional climate and chemical transport models to investigate potential changes in near-term U.S. air quality due to climate change.

Mycobacterium tuberculosis H37Rv has a single nucleotide polymorphism in PhoR which affects cell wall hydrophobicity and gene expression.

Mycobacterium tuberculosis is a successful pathogen that can adapt to multiple environmental niches. As part of its repertoire of adaptive responses, two-component regulatory systems play a major role in co-ordinating gene expression at the global level. The PhoPR system controls major cellular functions, including respiration, lipid metabolism, the immediate and enduring hypoxic responses, stress responses and persistence.

Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

Background: Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus.

Mycobacterium tuberculosis ClpP proteases are co-transcribed but exhibit different substrate specificities.

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions.

Deletion of SenX3-RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions.

Two component regulatory systems are key elements in the control of bacterial gene expression in response to environmental perturbations. The SenX3-RegX3 system is implicated in the control of phosphate uptake in Mycobacterium smegmatis and Mycobacterium tuberculosis. regX3 is reported to be essential in M.

The dUTPase enzyme is essential in Mycobacterium smegmatis.

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g.

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8).

The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally regulated by Dxs.

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1.

Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.

Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized.

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.

Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake.

Unlabelled: Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme 1, mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT).