Microwave-Generated Steam Decontamination of N95 Respirators Utilizing Universally Accessible Materials.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a severe, international shortage of N95 respirators, which are essential to protect health care providers from infection. Given the contemporary limitations of the supply chain, it is imperative to identify effective means of decontaminating, reusing, and thereby conserving N95 respirator stockpiles. To be effective, decontamination must result in sterilization of the N95 respirator without impairment of respirator filtration or user fit. Although numerous methods of N95 decontamination exist, none are universally accessible. In this work, we describe a microwave-generated steam decontamination protocol for N95 respirators for use in health care systems of all sizes, geographies, and means. Using widely available glass containers, mesh from commercial produce bags, a rubber band, and a 1,100-W commercially available microwave, we constructed an effective, standardized, and reproducible means of decontaminating N95 respirators. Employing this methodology against MS2 phage, a highly conservative surrogate for SARS-CoV-2 contamination, we report an average 6-log10 plaque-forming unit (PFU) (99.9999%) and a minimum 5-log10 PFU (99.999%) reduction after a single 3-min microwave treatment. Notably, quantified respirator fit and function were preserved, even after 20 sequential cycles of microwave steam decontamination. This method provides a valuable means of effective decontamination and reuse of N95 respirators by frontline providers facing urgent need.IMPORTANCE Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an increasing shortage of protective gear necessary to keep health care providers safe from infection. As of 9 April 2020, the CDC reported 9,282 cumulative cases of COVID-19 among U.S. health care workers (CDC COVID-19 Response Team, MMWR Morb Mortal Wkly Rep 69:477-481, 2020, https://doi.org/10.15585/mmwr.mm6915e6). N95 respirators are recommended by the CDC as the ideal method of protection from COVID-19. Although N95 respirators are traditionally single use, the shortages have necessitated the need for reuse. Effective methods of N95 decontamination that do not affect the fit or filtration ability of N95 respirators are essential. Numerous methods of N95 decontamination exist; however, none are universally accessible. In this study, we describe an effective, standardized, and reproducible means of decontaminating N95 respirators using widely available materials. The N95 decontamination method described in this work will provide a valuable resource for hospitals, health care centers, and outpatient practices that are experiencing increasing shortages of N95 respirators due to the COVID-19 pandemic.

Efficacy of pyrazinoic acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis.

New therapeutic strategies are needed to treat drug resistant tuberculosis (TB) and to improve treatment for drug sensitive TB. Pyrazinamide (PZA) is a critical component of current first-line TB therapy. However, the rise in PZA-resistant TB cases jeopardizes the future utility of PZA. To address this problem, we used the guinea pig model of TB and tested the efficacy of an inhaled dry powder combination, referred to as Pyrazinoic acid/ester Dry Powder (PDP), which is comprised of pyrazinoic acid (POA), the active moiety of PZA, and pyrazinoic acid ester (PAE), which is a PZA analog. Both POA and PAE have the advantage of being able to act on PZA-resistant Mycobacterium tuberculosis. When used in combination with oral rifampicin (R), inhaled PDP had striking effects on tissue pathology. Effects were observed in lungs, the site of delivery, but also in the spleen and liver indicating both local and systemic effects of inhaled PDP. Tissue granulomas that harbor M. tuberculosis in a persistent state are a hallmark of TB and they pose a challenge for therapy. Compared to other treatments, which preferentially cleared non-necrotic granulomas, R+PDP reduced necrotic granulomas more effectively. The increased ability of R+PDP to act on more recalcitrant necrotic granulomas suggests a novel mechanism of action. The results presented in this report reveal the potential for developing therapies involving POA that are optimized to target necrotic as well as non-necrotic granulomas as a means of achieving more complete sterilization of M. tuberculosis bacilli and preventing disease relapse when therapy ends.

The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation.

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

The Sec Pathways and Exportomes of Mycobacterium tuberculosis.

All bacteria utilize pathways to export proteins from the cytoplasm to the bacterial cell envelope or extracellular space. Many exported proteins function in essential physiological processes or in virulence. Consequently, the responsible protein export pathways are commonly essential and/or are important for pathogenesis. The general Sec protein export pathway is conserved and essential in all bacteria, and it is responsible for most protein export. The energy for Sec export is provided by the SecA ATPase. Mycobacteria and some Gram-positive bacteria have two SecA paralogs: SecA1 and SecA2. SecA1 is essential and works with the canonical Sec pathway to perform the bulk of protein export. The nonessential SecA2 exports a smaller subset of proteins and is required for the virulence of pathogens such as Mycobacterium tuberculosis. In this article, we review our current understanding of the mechanism of the SecA1 and SecA2 export pathways and discuss some of their better-studied exported substrates. We focus on proteins with established functions in M. tuberculosis pathogenesis and proteins that suggest potential roles for SecA1 and SecA2 in M. tuberculosis dormancy.

Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall.

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology.