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Tuberculosis (TB) is the archetypical chronic infection, with patients having months of symptoms before diagnosis. In the two years after successful therapy, survivors of TB have a three-fold increased risk of death.Guinea pigs were infected withGuinea pigs with TB demonstrated DNA hypermethylation and showed system-level similarity to humans with TB (In both guinea pigs and humans, TB perturbs epigenetic processes, promoting premature cellular aging and inflammation, a plausible means to explain the long-term detrimental health outcomes after TB.

Background Bacterial pathogen (Pseudomonas aeruginosa) could form biofilm that conveys multi-drug resistance. Bacteriophage as an alternative to antibacterial resistance is useful against biofilm complications. This study evaluated antibacterial and biofilm removal activities of lytic phage, specific against multi-drug-resistant clinical P. aeruginosa. Results The phage showed a wide range of pH (5–10) and heat (7–44 °C) stability. Electron microscopy showed ɸPauNE1 phage head (60 nm in diameter) and non-contractile tail (12 nm in length by 8 nm in width); hence, the family Podoviridae and the order Caudovirales. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed structured protein of 55 kDa and double-stranded DNA of 45 kb. The phage was species specific and had broad host range activity. It inhibited bacterial growth at multiplicity of infection (MOI) 1–0.000001 pfu/ml. Inhibition was maximal at both low (1 × 105) and high (1 × 109) bacterial CFU/ml. Biofilm removal test showed that the phage removed more than 60% cell biomass within CFU/ml of 1.5 × 108, 6.0 × 108 and l.0 × 109. Conclusion Phage (ɸPauNE1) was unique and had broad host range activity. The phage exhibited strong bacteriolytic activity against biofilm forming multi-drug-resistant strains. It had no lytic effect on the heterogeneous strains and so a promising bioagent.

Contains fulltext : 245732.pdf (Publisher’s version ) (Closed access) There is hope that host-directed therapy (HDT) for Tuberculosis (TB) can either shorten treatment duration, help cure drug resistant disease or limit the immunopathology. Many candidate HDT drugs have been proposed, however solid evidence only exists for a few select patient groups. The clinical presentation of TB is variable, with differences in severity, tissue pathology, and bacillary burden. TB clinical phenotypes likely determine the potential benefit of HDT. Underlying TB clinical phenotypes, there are TB "endotypes," defined as distinct molecular profiles, with specific metabolic, epigenetic, transcriptional, and immune phenotypes. TB endotypes can be characterized by either immunodeficiency or pathologic excessive inflammation. Additional factors, like comorbidities (HIV, diabetes, helminth infection), structural lung disease or Mycobacterial virulence also drive TB endotypes. Precise disease phenotyping, combined with in-depth immunologic and molecular profiling and multimodal omics integration, can identify TB endotypes, guide endotype-specific HDT, and improve TB outcomes, similar to advances in cancer medicine.

Bacillus Calmette–Guérin (BCG) is the most widely used vaccine worldwide and has been used to prevent tuberculosis for a century. BCG also stimulates an anti-tumour immune response, which urologists have harnessed for the treatment of non-muscle-invasive bladder cancer. A growing body of evidence indicates that BCG offers protection against various non-mycobacterial and viral infections. The non-specific effects of BCG occur via the induction of trained immunity and form the basis for the hypothesis that BCG vaccination could be used to protect against the severity of coronavirus disease 2019 (COVID-19). This Perspective article highlights key milestones in the 100-year history of BCG and projects its potential role in the COVID-19 pandemic., Bacillus Calmette–Guérin (BCG) has been used to prevent tuberculosis for a century and is also a standard approach for the treatment of non-muscle-invasive bladder cancer. However, BCG also has a plethora of non-specific effects that occur via the induction of trained immunity and have raised the hypothesis that BCG vaccination could be used to protect against coronavirus disease 2019 (COVID-19). In this Perspective, the authors describe the history of BCG, discuss the mechanisms of its effects, and consider its potential role during the COVID-19 pandemic.

Dr Deborah Birx, the White House Coronavirus Task Force coordinator, told NBC News on "Meet the Press"that "[T]he U S needs a 'breakthrough' in coronavirus testing to help screen Americans and get a more accurate picture of the virus' spread "We have been involved with biopathogen detection since the 2001 anthrax attacks and were the first to detect anthrax in real-time A variation on the laser spectroscopic techniques we developed for the rapid detection of anthrax can be applied to detect the Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2 virus) In addition to detecting a single virus, this technique allows us to read its surface protein structure In particular, we have been conducting research based on a variety of quantum optical approaches aimed at improving our ability to detect Corona Virus Disease-2019 (COVID-19) viral infection Indeed, the detection of a small concentration of antibodies, after an infection has passed, is a challenging problem Likewise, the early detection of disease, even before a detectible antibody population has been established, is very important Our team is researching both aspects of this problem The paper is written to stimulate the interest of both physical and biological scientists in this important problem It is thus written as a combination of tutorial (review) and future work (preview) We join Prof Federico Capasso and Editor Dennis Couwenberg in expressing our appreciation to all those working so heroically on all aspects of the COVID-19 problem And we thank Drs Capasso and Couwenberg for their invitation to write this paper © 2020 Navid Rajil et al , published by De Gruyter, Berlin/Boston 2020

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly- N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (

COVID-19 is characterized by hyperactivation by inflammatory cytokines and recruitment of macrophages, neutrophils, and other immune cells, all hallmarks of a strong inflammatory response that can lead to severe complications and multi-organ damage. Mortality in COVID-19 patients is associated with a high prevalence of neutrophil extracellular trap (NET) formation and microthrombosis that are exacerbated by hyperglycemia, diabetes, and old age. SARS-CoV-2 infection in humans and non-human primates have revealed long-term neurological consequences of COVID-19, possibly concomitant with the formation of Lewy bodies in the brain and invasion of the nervous system via the olfactory bulb. In this paper, we review the relevance of the human cathelicidin LL-37 in SARS-CoV-2 infections. LL-37 is an immunomodulatory, host defense peptide with direct anti-SARS-CoV-2 activity, and pleiotropic effects on the inflammatory response, neovascularization, Lewy body formation, and pancreatic islet cell function. The bioactive form of vitamin D and a number of other compounds induce LL-37 expression and one might predict its upregulation, could reduce the prevalence of severe COVID-19. We hypothesize upregulation of LL-37 will act therapeutically, facilitating efficient NET clearance by macrophages, speeding endothelial repair after inflammatory tissue damage, preventing α-synuclein aggregation, and supporting blood-glucose level stabilization by facilitating insulin release and islet β-cell neogenesis. In addition, it has been postulated that LL-37 can directly bind the S1 domain of SARS-CoV-2, mask angiotensin converting enzyme 2 (ACE2) receptors, and limit SARS-CoV-2 infection. Purposeful upregulation of LL-37 could also serve as a preventative and therapeutic strategy for SARS-CoV-2 infections.

Tuberculosis (TB) is among the greatest public health and safety concerns in the 21(st) century, Mycobacterium tuberculosis, which causes TB, infects alveolar macrophages and uses these cells as one of its primary sites of replication. The current TB treatment regimen, which consist of chemotherapy involving a combination of 3-4 antimicrobials for a duration of 6-12 months, is marked with significant side effects, toxicity, and poor compliance. Targeted drug delivery offers a strategy that could overcome many of the problems of current TB treatment by specifically targeting infected macrophages. Recent advances in nanotechnology and material science have opened an avenue to explore drug carriers that actively and passively target macrophages. This approach can increase the drug penetration into macrophages by using ligands on the nanocarrier that interact with specific receptors for macrophages. This review encompasses the recent development of drug carriers specifically targeting macrophages actively and passively. Future directions and challenges associated with development of effective TB treatment is also discussed.

Most phages of Gram-negative bacteria hosts encode spanins for disruption of the outer membrane, which is the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the outer membrane (OM). Here, we show that the T7-like coliphage phiKT causes an explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in Escherichia coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein before lysis. The protein gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similarly to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and, thus, establishes a new class of phage lysis proteins, the disruptins. IMPORTANCE We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

BACKGROUNDThe objective is to determine the impact of the Bacillus Calmette-Guérin (BCG) vaccine compared to placebo or no vaccine on COVID-19 infections and hospitalisations in healthcare workers. We are using a living and prospective approach to Individual-Participant-Data (IPD) meta-analysis of ongoing studies based on the Anytime Live and Leading Interim (ALL-IN) meta-analysis statistical methodology.METHODSPlanned and ongoing randomised controlled trials were identified from trial registries and by snowballing (final elicitation: Oct 3 2022). The methodology was specified prospectively – with no trial results available – for trial inclusion as well as statistical analysis. Inclusion decisions were made collaboratively based on a risk-of-bias assessment by an external protocol review committee (Cochrane risk-of-bias tool adjusted for use on protocols), expected homogeneity in treatment effect, and agreement with the predetermined event definitions. The co-primary endpoints were incidence of COVID-19 infection and COVID-19-related hospital admission. Accumulating IPD from included trials was analysed sequentially using the exacte-value logrank test (at level α = 0.5% for infections and level α = 4.5% for hospitalisations) and anytime-valid 95%-confidence intervals (CIs) for the hazard ratio (HR) for a predetermined fixed-effects approach to meta-analysis (no measures of statistical heterogeneity). Infections were included if demonstrated by PCR tests, antigen tests or suggestive lung CTs. Participants were censored at date of first COVID-19-specific vaccination and two-stage analyses were performed in calendar time, with a stratification factor per trial.RESULTSSix trials were included in the primary analysis with 4 433 participants in total. Thee-values showed no evidence of a favourable effect of minimal clinically relevance (HR < 0.8) in comparison to the null (HR = 1) for COVID-19 infections, nor for COVID-19 hospitalisations (HR < 0.7 vs HR = 1). COVID-19 infection was observed in 251 participants receiving BCG and 244 participants not receiving BCG, HR 1.02 (anytime-valid 95%-CI 0.78-1.35). COVID-19 hospitalisations were observed in 13 participants receiving BCG and 7 not receiving BCG, resulting in an uninformative estimate (HR 1.88; anytime-valid 95%-CI 0.26-13.40).DISCUSSIONIt is highly unlikely that BCG has a clinically relevant effect on COVID-19 infections in healthcare workers. With only limited observations, no conclusion could be drawn for COVID-19 related hospitalisation. Due to the nature of ALL-IN meta-analysis, emerging data from new trials can be included without violating type-I error rates or interval coverage. We intend to keep this meta-analysis alive and up-to-date, as more trials report. For COVID-19 related hospitalisations, we do not expect enough future observations for a meaningful analysis. For BCG-mediated protection against COVID-19 infections, on the other hand, more observations could lead to a more precise estimate that concludes the meta-analysis for futility, meaning that the current interval excludes the HR of 0.8 predetermined as effect size of minimal clinical relevance.OTHERNo external funding. Preregistered at PROSPERO: CRD42021213069.

Soon after commencement of the SARS-CoV-2 disease outbreak of 2019 (COVID-19), it became evident that the receptor-binding domain of the viral spike protein is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. This study addresses the relative lack of information regarding actual antibody concentrations in convalescent plasma samples from COVID-19 patients and extends these analyses to post-vaccination samples to estimate protective IgG antibody (Ab) levels. Both sample populations were similar and a protective Ab level of 7.5 µg/ml was determined, based on 95% of the normal distribution of the post-vaccination population. The results of this study have implications for future vaccine development, projection of protective efficacy duration, and understanding of the immune response to SARS-CoV-2 infection.One-Sentence SummaryUsing two quantitative immunoassays, we have found similar IgG antibody responses to the SARS-CoV-2 spike protein in populations of COVID-19 survivors and vaccine recipients that indicate a protective antibody concentration.

Bacille Calmette–Guérin (BCG) vaccination, widely used throughout the world to protect against infant tuberculous meningitis and miliary tuberculosis (TB), can provide broad non-specific protection against infectious respiratory diseases in certain groups. Interest in BCG has seen a resurgence within the scientific community as the mechanisms for non-specific protection have begun to be elucidated. The impact of the COVID-19 pandemic on nearly every aspect of society has profoundly illustrated the pressure that respiratory infections can place on a national healthcare system, further renewing interest in BCG vaccination as a public health policy to reduce the burden of those illnesses. However, the United States does not recommend BCG vaccination due to its variable effectiveness against adult TB, the relatively low risk of Mycobacterium tuberculosis infection in most of the United States, and the vaccine’s interference with tuberculin skin test reactivity that complicates TB screening. In this review, we explore the broad immune training effects of BCG vaccination and literature on the effects of BCG vaccination on COVID-19 spread, disease severity, and mortality. We further discuss barriers to scheduled BCG vaccination in the United States and how those barriers could potentially be overcome.

Aims: To demonstrate the efficacy of aromatic hydrazide compounds to inhibit growth of Mycobacterium tuberculosis. Study Design: To synthesize tuberculostats and test their antibacterial activity in-vitro. Place and Duration of Study: University of Nebraska, Durham Science Center, 6001 Dodge Street, Omaha NE 68182, and Texas A&M Health Science Center, Department of Microbial Pathogenesis and Immunology, 8447 State Hwy 47, Medical Research and Education Building, Room #3012, Bryan, TX 7780. From March 2019 to October 2019. Methodology: Hydrazide functional groups were formed by covalently bonding hydrazine onto a carbonyl carbon that is a substituent of a single aromatic ring. Microwave excitation was utilized for synthesis, followed by evaluation of antibacterial activity. These compounds were placed into tissue culture media at various concentrations and then Mycobacterium tuberculosis bacteria was added, in order to determine the level of growth inhibition. Growth inhibition of the bacteria was measured as a function of compound concentration versus growth inhibition. Results: Compounds A, B, C, and D carry hydrazide groups as a substituent to a single aromatic ring. All four compounds show zero violations of Rule of 5, indicating favorable drug-likeness. All four compounds showed greater than 50% growth inhibition of bacteria at concentrations below 50 micrograms per milliliter. Growth inhibition was measured by colony forming units and luminescence. Polar surface area, Log P, molecular volume, and other molecular properties were determined for these four compounds. Conclusion: These four hydrazide compounds induced substantial inhibition of bacterial growth. Microwave excitation for the synthesis of hydrazide compounds is effective. These compounds have favorable drug-likeness properties and are highly effective inhibiting the growth of Mycobacterium tuberculosis.

Multidrug resistance in pathogenic bacteria is an increasing problem in patient care and public health. Molecular nanomachines (MNMs) have the ability to open cell membranes using nanomechanical action. We hypothesized that MNMs could be used as antibacterial agents by drilling into bacterial cell walls and increasing susceptibility of drug-resistant bacteria to recently ineffective antibiotics. We exposed extensively drug-resistant Klebsiella pneumoniae to light-activated MNMs and found that MNMs increase the susceptibility to Meropenem. MNMs with Meropenem can effectively kill K. pneumoniae that are considered Meropenem-resistant. We examined the mechanisms of MNM action using permeability assays and transmission electron microscopy, finding that MNMs disrupt the cell wall of extensively drug-resistant K. pneumoniae, exposing the bacteria to Meropenem. These observations suggest that MNMs could be used to make conventional antibiotics more efficacious against multi-drug-resistant pathogens.

BackgroundLegionella can cause Legionnaires’ disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100.MethodsWe evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs.ResultsWe found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death.ConclusionsThese observations suggest a mechanism of Legionnaires’ disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.

The human immunodeficiency virus (HIV) pandemic is driving the re-emergence of tuberculosis (TB) as a global health threat, both by increasing the susceptibility of HIV-infected people to infection with Mycobacterium tuberculosis (Mtb), and increasing the rate of emergence of drug-resistant Mtb. There are several other clinical challenges for treatment of co-infected patients including: expense, pill burden, toxicity, and malabsorption that further necessitate the search for new drugs that may be effective against both pathogens simultaneously. The anti-helminthic niclosamide has been shown to have activity against a laboratory strain of Mtb in liquid culture while bacteriostatic activity against non-replicating M. abscessus was also recently described. Here we extend these findings to further demonstrate that niclosamide inhibits mycobacterial growth in infected human macrophages and mediates potent bacteriostatic activity against the virulent Mtb Beijing strain. Importantly, we provide the first evidence that niclosamide inhibits HIV replication in human macrophages and Jurkat T cells through post-integration effects on pro-virus transcription. The dual antiviral and anti-mycobacterial activity was further observed in an in vitro model of HIV and Mtb co-infection using human primary monocyte-derived macrophages. These results support further investigation of niclosamide and derivatives as anti-retroviral/anti-mycobacterial agents that may reduce clinical challenges associated with multi-drug regimens and drug resistance.

In the SARS-CoV-2 coronavirus pandemic of 2019 (COVID-19), it has become evident that the ACE-2 receptor-binding domain (RBD) of the viral spike protein (SP) is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. The most definitive confirmation of this contention is that the two mRNA COVID-19 vaccines in general use, which elicit antibodies specific for the RBD, exhibit approximately 95% protective efficacy against COVID-19. A potential challenge to vaccine efficacy is the emergence of SARS-CoV-2 variants possessing multiple mutations affecting amino acid residues in the RBD. Of concern are variants that arose in the United Kingdom, Brazil and South Africa. One of the variants, designated B.1.351, has shown a higher transmissibility due to greater affinity for the ACE-2 receptor and decreased neutralization by convalescent plasma, therapeutic monoclonal antibodies, and post-vaccination plasma. Common to several of the variants is the N501Y mutation in the RBD, which may be responsible for at least part of the observed variant properties. To test this hypothesis, we measured the ability of the Y501 RBD to inhibit binding of the wild type RBD and full SP (S1 + S2) to the ACE-2 protein and a human monoclonal IgG antibody elicited to the wild type RBD, relative to the wild type RBD in two enzyme-linked immunosorbent assays (ELISAs). We found no significant difference in the IC50 of the two RBD species’ inhibition of ACE-2 binding, but unexpectedly found that the IC50 of the wild type RBD inhibition of antibody binding was nearly twice that of the Y501 RBD, reflecting a lower affinity. These results suggest that the individual N501Y mutation does not contribute to altered viral properties by itself, but may contribute to a collective conformational shift produced by multiple mutations.

In vitro, animal model and anecdotal clinical evidence suggests that tuberculosis (TB) is not a monomorphic disease, and that host response to TB is protean with multiple distinct molecular pathways and pathologies (endotypes). We used unbiased clustering to identify separate TB endotypes with distinctive gene expression patterns and different clinical outcomes. A discovery cohort that included 443 TB patients and 527 asymptomatic controls identified two TB endotypes exhibiting distinct gene expression in proliferation, metabolism, and immune pathways. Specifically, TB patient endotype A was characterized by increased expression of genes related to inflammation and immunity and decreased metabolism and proliferation; in contrast, endotype B expressed increased pathways related to metabolism and proliferation. The endotype signatures were validated using an independent RNA-seq validation cohort consisting of two studies that included 118 TB patients and 208 controls. A second validation cohort confirmed differential pathway activity between TB endotypes and demonstrated that TB patients in endotype A had slower time to culture conversion, increased risk of death, and increased risk of poor clinical outcomes. These observations suggest that endotypes reflect different functional immunity; this postulate was further supported by the observation that TB patients with the hyperinflammatory endotype showed hyporesponsive cytokine and chemokine responses. Collectively, these findings provide further evidence that metabolic and immune profiling could inform and optimize targeted endotype-specific therapies for TB.

Lateral flow assay (LFA) has long been used as a biomarker detection technique. It has advantages such as low cost, rapid readout, portability, and ease of use. However, its qualitative readout process and lack of sensitivity are limiting factors. We report a photon-counting approach to accurately quantify LFAs while enhancing sensitivity. In particular, we demonstrate that the density of SARS-CoV-2 antibodies can be quantified and measured with an enhanced sensitivity using this simple laser optical analysis.

Nontyphoidal Salmonella bacteria induce a classic inflammatory diarrhea by eliciting a large influx of neutrophils, producing a robust oxidative burst. Despite substantial progress understanding the benefits to the host of the inflammatory response to Salmonella, little is known regarding how Salmonella can simultaneously resist the damaging effects of the oxidative burst. The multidrug efflux pump MacAB is important for survival of oxidative stress both in vitro and during infection. We describe a new pathway used by Salmonella Typhimurium to detoxify extracellular reactive oxygen species using a multidrug efflux pump (MacAB) to secrete a linear siderophore, a metabolite of enterobactin. The natural substrates of many multidrug efflux pumps are unknown, and functional roles of the linear metabolites of enterobactin are unknown. We bring two novel discoveries together to highlight an important mechanism used by Salmonella to survive under the oxidative stress conditions that this organism encounters during the classic inflammatory diarrhea that it also induces., Nontyphoidal salmonellae (NTS) are exposed to reactive oxygen species (ROS) during their residency in the gut. To survive oxidative stress encountered during infection, salmonellae employ several mechanisms. One of these mechanisms involves the multidrug efflux pump MacAB, although the natural substrate of this pump has not been identified. MacAB homologs in pseudomonads secrete products of nonribosomal peptide synthesis (NRPS). In Salmonella enterica serovar Typhimurium, the siderophore enterobactin is produced by NRPS in response to iron starvation and this molecule can be processed into salmochelin and several linear metabolites. We found that Salmonella mutants lacking the key NRPS enzyme EntF are sensitive to peroxide mediated killing and cannot detoxify extracellular H2O2. Moreover, EntF and MacAB function in a common pathway to promote survival of Salmonella during oxidative stress. We further demonstrated that S. Typhimurium secretes siderophores in iron-rich media when peroxide is present and that these MacAB-secreted metabolites participate in protection of bacteria against H2O2. We showed that secretion of anti-H2O2 molecules is independent of the presence of the known siderophore efflux pumps EntS and IroC, well-described efflux systems involved in secretion of enterobactin and salmochelin. Both salmochelin and enterobactin are dispensable for S. Typhimurium protection against ROS; however, linear metabolites of enterobactin produced by esterases IroE and Fes are needed for bacterial survival in peroxide-containing media. We determined that linearized enterobactin trimer protects S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion. Thus, we suggest that linearized enterobactin trimer is a natural substrate of MacAB and that its purpose is to detoxify extracellular reactive oxygen species.

BackgroundCOVID-19 is a major pandemic that has killed more than 196,000 people. The COVID-19 disease course is strikingly divergent. Approximately 80-85% of patients experience mild or no symptoms, while the remainder develop severe disease. The mechanisms underlying these divergent outcomes are unclear. Emerging health disparities data regarding African American and homeless populations suggest that vitamin D insufficiency (VDI) may be an underlying driver of COVID-19 severity. To better define the VDI-COVID-19 link, we determined the prevalence of VDI among our COVID-19 intensive care unit (ICU) patients.MethodsIn an Institutional Review Board approved study performed at a single, tertiary care academic medical center, the medical records of COVID-19 patients were retrospectively reviewed. Subjects were included for whom serum 25-hydroxycholecalcifoerol (25OHD) levels were determined. COVID-19-relevant data were compiled and analyzed. We determined the frequency of VDI among COVID-19 patients to evaluate the likelihood of a VDI-COVID-19 relationship.ResultsTwenty COVID-19 patients with serum 25OHD levels were identified; 65.0% required ICU admission.The VDI prevalence in ICU patients was 84.6%, vs. 57.1% in floor patients. Strikingly, 100% of ICU patients less than 75 years old had VDI. Coagulopathy was present in 62.5% of ICU COVID-19 patients, and 92.3% were lymphocytopenic.ConclusionsVDI is highly prevalent in severe COVID-19 patients. VDI and severe COVID-19 share numerous associations including hypertension, obesity, male sex, advanced age, concentration in northern climates, coagulopathy, and immune dysfunction. Thus, we suggest that prospective, randomized controlled studies of VDI in COVID-19 patients are warranted.

Rhodococcus equi is an opportunistic, intracellular pathogen that causes pyogranulomatous pneumonia in foals and immunocompromised people. Currently, there is no experimental model of R. equi pneumonia other than intra-bronchial experimental infection of foals with R. equi, which is labor-intensive and costly. This study’s objective was to develop a guinea pig (GP) model of R. equi pneumonia that would facilitate development of novel approaches for controlling and preventing this disease. Guinea pigs were infected with either 101, 102, 103, or 104 colony forming units (CFUs) of a virulent strain of R. equi using a Madison aerosol chamber, or 106 or 107 CFUs of this strain intratracheally. Animals were monitored daily for clinical signs of pneumonia, and were euthanized and necropsied on days 1, 3, 7, or 35 post-infection (PI). Lung homogenates were plated onto selective agar to determine bacterial load. No clinical signs of disease were observed regardless of the inoculum dose or infection method. No bacteria were recovered from GPs euthanized at 35 days PI. Histology and immunostaining of T-cells, B-cells, and macrophages in lungs showed that inflammatory responses in infected GPs were similarly unremarkable irrespective of dose or route of infection. Guinea pigs appear to be resistant to pulmonary infection with virulent R. equi even at doses that reliably produce clinical pneumonia in foals.

Worldwide, tuberculosis (TB) remains one of the most prevalent infectious diseases causing morbidity and death in >1.5 million patients annually. Mycobacterium tuberculosis (Mtb), the etiologic agent of TB, usually resides in the alveolar macrophages. Current tuberculosis treatment methods require more than six months, and low compliance often leads to therapeutic failure and multidrug resistant strain development. Critical to improving TB-therapy is shortening treatment duration and increasing therapeutic efficacy. In this study, we sought to determine if lung hemodynamics and pathological changes in Mtb infected cells can be used for the selective targeting of microparticles to infected tissue(s). Thioaptamers (TA) with CD44 (CD44TA) targeting moiety were conjugated to discoidal silicon mesoporous microparticles (SMP) to enhance accumulation of these agents/carriers in the infected macrophages in the lungs. In vitro, CD44TA-SMP accumulated in macrophages infected with mycobacteria efficiently killing the infected cells and decreasing survival of mycobacteria. In vivo, increased accumulations of CD44TA-SMP were recorded in the lung of M. tuberculosis infected mice as compared to controls. TA-targeted carriers significantly diminished bacterial load in the lungs and caused recruitment of T lymphocytes. Proposed mechanism of action of the designed vector accounts for a combination of increased uptake of particles that leads to infected macrophage death, as well as, activation of cellular immunity by the TA, causing increased T-cell accumulation in the treated lungs. Based on our data with CD44TA-SMP, we anticipate that this drug carrier can open new avenues in TB management.

Light-activated molecular nanomachines (MNMs) can be used to drill holes into prokaryotic (bacterial) cell walls and the membrane of eukaryotic cells, including mammalian cancer cells, by their fast rotational movement, leading to cell death. We examined how these MNMs function in multicellular organisms and investigated their use for treatment and eradication of specific diseases by causing damage to certain tissues and small organisms. Three model eukaryotic species, Caenorhabditis elegans, Daphnia pulex, and Mus musculus (mouse), were evaluated. These organisms were exposed to light-activated fast-rotating MNMs and their physiological and pathological changes were studied in detail. Slow rotating MNMs were used to control for the effects of rotation rate. We demonstrate that fast-rotating MNMs caused depigmentation and 70% mortality in C. elegans while reducing the movement as well as heart rate and causing tissue damage in Daphnia. Topically applied light-activated MNMs on mouse skin caused ulceration and microlesions in the epithelial tissue, allowing MNMs to localize into deeper epidermal tissue. Overall, this study shows that the nanomechanical action of light-activated MNMs is effective against multicellular organisms, disrupting cell membranes and damaging tissue in vivo. Customized MNMs that target specific tissues for therapy combined with spatial and temporal control could have broad clinical applications in a variety of benign and malignant disease states including treatment of cancer, parasites, bacteria, and diseased tissues., Graphical Abstract

Mycobacterium tuberculosis is primarily a respiratory pathogen. However, 15% of infections worldwide occur at extrapulmonary sites causing additional complications for diagnosis and treatment of the disease. In addition, dissemination of M. tuberculosis out of the lungs is thought to be more than just a rare event leading to extrapulmonary tuberculosis, but rather a prerequisite step that occurs during all infections, producing secondary lesions that can become latent or productive. In this review we will cover the clinical range of extrapulmonary infections and the process of dissemination including evidence from both historical medical literature and animal experiments for dissemination and subsequent reseeding of the lungs through the lymphatic and circulatory systems. While the mechanisms of M. tuberculosis dissemination are not fully understood, we will discuss the various models that have been proposed to address how this process may occur and summarize the bacterial virulence factors that facilitate M. tuberculosis dissemination.

Photodynamic inactivation (PDI) is a promising alternative for combating infections caused by antimicrobial resistant bacteria. Pneumonias are among the most worrisome infections because of their high-mortality rate. Previous studies have demonstrated the feasibility of using PDI with extracorporeal light to treat pneumonia. In this study, we analyzed key parameters for the viability of this treatment, including the selectivity of the photodynamic response for pathogens over host cells. Our results showed that PDI can induce killing of Staphylococcus aureus (of up to 4.18 log for the strain Xen29 and 3.62 log for Xen36) under conditions where little or no toxicity for host cells is observed. We validated pulmonary delivery of the photosensitizer and light in mice, using photobleaching as an indicator, and demonstrated preservation of healthy tissues as evidence of the safety of the protocol. Overall, PDI displays low toxicity on host tissues, making it a promising tool for treatment of pneumonias caused by S. aureus and other important pathogens.

Contains fulltext : 220633.pdf (Publisher’s version ) (Closed access) Mycobacterium tuberculosis (M. tuberculosis) has coevolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to improve outcomes and potentially shorten existing therapy will require identification of the full complement by which host immunity is inhibited. Perturbation of host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, lymphocytic choriomeningitis virus (LCMV), and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of patients with tuberculosis (TB) and their asymptomatic household contacts and found that the patients with TB have DNA hypermethylation of the IL-2/STAT5, TNF/NF-kappaB, and IFN-gamma signaling pathways. We performed methylation-sensitive restriction enzyme-quantitative PCR (MSRE-qPCR) and observed that multiple genes of the IL-12/IFN-gamma signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1, and JAK2) were hypermethylated in patients with TB. The DNA hypermethylation of these pathways was associated with decreased immune responsiveness with decreased mitogen-induced upregulation of IFN-gamma, TNF, IL-6, CXCL9, CXCL10, and IL-1beta production. The DNA hypermethylation of the IL-12/IFN-gamma pathway was associated with decreased IFN-gamma-induced gene expression and decreased IL-12-inducible upregulation of IFN-gamma. This study demonstrates that immune cells from patients with TB are characterized by DNA hypermethylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and nonspecific immune responsiveness.

Multidrug resistance in pathogenic bacteria is an increasing problem in patient care and public health. Molecular nanomachines (MNMs) have the ability to open cell membranes using nanomechanical action. We hypothesized that MNMs could be used as antibacterial agents by drilling into bacterial cell walls and increasing susceptibility of drug-resistant bacteria to recently ineffective antibiotics. We exposed extensively drug-resistant Klebsiella pneumoniae to light-activated MNMs and found that MNMs increase the susceptibility to Meropenem. MNMs with Meropenem can effectively kill K. pneumoniae that are considered Meropenem-resistant. We examined the mechanisms of MNM action using permeability assays and transmission electron microscopy, finding that MNMs disrupt the cell wall of extensively drug-resistant K. pneumoniae, exposing the bacteria to Meropenem. These observations suggest that MNMs could be used to make conventional antibiotics more efficacious against multi-drug-resistant pathogens.

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for millennia and developed multiple mechanisms to evade host immunity. Restoring host immunity in order to shorten existing therapy and improve outcomes will require identifying the full complement by which host immunity is inhibited. Perturbing host DNA methylation is a mechanism induced by chronic infections such as HIV, HPV, LCMV and schistosomiasis to evade host immunity. Here, we evaluated the DNA methylation status of TB patients and their asymptomatic household contacts demonstrating that TB patients have DNA hyper-methylation of the IL-2-STAT5, TNF-NF-ϰB and IFN-γ signaling pathways. By MSRE-qPCR, multiple genes of the IL-12-IFN-γ signaling pathway (IL12B, IL12RB2, TYK2, IFNGR1, JAK1 and JAK2) were hyper-methylated in TB patients. The DNA hyper-methylation of these pathways is associated with decreased immune responsiveness with decreased mitogen induced upregulation of IFN-γ, TNF, IL-6 and IL-1β production. The DNA hyper-methylation of the IL-12-IFN-γ pathway was associated with decreased IFN-γ induced gene expression and decreased IL-12 inducible up-regulation of IFN-γ. This work demonstrates that immune cells from TB patients are characterized by DNA hyper-methylation of genes critical to mycobacterial immunity resulting in decreased mycobacteria-specific and non-specific immune responsiveness.

Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise., Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% for smear-positive, culture-positive samples and 88% for smear-negative, culture-positive samples with a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93%, and the positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

Summary Although tuberculosis (TB) is one of the most common causes of morbidity and mortality in humans worldwide and diagnostic methods have been in place for more than 100 years, diagnosis remains a challenge. The main problems with diagnosis relate to the time needed to obtain a definitive result, difficulty in obtaining sputum, the primary clinical material used, and the ability of the causative agent, Mycobacterium tuberculosis , to cause disease in nearly any tissue within the body. In order to decrease incidence of TB, discovery of a novel interventions will be required, since current technologies have only been able to control numbers of infections, not reduce them. Diagnostic innovation is particularly needed because there are no effective pediatric or extrapulmonary TB diagnostic methods and multiple-drug resistance is only identified in less than 25% of those patients that are thought to have it. The most common diagnostic method worldwide remains acid-fast stain on sputum, with a threshold of ∼10,000 bacteria/ml that is only reached ∼5–6 months after development of symptoms. In order to obtain definitive diagnostic results earlier during the disease process, we have developed a diagnostic method designated reporter enzyme fluorescence (REF) that utilizes BlaC produced by M. tuberculosis and custom substrates to produce a specific fluorescent signal with as few as 10 bacteria/ml in clinical samples. We believe that the unique biology of the REF technique will allow it to contribute new diagnostic information that is complementary to all existing diagnostic tests as well as those currently known to be in development.

Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.