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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 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 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 dissemination.

Background Diseases such as tuberculosis (TB) have always had a large impact on human health. Bacillus Calmette-Guérin (BCG) is used as a surrogate for TB during the development of anti-TB drugs. Nanoparticles (NPs) have attracted great interest in drug development. The purpose of this study was to examine the potential of NPs as anti-TB compounds by studying the interacting mechanisms between NPs and bacteria. Results We investigated effects of gold and silver NPs on BCG and Escherichia coli . Experimentally, particle size and shape were characterized using transmission electron microscopy (TEM). Different concentrations of NPs were applied in bacterial culture. The growth of E. coli was monitored through colony forming units (CFU). The mechanism of interaction between NPs and bacteria was analyzed through bacterial thin sections followed by TEM and scanning electron microscopy. Antibacterial effects on BCG were observed by recording fluorescent protein expression levels. Conclusions The results suggest NPs have potential applications as anti-TB compounds. The antibacterial effects and mechanism of action for NPs were dependent upon composition and surface modifications.

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.

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for β-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 ± 2 x 10² colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10⁴ CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

Mycobacterium tuberculosis ( Mtb ) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn 2+ ) availability as a likely driver of bacterial phenotypic heterogeneity in vivo . Zn 2+ sequestration is part of “nutritional immunity”, where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn 2+ -limitation is an environmental cue sensed by Mtb , as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn 2+ -limited Mtb in vivo . Prolonged Zn 2+ limitation leads to numerous physiological changes in vitro , including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn 2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn 2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn 2+ -limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn 2+ -limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn 2+ availability likely plays a key role during early interactions with host cells.

Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis ( Mtb ), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe ( CDG-OMe ) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens. Rapid diagnostic methods that can be applied in resource-limited settings are important in the fight against tuberculosis. Here, fluorogenic probes are described that are activated by BlaC — an enzyme secreted by tubercle bacilli. The probes have enabled detection in unprocessed human sputum of live pathogen in less than 10 min.

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.

Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc(null) mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34(+) fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45(+)) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2-8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

Summary Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.