An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis.

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.

Rifaximin potentiates clarithromycin against Mycobacterium abscessus in vitro and in zebrafish.

Background: Mycobacterium abscessus is a non-tuberculous mycobacterium (NTM) that causes chronic pulmonary infections. Because of its extensive innate resistance to numerous antibiotics, treatment options are limited, often resulting in poor clinical outcomes. Current treatment regimens usually involve a combination of antibiotics, with clarithromycin being the cornerstone of NTM treatments. Objectives: To identify drug candidates that exhibit synergistic activity with clarithromycin against M. abscessus. Methods: We performed cell-based phenotypic screening of a compound library against M. abscessus induced to become resistant to clarithromycin. Furthermore, we evaluated the toxicity and efficacy of the top compound in a zebrafish embryo infection model. Results: The screen revealed rifaximin as a clarithromycin potentiator. The combination of rifaximin and clarithromycin was synergistic and bactericidal in vitro and potent in the zebrafish model. Conclusions: The data indicate that the rifaximin/clarithromycin combination is promising to effectively treat pulmonary NTM infections.

Design of Biodegradable, Climate-Specific Packaging Materials That Sense Food Spoilage and Extend Shelf Life.

The AgriFood systems in tropical climates are under strain due to a rapid increase in human population and extreme environmental conditions that limit the efficacy of packaging technologies to extend food shelf life and guarantee food safety. To address these challenges, we rationally designed biodegradable packaging materials that sense spoilage and prevent molding. We nanofabricated the interface of 2D covalent organic frameworks (COFs) to reinforce silk fibroin (SF) and obtain biodegradable membranes with augmented mechanical properties and that displayed an immediate colorimetric response (within 1 s) to food spoilage, using packaged poultry as an example. Loading COF with antimicrobial hexanal also mitigated biotic spoilage in high-temperature and -humidity conditions, resulting in a four-order of magnitude decrease in the total amount of mold growth in soybeans packaged in silk-COF, when compared to cling film (i.e., polyethylene). Together, the integration of sensing, structural reinforcement, and antimicrobial agent delivery within a biodegradable nanocomposite framework defines climate-specific packaging materials that can decrease food waste and enhance food safety.

Erythematous scaly plaques and nodules on scalp.

Cutaneous manifestations of syphilis are varied and may present with non-specific features. We describe a 45-year-old man who presented with erythematous scaly plaques and nodules on his scalp. In previously reported cases, there were only descriptions of nodules as well as tumors. However, in our case, the patient presented with plaques and nodules on his scalp that quickly resolved with treatment for syphilis. It is important to recognize and treat syphilis at an early stage.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

Clinical Characteristics and Long-Term Outcomes of Patients With Differing Haemoglobin Levels Undergoing Semi-Urgent and Elective Percutaneous Coronary Intervention in an Asian Population.

Introduction: This study aimed to investigate the impact of anaemia on long-term clinical outcomes in patients who underwent semi-urgent and elective percutaneous coronary intervention (PCI) in an Asian population. Although the effects of anaemia on outcomes in Asian patients are well studied for acute coronary syndrome, its impact on Asian patients undergoing semi-urgent and elective PCI is unclear. Methods: This was a retrospective cohort study of patients who underwent semi-urgent and elective PCI from January 1, 2014, to December 31, 2015, at a tertiary academic centre. A total of 1,685 patients were included. They were stratified into three groups: normal (≥12 g/dL), intermediate (10-11.9 g/dL), and low (<10 g/dL) haemoglobin levels. Demographics, risk factors, and end-points including the 5-point major adverse cardiac and cerebrovascular events (MACCE) (all-cause death, subsequent stroke, myocardial infarction, congestive cardiac failure, and target lesion revascularisation), cardiovascular death, and bleeding events were analysed. Results: Patients in intermediate and low haemoglobin level groups were older with more comorbidities. Compared to the normal haemoglobin level group, low haemoglobin level group patients were associated with an increased risk of composite endpoints of all-cause death, subsequent stroke, myocardial infarction, congestive cardiac failure, and target lesion revascularisation [adjusted hazard ratio (aHR) 1.89, 95% confidence interval (CI):1.22, 2.92; p = 0.004]. This was driven by the increased risk of target lesions revascularisation observed in the low haemoglobin level group compared to the normal haemoglobin level group (aHR 17.74, 95% CI: 1.74, 180.80; p = 0.015). The patients in the low haemoglobin level group were also associated with a higher risk of bleeding events compared to the normal haemoglobin level group (aHR 7.18, 95% CI: 1.13, 45.40; p = 0.036). Conclusion: In our Asian cohort, patients with anaemia undergoing PCI were associated with a higher comorbid burden. Despite adjustments for comorbidities, these patients had higher mortality and worse cardiovascular outcomes following contemporary PCI.

The impact of chronic kidney disease on long-term outcomes following semi-urgent and elective percutaneous coronary intervention.

INTRODUCTION: The effects of chronic kidney disease (CKD) on outcomes in patients undergoing semi-urgent and elective percutaneous coronary intervention (PCI) are unclear. This study aims to investigate impact of CKD on long-term outcomes of this population. METHODS: This was a retrospective cohort study of patients who underwent semi-urgent and elective PCI from 1 January 2014 to 31 December 2015 at a tertiary academic center. They were stratified into five groups - group 1 [estimated glomerular filtration rate (eGFR) ≥90 ml/min/1.73m2], group 2 (eGFR 60-89 ml/min/1.73m2), group 3 (eGFR 30-59 ml/min/1.73 m2), group 4 (eGFR <30 ml/min/1.73m2), and group 5 (dialysis). Demographics, risk factors in relation to endpoints of all-cause mortality, contrast-induced nephropathy (CIN), three-point major adverse cardiac events (MACE) (cardiac death, subsequent myocardial infarction, subsequent stroke), and four-point MACE (including target lesion revascularization) were analyzed. RESULTS: One thousand six hundred nine patients were included. Advanced CKD patients were more likely to be female and older, with higher prevalence of co-morbidities. Compared to group 1, group 4 patients were associated with increased risk of three-point [adjusted hazard ratio (aHR) 1.94, 95% confidence interval (CI): 1.06-3.55; P = 0.031] and four-point MACE (aHR 2.15, 95% CI: 1.21-3.80; P = 0.009). However, higher contrast volume usage [odds ratio (OR) 2.20, 95% CI: 1.04-4.68; P = 0.040) was associated with increased CIN risk but not reduced eGFR (OR 1.62, 95% CI: 0.57-4.65; P = 0.369). CONCLUSION: Advanced CKD patients undergoing PCI were associated with higher co-morbid burden. Despite adjustments for co-morbidities, these patients had higher mortality and worse cardiovascular outcomes at 3 years following contemporary PCI.

Hydrogen Sulfide Sensitizes Acinetobacter baumannii to Killing by Antibiotics.

The production of endogenous hydrogen sulfide (H2S) has been shown to confer antibiotic tolerance in all bacteria studied to date. Therefore, this mediator has been speculated to be a universal defense mechanism against antibiotics in bacteria. This is assuming that all bacteria produce endogenous H2S. In this study, we established that the pathogenic bacteria Acinetobacter baumannii does not produce endogenous H2S, giving us the opportunity to test the effect of exogenous H2S on antibiotic tolerance in a bacterium that does not produce it. By using a H2S-releasing compound to modulate the sulfide content in A. baumannii, we demonstrated that instead of conferring antibiotic tolerance, exogenous H2S sensitized A. baumannii to multiple antibiotic classes, and was able to revert acquired resistance to gentamicin. Exogenous H2S triggered a perturbation of redox and energy homeostasis that translated into hypersensitivity to antibiotic killing. We propose that H2S could be used as an antibiotic-potentiator and resistance-reversion agent in bacteria that do not produce it.

Allosteric pyruvate kinase-based "logic gate" synergistically senses energy and sugar levels in Mycobacterium tuberculosis.

Pyruvate kinase (PYK) is an essential glycolytic enzyme that controls glycolytic flux and is critical for ATP production in all organisms, with tight regulation by multiple metabolites. Yet the allosteric mechanisms governing PYK activity in bacterial pathogens are poorly understood. Here we report biochemical, structural and metabolomic evidence that Mycobacterium tuberculosis (Mtb) PYK uses AMP and glucose-6-phosphate (G6P) as synergistic allosteric activators that function as a molecular "OR logic gate" to tightly regulate energy and glucose metabolism. G6P was found to bind to a previously unknown site adjacent to the canonical site for AMP. Kinetic data and structural network analysis further show that AMP and G6P work synergistically as allosteric activators. Importantly, metabolome profiling in the Mtb surrogate, Mycobacterium bovis BCG, reveals significant changes in AMP and G6P levels during nutrient deprivation, which provides insights into how a PYK OR gate would function during the stress of Mtb infection.

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis, and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.

Regulatory T cells induced by Mycobacterium chelonae sensitization influence murine responses to bacille Calmette-Guerin.

The efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is highly varied globally. Differential sensitization to environmental mycobacteria prior to BCG vaccination may prime immune effects leading to this variation, but the precise immune mechanisms and cell types involved in this phenomenon are unknown. We hypothesized that pre-vaccination sensitization to environmental mycobacteria induces mycobacterium-specific Tregs that suppress responses to BCG. This was investigated by testing Treg responses following priming of BALB/c mice by i.p. immunization with heat-killed CHE. Such mice produced higher levels of IL-10 before and after intranasal, live BCG administration and had fewer lung inflammatory cells post-BCG, relative to nonsensitized mice. In CHE-sensitized mice, the percentage of splenic CD4+CD25+ cells expressing Foxp3 amongst total lymphocytes was not elevated significantly, but these cells limited nonspecific proliferation of CD4+CD25⁻ effector cells upon coculture and promoted higher expression levels of CD103 and Foxp3 in response to BCG antigen stimulation than CD4+CD25+ cells from nonsensitized mice. In adoptive transfer experiments, naïve, WT mice receiving CD4+CD25+ cells from CHE-sensitized mice and then given live BCG intranasally had significantly elevated lung IL-10 levels, reduced frequencies of lung IL-2-producing cells, and lower lymphocyte numbers in the BAL. Therefore, CHE sensitization induced CD4+CD25+ Tregs with functional, suppressive activity on BCG responses in vitro and in vivo. Treg induction could therefore be one mechanism underlying how environmental mycobacteria priming modulates host responses to the BCG vaccine.

Mycobacterium chelonae sensitisation induces CD4(+)-mediated cytotoxicity against BCG.

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre-vaccination sensitisation to non-tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env-specific T cells exerting cytotoxicity against BCG-infected host cells. After murine sensitisation with heat-killed antigens of different Env species, splenocytes from M. chelonae (CHE)-sensitised mice exerted the strongest cytotoxicity against autologous BCG-infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4(+), but not CD8(+) or CD56(+) cells, and CD4(+) cells showed higher percentage of cytotoxicity than CD4(-) cells, supporting a role for CD4(+) cells in CHE-induced, BCG-specific cytotoxicity. Additionally, this cytotoxicity was IFN-gamma, perforin and FasL dependent. After CHE-sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4(+) cells, the main cell type producing IFN-gamma. This was associated with 2- and 6-fold reductions in lung BCG counts 1 and 3 wk, respectively post- infection, relative to non-sensitised mice. This is the first report describing cytotoxicity against BCG-infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.

Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats.

The role of hydrogen sulfide (H(2)S) in myocardial infarction (MI) has not been previously studied. We therefore investigated the effect of H(2)S in a rat model of MI in vivo. Animals were randomly divided into three groups (n = 80) and received either vehicle, 14 micromol/kg of sodium hydrosulfide (NaHS), or 50 mg/kg propargylglycine (PAG) everyday for 1 wk before surgery, and the treatment was continued for a further 2 days after MI when the animals were killed. The mortality was 35% in vehicle-treated, 40% in PAG-treated, and 27.5% in NaHS-treated (P < 0.05 vs. vehicle) groups. Infarct size was 52.9 +/- 3.5% in vehicle-treated, 62.9 +/- 7.6% in PAG-treated, and 43.4 +/- 2.8% in NaHS-treated (P < 0.05 vs. vehicle) groups. Plasma H(2)S concentration was significantly increased after MI (59.2 +/- 7.16 microM) compared with the baseline concentration (i.e., 38.2 +/- 2.07 microM before MI; P < 0.05). Elevated plasma H(2)S after MI was abolished by treatment of animals with PAG (39.2 +/- 5.02 microM). We further showed for the first time cystathionine-gamma-lyase protein localization in the myocardium of the infarct area by using immunohistochemical staining. In the hypoxic vascular smooth muscle cells, we found that cell death was increased under the stimuli of hypoxia but that the increased cell death was attenuated by the pretreatment of NaHS (71 +/- 1.2% cell viability in hypoxic vehicle vs. 95 +/- 2.3% in nonhypoxic control; P < 0.05). In conclusion, endogenous H(2)S was cardioprotective in the rat model of MI. PAG reduced endogenous H(2)S production after MI by inhibiting cystathionine-gamma-lyase. The results suggest that H(2)S might provide a novel approach to the treatment of MI.