Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Targeting Mycobacterium tuberculosis response to environmental cues for the development of effective antitubercular drugs.

Sensing and response to environmental cues, such as pH and chloride (Cl-), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl- levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure-activity relationship studies on the hit compound "C6," or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl- response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl- response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl--responsive genes in the simultaneous presence of cholesterol and high external Cl- concentration, versus transcript levels observed during exposure to high external Cl- concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.

Hub Genes and Pathways Related to Lemon (Citrus limon) Leaf Response to Plenodomus tracheiphilus Infection and Influenced by Pseudomonas mediterranea Biocontrol Activity.

The lemon industry in the Mediterranean basin is strongly threatened by "mal secco" disease (MSD) caused by the fungus Plenodomus tracheiphlilus. Leaf pretreatments with Pseudomonas mediterranea 3C have been proposed as innovative tools for eco-sustainable interventions aimed at controlling the disease. In this study, by exploiting the results of previously performed RNAseq analysis, WCGNA was conducted among gene expression patterns in both inoculated (Pt) and pretreated and fungus-inoculated lemon plants (Citrus limon L.) (3CPt), and two indicators of fungal infection, i.e., the amount of fungus DNA measured in planta and the disease index (DI). The aims of this work were (a) to identify gene modules significantly associated with those traits, (b) to construct co-expression networks related to mal secco disease; (c) to define the effect and action mechanisms of P. mediterranea by comparing the networks. The results led to the identification of nine hub genes in the networks, with three of them belonging to receptor-like kinases (RLK), such as HERK1, CLAVATA1 and LRR, which play crucial roles in plant-pathogen interaction. Moreover, the comparison between networks indicated that the expression of those receptors is not induced in the presence of P. mediterranea, suggesting how powerful WCGNA is in discovering crucial genes that must undergo further investigation and be eventually knocked out.

Embolic Stroke of Undetermined Source: Role of Implantable Loop Recorder in Secondary Prevention.

BACKGROUND AND AIMS: Atrial fibrillation (AF) is the most important cause of embolic stroke of undetermined source (ESUS). Implantable loop recorder (ILR) demonstrated the highest sensitivity for detecting it. This register was created to confirm the high prevalence of AF in patients after ESUS and to verify possible benefits on clinical outcomes such as TIA (Transient Ischaemic Attack)/stroke recurrence and death using ILR. METHODS: A total of 278 patients admitted to "Molinette" Hospital in Stroke Unit department between 2011 and 2016, diagnosed with ESUS, underwent ILR implantation if they had at least one risk factor for AF. A total of 165 patients admitted to other departments in the same center for the same pathology, without ILR, represent the control group. We used propensity score to select 132 patients from each group (matching age, sex, CHADS-VASC, and HAS-BLEED baseline characteristics). RESULTS: The detection rate of AF episodes was significantly higher in the ILR group (p < 0.001). No significant protective role of ILR for clinical endpoints was found on univariate analysis, although a trend towards significance has been pointed for the composite outcome of death and ischemic events recurrence (OR 0.52, CI 0.26-1.04, p = 0.06). A protective role of ILR was found for deaths (OR 0.4, CI 0.17-0.94, p 0.03) and for the composite outcome (OR 0.41, CI 0.19-0.87, p 0.02) on multivariate analysis in the best subsets. CONCLUSION: With our statistical models, we identified a significant clinical benefit from ILR monitoring, evidenced by a trend of less death and TIA/stroke recurrence and relevant ILR protection for prediction of TIA/stroke recurrence.

A Novel Oral GyrB/ParE Dual Binding Inhibitor Effective against Multidrug-Resistant Neisseria gonorrhoeae and Other High-Threat Pathogens.

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

Silk Vista Baby flow diverter stent for ruptured intracranial aneurysms: a retrospective observational study.

PURPOSE: Flow diversion changed the approach to complex intracranial aneurysms, leading to a widespread use and a rapid technological evolution. Indeed, indications continued to expand, including ruptured intracranial aneurysms in selected cases. Recently, new devices have been designed specifically to target smaller vessels. Therefore, we conducted a multicenter study to evaluate clinical outcome, complications, and occlusion rate of patients with ruptured aneurysms treated with new generation low profile Silk Vista Baby (SVB) flow diverter stent (FD). METHODS: We performed a retrospective observational study on consecutive patients who underwent treatment with SVB for ruptured aneurysms at 12 Italian centers. Primary end point was favorable clinical outcome rate, defined as modified ranking score (mRS) of 0-2 at the 3 months. Secondary outcomes were complication rate, aneurysm re-rupture, and complete aneurysm occlusion at last radiological follow-up. RESULTS: Twenty-five patients were included; at 3 months' follow-up, 19 patients (79.1%) had favorable clinical outcome (mRS 0-2). Three patients (12.5%) died during follow-up. In-stent thrombosis occurred in two cases (8.3%), managed with glycoprotein IIb/IIIA and intra-stent angioplasty, without clinical consequences. In 18 (85.7%) patients, complete occlusion at 3 months was demonstrated. No rebleeding occurred during follow-up. Presentation with unfavorable World Federation of Neurosurgical Societies grading system (WFNS) and posterior circulation location were both significantly correlated with unfavorable clinical outcome (p = 0.005 and p = 0.02). CONCLUSIONS: Our data suggests that low profile FD treatment of ruptured intracranial aneurysms located distally of the circle of Willis is feasible. New generation low profile FD may represent an alternative option in carefully selected cases.

Evidence that Maackia amurensis seed lectin (MASL) exerts pleiotropic actions on oral squamous cells with potential to inhibit SARS-CoV-2 infection and COVID-19 disease progression.

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 2.7 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.

Automatic User Preferences Selection of Smart Hearing Aid Using BioAid.

Noisy environments, changes and variations in the volume of speech, and non-face-to-face conversations impair the user experience with hearing aids. Generally, a hearing aid amplifies sounds so that a hearing-impaired person can listen, converse, and actively engage in daily activities. Presently, there are some sophisticated hearing aid algorithms available that operate on numerous frequency bands to not only amplify but also provide tuning and noise filtering to minimize background distractions. One of those is the BioAid assistive hearing system, which is an open-source, freely available downloadable app with twenty-four tuning settings. Critically, with this device, a person suffering with hearing loss must manually alter the settings/tuning of their hearing device when their surroundings and scene changes in order to attain a comfortable level of hearing. However, this manual switching among multiple tuning settings is inconvenient and cumbersome since the user is forced to switch to the state that best matches the scene every time the auditory environment changes. The goal of this study is to eliminate this manual switching and automate the BioAid with a scene classification algorithm so that the system automatically identifies the user-selected preferences based on adequate training. The aim of acoustic scene classification is to recognize the audio signature of one of the predefined scene classes that best represent the environment in which it was recorded. BioAid, an open-source biological inspired hearing aid algorithm, is used after conversion to Python. The proposed method consists of two main parts: classification of auditory scenes and selection of hearing aid tuning settings based on user experiences. The DCASE2017 dataset is utilized for scene classification. Among the many classifiers that were trained and tested, random forests have the highest accuracy of 99.7%. In the second part, clean speech audios from the LJ speech dataset are combined with scenes, and the user is asked to listen to the resulting audios and adjust the presets and subsets. A CSV file stores the selection of presets and subsets at which the user can hear clearly against the scenes. Various classifiers are trained on the dataset of user preferences. After training, clean speech audio was convolved with the scene and fed as input to the scene classifier that predicts the scene. The predicted scene was then fed as input to the preset classifier that predicts the user's choice for preset and subset. The BioAid is automatically tuned to the predicted selection. The accuracy of random forest in the prediction of presets and subsets was 100%. This proposed approach has great potential to eliminate the tedious manual switching of hearing assistive device parameters by allowing hearing-impaired individuals to actively participate in daily life by automatically adjusting hearing aid settings based on the acoustic scene.

Biosynthetic Interrogation of Soil Metagenomes Reveals Metamarin, an Uncommon Cyclomarin Congener with Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the deadliest infectious diseases. Unfortunately, the development of antibiotic resistance threatens our current therapeutic arsenal, which has necessitated the discovery and development of novel antibiotics against drug-resistant Mycobacterium tuberculosis (Mtb). Cyclomarin A and rufomycin I are structurally related cyclic heptapeptides assembled by nonribosomal peptide synthetases (NRPSs), which show potent anti-Mtb activity with a new cellular target, the caseinolytic protein ClpC1. An NRPS adenylation domain survey using DNA extracted from ∼2000 ecologically diverse soils found low cyclomarin/rufomycin biosynthetic diversity. In this survey, a family of cyclomarin/rufomycin-like biosynthetic gene clusters (BGC) that encode metamarin, an uncommon cyclomarin congener with potent activity against both Mtb H37Rv and multidrug-resistant Mtb clinical isolates was identified. Metamarin effectively inhibits Mtb growth in murine macrophages and increases the activities of ClpC1 ATPase and the associated ClpC1/P1/P2 protease complex, thus causing cell death by uncontrolled protein degradation.

De Novo Transcriptome Sequencing of Rough Lemon Leaves (Citrus jambhiri Lush.) in Response to Plenodomus tracheiphilus Infection.

Mal secco is one of the most severe diseases of citrus, caused by the necrotrophic fungus Plenodomus tracheiphilus. With the main aim of identifying candidate genes involved in the response of citrus plants to "Mal secco", we performed a de novo transcriptome analysis of rough lemon seedlings subjected to inoculation of P. tracheiphilus. The analysis of differential expressed genes (DEGs) highlighted a sharp response triggered by the pathogen as a total of 4986 significant DEGs (2865 genes up-regulated and 2121 down-regulated) have been revealed. The analysis of the most significantly enriched KEGG pathways indicated that a crucial role is played by genes involved in "Plant hormone signal transduction", "Phenylpropanoid biosynthesis", and "Carbon metabolism". The main findings of this work are that under fungus challenge, the rough lemon genes involved both in the light harvesting and the photosynthetic electron flow were significantly down-regulated, thus probably inducing a shortage of energy for cellular functions. Moreover, the systemic acquired resistance (SAR) was activated through the induced salicylic acid cascade. Interestingly, RPM1 interacting protein 4, an essential positive regulator of plant defense, and BIR2, which is a negative regulator of basal level of immunity, have been identified thus representing useful targets for molecular breeding.

Neuroform Atlas stent-assisted coiling of ruptured intracranial aneurysms: A multicenter study.

PURPOSE: To assess efficacy, safety and to discuss optimal medical therapy of stent-assisted coiling of ruptured intracranial aneurysms. METHODS: Ruptured intracranial aneurysms treated with stent-assisted coiling in eight different institutions were retrospectively reviewed. Medical treatment regimens varied among the centers, mainly regarding heparin administration and post-procedural single or double antiplatelet therapy. Clinical and angiographic results, including complications and outcomes were analyzed and related to the different therapies. RESULTS: Sixty-one consecutive patients (male/female 23/38), aged 59.1 years (36-86) underwent stent-assisted coiling for ruptured intracranial aneurysm without antiplatelet pre-medication. Intravenous acetylsalicylic acid (ASA) 500mg was administered to all patients immediately after stent deployment. At the same time heparin was given as bolus in 15 patients (24.6%) as part of local protocol. Intravenous glycoprotein 2b/3a inhibitors (antiGP2b3a) were used as bail-out therapy for stent thrombosis. Stent thrombosis occurred in 22 patients (36.1%), of which 4 (6.5%) lead to incomplete and 18 (29.6) to complete occlusion of the stent. Heparin administration had no effect on thrombosis rate. Thrombosis resolution occurred in all cases with intravenous antiGP2b3a (7 tirofiban, 15 abciximab), without increasing overall complication rate. Single antiplatelet therapy with ASA (28 patients, 45.9%) or double antiplatelet therapy including ASA and clopidogrel (33 patients, 54.1%) were administered after procedure, depending on local protocols and on neurointerventionists' experience. Overall complication rate, including ischemia and hemorrhage was higher in patients in which only ASA was administered (21.4% vs. 12.1%). No late stent thrombosis was seen, regardless of whether a single or double antiplatelet regimen was used. Nevertheless, the small sample size suggests caution in interpreting these results. Moreover, a possible bias may arise from the decision whether to modify the maintenance therapy or not depending on the severity of the intracranial hemorrhage in a case-by-case assessment. At three months, 34 out of 38 patients with HH grade 1-2 (89.4%), and 11 out of 23 with Hunt-Hess grade of 3-4 (47.8%) were independent (Modified Ranking Scale 0-2). CONCLUSION: Stent assisted coiling of ruptured intracranial aneurysms is a feasible option when simple coiling is not possible. Optimal medical treatment is still controversial because balance between hemorrhagic and ischemic risks is difficult to evaluate. In our series, heparin bolus had no effect on subsequent stent thrombosis. In all cases peri-operative stent thrombosis was successfully managed using bail-out intravenous antiGP2b3a, which did not increase post-procedural hemorrhage rates. A non-significant trend towards increased complications rate was noticed in patients treated with single antiplatelet therapy versus double antiplatelet therapy.

Discovery of Ubonodin, an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex.

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.

Machine Learning Platform to Discover Novel Growth Inhibitors of Neisseria gonorrhoeae.

PURPOSE: To advance fundamental biological and translational research with the bacterium Neisseria gonorrhoeae through the prediction of novel small molecule growth inhibitors via naïve Bayesian modeling methodology. METHODS: Inspection and curation of data from the publicly available ChEMBL web site for small molecule growth inhibition data of the bacterium Neisseria gonorrhoeae resulted in a training set for the construction of machine learning models. A naïve Bayesian model for bacterial growth inhibition was utilized in a workflow to predict novel antibacterial agents against this bacterium of global health relevance from a commercial library of >105 drug-like small molecules. Follow-up efforts involved empirical assessment of the predictions and validation of the hits. RESULTS: Specifically, two small molecules were found that exhibited promising activity profiles and represent novel chemotypes for agents against N. gonorrrhoeae. CONCLUSIONS: This represents, to the best of our knowledge, the first machine learning approach to successfully predict novel growth inhibitors of this bacterium. To assist the chemical tool and drug discovery fields, we have made our curated training set available as part of the Supplementary Material and the Bayesian model is accessible via the web. Graphical Abstract.

Design, organocatalytic synthesis, and bioactivity evaluation of enantiopure fluorinated LpxC inhibitors.

Enantiopure compounds with a strategically incorporated fluorine atom intended to enhance LpxC inhibition have been synthesized using an organocascade fluorination reaction as the key step. These are the first low molecular weight LpxC inhibitors to contain a fluorine atom on a critically important chiral center that is substituted with two pharmacophoric moieties, and were thusly designed to provide new SAR data for this class of compounds. Fluorinated compounds were evaluated against ESKAPE pathogens and exhibited MICs of ≤12.5 µg mL-1 against Pseudomonas aeruginosa.

Optimization of N-benzyl-5-nitrofuran-2-carboxamide as an antitubercular agent.

The optimization campaign for a nitrofuran antitubercular hit (N-benzyl-5-nitrofuran-2-carboxamide; JSF-3449) led to the design, synthesis, and biological profiling of a family of analogs. These compounds exhibited potent in vitro antitubercular activity (MIC = 0.019-0.20 µM) against the Mycobacterium tuberculosis H37Rv strain and low in vitro cytotoxicity (CC50 = 40->120 µM) towards Vero cells. Significant improvements in mouse liver microsomal stability and mouse pharmacokinetic profile were realized by introduction of an α, α-dimethylbenzyl moiety. Among these compounds, JSF-4088 is highlighted due to its in vitro antitubercular potency (MIC = 0.019 µM) and Vero cell cytotoxicity (CC50 > 120 µM). The findings suggest a rationale for the continued evolution of this promising series of antitubercular small molecules.

Novel Pyrimidines as Antitubercular Agents.

Mycobacterium tuberculosis infection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused series of these analogs facilitated exploration of their in vitro activity, in vitro cytotoxicity, and physiochemical and absorption-distribution-metabolism-excretion properties. Select pyrimidines were then evaluated for their pharmacokinetic profiles in mice. The findings suggest a rationale for the further evolution of this promising series of antitubercular small molecules, which appear to share some similarities with the clinical compound PA-824 in terms of activation, while highlighting more general guidelines for the optimization of small-molecule antitubercular agents.

Naïve Bayesian Models for Vero Cell Cytotoxicity.

PURPOSE: To advance translational research of potential therapeutic small molecules against infectious microbes, the compounds must display a relative lack of mammalian cell cytotoxicity. Vero cell cytotoxicity (CC50) is a common initial assay for this metric. We explored the development of naïve Bayesian models that can enhance the probability of identifying non-cytotoxic compounds. METHODS: Vero cell cytotoxicity assays were identified in PubChem, reformatted, and curated to create a training set with 8741 unique small molecules. These data were used to develop Bayesian classifiers, which were assessed with internal cross-validation, external tests with a set of 193 compounds from our laboratory, and independent validation with an additional diverse set of 1609 unique compounds from PubChem. RESULTS: Evaluation with independent, external test and validation sets indicated that cytotoxicity Bayesian models constructed with the ECFP_6 descriptor were more accurate than those that used FCFP_6 fingerprints. The best cytotoxicity Bayesian model displayed predictive power in external evaluations, according to conventional and chance-corrected statistics, as well as enrichment factors. CONCLUSIONS: The results from external tests demonstrate that our novel cytotoxicity Bayesian model displays sufficient predictive power to help guide translational research. To assist the chemical tool and drug discovery communities, our curated training set is being distributed as part of the Supplementary Material. Graphical Abstract Naive Bayesian models have been trained with publically available data and offer a useful tool for chemical biology and drug discovery to select for small molecules with a high probability of exhibiting acceptably low Vero cell cytotoxicity.

Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System.

Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, the assay detected F. tularensis on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect F. tularensis in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

MntR(Rv2788): a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis.

The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis.