The DmsABC Sulfoxide Reductase Supports Virulence in Non-typeable Haemophilus influenzae.

Although molybdenum-containing enzymes are well-established as having a key role in bacterial respiration, it is increasingly recognized that some may also support bacterial virulence. Here, we show that DmsABC, a putative dimethylsulfoxide (DMSO) reductase, is required for fitness of the respiratory pathogen Haemophilus influenzae (Hi) in different models of infection. Expression of the dmsABC operon increased with decreasing oxygen availability, but despite this, a Hi2019Δd msA strain did not show any defects in anaerobic growth on chemically defined medium (CDM), and viability was also unaffected. Although Hi2019Δd msA exhibited increased biofilm formation in vitro and greater resistance to hypochlorite killing compared to the isogenic wild-type strain, its survival in contact with primary human neutrophils, in infections of cultured tissue cells, or in a mouse model of lung infection was reduced compared to Hi2019WT. The tissue cell infection model revealed a two-fold decrease in intracellular survival, while in the mouse model of lung infection Hi2019Δd msA was strongly attenuated and below detection levels at 48 h post-inoculation. While Hi2019WT was recovered in approximately equal numbers from bronchoalveolar lavage fluid (BALF) and lung tissue, survival of Hi2019Δd msA was reduced in lung tissue compared to BALF samples, indicating that Hi2019Δd msA had reduced access to or survival in the intracellular niche. Our data clearly indicate for the first time a role for DmsABC in H. influenzae infection and that the conditions under which DmsABC is required in this bacterium are closely linked to interactions with the host

Applying MDA and OMG Robotic Specification for Developing Robotic Systems

Robotics systems have special needs often related with their realtime nature and environmental properties. Often, control and communication paths within the system are tightly coupled to the actual physical configuration of the robot. As a consequence, these robots can only be assembled, configured, and programmed by robot experts. Traditional approaches, based on mainly writing the code without using software engineering techniques, are still used in the development process of these systems. Even when these robotic systems are successfully used, several problems can be identified and it is widely accepted that new approaches should be explored. The contribution of this research consists in delineating guidelines for the construction of robotic software systems, taking advantage of the application of the OMG standard robotic specifications which adhere to the model-driven approach MDA. Thereby the expert knowledge is captured in standard abstract models that can then be reused by other less experienced developers. In addition part of the code is automatically generated, reducing costs and improving quality

A case of coexisting Warthin tumor and langerhans cell histiocytosis associated with necrosis, eosinophilic abscesses and a granulomatous reaction in intraparotid lymph nodes

We present a patient (50-year-old male) with coexisting Warthin tumor and involvement of two intraparotid lymph nodes by Langerhans cell histiocytosis associated with necrosis, eosinophilic abscesses and a granulomatous reaction. This is the second documented case of this unusual combination of histological changes in nodal Langerhans cell histiocytosis and the first case involving intraparotid lymph nodes occurring together with an ipsilateral Warthin tumor

Nitroxide functionalized antibiotics are promising eradication agents against <i>Staphylococcus aureus</i> biofilms

ABSTRACTTreatment of Staphylococcus aureus biofilm-related infections represents an important medical challenge worldwide, as biofilms, even of drug-susceptible S. aureus strains, are highly refectory to conventional antibiotic therapy. Nitroxides were recently shown to induce dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Co-administration of the nitroxide CTEMPO with ciprofloxacin significantly improved the antibiotic’s biofilm-eradication activity against S. aureus. Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced ciprofloxacin’s minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter into cells localising at the surface and base of the biofilm structure. No toxicity was observed for the nitroxide CTEMPO and the nitroxide-antibiotic hybrids against human cells. Taken together, our results show that nitroxides can mediate dispersal of Gram-positive biofilms and that dual-acting biofilm-eradication antibiotics could provide broad-spectrum therapies for the treatment of biofilm-related infections.</jats:p

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics

AbstractAntibiotics are failing fast, and the development pipeline is alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which are inhibitors of bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is often stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors. Inhibitors of DsbA block bacterial oxidative protein folding and were previously identified by biophysical and biochemical assays. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that a combination of a cell-based AssT sulfotransferase assay and the UPEC motility assay, modified for a higher throughput format, can provide a robust and target-specific platform for the evaluation of DsbA inhibitors. Our pipeline could also be used in fragment and compound screening for the identification of new DsbA inhibitor classes or hits with a broad spectrum of activity. In conclusion, the establishment of accurate, high-throughput microbiological assays for antivirulence drug identification and early preclinical development, is a significant first step towards their translation into effective therapeutics.ImportanceThe safety net of last resort antibiotics is quickly vanishing as bacteria become increasingly resistant to most available drugs. If no action is taken, we will likely enter a post-antibiotic era, where common infections and minor injuries are once again lethal. The paucity in new antibiotic discovery of the past decades has compounded the problem of increasing antibiotic resistance, to the point that it now constitutes a global health crisis that demands global action. There is currently an urgent need for new antibacterial drugs with new targets and modes of action. To address this, research and development efforts into antivirulence drugs, such as DsbA inhibitors, have been ramping up globally. However, the development of microbiological assays as tools for effectively identifying and evaluating antivirulence drugs is lagging behind. Here, we present a high-throughput cell-based screening and evaluation pipeline, which could significantly advance development of DsbA inhibitor as antivirulence therapeutics.</jats:sec

Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms

Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro , but their action against Gram-positive bacterial biofilms remains unknown. </jats:p