A new innovative method, MICA Legionella , allows for the automatic enumeration of Legionella pneumophila in domestic water samples in 2 days, with a detection limit of 2 CFU per test portion. Here we show that it gives equivalent results to those obtained by the French standard method NF T90 -431 in 7 to 15 days.

Background Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires' disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. Objective This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. Methods MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. Results Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. Conclusion MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months.

Click-chemistry is a growing technology in the field of life sciences. In short, this technology makes it possible, under physiological conditions and in a non-lethal manner, to generate a covalent link between two functionalized molecules. In other terms, the functionalized molecules both present a bio-orthogonal function, which get connected together by a chemical reaction called “click-chemistry”. Since 2004, click chemistry has been used to combine a variety of molecules with Eucaryotic cells. In 2012 and 2014, two publications in Angewandte Chemie enlarged this kind of cell engeniering to Prokaryotes. They highlighted the existence of two natural monosaccharides of the ulosonic acids family, allowing the specific marking of Gram-negative bacteria and Legionella pneumophila. Those two discoveries led to the creation of DIAMIDEX company in December 2014. DIAMIDEX “technology” consists in marking Prokaryotes cells (adding them a bio-orthogional function) and associating them to colorimetric, fluorescent or magnetic molecules via click-chemistry. This asllows to detect/count/concentrate or identify specific microorganisms of interest. DIAMIDEX has developed more than 10 different monosaccharides that allow to target a wide range of microorganisms, from specific species to all microorganisms present in a sample. During the marking process, only dividing microorganisms will be targeted by our technology, as the monosaccharides need to be metabolized by the cells. With this approach, DIAMIDEX technology is able to provide a large contribution to the microbiotas field. This strategy has been used by Wang et al., (Biochemistry, 2017, 56(30, 3889-3893) to imaging Gram-Negative and Gram-Positive microbiotas in mice guts.

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction-diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded ('abnormal') proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis-Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called 'foci'. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

We recently developed a mathematical model for predicting reactive oxygen species (ROS) concentration and macromolecules oxidation in vivo. We constructed such a model using Escherichia coli as a model organism and a set of ordinary differential equations. In order to evaluate the major defences relative roles against hydrogen peroxide (H2O2), we investigated the relative contributions of the various reactions to the dynamic system and searched for approximate analytical solutions for the explicit expression of changes in H2O2 internal or external concentrations. Although the key actors in cell defence are enzymes and membrane, a detailed analysis shows that their involvement depends on the H2O2 concentration level. Actually, the impact of the membrane upon the H2O2 stress felt by the cell is greater when micromolar H2O2 is present (9-fold less H2O2 in the cell than out of the cell) than when millimolar H2O2 is present (about 2-fold less H2O2 in the cell than out of the cell). The ratio between maximal external H2O2 and internal H2O2 concentration also changes, reducing from 8 to 2 while external H2O2 concentration increases from micromolar to millimolar. This non-linear behaviour mainly occurs because of the switch in the predominant scavenger from Ahp (Alkyl Hydroperoxide Reductase) to Cat (catalase). The phenomenon changes the internal H2O2 maximal concentration, which surprisingly does not depend on cell density. The external H2O2 half-life and the cumulative internal H2O2 exposure do depend upon cell density. Based on these analyses and in order to introduce a concept of dose response relationship for H2O2-induced cell death, we developed the concepts of "maximal internal H2O2 concentration" and "cumulative internal H2O2 concentration" (e.g. the total amount of H2O2). We predict that cumulative internal H2O2 concentration is responsible for the H2O2-mediated death of bacterial cells.

Imlay and Linn show that exposure of logarithmically growing Escherichia coli to hydrogen peroxide (H2O2) leads to two kinetically distinguishable modes of cell killing. Mode one killing is pronounced near 1 mM concentration of H2O2 and is caused by DNA damage, whereas mode-two killing requires higher concentration (> 10 mM). The second mode seems to be essentially due to damage to all macromolecules. This phenomenon has also been observed in Fenton in vitro systems with DNA nicking caused by hydroxyl radical (HO center dot).To our knowledge, there is currently no mathematical model for predicting mode one killing in vitro or in vivo after H2O2 exposure.We propose a simple model, using Escherichia coli as a model organism and a set of ordinary differential equations. Using this model, we show that available iron and cell density, two factors potentially involved in ROS dynamics, play a major role in the prediction of the experimental results obtained by our team and in previous studies. Indeed the presence of the mode one killing is strongly related to those two parameters.To our knowledge, mode-one death has not previously been explained. Imlay and Linn (Imlay and Linn, 1986) suggested that perhaps the amount of the toxic species was reduced at high concentrations of H2O2 because hydroxyl (or other) radicals might be quenched directly by hydrogen peroxide with the concomitant formation of superoxide anion (a less toxic species). We demonstrate (mathematically and numerically) that free available iron decrease is necessary to explain mode one killing which cannot appear without it and that H2O2 quenching or consumption is not responsible for mode-one death.We are able to follow ROS concentration (particularly responsible for mode one killing) after exposure to H2O2. This model therefore allows us to understand two major parameters involved in the presence or not of the first killing mode.

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N-3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.

Legionella pneumophila …︁ …︁, ein potenziell tödliches Pathogen, wurde 1976 nach einem Ausbruch auf einer Tagung der American Legion in Philadelphia entdeckt. In ihrer Zuschrift auf S. 1299 ff. beschreiben S. Dukan, B. Vauzeilles und Mitarbeiter ein einfaches Nachweisverfahren für lebende Legionella pneumophila durch metabolischen Einbau eines modifizierten Kohlenhydrats in deren Lipopolysaccharide und anschließende Konjugation durch eine Klick-Reaktion. (Photo von Dr. Feely: CDC/Stafford Smith. Titelbild: David Garino.) Legionella pneumophila …︁ …︁, ein potenziell tödliches Pathogen, wurde 1976 nach einem Ausbruch auf einer Tagung der American Legion in Philadelphia entdeckt. In ihrer Zuschrift auf S. 1299 ff. beschreiben S. Dukan, B. Vauzeilles und Mitarbeiter ein einfaches Nachweisverfahren für lebende Legionella pneumophila durch metabolischen Einbau eines modifizierten Kohlenhydrats in deren Lipopolysaccharide und anschließende Konjugation durch eine Klick-Reaktion. (Photo von Dr. Feely: CDC/Stafford Smith. Titelbild: David Garino.) Aufkonvertierende Nanobiosonden In ihrer Zuschrift auf S. 1276 ff. zeigen X. Y. Chen et al., dass Nanobiosonden auf der Basis Lanthanoid-dotierter LiLuF4-Kern/Schale-Architekturen zum Nachweis von β-hCG geeignet sind und hohe Aufkonvertierungs-Quantenausbeuten ergeben.1 Zweidimensionale Nanomaterialien In ihrer Zuschrift auf S. 1290 ff. stellen H. C. Choi et al. das Musterwachstum großer MoS2-Atomlagen auf Goldoberflächen vor. Dabei gehen sie von [Mo(CO)6]-Dampf und H2S aus. Die MoS2 entstehen spezifisch und können durch einen Ätzprozess isoliert werden.1 Heterogene Katalyse In ihrer Zuschrift auf S. 1366 ff. nutzen M. Claeys et al. Magnetometrie und Röntgenbeugung in speziell entwickelten Reaktionskammern, um die größenabhängige Oxidation eines industriellen Fischer-Tropsch-Cobaltkatalysators unter Arbeitsbedingungen zu studieren.1

BACKGROUND:Legionella pneumophila is a waterborne pathogen responsible for Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been detected, like many other bacteria, in a "viable but non culturable" state (VBNC). The physiological significance of the VBNC state is unclear and controversial: it could be an adaptive response favoring long-term survival; or the consequence of cellular deterioration which, despite maintenance of certain features of viable cells, leads to death; or an injured state leading to an artificial loss of culturability during the plating procedure. VBNC cells have been found to be resuscitated by contact with amoebae.RESULTS:We used quantitative microscopic analysis, to investigate this "resuscitation" phenomenon in L. pneumophila in a model involving amending solid plating media with ROS scavengers (pyruvate or glutamate), and co-culture with amoebae. Our results suggest that the restoration observed in the presence of pyruvate and glutamate may be mostly due to the capacity of these molecules to help the injured cells to recover after a stress. We report evidence that this extracellular signal leads to a transition from a not-culturable form to a culturable form of L. pneumophila, providing a technique for recovering virulent and previously uncultivated forms of L. pneumophila.CONCLUSION:These new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples.

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture‐based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O‐antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila , while other Legionella species are not labeled.

Angeklickte Bakterien: Metabolisch aktive Gram-negative Bakterien können ein azidmodifiziertes Analogon von 3-Desoxy-D-mannooctulosonsäure (1; siehe Schema) spezifisch in Lipopolysaccharide auf ihrer Membran einbauen. Dieser Prozess versieht die Zelloberfläche mit einem bioorthogonalen chemischen Reporter und ermöglicht die schnelle Fluoreszenzmarkierung lebensfähiger Zellen mithilfe der Klick-Chemie. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this assumption has never been demonstrated and is therefore a matter of debate. Indeed, simultaneous determination of cell viability and metabolic activity has never been performed on the same cells. Here, we developed a microfluidic device to investigate the viability and the metabolic activity of Escherichia coli cells at single-cell level. Cells were immobilized in a flow chamber in which different solutions were sequentially injected according to different scenarios. By using time-lapse microscopy combined with automated tracking procedures, we first successfully assessed the ability of cells to divide and their metabolic activity at single-cell level. Applying these two procedures on the same cells after a hypochlorous acid (HOCl) treatment, we showed that the ability of cells to divide and their metabolic activity were anticorrelated. These results indicate that the relation between CV6 uptake and cell viability may be partially incorrect. Care must be taken in using the terms "CV6-positive" and "viable" synonymously.

Adaptation to a solar saltern environment requires mechanisms providing tolerance not only to salinity but also to UV radiation (UVR) and to reactive oxygen species (ROS). We cultivated prokaryote halophiles from two different salinity ponds: the concentrator M1 pond (240 g·L(-1) NaCl) and the crystallizer TS pond (380 g·L(-1) NaCl). We then estimated UV-B and hydrogen peroxide resistance according to the optimal salt concentration for growth of the isolates. We observed a higher biodiversity of bacterial isolates in M1 than in TS. All strains isolated from TS appeared to be extremely halophilic Archaea from the genus Halorubrum. Culturable strains isolated from M1 included extremely halophilic Archaea (genera Haloferax, Halobacterium, Haloterrigena, and Halorubrum) and moderately halophilic Bacteria (genera Halovibrio and Salicola). We also found that archaeal strains were more resistant than bacterial strains to exposure to ROS and UV-B. All organisms tested were more resistant to UV-B exposure at the optimum NaCl concentration for their growth, which is not always the case for H(2)O(2). Finally, if these results are extended to other prokaryotes present in a solar saltern, we could speculate that UVR has greater impact than ROS on the control of prokaryote biodiversity in a solar saltern.

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.

The toxicity of carbon dioxide has been established for close to a century. A number of animal experiments have explored both acute and long-term toxicity with respect to the lungs, the cardiovascular system, and the bladder, showing inflammatory and possible carcinogenic effects. Carbon dioxide also induces multiple fetal malformations and probably reduces fertility in animals. The aim of the review is to recapitulate the physiological and metabolic mechanisms resulting from CO(2) inhalation. As smokers are exposed to a high level of carbon dioxide (13%) that is about 350 times the level in normal air, we propose the hypothesis that carbon dioxide plays a major role in the long term toxicity of tobacco smoke.

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.