Persistence of microcystin production by Planktothrix agardhii (Cyanobacteria) exposed to different salinities

International audienceRecent reports predict increases in harmful cyanobacteria in water systems worldwide due to climatic and environmental changes, which would compromise water quality and public health. Among abiotic changes, higher salinities are expected to promote the growth of certain harmful species, such as Planktothrix agardhii, which forms blooms in brackish waters. Since P. agardhii is a common producer of cyanotoxin, we investigated the growth and tolerance of this species when exposed in vitro to a range of salinities, while assessing variations in its microcystin diversity and production in batch cultures during a time-frame experiment spanning 18 days. The study revealed salt acclimation of the brackish P. agardhii, which continued to produce microcystins in salty cultures, while maintaining its growth capacity at low to medium NaCl (ranging from 0 to 7.5 g l−1). With higher concentrations (10 to 15 g l−1) significantly less growth occurred, corresponding to the shortening of cyanobacterial filaments, which nevertheless maintained their metabolic functions, as revealed by the high intensity of chlorophyll auto-fluorescence and persistent microcystin production. These findings showed that moderate to high salt levels do not inhibit microcystin production by P. agardhii, at least for several weeks. This raises questions concerning the persistence of harmful cyanobacteria in shallow water systems more exposed to evaporation and consequently to an increase in salinity in the future, as predicted by various climate models

An Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways

A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism

Versatilité écologique de la cyanobactérie potentiellement toxique Planktothrix agardhii : influence de la salinité?

La recherche a été initiée par l'observation d'efflorescences de Planktothrix agardhii, une cyanobactérie dulçaquicole potentiellement toxique, dans deux étangs saumâtres, les étangs de l'Olivier et de Bolmon, avec dans ce dernier le déclin de l'espèce concomitamment à une augmentation de salinité. L'objectif de l'étude consistait à évaluer l'influence de la salinité du milieu sur la performance, l'hégémonie et la production de toxine de Planktothrix agardhii au sein de la communauté phytoplanctonique.La réalisation de suivis pluriannuels in situ couplés à des expérimentations en milieu contrôlé a permis de démontrer (i) la capacité d'acclimatation et d'adaptation à la salinité de Planktothrix agardhii, laquelle garantit sa suprématie et sa production toxinique en milieu saumâtre ; et (ii) la modification structurale et fonctionnelle de la communauté phytoplanctonique suite à une augmentation de salinité supérieure au seuil d'halotolérance de Planktothrix agardhii. La recherche témoigne ainsi de la versatilité des cyanobactéries qui renforce leur aptitude à être de bons compétiteurs, laissant présager leur persistance, la continuité de leurs nuisances, et leur expansion dans le futur.The research was launched by the observation of Planktothrix agardhii blooms, a potentially toxic freshwater cyanobacterium, in two brackish ponds, the Olivier and Bolmon ponds, with in the latter, the concomitantly collapse of P. agardhii with an increase in salinity. The goal of the study was to assess the salinity influence on the performance, the dominance and the toxin production of P. agardhii within the phytoplankton community.The achievement of a long-term monitoring in situ combined with batch cultures experiments has demonstrated (i) the ability of P. agardhii to acclimate and adapt to salinity, which ensure its supremacy and its toxin production in brackish areas, and (ii) the structural and functional changes of the phytoplankton community with the exceeding of the salt-tolerance threshold of P. agardhii .The research reflects the cyanobacteria versatility that enhances their suitability for being good performers, suggesting their persistence along with their nuisances, and their expansion in the future

An Intertwined Network of Regulation Controls Membrane Permeability Including Drug Influx and Efflux in Enterobacteriaceae

International audienceThe transport of small molecules across membranes is a pivotal step for controlling the drug concentration into the bacterial cell and it efficiently contributes to the antibiotic susceptibility in Enterobacteriaceae. Two types of membrane transports, passive and active, usually represented by porins and efflux pumps, are involved in this process. Importantly, the expression of these transporters and channels are modulated by an armamentarium of tangled regulatory systems. Among them, Helix-turn-Helix (HTH) family regulators (including the AraC/XylS family) and the two-component systems (TCS) play a key role in bacterial adaptation to environmental stresses and can manage a decrease of porin expression associated with an increase of efflux transporters expression. In the present review, we highlight some recent genetic and functional studies that have substantially contributed to our better understanding of the sophisticated mechanisms controlling the transport of small solutes (antibiotics) across the membrane of Enterobacteriaceae. This information is discussed, taking into account the worrying context of clinical antibiotic resistance and fitness of bacterial pathogens. The localization and relevance of mutations identified in the respective regulation cascades in clinical resistant strains are discussed. The possible way to bypass the membrane/transport barriers is described in the perspective of developing new therapeutic targets to combat bacterial resistance

A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

International audienceThe cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism

Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics

International audienceTo understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation which is defined by influx and efflux. Several methods exist to evaluate the membrane permeability and efflux pump activity but they present some disadvantages and limitations. An optimized spectrofluorimetric method using the intrinsic tryptophan fluorescence as internal standard as well as a complementary microfluorimetric assay following the time-course accumulation in intact individual cells have been developed. Comparing the latter population and single cell approaches can lead to the understanding of the phenotypic heterogeneity within a population. The two methodologies lead to the determination of parameters, concentration, accumulation rates, localization that contribute to emerging concepts (RTC2T, SICAR) with the aim to identify and detail the antibiotic chemotypes that are involved in influx/efflux

Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes

International audienceThe efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d

In-vivo loss of carbapenem resistance by extensively drug-resistant Klebsiella pneumoniae during treatment via porin expression modification

International audienceKlebsiella pneumoniae, an Enterobacteriaceae that mostly causes hospital-acquired infections, belongs to the recently published WHO's list of antibiotic-resistant pathogens that pose the greatest threat to human health. Indeed, K. pneumoniae is the enterobacterial species most concerned by both resistance to extended-spectrum cephalosporins, due to extended-spectrum β-lactamase (ESBL) production, and resistance to carbapenems, i.e. the β-lactams with the broadest activity. Carbapenem resistance is related not only to carbapenemase production, but also the production of ESBL or AmpC and the loss of general porins. Here, we characterized the mechanisms that deprived a urinary ESBL-producing, porin-deficient K. pneumoniae isolate, isolated 13 days after the end of a 40-day course of imipenem treatment, of its carbapenem resistance. These mechanisms were observed in two in-vivo derivatives of this isolate and consisted of mutations in genes encoding molecules that participate in the downregulation of the synthesis of PhoE, a porin specialized in phosphate transport. We obtained three new derivatives from one of the two original derivatives, following in-vitro antibiotic pressure, in which the carbapenem resistance was restored because of mutations in genes encoding molecules that participate in the upregulation of PhoE synthesis. Thus, we uncovered novel mechanisms of carbapenem resistance/susceptibility switching in K. pneumoniae