Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Screening for Non-polyenic Antifungal Produced by Actinobacteria from Moroccan Habitats: Assessment of Antimycin A19 Production by Streptomyces albidoflavus AS25.

Fungal diseases are currently a serious public health problem, due to the limited number of fact-based effective principles, and the emergence of resistant strains to the polyenic antifungals. The aim of this study was to screen, for non-polyenic antifungals production by Actinobacteria, and to validate the screening program by characterizingthe produced compounds.Actinobacteria isolates were tested against four clinic human-pathogenic fungi isolated from Hospital Mohammed V Rabat, Morocco. The production of non-polyenic antifungal metabolites by active isolates was investigated based on the yeast cell specificity as challenging targets, antibacterial activity, activity against resistant Candida tropicalis R2 and Pythium irregular (resistant to polyenes), inhibition of antifungal activity by the addition of exogenous ergosterol, and the UV-visible light spectrophotometric analysis of the active crude extracts.The antifungal compound produced was purified using various chromatographic techniques and the selected producing strain was identified using the polyphasic approach.Among 480 Actinobacteria isolates, 55 showed antifungal activity against all tested clinically derived fungi. After performing the screening program, 4 Actinobacteria that hadall the desired criteriawere selected. Using the polyphasic approach, the taxonomic position of the selected Streptomyces AS25, isolated from rhizospheric soil of Alyssum spinosum, showed that it belongs to Streptomyces genus with 100% partial 16S similarity with Streptomyces albidoflavus NBRC13010. On the basis of HPLC and mass spectrometry, the purified compound produced by Streptomyces AS25 was identified as a non-polyenic lactone, antimycin A19, which has been found for the first time to be produced by Streptomyces albidoflavus strain. Following the obtained results, it is important to note that our screening criteria for non-polyenic antifungals have been validated and the rhizospheric soil represents an interesting source to isolate Actinobacteria.

Glyphosate based- herbicide exposure affects gut microbiota, anxiety and depression-like behaviors in mice.

Recently, a number of studies have demonstrated the profound relationship between gut microbiota (GM) alterations and behavioral changes. Glyphosate-based herbicides (GBH) have been shown to induce behavioral impairments, and it is possible that they mediate the effects through an altered GM. In this study, we investigated the toxic effects of GBH on GM and its subsequent effects on the neurobehavioral functions in mice following acute, subchronic and chronic exposure to 250 or 500 mg/kg/day. The effect of these acute and repeated treatments was assessed at the behavioral level using the open field, the elevated plus maze, the tail suspension and splash tests. Then, mice were sacrificed and the intestinal samples were collected for GM analysis. Subchronic and chronic exposure to GBH induced an increase of anxiety and depression-like behaviors. In addition, GBH significantly altered the GM composition in terms of relative abundance and phylogenic diversity of the key microbes. Indeed, it decreased more specifically, Corynebacterium, Firmicutes, Bacteroidetes and Lactobacillus in treated mice. These data reinforce the essential link between GM and GBH toxicity in mice and suggest that observed intestinal dysbiosis could increase the prevalence of neurobehavioral alterations.