Adaptation in toxic environments: comparative genomics of loci carrying antibiotic resistance genes derived from acid mine drainage waters

International audienceSeveral studies have suggested the existence of a close relationship between antibiotic-resistant phenotypes and resistance to other toxic compounds such as heavy metals, which involve co-resistance or cross-resistance mechanisms. A metagenomic library was previously constructed in Escherichia coli with DNA extracted from the bacterial community inhabiting an acid mine drainage (AMD) site highly contaminated with heavy metals. Here, we conducted a search for genes involved in antibiotic resistance using this previously constructed library. In particular, resistance to antibiotics was observed among five clones carrying four different loci originating from CARN5 and CARN2, two genomes reconstructed from the metagenomic data. Among the three CARN2 loci, two carry genes homologous to those previously proposed to be involved in antibiotic resistance. The third CARN2 locus carries a gene encoding a membrane transporter with an unknown function and was found to confer bacterial resistance to rifampicin, gentamycin, and kanamycin. The genome of Thiomonas delicata DSM 16361 and Thiomonas sp. X19 were sequenced in this study. Homologs of genes carried on these three CARN2 loci were found in these genomes, two of these loci were found in genomic islands. Together, these findings confirm that AMD environments contaminated with several toxic metals also constitute habitats for bacteria that function as reservoirs for antibiotic resistance genes

Chemical Composition and Activity of Essential Oils of Carissa macrocarpa (Eckl.) A.DC. Cultivated in Tunisia and its Anatomical Features

This is the first study investigating the chemical composition of essential oils (EOs) isolated from different tissues of Carissa macrocarpa (Eckl.)A.DC, their antimicrobial activity and the anatomical characters of the aerial organs and the fruits. The main EO components were pentadecanal and tetradecan-1-ol (31.9 and 16.5% in fresh leaf EO, resp.), (E)-nerolidol and caryophyllene oxide (27.3 and 15.0% in fruit EO, resp.), linalool and hexahydrofarnesyl acetone (30.9 and 24.9% in stem EO, resp.), benzyl benzoate (24.3% in flower EO). The fruit EO was more active against Candida albicans (MIC=0.46 mg/mL) compared to the reference antibiotic (17.66 mg/mL). Furthermore, at this concentration it inhibited all the Gram-positive bacteria. Concerning the anatomical features, it is noteworth the presence of a large cluster of Ca oxalate crystals inside some parenchymatous cells. Large ducts corresponding to non articulated laticifers were identified in the cortex of leaf, stem and fruit pericarp. The laticifers categories and their distribution are taxonomically important to discriminate this species from others acclimated in different countries. Considering the obtained results, EOs of C. macrocarpa can be a good source of antimicrobial compounds, contributing to solve the problem of microbial resistance to antibiotics. This article is protected by copyright. All rights reserved

Spiralyde A, an Antikinetoplastid Dolabellane from the Brown Alga <i>Dictyota spiralis</i>

Bioassay-guided fractionation of the antikinetoplastid extract of the brown alga Dictyota spiralis has led to the isolation of spiralyde A (1), a new dolabellane aldehyde, along with other five known related diterpenes (2&#8211;6). Their structures were determined by HRESIMS, 1D and 2D NMR spectroscopy, and comparison with data reported in the literature. The antiparasitic activity of all compounds was evaluated. Spiralyde A (1) and the known compound 3,4-epoxy-7,18-dolabelladiene (2) were the most active compounds against Leishmania amazonensis and Trypanosoma cruzi. Spiralyde A (1) was the most potent compound, comparable to benznidazole, the reference drug for trypanocidal activity

Programmed cell death in Acanthamoeba castellanii Neff induced by several molecules present in olive leaf extracts.

Therapy against Acanthamoeba infections such as Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba Keratitis (AK), remains as an issue to be solved due to the existence of a cyst stage which is highly resistant to most chemical and physical agents. Recently, the activity of Olive Leaf Extracts (OLE) was demonstrated against Acanthamoeba species. However, the molecules involved in this activity were not identified and/or evaluated. Therefore, the aim of this study was to evaluate the activity of the main molecules which are present in OLE and secondly to study their mechanism of action in Acanthamoeba. Among the tested molecules, the observed activities ranged from an IC50 of 6.59 in the case of apigenine to an IC50 > 100 μg/ml for other molecules. After that, elucidation of the mechanism of action of these molecules was evaluated by the detection of changes in the phosphatidylserine (PS) exposure, the permeability of the plasma membrane, the mitochondrial membrane potential and the ATP levels in the treated cells. Vanillic, syringic and ursolic acids induced the higher permeabilization of the plasma membrane. Nevertheless, the mitochondrial membrane was altered by all tested molecules which were also able to decrease the ATP levels to less than 50% in IC90 treated cells after 24 h. Therefore, all the molecules tested in this study could be considered as a future therapeutic alternative against Acanthamoeba spp. Further studies are needed in order to establish the true potential of these molecules against these emerging opportunistic pathogenic protozoa

In Vitro Activity of Statins against Naegleria fowleri

Naegleria fowleri causes a deadly disease called primary amoebic meningoencephalitis (PAM). Even though PAM is still considered a rare disease, the number of reported cases worldwide has been increasing each year. Among the factors to be considered for this, awareness about this disease, and also global warming, as these amoebae thrive in warm water bodies, seem to be the key factors. Until present, no fully effective drugs have been developed to treat PAM, and the current options are amphotericin B and miltefosine, which present side effects such as liver and kidney toxicity. Statins are able to inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is a key enzyme for the synthesis of ergosterol of the cell membrane of these amoebae. Therefore, the in vitro activity of a group of statins was tested in this study against two types of strains of Naegleria fowleri. The obtained results showed that fluvastatin was the most effective statin tested in this study and was able to eliminate these amoebae at concentrations of 0.179 &plusmn; 0.078 to 1.682 &plusmn; 0.775 &micro;M depending on the tested strain of N. fowleri. Therefore, fluvastatin could be a potential novel therapeutic agent against this emerging pathogen

Effect of arsenite and growth in biofilm conditions on the evolution of Thiomonas sp. CB2

International audienceThiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjuga-tive element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments. DATA SUMMARY This study utilizes sequences previously generated from other studies. The accession number for the sequence data of the whole population genome is ERR3040228 (project number PRJEB29999)

Permeation of the <i>Acanthamoeba</i> Neff to the vital dye SYTOX^® green caused by addition of IC₉₀ of the bioactive molecules.

<p>Molecules were added to cells (10⁵ cells/ml) in the presence of 1 μM SYTOX^® green in PBS + 20 mM d-glucose and the increase in fluorescence (<i>λ</i>_exc = 485 nm, <i>λ</i>_em = 520 nm) monitored. Negative Control cells: cells labeled with the Sytox green in presence of 0.5% Methanol. Positive control contained 2.5% Triton X-100 (A). Confocal microscopy of <i>Acanthamoeba castellanii</i> Neff labeled with SYTOX^® Green. Amoeba were plated as above and incubated for 3 h with IC₉₀ of the vanillin (B), vanillic acid (C) syringic acid (D) and ursolic acid (E), Negative control (F). Cells were observed in a Leica TSC SPE- confocal microscope equipped with inverted optics (<i>λ</i>_exc = 482 nm and <i>λ</i>_em = 519 nm).</p

Effects of the bioactive molecules against cysts were evaluated by incubating 10⁴ cysts of <i>A</i>. <i>castellanii</i> Neff with the previously calculated IC₉₀ values of the selected drugs in PYG medium, and cells were counted with a Neubauer chamber at between 96 and 168 h.

<p>Troph: Number of Trophozoite emerged from the cyst in the PYC medium.</p

Screening of 24 molecules against <i>Acanthamoeba spp</i> (IC₅₀ and IC₉₀ expressed in μg/ml).

<p>Screening of 24 molecules against <i>Acanthamoeba spp</i> (IC₅₀ and IC₉₀ expressed in μg/ml).</p