Effect of Antimicrobial Consumption and Production Type on Antibacterial Resistance in the Bovine Respiratory and Digestive Tract

The aim of this study was to investigate the relationship between antimicrobial use and the occurrence of antimicrobial resistance in the digestive and respiratory tract in three different production systems of food producing animals. A longitudinal study was set up in 25 Belgian bovine herds (10 dairy, 10 beef, and 5 veal herds) for a 2 year monitoring of antimicrobial susceptibilities in E. coli and Pasteurellaceae retrieved from the rectum and the nasal cavity, respectively. During the first year of observation, the antimicrobial use was prospectively recorded on 15 of these farms (5 of each production type) and transformed into the treatment incidences according to the (animal) defined daily dose (TI_ADD) and (actually) used daily dose (TI_UDD). Antimicrobial resistance rates of 4,174 E. coli (all herds) and 474 Pasteurellaceae (beef and veal herds only) isolates for 12 antimicrobial agents demonstrated large differences between intensively reared veal calves (abundant and inconstant) and more extensively reared dairy and beef cattle (sparse and relatively stable). Using linear mixed effect models, a strong relation was found between antimicrobial treatment incidences and resistance profiles of 1,639 E. coli strains (p<0.0001) and 309 Pasteurellaceae (p≤0.012). These results indicate that a high antimicrobial selection pressure, here found to be represented by low dosages of oral prophylactic and therapeutic group medication, converts not only the commensal microbiota from the digestive tract but also the opportunistic pathogenic bacteria in the respiratory tract into reservoirs of multi-resistance.     

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.Eukaryotic plasma membranes (PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (Yetukuri et al., 2008; Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant PM contains an important number of sterols, among which β-sitosterol, stigmasterol, and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (SG) and acyl steryl glycosides (ASG) that represent up to approximately 15% of the tobacco (Nicotiana tabacum) PM (Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl inositol phosphorylceramides (GIPCs) are the major class of sphingolipids in plants, but they are absent in animals (Sperling and Heinz, 2003; Pata et al., 2010). Sphingolipidomic approaches identified up to 200 plant sphingolipids (for review, see Pata et al., 2010; Cacas et al., 2013).Although GIPCs belong to one of the earliest classes of plant sphingolipids that were identified in the late 1950s (Carter et al., 1958), only a few GIPCs have been structurally characterized to date because of their high polarity and a limited solubility in typical lipid extraction solvents. For these reasons, they were systematically omitted from published plant PM lipid composition. GIPCs are formed by the addition of an inositol phosphate to the ceramide moiety, the inositol headgroup of which can then undergo several glycosylation steps. The dominant glycan structure, composed of a hexose-GlcA linked to the inositol, is called series A. Polar heads containing three to seven sugars, so-called series B to F, have been identified and appeared to be species specific (Buré et al., 2011; Cacas et al., 2013; Mortimer et al., 2013). The ceramide moiety of GIPCs consists of a long-chain base (LCB), mainly t18:0 (called phytosphingosine) or t18:1 compounds (for review, see Pata et al., 2010), to which is amidified a very-long-chain fatty acid (VLCFA), the latter of which is mostly 2-hydroxylated (hVLCFA) with an odd or even number of carbon atoms. In plants, little is known about the subcellular localization of GIPCs. It is assumed, however, that they would be highly represented in the PM (Worrall et al., 2003; Sperling et al., 2005), even if this remains to be experimentally proven. The main argument supporting such an assumption is the strong enrichment of trihydroxylated LCB (t18:n) in detergent-insoluble membrane (DIM) fractions (Borner et al., 2005; Lefebvre et al., 2007), LCB being known to be predominant in GIPC’s core structure as aforementioned.In addition to this chemical complexity, lipids are not evenly distributed within the PM. Sphingolipids and sterols can preferentially interact with each other and segregate to form microdomains dubbed the membrane raft (Simons and Toomre, 2000). The membrane raft hypothesis suggests that lipids play a regulatory role in mediating protein clustering within the bilayer by undergoing phase separation into liquid-disordered and liquid-ordered phases. The liquid-ordered phase, termed the membrane raft, was described as enriched in sterol and saturated sphingolipids and is characterized by tight lipid packing. Proteins, which have differential affinities for each phase, may become enriched in, or excluded from, the liquid-ordered phase domains to optimize the rate of protein-protein interactions and maximize signaling processes. In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (Simons and Ikonen, 1997; Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see Mongrand et al., 2010; Simon-Plas et al., 2011; Cacas et al., 2012a).Moreover, lipids are not evenly distributed between the two leaflets of the PM. Within the PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer, whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. This asymmetrical distribution has been well established in human red blood cells, in which the outer leaflet contains sphingomyelin, phosphatidylcholine, and a variety of glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and their phosphorylated derivatives (Devaux and Morris, 2004). With regard to sphingolipids and glycerolipids, the asymmetry of the former is established during their biosynthesis and that of the latter requires ATPases such as the aminophospholipid translocase that transports lipids from the outer to the inner leaflet as well as multiple drug resistance proteins that transport phosphatidylcholine in the opposite direction (Devaux and Morris, 2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as proposed (Tjellstrom et al., 2010). Indeed, the authors showed that there is a pronounced transverse lipid asymmetry in root at the PM. Phospholipids and galactolipids dominate the cytosolic leaflet, whereas the apoplastic leaflet is enriched in sphingolipids and sterols.From such a high diversity of the plant PM thus arises the question of the respective contribution of lipids to membrane suborganization. Our group recently tackled this aspect by characterizing the order level of liposomes prepared from various plant lipids and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 2015). Fluorescence spectroscopy experiments showed that, among phytosterols, campesterol exhibits the strongest ability to order model membranes. In agreement with these data, spatial analysis of the membrane organization through multispectral confocal microscopy pointed to the strong ability of campesterol to promote liquid-ordered domain formation and organize their spatial distribution at the membrane surface. Conjugated sterols also exhibit a striking ability to order membranes. In addition, GIPCs enhance the sterol-induced ordering effect by emphasizing the formation and increasing the size of sterol-dependent ordered domains.The aim of this study was to reinvestigate the lipid composition and organization of the PM with a particular focus on GIPCs using tobacco leaves and cv Bright Yellow 2 (BY-2) cell cultures as models. Analyzing all membrane lipid classes at once, including sphingolipids, is challenging because they all display dramatically different chemical polarity, from very apolar (like free sterols) to highly polar (like polyglycosylated GIPCs) molecules. Most lipid extraction techniques published thus far use a chloroform/methanol mixture and phase partition to remove contaminants, resulting in the loss GIPCs, which remain in the aqueous phase, unextracted in the insoluble pellet, or at the interphase (Markham et al., 2006). In order to gain access to both glycerolipid and sphingolipid species at a glance, we developed a protocol whereby the esterifed or amidified fatty acids were hydrolyzed from the glycerol backbone (glycerolipids) or the LCB (sphingolipids) of membrane lipids, respectively. Fatty acids were then analyzed by gas chromatography-mass spectrometry (GC-MS) with appropriate internal standards for quantification. We further proposed that the use of methyl tert-butyl ether (MTBE) ensures the extraction of all classes of plant polar lipids. Our results indicate that GIPCs represent up to 40 mol % of total tobacco PM lipids. Interestingly, polyglycolyslated GIPCs are 5-fold enriched in DIMs of BY-2 cells when compared with the PM. Further investigation led us to develop a preparative purification procedure that allowed us to obtain enough material to raise antibodies against GIPCs. Using immunogold labeling on PM vesicles, it was found that polyglycosylated GIPCs cluster in membrane nanodomains, strengthening the idea that lateral nanosegregation of sphingolipids takes place at the PM in plants. Multispectral confocal microscopy was performed on vesicles prepared using GIPCs, phospholipids, and sterols and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite different fatty acid and polar head compositions, GIPCs extracted from tobacco leaves and BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together with sterols. Assuming that GIPCs are mostly present in the outer leaflet of the PM, interactions between sterols and sphingolipids were finally studied by the Langmuir monolayer technique, and the area of a single molecule of GIPC, or in interaction with phytosterols, was calculated. Using the calculation docking method, the energy of interaction between GIPCs and phytosterols was determined. A model was proposed in which GIPCs and phytosterols interact together to form liquid-ordered domains and in which the VLCFAs of GIPCs promote the interdigitation of the two membrane leaflets. The implications of domain formation and the asymmetrical distribution of lipids at the PM in plants are also discussed. Finally, we propose a model that reconsiders the intricate organization of the plant PM bilayer.     

Environmental drivers interactively affect individual tree growth across temperate European forests

Forecasting the growth of tree species to future environmental changes requires a better understanding of its determinants. Tree growth is known to respond to global‐change drivers such as climate change or atmospheric deposition, as well as to local land‐use drivers such as forest management. Yet, large geographical scale studies examining interactive growth responses to multiple global‐change drivers are relatively scarce and rarely consider management effects. Here, we assessed the interactive effects of three global‐change drivers (temperature, precipitation and nitrogen deposition) on individual tree growth of three study species (Quercus robur/petraea, Fagus sylvatica and Fraxinus excelsior). We sampled trees along spatial environmental gradients across Europe and accounted for the effects of management for Quercus. We collected increment cores from 267 trees distributed over 151 plots in 19 forest regions and characterized their neighbouring environment to take into account potentially confounding factors such as tree size, competition, soil conditions and elevation. We demonstrate that growth responds interactively to global‐change drivers, with species‐specific sensitivities to the combined factors. Simultaneously high levels of precipitation and deposition benefited Fraxinus, but negatively affected Quercus’ growth, highlighting species‐specific interactive tree growth responses to combined drivers. For Fagus, a stronger growth response to higher temperatures was found when precipitation was also higher, illustrating the potential negative effects of drought stress under warming for this species. Furthermore, we show that past forest management can modulate the effects of changing temperatures on Quercus’ growth; individuals in plots with a coppicing history showed stronger growth responses to higher temperatures. Overall, our findings highlight how tree growth can be interactively determined by global‐change drivers, and how these growth responses might be modulated by past forest management. By showing future growth changes for scenarios of environmental change, we stress the importance of considering multiple drivers, including past management and their interactions, when predicting tree growth.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

Recombinant human interleukins IL-1alpha, IL-1beta, IL-4, IL-6, and IL-7 show different and specific calcium-independent carbohydrate-binding properties

A method was developed for the determination of putative lectin activities of cytokines. It involved the immunoblotting measurement of the quantity of these cytokines unbound to a series of different immobilized glycoconjugates and displacement of the bound cytokines with oligosaccharides of known structures. This method allows demonstrating that the following interleukins specifically recognize different oligosaccharide structures in a calcium-independent mechanism: interleukin-1alpha binds to the biantennary disialylated N-glycan completed with two Neu5Acalpha2-3 residues; interleukin-1beta to a GM4 sialylated glycolipid Neu5Acalpha2-3Galbeta1-Cer having very long and unusual long-chain bases; interleukin-4 to the 1,7 intramolecular lactone of N-acetyl-neuraminic acid; interleukin-6 to compounds having N-linked and O-linked HNK-1-like epitopes; and interleukin-7 to the sialyl-Tn antigen. Because the glycan ligands are rare structures in human circulating cells, it is suggested that such activities could be essential for providing specific signaling systems to cells having both the receptors and the oligosaccharide ligands of the interleukin at their cell surface.     

Production of an S RNase with dual specificity suggests a novel hypothesis for the generation of new S alleles

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of self-incompatibility. The S(11) and S(13) RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S(11) or S(13) pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S(11) RNase to match those in the S(13) RNase was sufficient to completely replace the S(11) phenotype with the S(13) phenotype. We now show that an S(11) RNase in which only three amino acid residues were modified to match those in the S(13) RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S(11) and S(13) pollen). Thus, S(12)S(14) plants expressing this hybrid S RNase rejected S(11), S(12), S(13), and S(14) pollen yet allowed S(15) pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S(13) allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dual-specific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.