Abstract
Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance ...(AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436
Escherichia coli
were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.
To characterize bla(CTX-M) IncFII plasmids and clones of Escherichia coli from cats and dogs and to compare them with bla(CTX-M) IncFII plasmids reported in humans.
From December 2006 to April 2010, ...518 E. coli isolates from clinical infections in cats and dogs were screened for extended-spectrum β-lactamase (ESBL) production. Antimicrobial susceptibility was performed by disc diffusion and resistance genes were identified by PCR and sequencing. Plasmids were characterized using PCR-based replicon typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1-PFGE and Southern hybridization. Isolates were characterized by PFGE, phylogenetic grouping, O25b typing and multilocus sequence typing.
Nineteen E. coli isolates (3.7%) produced ESBLs, of which 14 (74%) carried bla(CTX-M) IncFII plasmids. The bla(CTX-M) gene was predominant and located on F31:A4:B1, F36:A4:B1 or F36:A1:B20 plasmids, abundantly reported in humans. The bla(CTX-M) F22:A1:B20 or F2:A2:B20 plasmids were also found. Different sequence types (STs) were identified, such as ST10, ST410, ST359, ST617 and ST224. Only one E. coli isolate belonged to the ST131 E. coli clone and carried a bla(CTX-M) F2:A2:B20 plasmid.
This is the first known extensive study on ESBL-producing E. coli isolates from pets in France. The ST131 clone was rare. However, the predominance of human-like bla(CTX-M) IncFII plasmids suggests exchanges of these plasmids with the human reservoir.
Food animals are a potential source of CTX-M resistance genes for human pathogens. We evaluated the transfer of the blaCTX-M-9 gene from animal strain of Salmonella enterica Virchow to ...Enterobacteriaceae of the human intestinal flora, using human flora-associated rats (HFA-rat) with and without cefixime treatment. In the absence of antibiotic, no transconjugant enterobacteria were found in the feces of HFA-rats. However, the transfer rate was high if Escherichia coli J5 recipient strains were co-inoculated orally with Salmonella. S. enterica Virchow persisted in the rat fecal flora, both during and after treatment with therapeutic doses of cefixime. The drug did not increase the transfer rate and E. coli J5-transconjugants were eliminated from the flora before the end of cefixime treatment. No cefixime was recovered in the rat feces. In the presence of recipient strains, the blaCTX-M-9 resistance gene was transferred from a strain of animal origin to the human intestinal flora, although transconjugant colonization was transient. Antibiotic use enhanced the persistence of donor strains, increasing the resistance gene pool and the risk of its spread.
Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with ...mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.
Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 ...transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.
•STOX1 overexpression drives drastic alterations of the l-malate/fumarate ratio and bioenergetic changes in trophoblasts.•Metabolic alterations upon STOX1 overexpression depend on iNOS uncoupling.•The NOS coupling agent and metabolite tetrahydrobiopterin (BH4) corrects STOX1-induced alterations including mitochondrial dysfunction in trophoblasts.•BH4 efficiently corrects the STOX1-induced preeclamptic/IUGR phenotype in mice and rats , and is a potential novel treatment.
Single sentence summary: Reduction of gestational preeclampsia/IUGR symptoms by tetrahydrobipterine is mediated by increased NOS coupling that restores l-malate levels.
Summary
Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full ...characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII‐mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live‐cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII‐mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer‐independent role for post‐Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1‐GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1‐coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1‐COPII system.
Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. ...Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.
The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid ...molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Δ⁵-sterols are replaced by 9δ,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Δ⁵-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.
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