Implementation of antibiotic stewardship programmes in French ICUs in 2018: a nationwide cross-sectional survey.

BACKGROUND: Antibiotic stewardship programmes have a pivotal role in ICUs, but the level of implementation of these programmes at the regional or national level is not well known. OBJECTIVES: The aim of our study was to assess the level of implementation of antibiotic stewardship programmes in French ICUs. METHODS: We conducted a nationwide cross-sectional survey from January to March 2018 using an online questionnaire sent as an E-mail link to ICU specialists (one questionnaire per ICU). RESULTS: Overall, 113 out of 206 (55%) ICUs participated. Access to local epidemiology regarding bacterial resistance and antibiotic consumption data was reported in 84% and 65% of ICUs, respectively. Local guidelines for antibiotic use were available in 54% of ICUs. The duration of empirical antibiotic therapy was limited in 46% of cases, following the recommendation of an external expert in 33%. An antibiotic stewardship programme leader was reported at the hospital level by 94% of respondents, being an infectious disease physician in 80%. His/her role in the ICU was mostly to discuss specific cases (50%) and to provide advice on antibiotic prescriptions (26%). Regarding microbiological diagnosis, blood cultures were not processed at night or during weekends in 57%. Molecular biology and MS techniques were available in 62% and 59% of cases, respectively. Therapeutic drug monitoring of ß-lactams was available in 46% of cases. Forty-three percent of respondents knew the expression 'antimicrobial/antibiotic stewardship'. CONCLUSIONS: Antibiotic stewardship programmes are not optimally implemented in French ICUs. Improvement efforts and regular monitoring of the level of implementation are needed.

MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria.

In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature-sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro.

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.

Amendment of soil by biochars and activated carbons to reduce chlordecone bioavailability in piglets.

Chlordecone (Kepone or CLD) is a highly persistent pesticide formerly used in French West Indies. Nowadays high levels of this pesticide are still found in soils which represent a subsequent source of contamination for outdoor-reared animals. In that context, sequestering matrices like biochars or activated carbons (ACs) are believed to efficiently decrease the bioavailability of such compounds when added to contaminated soils. The present study intends to test the respective efficiency of soil amendment strategies using commercial ACs or biochars (obtained by a 500 °C or 700 °C pyrolysis of 4 distinct type of wood). This study involved three experimental steps. The first one characterized specific surface areas of biochars and ACs. The second one assessed CLD-availability of contaminated artificial soils (50 µg g-1 of Dry Matter) amended with 5% of biochar or AC (mass basis). The third one assessed CLD bioavailability of those artificial soils through an in vivo assay. To limit ethically the number of animals, selections of the most promising media were performed between each experimental steps. Forty four castrated male 40-day-old piglets were exposed during 10 day by amended artificial soils according to their group (n = 4). Only treatment groups exposed through amended soil with AC presented a significant decrease of concentrations of CLD in liver and adipose tissue in comparison with the control group (p < 0.001). A non-significant decrease was obtained by amending artificial soil with biochars. This decrease was particularly high for a coconut shell activated carbon were relative bioavailability was found lower than 3.2% for both tissues. This study leads to conclude that AC introduced in CLD contaminated soil should strongly reduce CLD bioavailability.

Cystic fibrosis, lung infections, and a human tracheal antimicrobial peptide (hTAP).

In order to understand how lungs of healthy people, unlike those of cystic fibrosis (CF) patients, are protected against bacterial infections such as Pseudomonas aeruginosa, the following three key findings were made. First, P. aeruginosa do not multiply when planted onto tracheal epithelial cells from healthy humans but do so profusely on cells from deltaF508 CF patients. Second, some bacteria bind, and gain entrance into CF cells, even at a physiological salt concentration (104 mM). Third, human tracheal epithelial cells express an approximately 4 kDa peptide (hTAP), which is known in its bovine form to exhibit bactericidal action against P. aeruginosa. A model is proposed depicting both how normal epithelial cells, in a first-line self defense mechanism, may be protected against bacterial infection and how this mechanism may fail during the initial stages of CF.

[Metrorrhagia during the second trimester of pregnancy: obstetrical and perinatal outcome. A retrospective study including 85 cases]. / Métrorragies du deuxième trimestre de la grossesse: devenir obstétrical et périnatal.

OBJECTIVE OF THE STUDY: To examine pregnancy outcome in patients with second-trimester vaginal bleeding and to determine a relationship between presumed etiology and perinatal outcome. MATERIAL: and methods: A retrospective study performed in Toulouse La Grave CHU between January 1993 and December 1997 including 85 cases of vaginal bleeding from 15 to 27 week's amenorrhea (90 fetuses). Results are compared to overall deliveries during the same period (14941 deliveries). RESULTS: Mean age at diagnosis is 29.8 years (SD: 5.3 years). Vaginal bleeding in the midtrimester concern 0.57% of deliveries. Abnormal ultrasonographic findings are observed in 73% of patients (placenta previa, subchorionic hematoma, placental abruption) and 81% were hospitalized (mean hospital duration: 18 days). Perinatal mortality was 17.04% and preterm delivery rate 30.6% (vs 11% in the overall patients). Perinatal complications are significatively increased compared with the overall population especially if ultrasonographic examination was abnormal. Second-trimester placental abruption had the worse prognosis. On the other hand, perinatal outcome was comparable when the origin of bleeding was unknown and ultrasonographic examination normal. 41% patients underwent cesarean section. CONCLUSION: Preterm delivery, perinatal mortality and morbidity are increased in patients with second-trimester vaginal bleeding. The risk is higher when abnormalities are detected at ultrasonography making ultrasonography a useful tool for predicting perinatal outcome.

Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract.

Chicory is a major source of fructans with reported prebiotic-bifidogenic properties. In the present study, the potential anti-inflammatory activities of chicory were investigated. Ethyl acetate chicory root extract produced a marked inhibition of prostaglandin E(2) (PGE(2)) production in human colon carcinoma HT29 cells treated with the pro-inflammatory agent TNF-alpha. Two independent mechanisms of action were identified: (1) a drastic inhibition of the induction by TNF-alpha of cyclooxygenase 2 (COX-2) protein expression and (2) a direct inhibition of COX enzyme activities with a significantly higher selectivity for COX-2 activity. The inhibition of TNF-alpha-dependent induction of COX-2 expression was mediated by an inhibition of NF-kappaB activation. A major sesquiterpene lactone of chicory root, the guaianolide 8-deoxylactucin, was identified as the key inhibitor of COX-2 protein expression present in chicory extract. Altogether, the data presented strongly support chicory root as a promising source of functional food ingredient, combining prebiotic and anti-inflammatory properties.

Cell body responses to axonal injury: traumatic axotomy versus toxic neuropathy.

To compare the evolution of cell body responses to two different types of axonal injuries--sciatic nerve crush (axotomy) and chronic 2,5-hexanedione-induced neuropathy--we studied rat lumbar dorsal root ganglion neurons with light microscopy and morphometry. Compared with control neurons, axotomized cells showed early (1 day) increases in the frequencies of two responses, nuclear eccentricity and Nissl body displacement, and later (4 day) increases in average satellite cell nuclei and decreases in perikaryal diameters. In toxin-induced axonal degeneration, there were similar patterns of defined alterations, although the evolution progressed over weeks and the response magnitudes were smaller. We conclude that the two experimental conditions show basic morphologic similarities, implying cell body reorganization in toxic axonopathy may be a response to axonal dysfunction or degeneration.

Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function.

Most cases of cystic fibrosis are caused by a single deletion mutation (deltaF508) within the first nucleotide binding fold (NBF1) of the CFTR protein (cystic fibrosis transmembrane conductance regulator). NBF1 is classically defined as amino acid residues phenylalanine 433 through serine 589, encoded by exons 10-12, and only part of exon 9, of the CFTR gene. This assignment is based on sequence homology of this region of the CFTR protein with that of other nucleotide binding proteins. Here, we report that when the complete modular unit encoded precisely by exons 9-12 is expressed in Escherichia coli as glycine 404 through serine 589, i.e., as [G404-N432]NBF1 or as deltaF508[G404-N432]NBF1, the resultant proteins target the cytoplasmic membrane. Significantly, [G404-N432]NBF1 is readily labeled from the outside of intact E. coli spheroplasts with the water soluble, membrane impermeable probe Biotin-X-NHS, sulfosuccinimidyl-6-(biotinamido)-hexanoate. Similar findings were observed with the disease causing mutant deltaF508[G404-N432]NBF1. Three different control experiments which involved (1) assays for known cytosolic E. coli enzymes, (2) immuno-gold electron microscopy with antibody having an epitope for the biotin moiety, and (3) tests for biotinylation of the cytosolic component, Enzyme 1 of the glucose phosphotransferase system, demonstrated that the spheroplasts used in this study are neither leaky nor permeable to Biotin-X-NHS. In addition, membrane-associated [G404-N432]NBF1, upon solubilization with Triton X-100, was found to bind to an ATP-agarose column and be released therefrom by elution with ATP, emphasizing retention of a native-like structure. In sharp contrast, NBF1 localizes to the cytosol when the [G404-N432]-N-terminal region is replaced with the maltose binding protein. The novel findings reported here implicate a role of the N-terminal region of NBF1 in its subcellular localization and are directly relevant to our understanding of the membrane structure, function, and trafficking of CFTR.

Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization.

We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA, which have known half-lives. In particular, a probe for an extremely short-lived 5' region that is not found separate of the pre-rRNA identifies nascent transcripts within the nucleolus of an intact active cell, while other characterized probes identify molecules at different stages in the rRNA processing pathway. Through these studies, visualized by confocal and normal light microscopy, we (1) confirm that rDNA transcription occurs in small foci within nucleoli, (2) show that the nascent pre-rRNA transcripts and most likely also the rDNA templates are surprisingly extended in the nucleolus, (3) provide evidence that the 5' end of the nascent rRNA transcript moves more rapidly away from the template DNA than does the 3' end of the newly released transcript, and (4) demonstrate that the various subsequent rRNA processing steps occur sequentially further from the transcription site, with each early processing event taking place in a distinct nucleolar subdomain. These last three points are contrary to the generally accepted paradigms of nucleolar organization and function. Our findings also imply that the nucleolus is considerably more complex than the conventional view, inferred from electron micrographs, of only three kinds of regions - fibrillar centers, dense fibrillar components, and granular components - for the dense fibrillar component evidently consists of several functionally distinct sub-domains that correlate with different steps of ribosome biogenesis.

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis.

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

The Barr body is a looped X chromosome formed by telomere association.

We examined Barr bodies formed by isodicentric human X chromosomes in cultured human cells and in mouse-human hybrids using confocal microscopy and DNA probes for centromere and subtelomere regions. At interphase, the two ends of these chromosomes are only a micron apart, indicating that these inactive X chromosomes are in a nonlinear configuration. Additional studies of normal X chromosomes reveal the same telomere association for the inactive X but not for the active X chromosome. This nonlinear configuration is maintained during mitosis and in a murine environment.

The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein.

The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP). The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1. Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate). Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns. Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea. Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane. Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals. Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction. The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed.

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene.

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The cells produce both proinsulin I and II and efficiently process each into mature insulin, in a manner comparable to normal beta cells in isolated islets. Electron microscopy reveals typical beta-cell type secretory granules, in which insulin is stored. Insulin secretion is inducible up to 30-fold by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype.