Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

Inoculum effect on the efficacies of amoxicillin-clavulanate, piperacillin-tazobactam, and imipenem against extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing Escherichia coli in an experimental murine sepsis model.

Escherichia coli is commonly involved in infections with a heavy bacterial burden. Piperacillin-tazobactam and carbapenems are among the recommended empirical treatments for health care-associated complicated intra-abdominal infections. In contrast to amoxicillin-clavulanate, both have reduced in vitro activity in the presence of high concentrations of extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing E. coli bacteria. Our goal was to compare the efficacy of these antimicrobials against different concentrations of two clinical E. coli strains, one an ESBL-producer and the other a non-ESBL-producer, in a murine sepsis model. An experimental sepsis model {~5.5 log10 CFU/g [low inoculum concentration (LI)] or ~7.5 log(10) CFU/g [high inoculum concentration (HI)]} using E. coli strains ATCC 25922 (non-ESBL producer) and Ec1062 (CTX-M-14 producer), which are susceptible to the three antimicrobials, was used. Amoxicillin-clavulanate (50/12.5 mg/kg given intramuscularly [i.m.]), piperacillin-tazobactam (25/3.125 mg/kg given intraperitoneally [i.p.]), and imipenem (30 mg/kg i.m.) were used. Piperacillin-tazobactam and imipenem reduced spleen ATCC 25922 strain concentrations (-2.53 and -2.14 log10 CFU/g [P < 0.05, respectively]) in the HI versus LI groups, while amoxicillin-clavulanate maintained its efficacy (-1.01 log10 CFU/g [no statistically significant difference]). Regarding the Ec1062 strain, the antimicrobials showed lower efficacy in the HI than in the LI groups: -0.73, -1.89, and -1.62 log10 CFU/g (P < 0.05, for piperacillin-tazobactam, imipenem, and amoxicillin-clavulanate, respectively, although imipenem and amoxicillin-clavulanate were more efficacious than piperacillin-tazobactam). An adapted imipenem treatment (based on the time for which the serum drug concentration remained above the MIC obtained with a HI of the ATCC 25922 strain) improved its efficacy to -1.67 log10 CFU/g (P < 0.05). These results suggest that amoxicillin-clavulanate could be an alternative to imipenem treatment of infections caused by ESBL- and non-ESBL-producing E. coli strains in patients with therapeutic failure with piperacillin-tazobactam.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri(CCBH11880), Enterobacter hormaecheisubsp. oharae(CCBH10892) and Klebsiella pneumoniae(CCBH13327), isolated in Brazil. BesidesblaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2,aac(6’)-Ib-cr] and quinolones (qnrA1,qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to theblaNDM-1 gene in all strains.

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Fosfomycin versus meropenem in bacteraemic urinary tract infections caused by extended-spectrum β-lactamase-producing Escherichia coli (FOREST): study protocol for an investigator-driven randomised controlled trial.

INTRODUCTION Finding therapeutic alternatives to carbapenems in infections caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) is imperative. Although fosfomycin was discovered more than 40 years ago, it was not investigated in accordance with current standards and so is not used in clinical practice except in desperate situations. It is one of the so-called neglected antibiotics of high potential interest for the future. METHODS AND ANALYSIS The main objective of this project is to demonstrate the clinical non-inferiority of intravenous fosfomycin with regard to meropenem for treating bacteraemic urinary tract infections (UTI) caused by ESBL-EC. This is a 'real practice' multicentre, open-label, phase III randomised controlled trial, designed to compare the clinical and microbiological efficacy, and safety of intravenous fosfomycin (4 g/6 h) and meropenem (1 g/8 h) as targeted therapy for this infection; a change to oral therapy is permitted after 5 days in both arms, in accordance with predetermined options. The study design follows the latest recommendations for designing trials investigating new options for multidrug-resistant bacteria. Secondary objectives include the study of fosfomycin concentrations in plasma and the impact of both drugs on intestinal colonisation by multidrug-resistant Gram-negative bacilli. ETHICS AND DISSEMINATION Ethical approval was obtained from the Andalusian Coordinating Institutional Review Board (IRB) for Biomedical Research (Referral Ethics Committee), which obtained approval from the local ethics committees at all participating sites in Spain (22 sites). Data will be presented at international conferences and published in peer-reviewed journals. DISCUSSION This project is proposed as an initial step in the investigation of an orphan antimicrobial of low cost with high potential as a therapeutic alternative in common infections such as UTI in selected patients. These results may have a major impact on the use of antibiotics and the development of new projects with this drug, whether as monotherapy or combination therapy. TRIAL REGISTRATION NUMBER NCT02142751. EudraCT no: 2013-002922-21. Protocol V.1.1 dated 14 March 2014.

The Times They Are a-Changin': Carbapenems for Extended-Spectrum-β-Lactamase-Producing Bacteria.

Several antimicrobial agents are being investigated as alternatives to carbapenems in the treatment of infections caused by ESBL-producing Enterobacteriaceae, which may be useful in avoiding overuse of carbapenems in the context of recent global spread of carbapenem-resistant Enterobacteriaceae. The most promising candidates for invasive infections so far are β-lactam/β-lactamase inhibitor combinations and cephamycins.

Epiphyseal fracture of distal humerus in children, treated with bio-absorbable materials: one year follow-up

Introduction: The use of bioabsorbable materials for orthopaedic useand traumatic fracture fixation in children has been poorly investigatedin the litterature and the effects on growing bones seem contradictory.The aim of the study is to compare the clinical and radiological resultsand evolution between bioabsorbable and traditional K-Wires for thetreatment of elbow epiphyseal fractures in children.Method: From jan. 2008 to Dec. 2009 21 children with similar fracturesand age were separated in two groups according to the way of fracturefixation: bioabsorbable K-Wire group and traditional K-Wire group.Follow-up was done at 3, 6 and 12 month post-operatively. Range ofmotion and elbow stability were measured for all patients. Theradiological evolution of the two groups were compared in term ofconsolidation, ossous resorption and radiolucencies. The clinicalresults were compared according to the Mayo Elbow Peformancescore. Controlateral elbow is compared with injured elbow in the twogroups.Results: In the bioabsorbable K-wire group, there were 10 children,including 5 girles and 5 boys with an average age of 9.5 years, rangingfrom 5 to 14 years. They were 7 external condylar fractures and3 epitrochlear fractures. In the traditional K-Wire group there were11 children, 2 girls and 9 boys with an average age of 7.6 years,ranging from 4 to 14 years. There were 10 external condylar fracturesand 1 epitrochlear fracture. At first follow up. The Mayo ElbowPerformance score was 93.8 (85-100 )for the bioabsorbable K-Wiregroup and 95.5 (85-100) for the traditional K-Wire group. In twochildren from the bioabsorbable K-Wire group there were transitoryradiolucencies along the wire tract on the x-ray, without clinicalmanifestation of it.We didn't see any premature closure of growingcartilage.Discussion: There is no significant differencies in term of clinical andradiological outcome between the two groups. The use ofbioabsorbable pins seems to be a good alternative to removabletraditional materials, avoiding a second operation.

Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases in Spain: microbiological and clinical features.

Extended-spectrum β-lactamases (ESBL) of the CTX-M, SHV, and TEM families were recognized in 76 (67%), 31 (27%), and 6 (5%) isolates, respectively, among 162 ESBL-producing Klebsiella pneumoniae (ESBL-Kp) strains obtained in a multicenter study in Spain. Predisposing factors for ESBL-Kp acquisition included invasive procedures, mechanical ventilation, and previous antimicrobial use.

Four main virotypes among extended-spectrum-β-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

Impact of the MIC of piperacillin-tazobactam on the outcome of patients with bacteremia due to extended-spectrum-β-lactamase-producing Escherichia coli.

We investigated the impact of the piperacillin-tazobactam MIC in the outcome of 39 bloodstream infections due to extended-spectrum-β-lactamase-producing Escherichia coli. All 11 patients with urinary tract infections survived, irrespective of the MIC. For other sources, 30-day mortality was lower for isolates with a MIC of ≤ 2 mg/liter than for isolates with a higher MIC (0% versus 41.1%; P = 0.02).

Pathways for the synthesis of polyesters in plants; cutin, suberin, and polyhydroxyalkanoates

Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Erkrankungen durch verotoxinbildende Escherichia coli beim Menschen [Verotoxin-producing Escherichia coli in human disease]

Les colibacilles producteurs de vérotoxines sont impliqués dans la pathogénie de syndromes diarrhéiques et dans celle de certains syndromes hémolytiques et urémiques. Le syndrome diarrhéique est caractérisé par l'apparition soudaine de douleurs abdominales sévères à type de crampes, suivies d'une diarrhée aqueuse qui, ensuite, devient sanglante. Les diarrhées peuvent être accompagnées de vomissements et d'une fièvre modérée. La période d'incubation varie entre 3 et 9 jours. Le syndrome hémolytique et urémique, la première cause d'insuffisance rénale aiguë du nourrisson et de l'enfant, est caractérisé par une triade typique: anémie hémolytique microangiopathique avec thrombocytopénie et insuffisance rénale glomérulonéphritique aiguë. La cause du syndrome hémolytique et urémique avec diarrhées prodromique a été attribuée aux vérotoxines. Les toxines produites par les colibacilles seraient à l'origine de lésions vasculaires endothéliales prédominant au niveau rénal et induisant le syndrome hémolytique et urémique.