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.

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).

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.

Compact portable biosensor for arsenic detection in aqueous samples with Escherichia coli bioreporter cells.

We present a compact portable biosensor to measure arsenic As(III) concentrations in water using Escherichia coli bioreporter cells. Escherichia coli expresses green fluorescent protein in a linearly dependent manner as a function of the arsenic concentration (between 0 and 100 μg/L). The device accommodates a small polydimethylsiloxane microfluidic chip that holds the agarose-encapsulated bacteria, and a complete optical illumination/collection/detection system for automated quantitative fluorescence measurements. The device is capable of sampling water autonomously, controlling the whole measurement, storing and transmitting data over GSM networks. We demonstrate highly reproducible measurements of arsenic in drinking water at 10 and 50 μg/L within 100 and 80 min, respectively.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares ( CORBIA et al., 2000; MELO, 2009 ) . A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC nº 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares (CORBIA et al., 2000; MELO, 2009). A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC no 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Escherchia coli ribose binding protein based bioreporters revisited.

Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system.

Vuosina 1992-95 sioista eristettyjen E. coli -bakteerikantojen mikrobilääkeherkkyys ja kahden eri herkkyysmääritysmenetelmän vertailu käyttäen oksitetrasykliiniä

Virpi Ala-Risku, Marja Fossi ja Matti Nyberg

Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Enterohemorraginen Escherichia coli (EHEC) ja sen osoittaminen PCR-menetelmällä

Summary: Enterohemorrhagic Escherichia coli (EHEC) and its detection with a PCR method

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).