Germinant Synergy Facilitates Clostridium difficile Spore Germination under Physiological Conditions.

Clostridium difficile is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. C. difficile is the leading cause of nosocomial infectious diarrhea worldwide. C. difficile infection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step in C. difficile pathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest that C. difficile germination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficile is an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of a C. difficile infection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitate C. difficile spore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, but in vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulate C. difficile spore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

Cysteine Desulfurase IscS2 Plays a Role in Oxygen Resistance in Clostridium difficile.

Clostridium difficile is an anaerobic, spore-forming bacterium capable of colonizing the gastrointestinal tract of humans following disruption of the normal microbiota, typically from antibiotic therapy for an unrelated infection. With approximately 500,000 confirmed infections leading to 29,000 deaths per year in the United States, C. difficile infection (CDI) is an urgent public health threat. We previously determined that C. difficile survives in up to 3% oxygen. Low levels of oxygen are present in the intestinal tract, with the higher concentrations being associated with the epithelial cell surface. Additionally, antibiotic treatment, the greatest risk factor for CDI, increases the intestinal oxygen concentration. Therefore, we hypothesized that the C. difficile genome encodes mechanisms for survival during oxidative stress. Previous data have shown that cysteine desulfurases involved in iron-sulfur cluster assembly are involved in protecting bacteria from oxidative stress. In this study, deletion of a putative cysteine desulfurase (Cd630_12790/IscS2) involved in the iron-sulfur cluster (Isc) system caused a severe growth defect in the presence of 2% oxygen. Additionally, this mutant delayed colonization in a conventional mouse model of CDI and failed to colonize in a germfree model, which has higher intestinal oxygen levels. These data imply an undefined role for this cysteine desulfurase in protecting C. difficile from low levels of oxygen in the gut.

Experiencia en el uso de tromboprofilaxis farmacológica en un hospital colombiano de tercer nivel / Experience with the use of pharmacological thromboprophylaxis in a tertiary care Colombian hospital

Objetivo: Describir las características de la población con patologías no quirúrgicas en quien se indicó manejo de tromboprofilaxis farmacológica en un hospital de tercer nivel y determinar la indicación según riesgo de tromboembolia venosa (VTE) y sangrado con las escalas Padua e IMPROVE Bleeding Score. Materiales y métodos: estudio observacional, descriptivo, de corte transversal que incluyó a pacientes > 18 años, con patología no quirúrgica del servicio de medicina interna, con estancia hospitalaria mínima de 48 horas, atendidos entre el 1 de junio de 2015 a 1 de junio de 2016. En la valoración de riesgo de VTE y sangrado se aplicaron las escalas Padua e IMPROVE Bleeding Score, respectivamente. Resultados: se incluyeron 531 pacientes, de los que el 51,78% fueron hombres, con una edad promedio de 66 años y hospitalización de 37,2 días. La indicación farmacológica más empleada fue la heparina de bajo peso molecular (76,3%). De acuerdo a la escala de Padua, solo 225 pacientes (42,37%) tenían un alto riesgo de tener VTE; según la IMPROVE Bleeding Score, 447 (84,18%) pacientes presentaban bajo riesgo de sangrado y 84 pacientes (15,81%) alto riesgo. Además, se observó que los pacientes con puntaje PADUA ≥ 4, el 21,33% tenía puntaje IMPROVE Bleeding Score ≥7, adicionalmente el 20,23% de los pacientes con alto riesgo de sangrado según esta última escala presentó un episodio de sangrado. Conclusión: se evidenció un sobreuso de tromboprofilaxis farmacológica, lo cual se ve reflejado en episodios de sangrado en paciente que no tenían indicado su uso

Objective: To describe the characteristics of the population with non-surgical conditions who were prescribed pharmacological thromboprophylaxis in a tertiary care hospital, and to determine the indication according to the risk for developing venous thromboembolism (VTE) and bleeding using the Padua Scale and the IMPROVE Bleeding Score. Materials and Methods: This is an observational, descriptive, and cross-sectional study that included patients >18 years old from the internal medicine ward who had no surgical conditions, who were hospitalized for 48 hours as a minimum, between June 1st, 2015 and June 1st, 2016. When assessing the risk for VTE and bleeding the Padua scale and the IMPROVE Bleeding Score were used, respectively. Results: Five-hundred and thirty-one patients were included, half of them (51.78%) were male, their mean age was 66 years, and their average hospitalization time was 37.2 days. The most frequently used pharmacological indication was low molecular weight heparin (76.3%). According to the Padua scale, only 225 patients (42.37%) were at high risk for developing VTE; according to the IMPROVE Bleeding Score, 447 (84.18%) patients were at low risk for bleeding, and 84 patients (15.81%) were at high risk. Also, it was observed that patients with a >4 score in the PADUA scale, 21.33% had >7 points in the IMPROVE Bleeding Score; additionally, 20.23% of patients at high risk for bleeding according to the latter instrument had a bleeding episode. Conclusion: There was overuse of pharmacological thromboprophylaxis, which was reflected in the occurrence of bleeding episodes in patients who did not qualify for such indication

Aminocatalysis-Mediated on-Resin Ugi Reactions: Application in the Solid-Phase Synthesis of N-Substituted and Tetrazolo Lipopeptides and Peptidosteroids.

A new solid-phase protocol for the synthesis of N-substituted and tetrazolo peptides is described. The strategy relies on the combination of aminocatalysis-mediated on-resin Ugi reactions and peptide couplings for the N-alkylation of peptides at selected sites, including the N-terminal double lipidation, the simultaneous lipidation/biotinylation, and the steroid/lipid conjugation via tetrazole ring formation. The solid-phase Ugi four-component reactions were enabled by on-resin transimination steps prior to addition of the acid and isocyanide components. The strategy proved to be suitable for the feasible incorporation of complex N-substituents at both termini and at internal positions, which is not easily achievable by other solid-phase methods.

High-throughput analysis of meiotic crossover frequency and interference via flow cytometry of fluorescent pollen in Arabidopsis thaliana.

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.

Deep genome-wide measurement of meiotic gene conversion using tetrad analysis in Arabidopsis thaliana.

Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3.5×10(-4) conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions.