Burden and risk factors for Pseudomonas aeruginosa community-acquired pneumonia:a Multinational Point Prevalence Study of Hospitalised Patients

Pseudornonas aeruginosa is a challenging bacterium to treat due to its intrinsic resistance to the antibiotics used most frequently in patients with community-acquired pneumonia (CAP). Data about the global burden and risk factors associated with P. aeruginosa-CAP are limited. We assessed the multinational burden and specific risk factors associated with P. aeruginosa-CAP. We enrolled 3193 patients in 54 countries with confirmed diagnosis of CAP who underwent microbiological testing at admission. Prevalence was calculated according to the identification of P. aeruginosa. Logistic regression analysis was used to identify risk factors for antibiotic-susceptible and antibiotic-resistant P. aeruginosa-CAP. The prevalence of P. aeruginosa and antibiotic-resistant P. aeruginosa-CAP was 4.2% and 2.0%, respectively. The rate of P. aeruginosa CAP in patients with prior infection/colonisation due to P. aeruginosa and at least one of the three independently associated chronic lung diseases (i.e. tracheostomy, bronchiectasis and/or very severe chronic obstructive pulmonary disease) was 67%. In contrast, the rate of P. aeruginosa-CAP was 2% in patients without prior P. aeruginosa infection/colonisation and none of the selected chronic lung diseases. The multinational prevalence of P. aeruginosa-CAP is low. The risk factors identified in this study may guide healthcare professionals in deciding empirical antibiotic coverage for CAP patients

Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in β-D(1-2) glucan synthesis

Fil:Zorreguieta, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ugalde, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon

Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (PvirB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that PvirB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind PvirB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with PvirB and induces a DNA bending with an angle of 50.36°. DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with PvirB-lacZ fusions showed that in B. abortus IHF participates in the regulation of PvirB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.Fil:Ugalde, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A marr-type regulator directly activates transcription from the Brucella abortus virB promoter by sharing a redundant role with HutC

Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals. © 2012, American Society for Microbiology.Fil:Zorreguieta, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ugalde, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Engineered ACC deaminase-expressing free-living cells of Mesorhizobium loti show increased nodulation efficiency and competitiveness on Lotus spp.

Ethylene inhibits the establishment of symbiosis between rhizobia and legumes. Several rhizobia species express the enzyme ACC deaminase, which degrades the ethylene precursor 1-cyclopropane-1-carboxilate (ACC), leading to reductions in the amount of ethylene evolved by the plant. M. loti has a gene encoding ACC deaminase, but this gene is under the activity of the NifA-RpoN- dependent promoter; thus, it is only expressed inside the nodule. The M. loti structural gene ACC deaminase (acdS) was integrated into the M. loti chromosome under a constitutive promoter activity. The resulting strain induced the formation of a higher number of nodules and was more competitive than the wild-type strain on Lotus japonicus and L. tenuis. These results suggest that the introduction of the ACC deaminase activity within M. loti in a constitutive way could be a novel strategy to increase nodulation competitiveness of the bacteria, which could be useful for the forage inoculants industry.Fil:Conforte, V.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Echeverria, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ugalde, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Menéndez, A.B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Lepek, V.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Collecting Phaseolus in Costa Rica

A survey was carried out in Central Costa Rica in Jan. 1987, in an attempt to find web blight resistance in native var.; 43 samples representing 10 different taxa were found. Wild P. vulgaris was found in 2 different places in the province of San Jose; both populations presented truly wild characteristics as to growth habit (climbing vines on thickets, 2-3 m high) and seed characteristics (small, flat, roundish, shiny, grey-mottled, and 100-seed wt. of 4.9 g). A species called P. costaricensis was also found; in all populations, it presented strong climbing stems up to 10 m high, strong large, upright and multiflowered racemes, brilliant pink fuschia flowers with a capitate extorse stigma, and epigeal germination. Another species was apparently new: purplish flowers, large stipules, and large ovate, early caducous primary bracts. The name of P. talamancensis has been proposed. Symptoms of web blight were not observed in many of these wild populations (P. tuerckheimii and P. xanthotrichus), but these results are not conclusive since several of these populations were found outside the endemic areas. Further phytopathological assessments are required. (CIAT

Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium

CYP130 belongs to the subset of cytochrome P450s from Mycobacterium tuberculosis (Mtb) that have been structurally characterized. Despite several efforts for its functional characterization, CYP130 is still considered an orphan enzyme for which no endogenous or exogenous substrate has been identified. In addition, functional redox-partners for CYP130 have not been clearly established yet, hampering the elucidation of its physiological role. In the present study, a catalytically active fusion protein involving CYP130 and the NADPH reductase-domain of CYP102A1 from Bacillus megaterium was created. By screening a panel of known substrates of human P450s, dextromethorphan N-demethylation was identified as a reaction catalyzed by CYP130. The fusion enzyme showed higher catalytic activity, when compared to CYP130 reconstituted with a selection of non-native redox-partners. Molecular dynamics simulation studies based on the crystal structure of CYP130 revealed two primary docking poses of dextromethorphan within the active site consistent with the experimentally observed N-demethylation reaction during the entire molecular dynamics simulation. The dextromethorphan N-demethylation reaction was strongly inhibited by azole-drugs and maybe applied to identify mechanism-based inhibitors of CYP130. Furthermore, the present active CYP130-fusion protein may facilitate the identification of endogenous substrates from Mtb

Caracterización agronómica de líneas de frijol común seleccionadas por resistencia horizontal a patógenos de la mixteca poblana

La selección para resistencia horizontal se basa en la utilización de la variabilidad genética de un cultivo, natural o inducida, para acumular características que, en conjunto, permitan incrementar su rendimiento en presencia de los patógenos para los que se busca resistencia. Este proceso debe ir aparejado con el mejoramiento de características agronómicas como precocidad, hábito de crecimiento, calidad de semilla y rendimiento per se, con el objetivo de integrar en las variedades derivadas aspectos agronómicos y de resistencia. En el Colegio de Postgraduados, México, se ha conducido un programa de mejoramiento genético por resistencia horizontal basado en la selección y recombinación recurrente de individuos resistentes al tizón común y al virus del mosaico común del frijol (VMCF), que son patógenos de importancia económica en la Región Mixteca de Puebla, México. Como resultado de dos ciclos de selección se han obtenido 30 líneas recombinantes escogidas con base en rendimiento, estabilidad, características agronómicas y supervivencia al ataque de patógenos, que serían usadas como progenitores del 3er. ciclo. Para conocer las características agronómicas del germoplasma base del 3er. ciclo de selección recurrente, se comparó el comportamiento agronómico de 42 líneas recombinantes derivadas de dos ciclos de recombinación agrupadas en cuatro poblaciones, con el de cinco de siete de los progenitores iniciales y dos genotipos ("landraces") regionales, en Tepexi de Rodríguez, Puebla, México, que se localiza en la región Mixteca Alta de Puebla. Los resultados indican que el esquema de selección utilizado en el mejoramiento genético por resistencia horizontal a patógenos ha sido útil para incrementar el rendimiento y mejorar las características agronómicas relacionadas con precocidad y arquitectura, bajo condiciones de temporal limitante. (RA