Fernando Vázquez

Bioremediation of diesel-contaminated soils: Evaluation of potential in situ techniques by study of bacterial degradation

The development of a simple laboratory methodology allows theimplementation of in situbioremediation of polluted soils with diesel fuel. In thisinvestigation microbiological and chemical analyses and a suitable bioreactor design, were veryuseful for suggesting the best ways to improve biodegradation extents in a diesel-enrichedsoil. Biostimulation with inorganic nitrogen and phosphorus produced the best resultsin a simple bioreactor, with biodegradation extents higher than 90% after 45 days. Also,the addition of activated sludge from a domestic wastewater plant increased the degradationrate to a great extent. In both cases, microbiological studies showed the presence ofAcinetobacter sp. degrading most of thehydrocarbons. Simultaneously, a diesel fuel release(approximately 400,000 l) was studied. Samples taken in polluted soil and water revealed thatbacteria from the genus Acinetobacterwere predominant. In plate studies, Acinetobacter coloniesproduced a whitish substance with the characteristics of a biosurfactant. Remarkably, thepresence of this product was evident at the field site, both in the riverbanks and in the physicalrecovery plant. The study of the similarities between laboratory results and the diesel spillsite strongly suggested that natural conditions at the field site allowed the implementationof in situ bioremediation after physical removal of LNAPL (light nonaqueous-phase liquids).

Characterization of indigenous vaginal lactobacilli from healthy women as probiotic candidates

The probiotic relevant characteristics of 45 strains of vaginal Lactobacillus isolated from healthy women were analyzed. Of these, 21 strains were classified as L. crispatus, 17 as L. jensenii, six as L. gasseri, and one as L. plantarum. The rate of acidification varied significantly between the strains as did their ability to form biofilms. None used glycogen as a fermentable carbohydrate. H2O2 generation was common, especially among L. jensenii isolates (88%). No bacteriocinogenic strains were detected. Most strains harbored plasmids (from 1 to 7) of various sizes, those in excess of 50 kb being frequent. One of these plasmids was found to be promiscuous since it hybridized with extrachromosomal bands of 15 isolates. All strains were resistant to metronidazole, ciprofloxacin, gentamicin, clindamycin, trimethoprim, and sulfametoxazole and susceptible to a series of beta-lactams, erythromycin, tetracycline, and benzalkonium chloride. Almost half of the strains were highly resistant to nonoxinol 9, which is commonly used as a spermicide. Based on these analyses, strains of all three common species are proposed as new probiotic candidates.

Protection against Pneumococcal Pneumonia in Mice by Monoclonal Antibodies to Pneumolysin

ABSTRACT Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

Epidemiological study of Streptococcus pneumoniae carriers in healthy primary-school children

Abstract To obtain information on the Streptococcus pneumoniae carrier state in Spanish children, 332 healthy 6-year-old children from nine primary schools in northern Spain were screened. Thirty-six percent of the children had positive cultures yielding 128 strains. Seventy-one strains belonged to 14 serogroup/serotypes, the most frequent being 19, 23, 3, 24 and 11. Fifty-seven strains were non-typeable. The identification of strains with equivocal results was confirmed at species level by means of hybridisation with a specific probe, pneumolysin-mediated agglutination and a pathogenicity test in mice. Sixty-four percent of strains showed resistance to penicillin, 22% of these also being resistant to cefotaxime. More than 40% of the strains were resistant to trimethoprim-sulfamethoxazole, tetracycline and erythromycin. Twenty percent of the erythromycin-resistant strains were susceptible to clindamycin. Two strains were resistant to rifampicin and one strain was resistant to ofloxacin. All strains were susceptible to vancomycin. Previous antibiotic administration and having siblings under the age of 2 years correlated with the carriage of pneumococcus. There was no correlation with the carriage of antibiotic-resistant strains, or a record of previous infections, previous hospital admissions or having relatives with chronic respiratory disease.

The conserved undecapeptide shared by thiol‐activated cytolysins is involved in membrane binding

Thiol‐activated cytolysins share a conserved hydrophobic, Trp‐rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY‐5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BackgroundIntranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia.MethodsTo examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice.ResultsIn bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues.ConclusionThe data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival.

La microbiota vaginal: composición, papel protector, patología asociada y perspectivas terapéuticas

La microbiota vaginal, dominada por Lactobacillus crispatus, L. jensenii y L. gasseri, protege a la mucosa frente al establecimiento de microorganismos patogenos mediante tres mecanismos complementarios: a) la adherencia especifica al epitelio, que bloquea su asentamiento, b) la produccion de compuestos antimicrobianos y c) la coagregacion con los patogenos, que potencia su efecto microbiocida. A pesar de ello, en ocasiones se ve desplazada por microorganismos indeseables, lo que se asocia con la aparicion de vaginosis bacteriana, vaginitis por Candida spp., tricomoniasis e infecciones del tracto urinario inferior. Muy raramente, los lactobacilos causan patologia, invariablemente en pacientes inmunodeprimidos. Los cuadros dominantes son bacteriemias (alrededor del 50% de los casos) y endocarditis (30%). Sin embargo, no se ha descrito patologia genital por lactobacilos. El efecto mutualista de los lactobacilos sugiere que su instilacion podria regenerar el ecosistema vaginal, eliminando las recidivas asociadas al tratamiento de la infeccion.

Streptococcus pneumoniae virulence factors and their clinical impact: an update

The morbidity and mortality rates associated with Streptococcus pneumoniae remain very high worldwide. The virulence of this bacterium is largely dependent on its polysaccharide capsule, which is quite heterogeneous and represents a serious obstacle for designing effective vaccines. However, it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease. An important related finding from experimental animal models is that non-capsulated strains of pneumococci are protective against capsulated ones. Hence, new vaccine designs are focused on the surface proteins (e. g., PspA and PspC) and on the cytolysin, pneumolysin. Moreover, several virulence factors have potential value for pneumococcal diagnosis by urinalysis. In this paper, we review the virulence factors involved in bacteria-host interactions, and the new developments in vaccines and diagnostic methods.

A specific and ultrasensitive chemiluminescent sandwich ELISA test for the detection and quantitation of pneumolysin.

A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml−1, with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively. The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains; a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.

Actualización en infecciones de transmisión sexual: epidemiología, diagnóstico y tratamiento

immune-response modifier in the treatment of genital warts, and that are questions in the goal of this review. Viral hepatitis and HIV were no reviewed by space reasons.