Control biológico de patógenos fúngicos en precosecha de frutas: mejora de la tolerancia de agentes de biocontrol al estrés hídrico y térmico

El control biológico ha demostrado ser una posible alternativa a los productos químicos de síntesis en el control de plagas y enfermedades. Sin embargo, esta técnica se ve limitada en muchos casos por las condiciones fluctuantes del medio y por el estrecho margen de condiciones ambientales bajo las cuales los agentes de biocontrol son capaces de establecerse y controlar de forma efectiva, así como por la dificultad de obtener un producto formulado final con viabilidad y vida útil adecuadas. El presente proyecto se planteaba como objetivo principal el estudio de los mecanismos de supervivencia en condiciones de estrés ambiental de dos agentes de biocontrol (Candida sake CPA-1 y Pantoea agglomerans CPA-2), con la finalidad de mejorar su competencia ecológica y su efectividad, mediante manipulación fisiológica. El enfoque era de gran novedad en el campo del biocontrol y no obstante los resultados obtenidos han sido muchos y muy satisfactorios y suponen una vía abierta para poder hacer del control biológico una estrategia competitiva y comparable a los productos químicos de síntesis. A continuación se describen los resultados obtenidos en cada uno de los objetivos para los dos agentes de biocontrol objeto de estudio por separado.

Producción y Técnicas de formulación de agentes de biocontrol efectivos frente a las principales enfermedades de postcosechas de fruta dulce

El principal objetivo del presente SubProyecto era estudiar el proceso de desarrollo de la levadura Candida sake como agente de biocontrol de las principales enfermedades de postcosecha de fruta de pepita, para su futura utilización comercial. Para ello se plantearon los siguientes objetivos específicos: 1º.- Optimización del proceso de producción del agente de bicontrol 2º.- Estudio de las técnicas de formulación del agente de biocontrol 3º.- Evaluación de la efectividad como agente de biocontrol a escala comercial, de los mejores productos obtenidos con el sistema de producción y formulación óptimos.

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Immunomodulation by microbial ribosomes

Over the past twenty years, many authors have reported evidence of the immunoprotective capacity of ribosomes isolated from bacteria, fungi and parasites. Since 1971 we have explored the protective capacity of ribosomes isolated from a large variety of microorganisms responsible for human and animal diseases. More recently, using monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice, we have studied the mechanism of the protection induced by ribosomes. These studies, in parallel with the development of a technology for the large scale production of ribosomes, have allowed us to achieve a new regard for ribosomal vaccines for use in human. The general concept of ribosomal vaccines in presented and examples of two such vaccines are described with data on the specific protection that they induce in mice against experimental infections with Klebsiella peneumoniae, Streptococcus pneumoniae, S. pyogenes and Haemophilus influenzae for the first one, and against Candida albicans type A and type B for the second one. Because of their high immunogenicity and their innocuity these vaccines represent a decisive improvement over classical microbial vaccines.

The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia.

INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

Gas emission during laparoscopic colorectal surgery using a bipolar vessel sealing device: A pilot study on four patients.

BACKGROUND: Dissection during laparoscopic surgery produces smoke containing potentially toxic substances. The aim of the present study was to analyze smoke samples produced during laparoscopic colon surgery using a bipolar vessel sealing device (LigaSuretrade mark). METHODS: Four consecutive patients undergoing left-sided colectomy were enrolled in this pilot study. Smoke was produced by the use of LigaSuretrade mark. Samples (5,5l) were evacuated from the pneumoperitoneum in a closed system into a reservoir. Analysis was performed with CO2-laser-based photoacoustic spectroscopy and confirmed by a Fourier-transform infrared spectrum. The detected spectra were compared to the available spectra of known toxins. RESULTS: Samples from four laparoscopic sigmoid resections were analyzed. No relevant differences were noted regarding patient and operation characteristics. The gas samples were stable over time proven by congruent control measurements as late as 24 h after sampling. The absorption spectra differed considerably between the patients. One broad absorption line at 100 ppm indicating H2O and several unknown molecules were detected. With a sensitivity of alpha min ca 10-5 cm-1 no known toxic substances like phenol or indole were identified. CONCLUSION: The use of a vessel sealing device during laparoscopic surgery does not produce known toxic substances in relevant quantity. Further studies are needed to identify unknown molecules and to analyze gas emission under various conditions.

Life histories and distribution of ostracods with depth in western Lake Geneva (Petit-Lac), Switzerland: A reconnaissance study

Because environmental conditions within a given basin are different for each season and at different water depth, knowledge of the life history and depth distribution of target species is important for environmental and palaeoenvironmental interpretations based on ostracod species assemblages and/or the geochemical compositions of their valves. In order to determine the distribution of species with depth as well as the life history of species from Lake Geneva, a one year sampling campaign of living ostracods was conducted at five sites (2, 5, 13, 33 and 70 m water depth) on a monthly basis in the Petit-Lac (western basin of Lake Geneva, Switzerland). Based on the results, the different species can be classified into three groups. Littoral taxa are found at 2 and 5 m water depth and include, in decreasing numbers of individuals, Cypridopsis vidua (O. F.Müller, 1776), Pseudocandona compressa (Koch, 1838), Limnocythere inopinata (Baird, 1843), Herpetocypris reptans (Baird, 1835), Potamocypris smaragdina (Vávra, 1891), Potamocypris similis (G. W. Müller, 1912), Plesiocypridopsis newtoni (Brady & Robertson, 1870), Prionocypris zenkeri (Chyzer & Toth, 1858) and Ilyocypris sp. Brady & Norman, 1889. Sublittoral species are found in a majority at 13 m water depth and to a lesser extend at 33 m water depth and include, in decreasing numbers of individuals, Fabaeformiscandona caudata (Kaufmann, 1900), Limnocytherina sanctipatricii, Candona candida (O. F. Müller, 1776) and Isocypris beauchampi (Paris, 1920). Profundal species are found equally at 13, 33 and 70 m water depth and includes, in decreasing numbers of individuals, Cytherissa lacustris (Sars, 1863), Candona neglecta Sars, 1887 and Cypria lacustris Lilljeborg, 1890. The occurrence of Limnocytherina sanctipatricii (Brady & Robertson, 1869) is restricted from late winter to late spring when temperatures are low, while C. vidua, L. inopinata, P. smaragdina, P. similis, P. newtoni and Ilyocypris sp. occur predominantly from spring to early autumn when temperatures are high. Individuals of C. neglecta, C. candida, F. caudata, P. compressa, C. lacustris, H. reptans and Cp. lacustris occur throughout the year with juveniles and adults occurring during the same period (C. neglecta at 70 m, C. lacustris at 13, 33 and 70 m, and H. reptans at 2, 5 and 13 m water depth) or with juveniles occurring during a different period of the year than adults (C. neglecta at 13 and 33 m and C. candida, F. caudata and P. compressa at their respective depth of occurrence). Among the environmental parameters investigated, an estimate of the relationship between ostracod autoecology and environmental parameters suggests that in the Petit-Lac: (i) water temperature and substrate characteristics are important factors controlling the distribution of species with depth, (ii) water temperature is also important for determining the timing of species development and, hence, its specific life history, and (iii) water oxygen and sedimentary organic matter content is less important compared to the other environmental parameter monitored.

Candidiasis Peritoneal: prevalença i factors de risc. Estudi preliminar

Introducció: Un cultiu positiu de líquid peritoneal per a Càndida spp amb clínica associada, és diagnòstic de Candidiasis Peritoneal (CP). Objetius: L’objectiu primari és coneixer la prevalença de CP. Com objectius secundaris coneixer els possibles factors de risc. Tipus d’estudi: Prospectiu, observacional. Mètodes: S’agafa una mostra de 74 pacients amb diagnòstic de peritonitis (2007-2010). Durant la cirugía s’aspira líquid peritoneal lliure i és conrea. Resultats i conclusions: La prevalença va ser del 17.6% (46.15% C. albicans). L’ afectació del tracte gastro-intestinal-superior (OR 6.554) i l’aparició de fallada cardio-vascular durant la cirugia (OR 5.827), són factors de risc per a desenvolupar-la. És estudi preliminar.

New nonculture-based methods for the diagnosis of invasive candidiasis.

PURPOSE OF REVIEW: Invasive candidiasis is a severe infectious complication occurring mostly in onco-hematologic and surgical patients. Its conventional diagnosis is insensitive and often late, leading to a delayed treatment and a high mortality. The purpose of this article is to review recent contributions in the nonconventional diagnostic approaches of invasive candidiasis, both for the detection of the epidose and the characterization of the etiologic agent. RECENT FINDINGS: Antigen-based tests to detect invasive candidiasis comprise a specific test, mannan, as well as a nonspecific test, beta-D-glucan. Both have a moderate sensitivity and a high specificity, and cannot be recommended alone as a negative screening tool or a positive syndrome driven diagnostic tool. Molecular-based tests still have not reached the stage of rapid, easy to use, standardized tests ideally complementing blood culture at the time of blood sampling. New tests (fluorescence in-situ hybridization or mass spectrometry) significantly reduce the delay of identification of Candida at the species level in positive blood cultures, and should have a positive impact on earlier appropriate antifungal therapy and possibly on outcome. SUMMARY: Both antigen-based and molecular tests appear as promising new tools to complement and accelerate the conventional diagnosis of invasive candidiasis with an expected significant impact on earlier and more focused treatment and on prognosis.

Systemic antibody responses to gut commensal bacteria during chronic HIV-1 infection.

BACKGROUND: Human systemic antibody responses to commensal microbiota are not well characterised during health and disease. Of particular interest is the analysis of their potential modulation caused by chronic HIV-1 infection which is associated with sustained enteropathy and systemic B cell disturbances reflected by impaired B cell responses and chronic B cell hyperactivity. The mechanisms underlying B cell hyperactivation and the specificities of the resulting hypergammaglobulinaemia are only poorly understood. METHODS: By a technique referred to as live bacterial FACS (fluorescence-activated cell sorting), the present study investigated systemic antibody responses to several gut and skin commensal bacteria as well as Candida albicans in longitudinal plasma and serum samples from healthy donors, chronic HIV-1-infected individuals with or without diarrhoea and patients with inflammatory bowel disease (IBD). RESULTS: The data show that systemic antibody responses to the commensal microbiota were abundantly present in humans and remained remarkably stable over years. Overall systemic antibody responses to gut commensal bacteria were not affected during chronic HIV-1 infection, with titres decreasing when normalised to elevated plasma immunoglobulin G (IgG) levels found in patients with HIV. In contrast, increases in the titres of high affinity antimicrobiota antibodies were detected in patients with IBD, demonstrating that conditions with known increased intestinal permeability and aberrant mutualism can induce changes in antibody titres observed in these assays. CONCLUSION: Neither HIV-associated enteropathy nor B cell dysfunction impact on the high-affinity systemic antibody responses to gut commensal bacteria. HIV-associated hypergammaglobulinaemia is therefore unlikely to be driven by induction of antimicrobiota antibodies.

Is (1→3)-β-D-glucan the missing link from bedside assessment to pre-emptive therapy of invasive candidiasis?

ABSTRACT: Invasive candidiasis is a frequent life-threatening complication in critically ill patients. Early diagnosis followed by prompt treatment aimed at improving outcome by minimizing unnecessary antifungal use remains a major challenge in the ICU setting. Timely patient selection thus plays a key role for clinically efficient and cost-effective management. Approaches combining clinical risk factors and Candida colonization data have improved our ability to identify such patients early. While the negative predictive value of scores and predicting rules is up to 95 to 99%, the positive predictive value is much lower, ranging between 10 and 60%. Accordingly, if a positive score or rule is used to guide the start of antifungal therapy, many patients may be treated unnecessarily. Candida biomarkers display higher positive predictive values; however, they lack sensitivity and are thus not able to identify all cases of invasive candidiasis. The (1→3)-β-D-glucan (BG) assay, a panfungal antigen test, is recommended as a complementary tool for the diagnosis of invasive mycoses in high-risk hemato-oncological patients. Its role in the more heterogeneous ICU population remains to be defined. More efficient clinical selection strategies combined with performant laboratory tools are needed in order to treat the right patients at the right time by keeping costs of screening and therapy as low as possible. The new approach proposed by Posteraro and colleagues in the previous issue of Critical Care meets these requirements. A single positive BG value in medical patients admitted to the ICU with sepsis and expected to stay for more than 5 days preceded the documentation of candidemia by 1 to 3 days with an unprecedented diagnostic accuracy. Applying this one-point fungal screening on a selected subset of ICU patients with an estimated 15 to 20% risk of developing candidemia is an appealing and potentially cost-effective approach. If confirmed by multicenter investigations, and extended to surgical patients at high risk of invasive candidiasis after abdominal surgery, this Bayesian-based risk stratification approach aimed at maximizing clinical efficiency by minimizing health care resource utilization may substantially simplify the management of critically ill patients at risk of invasive candidiasis.

Antimicrobial activity in vitro of plumbagin isolated from Plumbago species

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 µg/ml and of Candida albicans at a concentration of 0.78 µg/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.

Antimicrobial activity and chemical investigation of Brazilian Drosera

The antimicrobial activity of three different extracts (hexanic, ethyl acetate, methanol) obtained from Brazilian Drosera species (D. communis, D. montana var. montana, D. brevifolia, D. villosa var. graomogolensis, D. villosa var. villosa, Drosera sp. 1, and Drosera sp. 2 ) were tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecium (ATCC23212), Pseudomonas aeruginosa (ATCC27853), Escherichia coli (ATCC11229), Salmonella choleraesuis (ATCC10708), Klebsiella pneumoniae (ATCC13883), and Candida albicans (a human isolate). Better antimicrobial activity was observed with D. communis and D. montana var. montana ethyl acetate extracts. Phytochemical analyses from D. communis, D. montana var. montana and D. brevifolia yielded 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin); long chain aliphatic hydrocarbons were isolated from D. communis and from D. villosa var. villosa, a mixture of long chain aliphatic alcohols and carboxylic acids, was isolated from D. communis and 3b-O-acetylaleuritolic acid from D. villosa var. villosa.