Institute Microbiologia

Lisbon, Portugal

Institute Microbiologia

Lisbon, Portugal
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De Barbosa C.B.,Federal University of Rio de Janeiro | Lazzarini L.C.O.,Federal University of Rio de Janeiro | Elias A.R.,Oswaldo Cruz Institute | Leung J.A.M.,Clementino Fraga Filho University Hospital | And 9 more authors.
International Journal of Tuberculosis and Lung Disease | Year: 2012

BACKGROUND: We recently described the Mycobacterium tuberculosis RD Rio genotype, a clonally derived sublineage within the Latin American-Mediterranean (LAM) family. Genetic diversity of M. tuberculosis likely affects the clinical aspects of tuberculosis (TB). Prospective studies that address this issue are scarce and remain controversial. OBJECTIVE: To determine the association of differential clinical features of pulmonary TB with the RD Rio M. tuberculosis etiology. METHODS: Culture-proven pulmonary TB patients (n = 272) were clinically evaluated, including history, physical examination, chest X-ray and anti-human immunodeficiency virus serology. Isolates were classified as RD Rio or non-RD Rio M. tuberculosis by multiplex polymerase chain reaction and further spoligotyped. Clinical and M. tuberculosis genotype data were analyzed. RESULTS: RD Rio M. tuberculosis caused disease in 26.5% (72/270) of all TB cases. The LAM genotype, of which RD Rio strains are members, was responsible for 46.0% of the TB cases. Demographic data, major signs and symptoms, radiographic presentation, microbiological features and clinical outcomes were not significantly different among patients with TB caused by RD Rio and non-RD Rio strains. CONCLUSIONS: Disease caused by M. tuberculosis RD Rio strains was not clinically distinctive or more severe than disease caused by non-RD Rio strains in this series of TB patients. Larger prospective studies specifically designed to disclose differential clinical characteristics of TB caused by specific M. tuberculosis lineages are needed. © 2012 The Union.

Hanscheid T.,Institute Microbiologia | Gresnigt T.,University of Amsterdam | Zoller T.,Charité - Medical University of Berlin | Melo-Cristino J.,Institute Microbiologia | And 3 more authors.
Malaria Journal | Year: 2014

Background: Haemolytic conditions may contribute to disease pathogenesis and severe clinical manifestations through the liberation of free haemoglobin (Hb) and production of toxic free haem. Thus, free Hb and haem should be associated with altered MetHb and COHb levels in malaria as in other conditions. Methods. This study comprises data collected at three different sites: (i) a retrospective analysis of the first arterial blood gas result (ABGS) of any patient during 2010 at the University Hospital in Lisbon; (ii) a retrospective analysis of ABGS from patients with severe malaria admitted to the intensive care unit in Berlin, Germany; and (iii) a prospective study of non-invasive MetHb measurements in children with and without malaria in Lambaréné, Gabon. Results: In Lisbon, the mean MetHb level was 1.4% (SD: 0.5) in a total of 17,834 ABGS. Only 11 of 98 samples with a MetHb level of >3.0 referred to infections. COHb levels showed no particular association with clinical conditions, including sepsis. In 13 patients with severe malaria in Berlin, the mean MetHb levels on admission was 1.29%, with 1.36% for cerebral malaria and 1.14% for non-cerebral malaria (P > 0.05). All COHb measurements were below 2.3%. In Lambaréné, Gabon, 132 healthy children had a mean MetHb level of 1.57%, as compared to 150 children with malaria, with a value of 1.77% and 2.05% in uncomplicated and complicated cases, respectively (P < 0.01). Conclusions: The data appears consistent with the methaemoglobin/haem hypothesis in malaria and sepsis pathogenesis. However, although MetHb was significantly different between healthy controls and children with malaria in Africa, the difference was rather small, also when compared to previous studies. Still, non-invasive bedside MetHb testing may warrant further evaluation as it could be a simple adjuvant tool for prognosis in resource poor settings. © 2014 Hänscheid et al.; licensee BioMed Central Ltd.

PubMed | University of Amsterdam, University of Tübingen, Institute Microbiologia, Makerere University and University of Cape Town
Type: Journal Article | Journal: PLoS neglected tropical diseases | Year: 2015

Foci of the HIV epidemic and helminthic infections largely overlap geographically. Treatment options for helminth infections are limited, and there is a paucity of drug-development research in this area. Limited evidence suggests that antiretroviral therapy (ART) reduces prevalence of helminth infections in HIV-infected individuals. We investigated whether ART exposure and cotrimoxazole preventive therapy (CTX-P) is associated with a reduced prevalence of helminth infections.This cross-sectional study was conducted at a primary HIV-clinic in Lambarn, Gabon. HIV-infected adults who were ART-nave or exposed to ART for at least 3 months submitted one blood sample and stool and urine samples on 3 consecutive days. Outcome was helminth infection with intestinal helminths, Schistosoma haematobium, Loa loa or Mansonella perstans. Multivariable logistic regression was used to assess associations between ART or CTX-P and helminth infection. In total, 408 patients were enrolled. Helminth infection was common (77/252 [30.5%]). Filarial infections were most prevalent (55/310 [17.7%]), followed by infection with intestinal helminths (35/296 [11.8%]) and S. haematobium (19/323 [5.9%]). Patients on CTX-P had a reduced risk of Loa loa microfilaremia (adjusted odds ratio (aOR) 0.47, 95% CI 0.23-0.97, P = 0.04), also in the subgroup of patients on ART (aOR 0.36, 95% CI 0.13-0.96, P = 0.04). There was no effect of ART exposure on helminth infection prevalence.CTX-P use was associated with a decreased risk of Loa loa infection, suggesting an anthelminthic effect of antifolate drugs. No relation between ART use and helminth infections was established.

Lobato L.S.,Oswalde Cruz Institute | Rosa P.S.,Lauro Of Sousa Lima Institute | Da Silva Ferreira J.,Oswalde Cruz Institute | Da Silva Neumann A.,Oswalde Cruz Institute | And 13 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2014

Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepards model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis, It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation. ©2014 American Society for Microbiology.

Neumann A.,University of Veterinary Medicine Hannover | Vollger L.,University of Veterinary Medicine Hannover | Berends E.T.M.,University Utrecht | Molhoek E.M.,TNO | And 10 more authors.
Journal of Innate Immunity | Year: 2014

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using im-munofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutro-phil peptide-1 showed similar protection of neutrophil-de-rived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-de-rived DNA or NETs against bacterial nuclease degradation. © 2014 S. Karger AG, Basel.

Vannini V.,CONICET | Rodriguez A.,CONICET | Vera J.L.,CONICET | de Valdez G.F.,CONICET | And 3 more authors.
Biotechnology Letters | Year: 2011

Purpose of work: To clone, express and characterize uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD) from Lactobacillus reuteri. Some strains of Lb. reuteri produce cobalamin (vitamin B 12). Cobalamin biosynthesis relies on the sequential action of more than 25 enzymes in a complex metabolic pathway. We have cloned, expressed and characterized the gene in Lb. reuteri that codes for the S-adenosy l-methionine uroprophyrinogen III methyltransferase/synthase (CobA/HemD), a key bifunctional enzyme in the biosynthesis of cobalamin and other tetrapyrrols. © 2011 Springer Science+Business Media B.V.

Dogi C.A.,CONICET | Weill F.,University of Buenos Aires | Perdigon G.,CONICET | Perdigon G.,Institute Microbiologia
Immunobiology | Year: 2010

The gut associated lymphoid tissue (GALT) is anatomical and functionally divided in inductive and effectors sites. In previous works we demonstrated that non-pathogenic bacteria with probiotic characteristics can improve the gut mucosal immune system, with an increase in the number of IgA and cytokines producing cells in the effector site of the intestine. In the present work we studied the effect of non-pathogenic Gram(+), Gram(-) bacteria and a Gram(+) probiotic strain on the inductor site (PP) after the oral administration to BALB/c mice. We also studied some signals induced by the assayed strain in the effectors site, such as the enzyme calcineurin and TLR-9 as a way to understand the mechanisms induced in such bacterial stimulation. The implicance of the lipoteichoic acid (LTA) in the immunostimulation was analyzed. All strains increased the number of IFN-γ and TNF-α(+) cells, but not of IL-10(+) cells in the total population of PP. The release of IFN-γ and TNF-α was only induced by LPS stimulation. All assayed strains increased the number of calcineurin(+) cells, while only Gram(+) strains increased the number of TLR-9(+) cells. The immunostimulatory properties of the purified LTA from Gram(+) strains was evaluated on a monocyte-macrophage U937 cell line. These cells showed capacity to release TNF-α and IL-10 in response to all LTA assayed in a dose-dependent way. Gram(+) strains induced signals through the calcineurin enzyme able to activate the transcriptional factor NFAT and through TLR-9. The LTA molecule from Gram(+) strains would not be the only structure involved in the immunostimulatory properties observed, specially for the probiotic strain. © 2009 Elsevier GmbH. All rights reserved.

Ramos A.N.,Institute Microbiologia | Gobbato N.,Institute Microbiologia | Rachid M.,Institute Microbiologia | Gonzalez L.,National University of Tucuman | And 3 more authors.
International Immunopharmacology | Year: 2010

In a previous study we determined that by-products of Lactobacillus plantarum inhibited pathogenicity of Pseudomonas aeruginosa and is effective in the treatment of infected wounds. This study assesses the cytotoxic activity of acetic acid (AA), supernatants of L. plantarum and P. aeruginosa, with and without signal acyl-homoserine-lactones (AHL), and mixtures of both bacterial supernatants on human neutrophils. Cytotoxicity was determined through viability using trypan blue, apoptosis by Annexin V, necrosis by propidium iodide and intracellular pH by SNARF-1. We found that supernatants of L. plantarum caused less cytotoxicity than AA at the same extracellular pH (p < 0.05). P. aeruginosa induced a remarkable drop in intracellular pH, which was independent of extracellular pH. This intracellular acidity was correlated with a significant decrease in viability and was higher than supernatants of AHL producing P. aeruginosa (p < 0.05). When supernatants were mixed, the quantity of AHL diminished (p < 0.001) and the cytotoxic effect induced by P. aeruginosa was ameliorated by L. plantarum supernatant (p < 0.001 vs p < 0.01). These results are in agreement with the inflammatory in vivo assays determined by intradermal inoculations in Balb/c mice. Our findings will be useful for the formulation of effective and inexpensive products to resolve infected chronic wounds in our hospitals. © 2009 Elsevier B.V. All rights reserved.

Ale C.E.,CONICET | Bru E.,CONICET | Strasser de Saad A.M.,Institute Microbiologia | Pasteris S.E.,CONICET
Journal of Applied Microbiology | Year: 2014

Aim: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). Methods and Results: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (109 CFU ml-1), while O. oeni reached 107 CFU ml-1. At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l-1 SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l-1 SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. Conclusions: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. Significance and Impact of the Study: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics. © 2014 The Society for Applied Microbiology.

Rebelo M.,Institute Medicina Molecular | Rebelo M.,Albert Schweitzer Hospital | Tempera C.,Institute Medicina Molecular | Fernandes J.F.,Albert Schweitzer Hospital | And 8 more authors.
Malaria Journal | Year: 2015

Background: In vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. Although different in vitro drug assays are available, some of their inherent characteristics limit their application, especially in the field. A recently developed approach based on the flow cytometric detection of haemozoin (Hz) allowed reagent-free monitoring of parasite maturation and detection of drug effects in culture-adapted parasites. In this study, the set-up, performance and usefulness of this novel assay were investigated under field conditions in Gabon. Methods: An existing flow cytometer (Cyflow Blue) was modified on site to detect light depolarization caused by Hz. Blood from malaria patients was incubated for 72 hrs with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing red blood cells (RBC) was used as maturation indicator and measured at 24, 48 and 72 hrs of incubation to determine parasite growth and drug effects. Results: The flow cytometer was easily adapted on site to detect light depolarization caused by Hz. Analysis of ex vivo cultures of parasites, obtained from blood samples of malaria patients, showed four different growth profiles. In 39/46 samples, 50% inhibitory concentrations (IC50) were successfully determined. IC50 values for chloroquine were higher than 200 nM in 70% of the samples, indicating the presence of chloroquine-resistant parasites. For artesunate and artemisinin, IC50 values ranged from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively, indicating fully sensitive parasites. Conclusion: Flow cytometric detection of Hz allowed the detection of drug effects in blood samples from malaria patients, without using additional reagents or complex protocols. Adjustment of the initial parasitaemia was not required, which greatly simplifies the protocol, although it may lead to different IC50 values. Further investigation of set-up conditions of the Hz assay, as well as future studies in various settings should be performed to further determine the usefulness of this assay as a tool for rapid resistance testing in malaria-endemic countries. © 2015 Rebelo et al.; licensee BioMed Central.

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