Enhancing the internal plant colonization rate with endophytic nitrogen-fixing bacteria

Several diazotrophic strains of Klebsiella oxytoca and K. terrigena that colonize the plant-host interior were able to produce the plant cell wall depolymerising enzyme pectate lyase (Pel). The activity of the K. oxytoca enzyme was weaker than that of phytopathogenic bacteria, and it was located mainly inside the cells. A small fraction of the cells (10⁻⁶ to 10⁻⁵) in populations grown in nonselective media was able to grow in a selective medium with polygalactorunate (PC) as sole carbon source. After passage through selective medium cells were converted to the Pet+ -phenotype, and total Pet-activity in population of K. oxytoca increased. The increased level of Pelactivity of K. oxytoca and K. terrigena correlated with a 10-fold higher rate of internal colonization of wheat roots. Cultures of K. oxytoca VN13 grown in selective medium with PG also showed increased stimulation of wheat growth. Seedlings inoculated with such cultures exhibited better development resulting in higher biomass.Декілька штамів Klebsiella oxytoca та K. terrigena, здатних колонізувати рослини зсередини, виділяли пектат ліазу (ПЛ) – фермент, який деполімеризус клітинну стінку. Ак­ тивність ПЛ була нижчою, ніж у фітопатогенних бактерій, і зосереджувалась всередині клітин. Невелика кількість клітин популяції (10⁻⁶ –10⁻⁵ ) спроможна рости на селективному се­редовищі з полігалактуронатом натрію (ПГ), який викори­стовували як джерело вуглецю. Після пасажу через селективне середовище всі клітини набували Реl+ -фенотипу, і загальна ПЛ-активність К. oxytoca зростала. Підвищена ПЛ-активність бактерій K. oxytoca та K. terrigena корелювала з посилен­ням у 10 разів внутрішньої колонізації коренів пшениці. Куль­тура бактерій, яка виростала в селективному середовищі з ПГ, краще стимулювала розвиток пшениці, що проявлялося у збільшенні її біомаси, ніж культура, зрощена без селекціїУ нескольких изученных штаммов Klebsiella oxytoca и K. terrigena, способных колонизировать растения изнутри, обна­ружена активность пектат лиазы (ПЛ) –фермента, кото­рый деполимеризует клеточную стенку. Активность ПЛ была ниже, чем у фитопатогенных бактерий, и сосредоточена внутри клетки. Только небольшая часть популяции клеток (10⁻⁶ –10⁻⁵ ) способна расти на селективной среде с полигалактуронатом, который использовали в качестве источника уг­лерода. После пассажа через селективную среду все клетки приобретают Pel+ фенотип, и при этом общая ПЛ-активность K. oxytoca и K. terrigena возрастает Повышенная ПЛ-активность бактерий коррелировала с уусилением в 10 раз внутренней колонизации корней пшеницы. Культура бактерий K. oxytoca VN13, выросшая в селективных условиях, лучше стимулировала развитие пшеницы, что проявлялось в увеличении ее биомассы

A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens

A specific detection method was developed to discriminate Klebsiella oxytoca between other species of the genus Klebsiella on the basis of PCR amplification of the unique DNA sequences within the polygalacturonase-encoding (pehX) gene. Four primers have been designed for performing PCRs gaining amplicons of 282, 344, 451 and 513 bp. The specificity of the test was verified by the lack of PCR products in case of related K. pneumoniae, K. planticola, and polygalacturonate-degrading species of the genus Erwinia. The PCR-mediated test gives a rapid answer, concerning the presence of K. oxytoca in a sample, or in differentiating this bacterium from other species, such as K. pneumoniae, with which they can be confused. The diagnostic test can be used in ecological monitoring of K. oxytoca as well as in medical laboratories.На основі ПЛР-ампліфікації унікальних послідовностей ДНК гена, що кодує фермент полігалактуроназу (pehX), розроблено специфічний метод для вирізнення бактерії K. oxytoca серед інших бактерій роду Klebsiella. Чотири пари праймерів створе­но для отримання ампліконів 282, 344, 451 та 513 п. н. Специфічність тесту підтверджено відсутністю продуктів ПЛР у близьких бактерій K. pneumoniae, K. planticola та видів роду Erwinia, що розкладають полігалактуронат ПЛР-тест дозволяє швидко визначити наявність K. oxytoca у зразку або відрізнити цю бактерію від представників інших видів, на­приклад, від К. pneumoniae, яка дуже схожа на неї. Діагно­стичний тест може бути використано в екологічному моні­торингу K. oxytoca, а також у медичних лабораторіях.На основе ПЦР-амплификации уникальних последовательно­стей ДНК гена, кодирующего фермент полигалактуроназу (pehX), разработан метод для выявления бактерии K. oxytoca среди других бактерий рода Klebsiella. Четыре пары праймеров созданы для получения ампликонов размером 282, 344, 451 и 513 пар нуклеотидов. Специфичность теста подтверждена отсутствием продуктов ПЦР у близких бактерий K. pneu­moniae, K. planticola и видов рода Erwinia, разлагающих полигалактуронат ПЦР-тест позволяет быстро определить наличие К. oxytoca в образцах или отличить эту бактерию от представителей других видов, например, от K. pneumoniae, которая очень на нее похожа. Диагностический тест может быть использован в экологическом мониторинге K. oxytoca, а также в медицинских лабораториях

Fungal iron availability during deep seated candidiasis is defined by a complex interplay involving systemic and local events

Peer reviewedPublisher PD

АВТОМАТИЗАЦІЯ РОБОЧОГО МІСЦЯ ЛІКАРЯ-БІОХІМІКА ГЕНЕТИКА

In the laboratory of medical genetics NCSH «OKHMATDYT» to identify the reliability of laboratory results and control the number and frequency of errors, carry out internal quality control. In order to evaluate the results of measurements of samples and eliminate harmful analytical errors developed and introduced software that allows you to build a calibration curve and process the results of biochemical research activity of lysosomal enzymes.В лаборатории медицинской генетики НДСБ «ОХМАТДЕТ» для выявления достоверности результатов лабораторных исследований и контроля количества и частоты возникновения ошибок, осуществляют внутрилабораторный контроль качества. С целью оценки результатов измерений образцов и устранения недопустимых аналитических ошибок разработано и внедрено программное обеспечение, позволяющее строить калибровочный график и обрабатывать результаты биохимического исследования активности лизосомных ферментов.У лабораторії медичної генетики НДСЛ «ОХМАТДИТ» для виявлення достовірності результатів лабораторних досліджень та контролю кількості і частоти виникнення помилок, здійснюють внутрішньолабораторний контроль якості. З метою оцінки результатів вимірювань зразків та усунення неприпустимих аналітичних помилок розроблено та впроваджено програмне забезпечення, що дозволяє будувати калібрувальний графік та обробляти результати біохімічного дослідження активності лізосомних ферментів

Heme catabolism by heme oxygenase-1 confers host resistance to Mycobacterium infection

Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (M) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1(-/-)) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1(+/+)) controls. Furthermore, Hmox1(-/-) mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1(-/-) versus Hmox1(+/+) SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected M, an effect mimicked by exogenous heme administration to M. avium-infected wild-type M in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in M, contributing critically to host resistance to Mycobacterium infection.European Community 6th Framework grant: (LSH-2005-1.2.5-1), FCT fellowships: (SFRH/BD/29257/2006, SFRH/BPD/25436/2005), Instituto Gulbenkian de Ciência, Universidade do Minho, ICBAS

Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively

Turning the Table: Plants Consume Microbes as a Source of Nutrients

Interactions between plants and microbes in soil, the final frontier of ecology, determine the availability of nutrients to plants and thereby primary production of terrestrial ecosystems. Nutrient cycling in soils is considered a battle between autotrophs and heterotrophs in which the latter usually outcompete the former, although recent studies have questioned the unconditional reign of microbes on nutrient cycles and the plants' dependence on microbes for breakdown of organic matter. Here we present evidence indicative of a more active role of plants in nutrient cycling than currently considered. Using fluorescent-labeled non-pathogenic and non-symbiotic strains of a bacterium and a fungus (Escherichia coli and Saccharomyces cerevisiae, respectively), we demonstrate that microbes enter root cells and are subsequently digested to release nitrogen that is used in shoots. Extensive modifications of root cell walls, as substantiated by cell wall outgrowth and induction of genes encoding cell wall synthesizing, loosening and degrading enzymes, may facilitate the uptake of microbes into root cells. Our study provides further evidence that the autotrophy of plants has a heterotrophic constituent which could explain the presence of root-inhabiting microbes of unknown ecological function. Our discovery has implications for soil ecology and applications including future sustainable agriculture with efficient nutrient cycles

Garlic Accelerates Red Blood Cell Turnover and Splenic Erythropoietic Gene Expression in Mice: Evidence for Erythropoietin-Independent Erythropoiesis

Garlic (Allium sativum) has been valued in many cultures both for its health effects and as a culinary flavor enhancer. Garlic's chemical complexity is widely thought to be the source of its many health benefits, which include, but are not limited to, anti-platelet, procirculatory, anti-inflammatory, anti-apoptotic, neuro-protective, and anti-cancer effects. While a growing body of scientific evidence strongly upholds the herb's broad and potent capacity to influence health, the common mechanisms underlying these diverse effects remain disjointed and relatively poorly understood. We adopted a phenotype-driven approach to investigate the effects of garlic in a mouse model. We examined RBC indices and morphologies, spleen histochemistry, RBC half-lives and gene expression profiles, followed up by qPCR and immunoblot validation. The RBCs of garlic-fed mice register shorter half-lives than the control. But they have normal blood chemistry and RBC indices. Their spleens manifest increased heme oxygenase 1, higher levels of iron and bilirubin, and presumably higher CO, a pleiotropic gasotransmitter. Heat shock genes and those critical for erythropoiesis are elevated in spleens but not in bone marrow. The garlic-fed mice have lower plasma erythropoietin than the controls, however. Chronic exposure to CO of mice on garlic-free diet was sufficient to cause increased RBC indices but again with a lower plasma erythropoietin level than air-treated controls. Furthermore, dietary garlic supplementation and CO treatment showed additive effects on reducing plasma erythropoietin levels in mice. Thus, garlic consumption not only causes increased energy demand from the faster RBC turnover but also increases the production of CO, which in turn stimulates splenic erythropoiesis by an erythropoietin-independent mechanism, thus completing the sequence of feedback regulation for RBC metabolism. Being a pleiotropic gasotransmitter, CO may be a second messenger for garlic's other physiological effects

Relationship between Anaemia, Haemolysis, Inflammation and Haem Oxygenase-1 at Admission with Sepsis: a pilot study

Upregulation of haem oxygenase-1 (HO-1), due to haemolysis and/or inflammation, can lead to impaired immune function. Anaemia is common among sepsis patients, but the consequences of sepsis-associated anaemia are poorly understood. Here, our objective was to determine the prevalence and extent of anaemia, haemolysis, inflammation, and HO-1 induction after early hospital admission. We hypothesised that inflammation- or infection-induced haemolysis contributes to sepsis-associated anaemia and that this will lead to expression of HO-1. In this study, plasma obtained from seventy adult patients within 12 hours of admission to intensive care due to sepsis were analysed for anaemia, haemolysis and inflammatory markers by ELISA and microbead array. The majority (82.6%) of patients were anaemic with evidence of haemolysis (raised haem, haptoglobin, haemopexin, and HO-1 concentrations). Interestingly, concentrations of both haemoglobin and IL-10 were moderately positively correlated with HO-1 concentration (Hb: r = 0.32, p = 0.007; IL-10 r = 0.39, p = 0.0008) whereas HO-1 concentration was weakly negatively correlated with haemopexin (r = -0.23, p = 0.055). Anaemia, while common, was not associated with HO-1 concentration. After adjusting for confounding, HO-1 induction appears to be associated primarily with IL-10 concentration rather than haemolysis. Disease severity at diagnosis was correlated with early plasma IL-10 (r = 0.35, p = 0.003) and HO-1 (r = 0.24, p = 0.048) concentrations. Notably, admission levels of haem, HO-1, and IL-10 were indicators of survival