Distribution and Environmental Drivers of Fungal Denitrifiers in Global Soils.

The microbial process of denitrification is the primary source of the greenhouse gas nitrous oxide (N2O) from terrestrial ecosystems. Fungal denitrifiers, unlike many bacteria, lack the N2O reductase, and thereby are sources of N2O. Still, their diversity, global distribution, and environmental determinants, as well as their relative importance, compared to bacterial and archaeal denitrifiers, remain unresolved. Employing a phylogenetically informed approach to analyze 1,980 global soil and rhizosphere metagenomes for the denitrification marker gene nirK, which codes for the copper dependent nitrite reductase in denitrification, we show that fungal denitrifiers are sparse, yet cosmopolitan and that they are dominated by saprotrophs and pathogens. Few showed biome-specific distribution patterns, although members of the Fusarium oxysporum species complex, which are known to produce substantial amounts of N2O, were proportionally more abundant and diverse in the rhizosphere than in other biomes. Fungal denitrifiers were most frequently detected in croplands, but they were most abundant in forest soils when normalized to metagenome size. Nevertheless, the overwhelming dominance of bacterial and archaeal denitrifiers suggests a much lower fungal contribution to N2O emissions than was previously estimated. In relative terms, they could play a role in soils that are characterized by a high carbon to nitrogen ratio and a low pH, especially in the tundra as well as in boreal and temperate coniferous forests. Because global warming predicts the proliferation of fungal pathogens, the prevalence of potential plant pathogens among fungal denitrifiers and the cosmopolitan distribution of these organisms suggest that fungal denitrifier abundance may increase in terrestrial ecosystems. IMPORTANCE Fungal denitrifiers, in contrast to their bacterial counterparts, are a poorly studied functional group within the nitrogen cycle, even though they produce the greenhouse gas N2O. To curb soil N2O emissions, a better understanding of their ecology and distribution in soils from different ecosystems is needed. Here, we probed a massive amount of DNA sequences and corresponding soil data from a large number of samples that represented the major soil environments for a broad understanding of fungal denitrifier diversity at the global scale. We show that fungal denitrifiers are predominantly cosmopolitan saprotrophs and opportunistic pathogens. Fungal denitrifiers constituted, on average, 1% of the total denitrifier community. This suggests that earlier estimations of fungal denitrifier abundance, and, thereby, it is also likely that the contributions of fungal denitrifiers to N2O emissions have been overestimated. Nevertheless, with many fungal denitrifiers being plant pathogens, they could become increasingly relevant, as soilborne pathogenic fungi are predicted to increase with ongoing climate change.

Dynamics of the Apple Fruit Microbiome after Harvest and Implications for Fruit Quality.

The contribution of the apple microbiome to the production chain of apple was so far largely unknown. Here, we describe the apple fruit microbiome and influences on its composition by parameters such as storage season, storage duration, storage technology, apple variety, and plant protection schemes. A combined culturing and metabarcoding approach revealed significant differences in the abundance, composition, and diversity of the apple fruit microbiome. We showed that relatively few genera contribute a large portion of the microbiome on fruit and that the fruit microbiome changes during the storage season depending on the storage conditions. In addition, we show that the plant protection regime has an influence on the diversity of the fruit microbiome and on the dynamics of pathogenic fungal genera during the storage season. For the genus Neofabraea, the quantitative results from the metabarcoding approach were validated with real-time PCR. In conclusion, we identified key parameters determining the composition and temporal changes of the apple fruit microbiome, and the main abiotic driving factors of microbiome diversity on apple fruit were characterized.

A reduction of prothrombin conversion by cardiac surgery with cardiopulmonary bypass shifts the haemostatic balance towards bleeding.

Cardiac surgery with cardiopulmonary bypass (CPB) is associated with blood loss and post-surgery thrombotic complications. The process of thrombin generation is disturbed during surgery with CPB because of haemodilution, coagulation factor consumption and heparin administration. We aimed to investigate the changes in thrombin generation during cardiac surgery and its underlying pro- and anticoagulant processes, and to explore the clinical consequences of these changes using in silico experimentation. Plasma was obtained from 29 patients undergoing surgery with CPB before heparinisation, after heparinisation, after haemodilution, and after protamine administration. Thrombin generation was measured and prothrombin conversion and thrombin inactivation were quantified. In silico experimentation was used to investigate the reaction of patients to the administration of procoagulant factors and/or anticoagulant factors. Surgery with CPB causes significant coagulation factor consumption and a reduction of thrombin generation. The total amount of prothrombin converted and the rate of prothrombin conversion decreased during surgery. As the surgery progressed, the relative contribution of α2-macroglobulin-dependent thrombin inhibition increased, at the expense of antithrombin-dependent inhibition. In silico restoration of post-surgical prothrombin conversion to pre-surgical levels increased thrombin generation excessively, whereas co-administration of antithrombin resulted in the normalisation of post-surgical thrombin generation. Thrombin generation is reduced during surgery with cardiopulmonary bypass because of a balance shift between prothrombin conversion and thrombin inactivation. According to in silico predictions of thrombin generation, this new balance increases the risk of thrombotic complications with prothrombin complex concentrate administration, but not if antithrombin is co-administered.

Quantitative Selection Analysis of Bacteriophage φCbK Susceptibility in Caulobacter crescentus.

Classical molecular genetics uses stringent selective conditions to identify mutants with distinct phenotypic responses. Mutations giving rise to less pronounced phenotypes are often missed. However, to gain systems-level insights into complex genetic interaction networks requires genome-wide assignment of quantitative phenotypic traits. In this paper, we present a quantitative selection approach coupled with transposon sequencing (QS-TnSeq) to globally identify the cellular components that orchestrate susceptibility of the cell cycle model bacterium Caulobacter crescentus toward bacteriophage φCbK infection. We found that 135 genes representing 3.30% of the Caulobacter genome exhibit significant accumulation of transposon insertions upon φCbK selection. More than 85% thereof consist of new factors not previously associated with phage φCbK susceptibility. Using hierarchical clustering of dose-dependent TnSeq datasets, we grouped these genes into functional modules that correlate with different stages of the φCbK infection process. We assign φCbK susceptibility to eight new genes that represent novel components of the pilus secretion machinery. Further, we demonstrate that, from 86 motility genes, only seven genes encoding structural and regulatory components of the flagellar hook increase phage resistance when disrupted by transposons, suggesting a link between flagellar hook assembly and pili biogenesis. In addition, we observe high recovery of Tn5 insertions within regulatory sequences of the genes encoding the essential NADH:ubiquinone oxidoreductase complex indicating that intact proton motive force is crucial for effective phage propagation. In sum, QS-TnSeq is broadly applicable to perform quantitative and genome-wide systems-genetics analysis of complex phenotypic traits.

Measurement of thrombin generation intra-operatively and its association with bleeding tendency after cardiac surgery.

INTRODUCTION: Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are susceptible to haemostatic disturbances. Monitoring the haemostatic capacity by conventional clotting tests is challenging. MATERIALS AND METHODS: Thrombin generation (TG) by Calibrated Automated Thrombography, clotting tests and tissue factor pathway inhibitor (TFPI) measurements were performed to describe the relationship between haemostatic changes and alterations in these tests. Blood samples were collected before, during and after CPB. Furthermore, it was investigated whether TG measured intraoperatively, is associated with increased risk of bleeding postoperatively. RESULTS: TG diminished significantly (p<0.01) after heparinization in the presence and absence of platelets (37% and 50%) compared to baseline. After the start of CPB, TG elevated and persisted till the end of surgery but remained lower than preoperatively. Activated clotting time increased after heparinization and after the start of bypass compared to baseline (400% and 500%). Anti-FXa activity reduced on the start of CPB compared to the level after heparinization, to almost the baseline value following protamine reversal of heparin. The plasma levels of total and free TFPI elevated 9 and 14 fold during bypass and remained after protamine administration higher than preoperatively. Plasma D-dimer levels reduced (p<0.01) when bypass started. However, a marked elevation was observed in the following time points. TG in platelet-rich plasma measured after heparinization and after the start of CPB associated (p<0.05) with postoperative blood loss. CONCLUSIONS: TG can be determined during CPB despite the high heparinization level, it reflects the haemostatic capacity better than clotting-based assays and might better predict bleeding when performed intraoperatively.

Preoperative thrombin generation is predictive for the risk of blood loss after cardiac surgery: a research article.

BACKGROUND: In this study the value of thrombin generation parameters measured by the Calibrated Automated Thrombography for prediction of blood loss after cardiac surgery with cardiopulmonary bypass was investigated. METHODS: Thirty male patients undergoing first-time coronary artery bypass grafting were enrolled. Blood samples were taken pre-bypass before heparinisation (T1) and 5 min after protamine administration (T2). Thrombin generation was measured both in platelet-rich plasma and in platelet-poor plasma. Besides thrombin generation measurements, activated clotting time, haematocrit, haemoglobin, platelet number, fibrinogen, antithrombin, D-dimers, prothrombin time and activated partial thromboplastin time were determined. Blood loss was measured and the amount of transfusion products was recorded postoperatively until 20 hours after surgery. Patients were divided into two groups based on the median volume of postoperative blood loss (group 1: patients with median blood loss <930 ml; group 2: patients with median blood loss ≥930 ml). RESULTS: On T1, patients of group 2 had a significantly lower endogenous thrombin potential and peak thrombin (p<0.001 and p=0.004 respectively) in platelet-rich plasma, a significantly lower endogenous thrombin potential (p=0.004) and peak thrombin (p=0.014) in platelet-poor plasma, and a lower platelet count (p=0.002). On T2 both endogenous thrombin potential and peak thrombin remain significantly lower (p=0.011 and p=0.010) in group 2, measured in platelet-rich plasma but not in platelet-poor plasma. In addition, platelet number remains lower in group 2 after protamine administration (p=0.002). CONCLUSIONS: The key finding is that the Calibrated Automated Thrombography assay, performed preoperatively, provides information predictive for blood loss after cardiac surgery.

An evaluation of factors affecting activated coagulation time.

OBJECTIVE: Although failure to achieve an adequate activated coagulation time (ACT) after full heparinization before cardiopulmonary bypass often is attributed to antithrombin (AT) deficiency, it remains unclear if this is a causative mechanism of decreased heparin responsiveness. Therefore, the authors determined the relationship between AT and other coagulation-related factors that affect the ACT measurement and heparin sensitivity index before the establishment of cardiopulmonary bypass. DESIGN: Observational study. SETTING: University medical center. PARTICIPANTS: Adult elective cardiac surgical patients. INTERVENTIONS: Preoperative data collection included demographics, type of preoperative medical therapy, hemoglobin, platelet count, and AT. Intraoperative measurements included ACT and anti-Xa activity. RESULTS: Of the 203 patients enrolled in this study, 10% (n = 21) did not achieve an adequate ACT (≥400 seconds) after full heparinization. Subnormal AT activity (55%-79%) was not related to a low ACT and a low heparin sensitivity index. Preoperative low-molecular-weight heparin therapy did not cause a decreased ACT response. However, preoperative low hemoglobin levels and high platelet counts were associated with a decreased ACT. CONCLUSIONS: All these observations suggest that failure to achieve an adequate ACT is, in general, not an indicator of AT deficiency but could be affected by high platelet counts and low hemoglobin levels.