Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.     

Structural studies on yeast 3-phosphoglycerate kinase. Isolation by affinity chromatography and characterization of the peptides produced by cyanogen bromide cleavage. Location of the single cysteinyl residue in the primary structure.

Cyanogen bromide cleavage of yeast 3-phosphoglycerate kinase yielded four fragments which account for the amino acid composition of the entire molecule. These results are consistent with a single polypeptide chain of molecular weight 42 000. Affinity chromatography on Sepharose-mercurial followed by gel filtration on Sephadex was used with success for separation of peptides. The carboxyl and N-terminal fragments were characterized. The N-terminal fragment contained the single cysteinyl residue of the protein. After cyanylation and subsequent cleavage, this cysteinyl residue was located near position 100.     

Improved Visualization of Intracranial Vessels with Intraoperative Coregistration of Rotational Digital Subtraction Angiography and Intraoperative 3D Ultrasound

Introduction

Ultrasound can visualize and update the vessel status in real time during cerebral vascular surgery. We studied the depiction of parent vessels and aneurysms with a high-resolution 3D intraoperative ultrasound imaging system during aneurysm clipping using rotational digital subtraction angiography as a reference.

Methods

We analyzed 3D intraoperative ultrasound in 39 patients with cerebral aneurysms to visualize the aneurysm intraoperatively and the nearby vascular tree before and after clipping. Simultaneous coregistration of preoperative subtraction angiography data with 3D intraoperative ultrasound was performed to verify the anatomical assignment.

Results

Intraoperative ultrasound detected 35 of 43 aneurysms (81%) in 39 patients. Thirty-nine intraoperative ultrasound measurements were matched with rotational digital subtraction angiography and were successfully reconstructed during the procedure. In 7 patients, the aneurysm was partially visualized by 3D-ioUS or was not in field of view. Post-clipping intraoperative ultrasound was obtained in 26 and successfully reconstructed in 18 patients (69%) despite clip related artefacts. The overlap between 3D-ioUS aneurysm volume and preoperative rDSA aneurysm volume resulted in a mean accuracy of 0.71 (Dice coefficient).

Conclusions

Intraoperative coregistration of 3D intraoperative ultrasound data with preoperative rotational digital subtraction angiography is possible with high accuracy. It allows the immediate visualization of vessels beyond the microscopic field, as well as parallel assessment of blood velocity, aneurysm and vascular tree configuration. Although spatial resolution is lower than for standard angiography, the method provides an excellent vascular overview, advantageous interpretation of 3D-ioUS and immediate intraoperative feedback of the vascular status. A prerequisite for understanding vascular intraoperative ultrasound is image quality and a successful match with preoperative rotational digital subtraction angiography.     

Analysis of Transmission of MRSA and ESBL-E among Pigs and Farm Personnel

Livestock-associated bacteria with resistance to two or more antibiotic drug classes have heightened our awareness for the consequences of antibiotic consumption and spread of resistant bacterial strains in the veterinary field. In this study we assessed the prevalence of concomitant colonization with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and enterobacteriaceae expressing extended-spectrum betalactamases (ESBL-E) in farms at the German-Dutch border region. Nasal colonization of pigs with MRSA (113/547 (20.7%)) was less frequent than rectal colonization with ESBL-E (163/540 (30.2%)). On the individual farm level MRSA correlated with ESBL-E recovery. The data further provide information on prevalence at different stages of pig production, including abattoirs, as well as in air samples and humans living and working on the farms. Notably, MRSA was detected in stable air samples of 34 out of 35 pig farms, highlighting air as an important MRSA transmission reservoir. The majority of MRSA isolates, including those from humans, displayed tetracycline resistance and spa types t011 and t034 characteristic for LA-MRSA, demonstrating transmission from pigs to humans. ESBL-E positive air samples were detected on 6 out of 35 farms but no pig-to-human transmission was found. Detection of ESBL-E, e.g. mostly Escherichia coli with CTX-M-type ESBL, was limited to these six farms. Molecular typing revealed transmission of ESBL-E within the pig compartments; however, related strains were also found on unrelated farms. Although our data suggest that acquisition of MRSA and ESBL-E might occur among pigs in the abattoirs, MRSA and ESBL-E were not detected on the carcasses. Altogether, our data define stable air (MRSA), pig compartments (ESBL-E) and abattoir waiting areas (MRSA and ESBL-E) as major hot spots for transmission of MRSA and/or ESBL-E along the pig production chain.     

Non-Adrenergic Vasopressors in Patients with or at Risk for Vasodilatory Shock. A Systematic Review and Meta-Analysis of Randomized Trials

Introduction

Hypotensive state is frequently observed in several critical conditions. If an adequate mean arterial pressure is not promptly restored, insufficient tissue perfusion and organ dysfunction may develop. Fluids and catecholamines are the cornerstone of critical hypotensive states management. Catecholamines side effects such as increased myocardial oxygen consumption and development of arrhythmias are well known. Thus, in recent years, interest in catecholamine-sparing agents such as vasopressin, terlipressin and methylene blue has increased; however, few randomized trials, mostly with small sample sizes, have been performed. We therefore conducted a meta-analysis of randomized trials to investigate the effect of non-catecholaminergic vasopressors on mortality.

Methods

PubMed, BioMed Central and Embase were searched (update December 31^st, 2014) by two independent investigators. Inclusion criteria were: random allocation to treatment, at least one group receiving a non-catecholaminergic vasopressor, patients with or at risk for vasodilatory shock. Exclusion criteria were: crossover studies, pediatric population, non-human studies, studies published as abstract only, lack of data on mortality. Studied drugs were vasopressin, terlipressin and methylene blue. Primary endpoint was mortality at the longest follow-up available.

Results

A total of 1,608 patients from 20 studies were included in our analysis. The studied settings were sepsis (10/20 studies [50%]), cardiac surgery (7/20 [35%]), vasodilatory shock due to any cause (2/20 [19%]), and acute traumatic injury (1/20 [5%]). Overall, pooled estimates showed that treatment with non-catecholaminergic agents improves survival (278/810 [34.3%] versus 309/798 [38.7%], risk ratio = 0.88, 95% confidence interval = 0.79 to 0.98, p = 0.02). None of the drugs was associated with significant reduction in mortality when analyzed independently. Results were not confirmed when analyzing studies with a low risk of bias.

Conclusions

Catecholamine-sparing agents in patients with or at risk for vasodilatory shock may improve survival. Further researches on this topic are needed to confirm the finding.     

α-Thalassemia Associated with Hb Instability: A Tale of Two Features. The Case of Hb Rogliano or α1 Cod 108(G15)Thr→Asn and Hb Policoro or α2 Cod 124(H7)Ser→Pro.

We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and β1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1β1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.     

Variant rs1801157 in the 3’UTR of SDF-1? Does Not Explain Variability of Healthy-Donor G-CSF Responsiveness

The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3’UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3’UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3’UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3’ UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.     

Sex- and Time-Dependent Patterns in Risk Factors of End-Stage Renal Disease: A Large Austrian Cohort with up to 20 Years of Follow-Up

Objective

We investigated the association between metabolic factors and End-Stage Renal Disease (ESRD) and quantified the magnitude of their influence dependent on sex and time of exposure up to 20 years.

Material and Methods

A prospective cohort study was conducted to determine risk factors for the development of ESRD. From 1988 to 2005 185,341 persons (53.9% women) participated in the “Vorarlberg Health Monitoring and Promotion Programme” (VHM&PP). Data on body mass index (BMI), fasting blood glucose (FBG), systolic (BPsys) and diastolic (BPdia) blood pressure, total cholesterol (TC), triglycerides (TG), gamma-glutamyltransferase (GGT) and smoking status were collected. Data of the population-based VHM&PP were merged with the Austrian Dialysis and Transplant Registry. Cox proportional hazards models were applied to calculate hazard ratios (HRs) for ESRD, stratified by sex and 5-year time intervals.

Results

During a mean follow-up of 17.5 years 403 patients (39.1% women) developed ESRD. Significant risk factors were: BMI (per 1 kg/m²) HR 1.04 (95% CI 1.01–1.06), FBG (per 1 mmol/L) HR 1.09 (1.05–1.12), BPsys (per 5 mmHg) HR 1.10 (1.07–1.14), BPdia (per 5 mmHg) HR 1.09 (1.03–1.15), TG (per 1 mmol/L) HR 1.07 (1.02–1.13), TC (per 1 mmol/L) HR 1.22 (1.13–1.32). We observed a sex-specific risk pattern with an increased ESRD risk for men for increasing TG and smoking, and for women for increasing BMI and GGT. In time interval analyses BPsys and TC were associated with early ESRD onset, whereas BMI, FBG, BPdia and GGT were associated with later onset.

Conclusions

Anthropometric and metabolic factors are differentially associated with the long-term risk for ESRD in a sex- and time-dependent manner. Consideration of these patterns in preventive and therapeutic strategies could have an impact on ESRD incidence.     

CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice

Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimer''s disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy.