One shot-two pathogens blocked: Exposure of Arabidopsis to hexadecane,a long chain volatile organic compound,confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.     

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background

Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.

Methods and Principal Findings

To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.

Conclusions

We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.     

Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model

Background

Epidemiological studies suggest an association between exposure to volatile organic compounds (VOCs) and adverse allergic and respiratory symptoms. However, whether VOCs exhibit a causal role as adjuvants in asthma development remains unclear.

Methods

To investigate the effect of VOC exposure on the development of allergic airway inflammation Balb/c mice were exposed to VOCs emitted by new polyvinylchloride (PVC) flooring, sensitized with ovalbumin (OVA) and characterized in acute and chronic murine asthma models. Furthermore, prevalent evaporated VOCs were analyzed and mice were exposed to selected single VOCs.

Results

Exposure of mice to PVC flooring increased eosinophilic lung inflammation and OVA-specific IgE serum levels compared to un-exposed control mice. The increased inflammation was associated with elevated levels of Th2-cytokines. Long-term exposure to PVC flooring exacerbated chronic airway inflammation. VOCs with the highest concentrations emitted by new PVC flooring were N-methyl-2-pyrrolidone (NMP) and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). Exposure to NMP or TXIB also increased the allergic immune response in OVA-sensitized mice. In vitro or in vivo exposure to NMP or TXIB reduced IL-12 production in maturing dendritic cells (DCs) and enhanced airway inflammation after adoptive DC transfer into Balb/c mice. At higher concentrations both VOCs induced oxidative stress demonstrated by increased isoprostane and glutathione-S-transferase-pi1 protein levels in the lung of non-sensitized mice. Treatment of PVC flooring-exposed mice with N-acetylcysteine prevented the VOC-induced increase of airway inflammation.

Conclusions

Our results demonstrate that exposure to VOCs may increase the allergic immune response by interfering with DC function and by inducing oxidative stress and has therefore to be considerate as risk factor for the development of allergic diseases.     

Defining adult asthma endotypes by clinical features and patterns of volatile organic compounds in exhaled air

Background

Several classifications of adult asthma patients using cluster analyses based on clinical and demographic information has resulted in clinical phenotypic clusters that do not address molecular mechanisms. Volatile organic compounds (VOC) in exhaled air are released during inflammation in response to oxidative stress as a result of activated leukocytes. VOC profiles in exhaled air could distinguish between asthma patients and healthy subjects. In this study, we aimed to classify new asthma endotypes by combining inflammatory mechanisms investigated by VOC profiles in exhaled air and clinical information of asthma patients.

Methods

Breath samples were analyzed for VOC profiles by gas chromatography–mass spectrometry from asthma patients (n = 195) and healthy controls (n = 40). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate healthy from asthmatic subjects. 2-step cluster analysis based on clinical information and VOCs in exhaled air were used to form asthma endotypes.

Results

We identified 16 VOCs, which could distinguish between healthy and asthma subjects with a sensitivity of 100% and a specificity of 91.1%. Cluster analysis based on VOCs in exhaled air and the clinical parameters FEV1, FEV1 change after 3 weeks of hospitalization, allergic sensitization, Junipers symptoms score and asthma medications resulted in the formation of 7 different asthma endotype clusters. We identified asthma clusters with different VOC profiles but similar clinical characteristics and endotypes with similar VOC profiles, but distinct clinical characteristics.

Conclusion

This study demonstrates that both, clinical presentation of asthma and inflammatory mechanisms in the airways should be considered for classification of asthma subtypes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0136-8) contains supplementary material, which is available to authorized users.     

Host Deception: Predaceous Fungus,Esteya vermicola,Entices Pine Wood Nematode by Mimicking the Scent of Pine Tree for Nutrient

Background

A nematophagous fungus, Esteya vermicola, is recorded as the first endoparasitic fungus of pine wood nematode (PWN), Bursaphelenchus xylophilus, in last century. E. vermicola exhibited high infectivity toward PWN in the laboratory conditions and conidia spraying of this fungus on Japanese red pine, Pinus densiflora, seedlings in the field protected the pine trees from pine wilt disease to some extent, indicating that it is a potential bio-control agent against PWN. Previous research had demonstrated that the living fungal mycelia of E. vermicola continuously produced certain volatile organic compounds (VOCs), which were responsible for the PWN attraction. However, identity of these VOCs remains unknown.

Methodology/Principal Findings

In this study, we report the identification of α-pinene, β-pinene, and camphor produced by living mycelia of E. vermicola, the same volatile compounds emitted from PWN host pine tree, as the major VOCs for PWN attraction using gas chromatography-mass spectrometry (GC-MS). In addition, we also confirmed the host deception behavior of E. vermicola to PWN by using synthetic VOCs in a straightforward laboratory bioassay.

Conclusions/Significance

This research result has demonstrated that the endoparasitic nematophagous fungus, E. vermicola, mimics the scent of PWN host pine tree to entice PWN for the nutrient. The identification of the attractive VOCs emitted from the fungus E. vermicola is of significance in better understanding parasitic mechanism of the fungus and the co-evolution in the two organisms and will aid management of the pine wilt disease.     

Exhaled volatile organic compounds in patients with non-small cell lung cancer: cross sectional and nested short-term follow-up study

Background

Non-invasive diagnostic strategies aimed at identifying biomarkers of lung cancer are of great interest for early cancer detection. The aim of this study was to set up a new method for identifying and quantifying volatile organic compounds (VOCs) in exhaled air of patients with non-small cells lung cancer (NSCLC), by comparing the levels with those obtained from healthy smokers and non-smokers, and patients with chronic obstructive pulmonary disease. The VOC collection and analyses were repeated three weeks after the NSCLC patients underwent lung surgery.

Methods

The subjects'' breath was collected in a Teflon^® bulb that traps the last portion of single slow vital capacity. The 13 VOCs selected for this study were concentrated using a solid phase microextraction technique and subsequently analysed by means of gas cromatography/mass spectrometry.

Results

The levels of the selected VOCs ranged from 10^-12 M for styrene to 10^-9 M for isoprene. None of VOCs alone discriminated the study groups, and so it was not possible to identify one single chemical compound as a specific lung cancer biomarker. However, multinomial logistic regression analysis showed that VOC profile can correctly classify about 80 % of cases. Only isoprene and decane levels significantly decreased after surgery.

Conclusion

As the combination of the 13 VOCs allowed the correct classification of the cases into groups, together with conventional diagnostic approaches, VOC analysis could be used as a complementary test for the early diagnosis of lung cancer. Its possible use in the follow-up of operated patients cannot be recommended on the basis of the results of our short-term nested study.     

Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame

Sesame is an important vegetable crop for the production of oil in Korea. The main obstacle of sesame cultivation is the occurrence of damping-off diseases and wilt caused by a complex of soil-borne pathogens in fields cultivated for two or more successive years. To protect sesame seedlings against these diseases, Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium (PGPR) previously shown to suppress disease incidence and promote growth on cucumber and pepper in the greenhouse and field experiments, was evaluated for its capacity for biological control and growth promotion in vitro and in situ. Seed treatment with strain E681 alone did not show consistent protection. Therefore, seed pelleting with strain E681 was attempted to increase the seed size and improve the stability and effectiveness of biocontrol capacity by strain E681. Through screening of pelleting materials, a combination of clay and vermiculite was selected for further experiments to enhance seed germination and root colonization of strain E681 on sesame. In greenhouse trials, formulations of strain E681 reduced disease incidence in disease-conducive soil. In the field, pelleting of sesame seeds with strain E681 significantly reduced pre- and post-emergence damping-off compared to the non-treated or pelleting alone controls; pelleting also promoted the plant growth and the grain yield. Furthermore, the efficacy of strain E681 for biological control and plant growth promotion was improved by sesame seed pelleting compared to the treatment with strain E681 alone. Hence, the application of strain E681 via seed pelleting offers potential to overcome some of the problems associated with successive years of sesame cultivation.     

The Effects of Paenibacillus polymyxa E681 on Antifungal and Crack Remediation of Cement Paste

This study investigated the antifungal effects of cement paste containing Paenibacillus polymyxa E681 against Aspergillus niger, a deleterious fungus commonly found in cement buildings and structures. To test the antifungal effects, cement paste containing P. polymyxa E681 was neutralized by CO₂ gas, and the fungal growth inhibition was examined according to the clear zone around the cement specimen. In addition to the antifungal effects of the cement paste added with bacteria, calcium crystal precipitation of P. polymyxa E681 was examined by qualitative and quantitative analyses. The cement paste containing P. polymyxa E681 showed strong antifungal effects but fusA mutant (deficient in fusaricidin synthesis) showed no antifungal activity. Crack sealing of the cement paste treated with P. polymyxa E681 was captured by light microscope showed fungal growth inhibition and crack repairing in cement paste.     

High-Resolution Mechanical Imaging of Glioblastoma by Multifrequency Magnetic Resonance Elastography

Objective

To generate high-resolution maps of the viscoelastic properties of human brain parenchyma for presurgical quantitative assessment in glioblastoma (GB).

Methods

Twenty-two GB patients underwent routine presurgical work-up supplemented by additional multifrequency magnetic resonance elastography. Two three-dimensional viscoelastic parameter maps, magnitude |G*|, and phase angle φ of the complex shear modulus were reconstructed by inversion of full wave field data in 2-mm isotropic resolution at seven harmonic drive frequencies ranging from 30 to 60 Hz.

Results

Mechanical brain maps confirmed that GB are composed of stiff and soft compartments, resulting in high intratumor heterogeneity. GB could be easily differentiated from healthy reference tissue by their reduced viscous behavior quantified by φ (0.37±0.08 vs. 0.58±0.07). |G*|, which in solids more relates to the material''s stiffness, was significantly reduced in GB with a mean value of 1.32±0.26 kPa compared to 1.54±0.27 kPa in healthy tissue (P = 0.001). However, some GB (5 of 22) showed increased stiffness.

Conclusion

GB are generally less viscous and softer than healthy brain parenchyma. Unrelated to the morphology-based contrast of standard magnetic resonance imaging, elastography provides an entirely new neuroradiological marker and contrast related to the biomechanical properties of tumors.     

Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

Background

In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.

Methods

Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.

Results

Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.

Conclusions

MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.     

Systemic Resistance Induced by Volatile Organic Compounds Emitted by Plant Growth-Promoting Fungi in Arabidopsis thaliana

Volatile organic compounds (VOC) were extracted and identified from plant growth-promoting fungi (PGPF), Phoma sp., Cladosporium sp. and Ampelomyces sp., using gas chromatography–mass spectrometry (GC-MS). Among the three VOC extracted, two VOC blends (emitted from Ampelomyces sp. and Cladosporium sp.) significantly reduced disease severity in Arabidopsis plants against Pseudomonas syringae pv. tomato DC3000 (Pst). Subsequently, m-cresol and methyl benzoate (MeBA) were identified as major active volatile compounds from Ampelomyces sp. and Cladosporium sp., respectively, and found to elicit induced systemic resistance (ISR) against the pathogen. Molecular signaling for disease suppression by the VOC were investigated by treating different mutants and transgenic Arabidopsis plants impaired in salicylic acid (SA) or Jasmonic acid (JA)/ethylene (ET) signaling pathways with m-cresol and MeBA followed by challenge inoculation with Pst. Results show that the level of protection was significantly lower when JA/ET-impaired mutants were treated with MeBA, and in SA-, and JA/ET-disrupted mutants after m-cresol treatment, indicating the involvement of these signal transduction pathways in the ISR primed by the volatiles. Analysis of defense-related genes by real-time qRT-PCR showed that both the SA-and JA-signaling pathways combine in the m-cresol signaling of ISR, whereas MeBA is mainly involved in the JA-signaling pathway with partial recruitment of SA-signals. The ET-signaling pathway was not employed in ISR by the volatiles. Therefore, this study identified two novel volatile components capable of eliciting ISR that may be promising candidates in biological control strategy to protect plants from diseases.     

Serum Copeptin and Cortisol Do Not Accurately Predict Sickle Cell Anaemia Vaso-Occlusive Crisis as C-Reactive Protein

Objective

This study assessed the diagnostic performance and prognostic properties of C-reactive protein (CRP), copeptin and cortisol in individuals with sickle cell anaemia (SCA).

Design

Prospective case-control study

Methods

Sixty consecutive SCA subjects (18–40 years) comprising 30 subjects in the steady state and 30 subjects in vaso-occlusive crisis (VOC) were recruited into this study. Thirty (30) apparently healthy individuals with HbAA genotype served as controls. ELISA was used for the determination of serum levels of copeptin, CRP and cortisol. Data obtained were statistically analyzed using the Student’s t-test and Mann Whitney U as appropriate and P<0.05 was considered significant.

Results

SCA subjects in VOC had significantly lower copeptin level and significantly higher CRP level compared with controls. However, serum levels of copeptin, cortisol and CRP were significantly higher in SCA subjects in VOC compared with SCA subjects in steady state. Furthermore, CRP had the widest Area under the ROC curve (AUROC) than copeptin and cortisol. No significant difference was observed in the levels of copeptin, CRP and cortisol when SCA subjects in VOC who were hospitalized for less ≤5 days were compared with subjects who had longer stays.

Conclusion

It could be concluded that C-reactive protein has a superior diagnostic performance for vaso-occlusive crisis in individuals with sickle cell anaemia and that C-reactive protein, cortisol and copeptin are not good prognostic markers in SCA subjects in vaso-occlusive crisis.     

Systemic resistance and lipoxygenase-related defence response induced in tomato by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>strain BTP1

Background  

Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes.     

Molecular epidemiology of a Pseudomonas aeruginosa hospital outbreak driven by a contaminated disinfectant-soap dispenser

Background and Objective

Pseudomonas aeruginosa infection represents a main cause of morbidity and mortality among immunocompromised patients. This study describes a fatal epidemic of P. aeruginosa that occurred in a hematology unit in Italy.

Methods

Retrospective cohort study, prospective surveillance, auditing, extensive testing on healthcare workers and environmental investigation were performed to define the dynamics and potential causes of transmission. RAPD, macrorestriction analyses and sequence typing were used to define relationships between P. aeruginosa isolates.

Results

Eighteen cases of infection were identified in the different phases of the investigation. Of these, five constitute a significant molecular cluster of infection. A P. aeruginosa strain with the same genetic fingerprint and sequence type (ST175) as clinical isolates strain was also isolated from a heavily contaminated triclosan soap dispenser.

Discussion and Conclusions

Our results are consistent with the hypothesis that patients became indirectly infected, e.g., during central venous catheter handling through contaminated items, and that the triclosan soap dispenser acted as a common continuous source of P. aeruginosa infection. Since P. aeruginosa is intrinsically unsusceptible to triclosan, the use of triclosan-based disinfectant formulations should be avoided in those healthcare settings hosting patients at high risk of P. aeruginosa infection.     

Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content

Background

The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.

Methodology

Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.

Principal Findings

Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM.

Conclusion

Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.     

Repression of Invasion Genes and Decreased Invasion in a High-Level Fluoroquinolone-Resistant Salmonella Typhimurium Mutant

Background

Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability.

Methodology/Principal Findings

Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 µg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant.

Conclusions/Significance

In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.