Evaluation of Phenolic Root Exudates as Stimulants of Saptrophic Fungi in the Rhizosphere.

The rhizosphere microbial community of crop plants in intensively managed arable soils is strongly dominated by bacteria, especially in the initial stages of plant development. In order to establish more diverse and balanced rhizosphere microbiomes, as seen for wild plants, crop variety selection could be based on their ability to promote growth of saprotrophic fungi in the rhizosphere. We hypothesized that this can be achieved by increasing the exudation of phenolic acids, as generally higher fungal abundance is observed in environments with phenolic-rich inputs, such as exudates of older plants and litter leachates. To test this, a rhizosphere simulation microcosm was designed to establish gradual diffusion of root exudate metabolites from sterile sand into arable soil. With this system, we tested the fungus-stimulating effect of eight phenolic acids alone or in combination with primary root metabolites. Ergosterol-based fungal biomass measurements revealed that most phenolic acids did not increase fungal abundance in the arable soil layer. These results were supported by comparison of fungal biomass in the rhizosphere of wild type Arabidopsis thaliana plants and mutants with altered phenolic acid metabolism. Salicylic acid was the only phenolic acid that stimulated a higher fungal biomass in the arable soil layer of microcosms, but only when combined with a background of primary root metabolites. However, such effect on rhizosphere fungi was not confirmed for a salicylic acid-impaired A. thaliana mutant. For three phenolic acid treatments (chlorogenic acid, salicylic acid, vanillic acid) fungal and bacterial community compositions were analyzed using amplicon sequencing. Despite having little effect on fungal biomass, phenolic acids combined with primary metabolites promoted a higher relative abundance of soil-borne fungi with the ability to invade plant roots (Fusarium, Trichoderma and Fusicolla spp.) in the simulated rhizosphere. Bacterial community composition was also affected by these phenolic acids. Although this study indicates that phenolic acids do not increase fungal biomass in the rhizosphere, we highlight a potential role of phenolic acids as attractants for root-colonizing fungi.

Exploring bacterial interspecific interactions for discovery of novel antimicrobial compounds.

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram-positive and Gram-negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram-positive Paenibacillus sp. AD87 and the Gram-negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5-bis(1-methylethyl)-pyrazine and an unknown Pederin-like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5-bis(1-methylethyl)-pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Impact of interspecific interactions on antimicrobial activity among soil bacteria.

Certain bacterial species produce antimicrobial compounds only in the presence of a competing species. However, little is known on the frequency of interaction-mediated induction of antibiotic compound production in natural communities of soil bacteria. Here we developed a high-throughput method to screen for the production of antimicrobial activity by monocultures and pair-wise combinations of 146 phylogenetically different bacteria isolated from similar soil habitats. Growth responses of two human pathogenic model organisms, Escherichia coli WA321 and Staphylococcus aureus 533R4, were used to monitor antimicrobial activity. From all isolates, 33% showed antimicrobial activity only in monoculture and 42% showed activity only when tested in interactions. More bacterial isolates were active against S. aureus than against E. coli. The frequency of interaction-mediated induction of antimicrobial activity was 6% (154 interactions out of 2798) indicating that only a limited set of species combinations showed such activity. The screening revealed also interaction-mediated suppression of antimicrobial activity for 22% of all combinations tested. Whereas all patterns of antimicrobial activity (non-induced production, induced production and suppression) were seen for various bacterial classes, interaction-mediated induction of antimicrobial activity was more frequent for combinations of Flavobacteria and alpha- Proteobacteria. The results of our study give a first indication on the frequency of interference competitive interactions in natural soil bacterial communities which may forms a basis for selection of bacterial groups that are promising for the discovery of novel, cryptic antibiotics.

Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans†Ter331.

The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A. niger, indicating functional variation within this group of Collimonas-produced polyyne-like 'collimomycins'. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.

Structural and functional variation in soil fungal communities associated with litter bags containing maize leaf.

Soil fungi are key players in the degradation of recalcitrant organic matter in terrestrial ecosystems. To examine the organisms and genes responsible for complex organic matter degradation in soil, we tracked changes in fungal community composition and expressed genes in soil adjacent to mesh bags containing maize leaves undergoing decomposition. Using high-throughput sequencing approaches, changes in fungal community composition were determined by targeting 18S rRNA gene sequences, whereas community gene expression was examined via a metatranscriptomic approach. The majority of the 93 000 partial 18S rRNA gene sequences generated, were affiliated with the Ascomycota and Basidiomycota. Fungal diversity was at least 224 operational taxonomic units at the 97% similarity cutoff level. During litter degradation, the relative proportion of Basidiomycota increased, with a decrease in Ascomycota : Basidiomycota ratios over time. The most commonly detected decomposition-associated fungi included Agaricomycetes and Tremellales as well as unclassified Mucoromycotina. The majority of protein families found in the metatranscriptomic data were affiliated to fungal groups described to degrade plant-derived cellulose, such as Mucoraceae, Chaetomiaceae, Sordariaceae, Sebacinaceae, Tremellaceae, Psathyrellaceae and Schizophyllaceae. The combination of high-throughput rRNA gene-based and metatranscriptomic approaches provided perspectives into the organisms and genes involved in complex organic matter in soil.

Dual transcriptional profiling of a bacterial/fungal confrontation: Collimonas fungivorans versus Aspergillus niger.

Interactions between bacteria and fungi cover a wide range of incentives, mechanisms and outcomes. The genus Collimonas consists of soil bacteria that are known for their antifungal activity and ability to grow at the expense of living fungi. In non-contact confrontation assays with the fungus Aspergillus niger, Collimonas fungivorans showed accumulation of biomass concomitant with inhibition of hyphal spread. Through microarray analysis of bacterial and fungal mRNA from the confrontation arena, we gained new insights into the mechanisms underlying the fungistatic effect and mycophagous phenotype of collimonads. Collimonas responded to the fungus by activating genes for the utilization of fungal-derived compounds and for production of a putative antifungal compound. In A. niger, differentially expressed genes included those involved in lipid and cell wall metabolism and cell defense, which correlated well with the hyphal deformations that were observed microscopically. Transcriptional profiles revealed distress in both partners: downregulation of ribosomal proteins and upregulation of mobile genetic elements in the bacteria and expression of endoplasmic reticulum stress and conidia-related genes in the fungus. Both partners experienced nitrogen shortage in each other's presence. Overall, our results indicate that the Collimonas/Aspergillus interaction is a complex interplay between trophism, antibiosis and competition for nutrients.

Identification and characterization of genes underlying chitinolysis in Collimonas fungivorans Ter331.

Through a combinatorial approach of plasposon mutagenesis, genome mining, and heterologous expression, we identified genes contributing to the chitinolytic phenotype of bacterium Collimonas fungivorans Ter331. One of five mutants with abolished ability to hydrolyze colloidal chitin carried its plasposon in the chiI gene coding for an extracellular endochitinase. Two mutants were affected in the promoter of chiP-II coding for an outer-membrane transporter of chitooligosaccharides. The remaining two mutations were linked to chitobiose/N-acetylglucosamine uptake. Thus, our model for the Collimonas chitinolytic system assumes a positive feedback regulation of chitinase activity by chitin degradation products. A second chitinase gene, chiII, coded for an exochitinase that preferentially released chitobiose from chitin analogs. Genes hexI and hexII showed coding resemblance to N-acetylglucosaminidases, and the activity of purified HexI protein towards chitin analogs suggested its role in converting chitobiose to N-acetylglucosamine. The hexI gene clustered with chiI, chiII, and chiP-II in one locus, while chitobiose/N-acetylglucosamine uptake genes colocalized in another. Both loci contained genes for conversion of N-acetylglucosamine to fructose-6-phosphate, confirming that C. fungivorans Ter331 features a complete chitin pathway. No link could be established between chitinolysis and antifungal activity of C. fungivorans Ter331, suggesting that the bacterium's reported antagonism towards fungi relies on other mechanisms.

Motilin receptor expression in smooth muscle, myenteric plexus, and mucosa of human inflamed and noninflamed intestine.

BACKGROUND: Besides regulation of upper gastrointestinal motility, motilin seems to play a role in the inflammatory response. Motilin receptor expression in human intestine has not been studied thoroughly. This study aimed to describe the intestinal distribution of motilin receptors in inflammatory bowel disease (IBD) and control patients. METHODS: Quantitative autoradiography, immunohistochemistry, and reverse-transcriptase polymerase chain reaction (RT-PCR) were used to detect motilin receptors in tissue of 25 IBD patients (13 Crohn's disease [CD], 12 ulcerative colitis [UC]) and 19 patients with a neoplasm (controls). RESULTS: Median muscular motilin binding was 3 and 8 fmol/g tissue in colon and ileum, respectively. In the gastroduodenal region the median was higher (93 fmol/g). In UC colonic muscular motilin binding was significantly increased compared to controls (7 vs. 3 fmol/g, P < or = 0.05). Expression in CD was similar to controls. Besides the binding found in the muscular compartment, motilin binding was also found in the mucosa, which was even higher than in the muscle (3 versus 11 and 8 versus 27 fmol/g for colon and ileum (P < or = 0.06), respectively). RT-PCR and immunohistochemistry confirmed the mucosal motilin receptor expression. The mucosal motilin receptors were located in the epithelial cells. In the muscular compartment receptors were strongly present in the myenteric plexus and weakly in the smooth muscle cells. In IBD tissue the expression pattern was not different. CONCLUSIONS: The motilin receptor is expressed in human colonic and ileal smooth muscle. Further, motilin receptor expression was also shown in the mucosa. Muscular binding in UC patients is increased but no different expression pattern was found.

Infliximab treatment influences the serological expression of matrix metalloproteinase (MMP)-2 and -9 in Crohn's disease.

BACKGROUND: Matrix metalloproteinases (MMPs) are actively involved in the pathogenesis of Crohn's disease (CD). We assessed the effect of the anti-tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody infliximab on the in vitro and in vivo expression of MMP-2 and MMP-9 in CD. METHODS: Infliximab-treated fistulizing (n = 10) or active disease (n = 7) CD patients, from an in-house study, and fistulizing CD patients (n = 42) and active CD patients (n = 24) from 2 placebo controlled studies were evaluated for serum MMP levels and clinical response. Biopsies were evaluated immunohistochemically for the MMPs. Whole blood cultures stimulated with lipopolysaccharide (LPS)/infliximab were evaluated for MMP mRNA and protein levels. RESULTS: Serum MMP-2 levels in CD patients increased during follow-up, similarly in responders and nonresponders, by infliximab. Immunohistochemistry showed no clear MMP-2 change in biopsies. Serum MMP-9 levels, however, showed a consistent pattern of decrease in most CD patients, particularly in those responding, and MMP-9-positive polymorphonuclear leukocytes in biopsies also decreased by infliximab. LPS stimulation of whole blood increased the MMP-9 levels in plasma significantly in CD patients and controls, but infliximab had no effect on the secretion. Long-term LPS stimulation raised leukocyte MMP-9 mRNA levels 16-fold and infliximab inhibited this induction by 80%. CONCLUSIONS: Infliximab treatment increases MMP-2 and decreases MMP-9 in serum of patients with CD, the latter also in the intestine, which extends and confirms our previous ex vivo explants observations. However, these changes were not strictly associated with the response to treatment. The enhanced leukocyte MMP-9 expression in CD seems to be regulated by TNF-alpha.

Role of tumor necrosis factor-alpha and interleukin-10 gene polymorphisms in irritable bowel syndrome.

OBJECTIVES: Imbalances in the genetically controlled pro- and anti-inflammatory cytokine production may promote ongoing low-grade inflammation after an acute gastroenteritis, and subsequently, irritable bowel syndrome (IBS) (post-infectious IBS, PI-IBS). We studied gene promoter single nucleotide polymorphisms (SNPs) of tumor necrosis factor-alpha (TNF-alpha, pro-inflammatory) and interleukin-10 (IL-10, anti-inflammatory) in IBS patients and controls. METHODS: DNA was extracted from peripheral blood leucocytes of 111 IBS patients and 162 healthy controls. Genotype and allele frequencies were assessed by analyzing SNPs at position -308 (TNF-alpha) and -1082 and -819 (IL-10). RESULTS: Homozygous high producers for TNF-alpha (A/A) were rare (overall prevalence 2.6%). The heterozygous TNF-alpha genotype (G/A, high producer) was significantly more prevalent in IBS compared to controls (41%vs 26%, p= 0.02). More patients (41%) than controls (30%) were positive for the A allele (p= 0.044; odds ratio (OR) 1.68, 95% confidence interval (CI) 1.01-2.79), with a similar trend for diarrhea (54%) versus constipation and alternating subtypes (<33%, p= 0.079), but not for subgroups according to a history of acute gastroenteritis. IL-10 genotypes were similarly distributed in patients and controls for both SNPs. Possession of a high producer TNF-alpha and a low producer IL-10 genotype were significantly more prevalent in IBS (9%) versus controls (3%, p= 0.035; OR 3.11, 95% CI 1.03-9.36) and in diarrhea (20%) compared to other IBS subtypes (<4%, p= 0.026). CONCLUSIONS: Our results support the emerging hypothesis that genetically determined immune activity plays a role in the pathophysiology of IBS. Future studies in larger, clinically relevant, IBS subgroups are warranted to establish definite associations with cytokine gene polymorphisms.

Souter-Strathclyde total elbow arthroplasty: medium-term results.

The Souter-Strathclyde unconstrained elbow prosthesis was prospectively studied in 36 patients (45 prostheses) with rheumatoid arthritis (Larsen grade 4 and grade 5). The mean age of the patients at the time of operation was 63 years (range: 39 to 75 years). Eight patients (9 prostheses) died within five years of implantation, from causes unrelated to the elbow arthroplasty. One patient was lost to follow-up, leaving 27 patients (35 prostheses) for review. The mean length of follow-up was 98 months (range: 60 to 174 months). At 8.2 years follow-up, the prosthesis showed a probability of survival of 76% (SD 9%) with revision of the humeral component as an end point; the percentage dropped to 67% (SD 9%) when radiographic loosening was taken as an end-point. Survival of the ulnar component was 98%. Loosening of the humeral component seems to be related to both the short humeral stem and a persistent extension deficit.