Antimicrobial and antibiofilm activities of 1-alkylquinolinium bromide ionic liquids

Quinoline derivatives are known to possess a range of bioactive and medicinal activities, which have been exploited in the design of antibacterial, antifungal and antimalarial compounds. In this study, we report on the microbiological toxicity of a series of 1-alkylquinolinium bromides against a range of clinically relevant microorganisms, in both planktonic and sessile (biofilm) cultures. A comparison of antimicrobial activity against planktonic bacteria and established biofilms is presented. In general, 1-alkylquinolinium ionic liquids possess excellent, broad spectrum antimicrobial activity against microorganisms grown in both the planktonic and sessile, or biofilm, mode of growth. Importantly, these compounds are potent against Gram positive and Gram negative bacteria, as well as fungi, with a clear dependency on length of the alkyl substituent for activity, with compounds containing twelve and fourteen carbons in the alkyl group exhibiting highest antimicrobial and antibiofilm activity. © 2010 The Royal Society of Chemistry.

Sexual division of antibacterial resource defence in breeding burying beetles, Nicrophorus vespilloides

A key component of parental care involves defending resources destined for offspring from a diverse array of potential interspecific competitors, such as social parasites, fungi and bacteria. 2. Just as with other aspects of parental care, such as offspring provisioning or brood defence, sexual conflict between parents may arise over how to share the costs of this form of care. There has been little previous work, however, to investigate how this particular burden might be shared. 3. Here, we describe a hitherto uncharacterized form of parental care in burying beetles Nicrophorus vespilloides, a species which prepares carrion for its young and faces competition from microbes for this resource. We found that parents defend the carcass with antibacterial anal exudates, and that the antibacterial activity of these exudates is only upregulated following the discovery of a corpse. At the same time, phenoloxidase activity in the anal exudates is downregulated, indicating parallels with the internal insect immune system. 4. In unmanipulated breeding pairs, females had higher antibacterial activity in their anal exudates than males, suggesting sex-specific roles in this aspect of parental care. 5. When we experimentally widowed males, we found that they increased levels of antibacterial activity in their anal exudates. Experimentally widowing females, however, led them to decrease levels of antibacterial activity in their anal exudates. Widowed beetles of each sex thus produced anal exudates of comparable antibacterial activity. We suggest that this flexible division of antibacterial activity may be coordinated by Juvenile Hormone. © 2009 British Ecological Society.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium.

Background
The identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.

Methods
Cystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24 h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.

Results
Treatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.

Conclusion
Our data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not.

Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review protocol

Background: There is growing interest in the potential utility of molecular diagnostics in improving the detection of life-threatening infection (sepsis). LightCycler® SeptiFast is a multipathogen probebased real-time PCR system targeting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here the protocol of the first systematic review of published clinical diagnostic accuracy studies of this technology when compared with blood culture in the setting of suspected sepsis. Methods/design: Data sources: the Cochrane Database of Systematic Reviews, the Database of Abstracts of Reviews of Effects (DARE), the Health Technology Assessment Database (HTA), the NHS Economic Evaluation Database (NHSEED), The Cochrane Library, MEDLINE, EMBASE, ISI Web of Science, BIOSIS Previews, MEDION and the Aggressive Research Intelligence Facility Database (ARIF). Study selection: diagnostic accuracy studies that compare the real-time PCR technology with standard culture results performed on a patient's blood sample during the management of sepsis. Data extraction: three reviewers, working independently, will determine the level of evidence, methodological quality and a standard data set relating to demographics and diagnostic accuracy metrics for each study. Statistical analysis/data synthesis: heterogeneity of studies will be investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in Receiver Operator Characteristic (ROC) space. Bivariate model method will be used to estimate summary sensitivity and specificity. The authors will investigate reporting biases using funnel plots based on effective sample size and regression tests of asymmetry. Subgroup analyses are planned for adults, children and infection setting (hospital vs community) if sufficient data are uncovered. Dissemination: Recommendations will be made to the Department of Health (as part of an open-access HTA report) as to whether the real-time PCR technology has sufficient clinical diagnostic accuracy potential to move forward to efficacy testing during the provision of routine clinical care.

A NOVEL LIFE-HISTORY IN AGLAOTHAMNION-DIAPHANUM-SP NOV (CERAMIACEAE, RHODOPHYTA) FROM BRITTANY AND THE BRITISH-ISLES

A diminutive species of Aglaothamnion (Ceramiaceae, Rhodophyta), A. diaphanum sp. nov., is described from Brittany (Atlantic France), the Isles of Scilly (off S.W. England) and western Ireland. Aglaothamnion diaphanum is confined to the sublittoral zone, where it grows almost exclusively on algae and sessile animals attached to hard substrata. Thalli are delicate, and branched distichously in one plane. The main axes are ecorticate but may form loose non-corticating rhizoidal filaments. The lateral branches bear a characteristic, regularly alternate distichous series of branchlets, the first of which is always adaxial. All vegetative cells are uninucleate. The majority of field-collected plants bear only bisporangia, but a few bisporangial plants also form spermatangia; some male plants and a single female specimen have been collected. The spermatangial branchlets consist of 3-5 spermatangial mother cells each bearing 2-4 spermatangia, which are constricted around a central nucleus. None of the U-shaped carpogonial branches showed any sign of fertilization, and the gametangia appear to be non-functional. The bisporangia are ovoid and contain two uninucleate spores separated by an oblique curved wall. The occurrence of bisporangia and the lack of adherent cortication distinguish A. diaphanum from two similar species, Aglaothamnion bipinnatum (P. Crouan et H. Crouan) Feldmann-Mazoyer and Aglaothamnion decompositum (J. Agardh) Halos. The life history in culture of French and Irish isolates of A. diaphanum consists of a series of bisporangial generations, a single plant of which also formed spermatangia. Apical cells of bisporophytes are haploid (n = c. 32), but the first division of meiosis, with chromosome pairing and crossing over, occurs in dividing bisporocytes. The germinating bispores are haploid. Endodiploidization may occur in the early stages of sporangium development, as in some phycomycete fungi, or in vegetative cells that subsequently give rise to bisporocytes. This is the first demonstration in the red algae of meiotic bisporangia on plants of which the apical cells, at least, are haploid.

Isolation of Alternaria alternata from an emollient cream:implications for public health

A specimen of emollient cream, which was observed to be contaminated peripherally with a filamentous fungus was examined for the presence of fungi and the resulting fungal colonies were examined phenotypically and genotypically. Subsequent DNA extraction and PCR amplification of the large internal transcribed spacer region [ITS1-5.8S-ITS2] yielded an amplicon of 512 bp. Sequence analysis identified this as Alternaria alternata at the 100% homology level with all 512/512 bases called. This organism has been previously reported as a cause of opportunistic infections involving skin and immunocompromised patients. This is the first report of an emollient cream as a source of this organism. It highlights the need for proper management of such preparations in order to minimize the potential spread of fungi to susceptible patient populations.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.

Stable isotope probing analysis of the influence of liming on root exudate utilization by soil microorganisms

Rhizosphere microorganisms play an important role in soil carbon flow, through turnover of root exudates, but there is little information on which organisms are actively involved or on the influence of environmental conditions on active communities. In this study, a (CO2)-C-13 pulse labelling field experiment was performed in an upland grassland soil, followed by RNA-stable isotope probing (SIP) analysis, to determine the effect of liming on the structure of the rhizosphere microbial community metabolizing root exudates. The lower limit of detection for SIP was determined in soil samples inoculated with a range of concentrations of C-13-labelled Pseudomonas fluorescens and was found to lie between 10(5) and 10(6) cells per gram of soil. The technique was capable of detecting microbial communities actively assimilating root exudates derived from recent photo-assimilate in the field. Denaturing gradient gel electrophoresis (DGGE) profiles of bacteria, archaea and fungi derived from fractions obtained from caesium trifluoroacetate (CsTFA) density gradient ultracentrifugation indicated that active communities in limed soils were more complex than those in unlimed soils and were more active in utilization of recently exuded C-13 compounds. In limed soils, the majority of the community detected by standard RNA-DGGE analysis appeared to be utilizing root exudates. In unlimed soils, DGGE profiles from C-12 and C-13 RNA fractions differed, suggesting that a proportion of the active community was utilizing other sources of organic carbon. These differences may reflect differences in the amount of root exudation under the different conditions.

The effects of multiple metal contamination on ectomycorrhizal Scots pine (Pinus sylvestris) seedlings

Experiments were conducted to investigate the effects of single and multiple metal contamination (Cd, Pb, Zn, Sb, Cu) on Scots pine seedlings colonised by ectomycorrhizal (ECM) fungi from natural soil inoculum. Seedlings were grown in either contaminated field soil from the site of a chemical accident, soils amended with five metals contaminating the site, or in soil from an uncontaminated control site. Although contaminated and metal-amended soil significantly inhibited root and shoot growth of the Scots pine seedlings, total root tip density was not affected. Of the five metals tested in amended soils, Cd was the most toxic to ECM Scots pine. Field-contaminated soil had a toxic effect on ECM fungi associated with Scots pine seedlings and caused shifts in ECM species composition on ECM seedlings. When compared to soils amended with only one metal, soils amended with a combination of all five metals tested had lower relative toxicity and less accumulation of Pb, Zn and Sb into seedlings. This would indicate that the toxicity of multiple metal contamination cannot be predicted from the individual toxicity of the metals investigated.

Wood degradation, and cellulase and ligninase production, by Trametes and other wood-inhabiting basidiomycetes from indigenous forests of Zimbabwe

Nine species of Trametes and five other wood inhabiting basidiomycetes, were collected from the indigenous forests of Zimbabwe and analysed for cellulases, ligninases, extracellular phenolases and wood degrading ability for the first time. Cellulase enzyme activities varied widely among the species. After 15 d growth exo-glucanase activity had increased in the majority of species whilst Biter paper activity showed the opposite trend, being greatly reduced in all species on day 15 compared to day IO. Endo-glucanase activity was relatively uniform at both sampling times. The fungi were more active against water soluble cellulose derivatives than filter paper cellulase. In all the fungi tested, cellulose activity on filter paper was significantly less than endo- and exo-glucanase activities. The highest cellulase activity was expressed by Cerrena meyenii (683 U mg(-1)) Phaeotrametes decipiens, Trametes modesta, and T. pocas also expressed relatively high cellulase activity on all types of cellulose tested. All Trametes species tested positive for extracellular phenol oxidases whilst Fomotopsis spragueii and Irpex stereoides tested negative. Ail but one of the Trametes species in the study were able to degrade two different lignin preparations in tests for lignin degradation. T. menziesii was unable to degrade both lignin preparations although it had tested positive for production of extracellular oxidase. The species in this study degraded hardwood to a greater extent than softwood. Eight of them caused more than 80% dry weight loss of wood blocks during 70 d incubation. Those fungi that expressed high cellulase activity also caused high weight loss on wood.

Enhanced antimicrobial activities of 1-alkyl-3-methyl imidazolium ionic liquids based on silver or copper containing anions

We have developed a series of 1-alkyl-3-methylimidazolium tetrachlorocuprate(II) and dibromoargentate(I) ionic liquids with enhanced antimicrobial activity when compared with 1-alkyl-3-methylimidazolium chloride ionic liquids. These new ionic liquids proved to be effective against a range of pathogenic bacteria and fungi.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.