Rugulactone and its analogues exert antibacterial effects through multiple mechanisms including inhibition of thiamine biosynthesis

Rugulactone is a dihydro-α-pyrone isolated from the plant Cryptocarya rugulosa in 2009. It has been reported to display IkB kinase (IKK) inhibitory activity, as well as antibiotic activity in several strains of pathogenic bacteria. However, its biological targets and mode of action in bacteria have not yet been explored. Here we present enantioselective syntheses of rugulactone and of some corresponding activity-based protein profiling (ABPP) probes. We found that the ABPP probes in this study are more potent than rugulactone against Staphyloccocus aureus NCTC 8325, S. aureus Mu50, Listeria welshimeri SLCC 5334 and Listeria monocytogenes EGD-e, and that molecules of this class probably exert their antibacterial effect through a combination of targets. These targets include covalent inhibition of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMPP) kinase (ThiD), which is an essential component of the thiamine biosynthesis pathway in bacteria. This represents the first example of a small-molecule inhibitor of ThiD.     

Similarities in effluent organic matter characteristics from Connecticut wastewater treatment plants

Effluent organic matter (EfOM) from five Connecticut (USA) municipal wastewater treatment plants was isolated with DAX8 (hydrophobic fraction) and XAD4 (transphilic fraction) resins. Isolate recoveries ranged from 18 to 42% of the total organic carbon for DAX8 resin and from 6 to 12% for XAD4 resin. Isolated EfOM was characterized by traditional organic geochemistry techniques. Weight-averaged molecular weights of extracted EfOM by size exclusion chromatography were 450-670 Da with higher weights observed for the hydrophobic fractions than the transphilic fractions. Fluorescence characterization showed both humic- and fulvic-like fluorescence, as well as tryptophan- and tyrosine-like fluorescence, the latter not commonly observed for terrestrial organic matter. Fluorescence indices were between 1.5 and 1.9 with lower values observed for hydrophobic EfOM fractions than for transphilic fractions. Specific ultraviolet absorbance was measured between 0.8 and 3.0 L mg⁻¹ m⁻¹ with higher values for the hydrophobic EfOM fractions. Together these results indicated that isolated EfOM is similar in characteristics to microbially derived organic matter from natural aquatic systems. Little variation in EfOM characteristics was observed between the five wastewater treatment plants, suggesting that the characteristics of EfOM are similar, regardless of treatment plant design.     

Determination of copper speciation in highway stormwater runoff using competitive ligand exchange - Adsorptive cathodic stripping voltammetry

Low concentrations of dissolved copper have been shown to adversely affect the olfactory system of salmonid species, impairing their ability to avoid predators and likely increasing mortality. These studies have resulted in increased regulatory scrutiny of stormwater discharges to surface waters inhabited by threatened and endangered salmonid species. Because it is primarily the free ionic (Cu²⁺) and weakly complexed forms of copper that are bioavailable, it is critical to understand the speciation of copper in stormwater. This paper reports on the characterization of copper binding ligands and copper speciation in composite samples of highway stormwater runoff collected at four sites in Oregon, USA using competitive ligand exchange - adsorptive cathodic stripping voltammetry (CLE-ACSV). Although the concentration and strength of copper binding ligands in stormwater varied considerable between sites and storms, the vast majority (>99.9%) of the total dissolved copper in composite samples was complexed by organic ligands in stormwater. Although total dissolved copper concentrations range from 2 to 20 μg/L, the analytically determined free ionic copper concentrations did not exceed 10⁻¹⁰ M (6.3 ng/L) in any of the fully characterized samples, suggesting that much of the copper in highway stormwater is not bioavailable. Analytically determined free ionic copper concentrations were compared with those predicted by a readily available chemical equilibrium models and found to be in reasonable agreement.     

Freshwater DOM quantity and quality from a two-component model of UV absorbance

We present a model that considers UV-absorbing dissolved organic matter (DOM) to consist of two components (A and B), each with a distinct and constant spectrum. Component A absorbs UV light strongly, and is therefore presumed to possess aromatic chromophores and hydrophobic character, whereas B absorbs weakly and can be assumed hydrophilic. We parameterised the model with dissolved organic carbon concentrations [DOC] and corresponding UV spectra for c. 1700 filtered surface water samples from North America and the United Kingdom, by optimising extinction coefficients for A and B, together with a small constant concentration of non-absorbing DOM (0.80 mg DOC L⁻¹). Good unbiased predictions of [DOC] from absorbance data at 270 and 350 nm were obtained (r² = 0.98), the sum of squared residuals in [DOC] being reduced by 66% compared to a regression model fitted to absorbance at 270 nm alone. The parameterised model can use measured optical absorbance values at any pair of suitable wavelengths to calculate both [DOC] and the relative amounts of A and B in a water sample, i.e. measures of quantity and quality. Blind prediction of [DOC] was satisfactory for 9 of 11 independent data sets (181 of 213 individual samples).     

Spatial and temporal variation in indicator microbe sampling is influential in beach management decisions

Fecal indicator microbes, such as enterococci, are often used to assess potential health risks caused by pathogens at recreational beaches. Microbe levels often vary based on collection time and sampling location. The primary goal of this study was to assess how spatial and temporal variations in sample collection, which are driven by environmental parameters, impact enterococci measurements and beach management decisions. A secondary goal was to assess whether enterococci levels can be predictive of the presence of Staphylococcus aureus, a skin pathogen. Over a ten-day period, hydrometeorologic data, hydrodynamic data, bather densities, enterococci levels, and S. aureus levels including methicillin-resistant S. aureus (MRSA) were measured in both water and sand. Samples were collected hourly for both water and sediment at knee-depth, and every 6 h for water at waist-depth, supratidal sand, intertidal sand, and waterline sand. Results showed that solar radiation, tides, and rainfall events were major environmental factors that impacted enterococci levels. S. aureus levels were associated with bathing load, but did not correlate with enterococci levels or any other measured parameters. The results imply that frequencies of advisories depend heavily upon sample collection policies due to spatial and temporal variation of enterococci levels in response to environmental parameters. Thus, sampling at different times of the day and at different depths can significantly impact beach management decisions. Additionally, the lack of correlation between S. aureus and enterococci suggests that use of fecal indicators may not accurately assess risk for some pathogens.     

Molecular studies to identify the Fusarium species responsible for HT-2 and T-2 mycotoxins in UK oats

High levels of Fusarium mycotoxins HT-2 and T-2 have been detected in UK oats since surveys started in 2002. Fusarium langsethiae and the closely related species F. sporotrichioides have previously been associated with the contamination of cereals with type A trichothecenes HT-2 and T-2 in Nordic countries. Preliminary microbiological analysis of UK oat samples with high concentrations of HT-2 and T-2 detected and isolated F. langsethiae and F. poae but not the other type A trichothecene producing species F. sporotrichioides, F. sibiricum and F. armeniacum. Two hundred and forty oat flour samples with a known mycotoxin profile were selected from a previous four year study (2002-2005) to cover the full concentration range from below the limit of quantification (<20 μg/kg) to 9,990 μg/kg HT-2+T-2 combined. All samples were analysed for the DNA of F. langsethiae, F. poae and F. sporotrichioides based on previously published PCR assays. F. langsethiae was detectable in nearly all samples; F. poae was detected in 90% of samples whereas F. sporotrichioides was not detected in any sample. A real-time PCR assay was developed to quantify F. langsethiae DNA in plant material. The assay could quantify as low as 10(-4)ngF. langsethiae DNA/μl. Based on this assay and a previously published assay for F. poae, both species were quantified in the oat flour samples with known HT-2+T-2 content. Results showed a good regression (P<0.001, r(2)=0.60) between F. langsethiae DNA and HT-2+T-2 concentration. F. poae DNA concentration was not correlated to HT2+T2 concentration (P=0.448) but was weakly correlated to nivalenol concentration (P<0.001, r(2)=0.09). Multiple regression with F. langsethiae and F. poae DNA as explanatory variates identified that both F. langsethiae and F. poae DNA were highly significant (P<0.001) but F. poae DNA only accounted for an additional 4% of the variance and the estimate was negative, indicating that higher concentrations of F. poae DNA were correlated with slightly lower concentrations of HT2+T2 detected. A stronger regression (P<0.001, r(2)=0.77) between F. langsethiae DNA and HT-2+T-2 was obtained after extraction and quantification of DNA and mycotoxins from individual oat grains. The results from this study provide strong evidence that F. langsethiae is the primary, if not sole, fungus responsible for high HT-2 and T-2 in UK oats.     

Metabolomic profiling of beer reveals effect of temperature on non-volatile small molecules during short-term storage

The effect of temperature on non-volatile compounds in beer has not been well characterised during storage. Here, a metabolomics approach was applied to characterise the effect of storage temperature on non-volatile metabolite variation after 16weeks of storage, using fresh beer as a control. The metabolite profile of room temperature stored (RT) and cold temperature stored (CT) beer differed significantly from fresh, with the most substantial variation observed between RT and fresh beer. Metabolites that changed during storage included prenylated flavonoids, purines, and peptides, and all showed reduced quantitative variation under the CT storage conditions. Corresponding sensory panel observations indicated significant beer oxidation after 12 and 16weeks of storage, with higher values reported for RT samples. These data support that temperature affected beer oxidation during short-term storage, and reveal 5-methylthioadenosine (5-MTA) as a candidate non-volatile metabolite marker for beer oxidation and staling.     

Comparative examination of Escherichia coli O157:H7 survival on romaine lettuce and in soil at two independent experimental sites

Little is known about the influence of abiotic factors such as climate and soil chemistry on the survival of Escherichia coli O157:H7 in field lettuce. We applied a nalidixic acid-resistant derivative of strain ATCC 700728 to field-grown romaine lettuce in two regions in Canada characterized by large variances in soil type and climate. Surviving populations in soil and on lettuce leaves were estimated on sorbitol MacConkey agar supplemented with nalidixic acid. Data were fitted with the Weibull decline function to permit comparison of decay rates in the two experimental sites. E. coli O157:H7 populations fell from 10? to <102 CFU/g on leaves, and <103 CFU/g in soil within 7 days after inoculation. Analysis revealed there was no significant difference between decay rates at the two experimental sites in either environment. The results of this study suggest that the inherent ecological fitness of E. coli O157:H7 ATCC 700728 determines the extent of survival in the production environment.     

Gas-phase and transpiration-driven mechanisms for volatilization through wetland macrophytes

Natural and constructed wetlands have gained attention as potential tools for remediation of shallow sediments and groundwater contaminated with volatile organic compounds (VOCs). Wetland macrophytes are known to enhance rates of contaminant removal via volatilization, but the magnitude of different volatilization mechanisms, and the relationship between volatilization rates and contaminant physiochemical properties, remain poorly understood. Greenhouse mesocosm experiments using the volatile tracer sulfur hexafluoride were conducted to determine the relative magnitudes of gas-phase and transpiration-driven volatilization mechanisms. A numerical model for vegetation-mediated volatilization was developed, calibrated with tracer measurements, and used to predict plant-mediated volatilization of common VOCs as well as quantify the contribution of different volatilization pathways. Model simulations agree with conclusions from previous work that transpiration is the main driver for volatilization of VOCs, but also demonstrate that vapor-phase transport in wetland plants is significant, and can represent up to 50% of the total flux for compounds with greater volatility like vinyl chloride.     

Sequence and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1 predict in vivo sensitivity to dioxins

There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324_Ser380 vs common tern--250-fold less sensitive than chicken; Val325_Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324_Ala380 and Val324_Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324_Ala380) and Japanese quail (Coturnix japonica; Val324_Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.