Isolation and characterization of a novel simazine-degrading bacterium from agricultural soil of central Chile, Pseudomonas sp MHP41

s-Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of mu = 0.10 h(-1), yielding a high biomass of 4.2 x 10(8) CFU mL(-1). Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans. This is the first s-triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s-triazine-contaminated environments.

Fumonisin B-1 as a Urinary Biomarker of Exposure in a Maize Intervention Study Among South African Subsistence Farmers

Background: The consumption of maize highly contaminated with carcinogenic fumonisins has been linked to high oesophageal cancer rates. The aim of this study was to validate a urinary fumonisin B-1 (UFB1) biomarker as a measure of fumonisin exposure and to investigate the reduction in exposure following a simple and culturally acceptable intervention.

Methods: At baseline home-grown maize, maize-based porridge, and first-void urine samples were collected from female participants (n = 22), following their traditional food practices in Centane, South Africa. During intervention the participants were trained to recognize and remove visibly infected kernels, and to wash the remaining kernels. Participants consumed the porridge prepared from the sorted and washed maize on each day of the two-day intervention. Porridge, maize, and urine samples were collected for FB1 analyses.

Results: The geometric mean (95% confidence interval) for FB1 exposure based on porridge (dry weight) consumption at baseline and following intervention was 4.84 (2.87-8.14) and 1.87 (1.40-2.51) mg FB1/kg body weight/day, respectively, (62% reduction, P < 0.05). UFB1C, UFB1 normalized for creatinine, was reduced from 470 (295-750) at baseline to 279 (202-386) pg/mg creatinine following intervention (41% reduction, P = 0.06). The UFB1C biomarker was positively correlated with FB1 intake at the individual level (r - 0.4972, P < 0.01). Urinary excretion of FB1 was estimated to be 0.075% (0.054%-0.104%) of the FB1 intake.

Conclusion: UFB1 reflects individual FB1 exposure and thus represents a valuable biomarker for future fumonisin risk assessment.

Impact: The simple intervention method, hand sorting and washing, could positively impact on food safety and health in communities exposed to fumonisins. Cancer Epidemiol Biomarkers Prev; 20(3); 483-9. (C)2011 AACR.

Simple intervention method to reduce fumonisin exposure in a subsistence maize-farming community in South Africa

In the Centane magisterial area of South Africa, high rates of oesophageal cancer have been associated with home-grown maize contaminated with fumonisins. The aim of this study was to implement a simple intervention method to reduce fumonisin exposure in a subsistence-farming community. The hand-sorting and washing procedures, based on traditional maize-based food preparation practices, were previously customised under laboratory-controlled conditions. Home-grown maize and maize-based porridge collected at baseline were analysed for fumonisin B1, B2 and B3. The geometric mean (95% confidence interval) of fumonisin contamination in the home-grown maize at baseline was 1.67 (1.21-2.32) mg kg-1 and 1.24 (0.75-2.04) mg kg -1 (dry weight) in the porridge. Fumonisin exposure was based on individual stiff porridge consumption and the specific fumonisin levels in the porridge (dry weight) consumed. Porridge (dry weight) consumption at baseline was 0.34 kg day-1 and fumonisin exposure was 6.73 (3.90-11.6) mu g kg-1 body weight day-1. Female participants (n = 22) were trained to recognise and remove visibly infected/damaged kernels and to wash the remaining maize kernels. The discarded kernels represented 3.9% by weight and the fumonisins varied from 17.1 to 76.9 mg kg-1. The customised hand-sorting and washing procedures reduced fumonisin contamination in the maize and porridge by 84 and 65%, respectively. The intervention reduced fumonisin exposure by 62% to 2.55 (1.94-3.35) mu g kg-1 body weight day-1. This simple intervention method has the potential to improve food safety and health in subsistence-farming communities consuming fumonisin-contaminated maize as their staple diet.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

The application of Near-Infrared Reflectance Spectroscopy (NIRS) to detect melamine adulteration of soya bean meal

Soya bean products are used widely in the animal feed industry as a protein based feed ingredient and
have been found to be adulterated with melamine. This was highlighted in the Chinese scandal of
2008. Dehulled soya (GM and non-GM), soya hulls and toasted soya were contaminated with melamine
and spectra were generated using Near Infrared Reflectance Spectroscopy (NIRS). By applying chemometrics
to the spectral data, excellent calibration models and prediction statistics were obtained. The coefficients
of determination (R2) were found to be 0.89–0.99 depending on the mathematical algorithm used,
the data pre-processing applied and the sample type used. The corresponding values for the root mean
square error of calibration and prediction were found to be 0.081–0.276% and 0.134–0.368%, respectively,
again depending on the chemometric treatment applied to the data and sample type. In addition, adopting
a qualitative approach with the spectral data and applying PCA, it was possible to discriminate
between the four samples types and also, by generation of Cooman’s plots, possible to distinguish
between adulterated and non-adulterated samples.

Arsenic in groundwater and its influence on exposure risks through traditionally cooked rice in Prey Vêng Province, Cambodia

Arsenic (As) contamination of communal tubewells in Prey Vêng, Cambodia, has been observed since 2000. Many of these wells exceed the WHO As in drinking water standard of 10 µg/L by a factor of 100. The aim of this study was to assess how cooking water source impacts dietary As intake in a rural community in Prey Vêng. This aim was fulfilled by (1) using geostatistical analysis techniques to examine the extent of As contaminated groundwater in Prey Vêng and identify a suitable study site, (2) conducting an on-site study in two villages to measure As content in cooked rice prepared with water collected from tubewells and locally harvested rainwater, and (3) determining the dietary intake of As from consuming this rice. Geostatistical analysis indicated that high risk tubewells (>50 µg As/L) are concentrated along the Mekong River's east bank. Participants using high risk tubewells are consuming up to 24 times more inorganic As daily than recommended by the previous FAO/WHO provisional tolerable daily intake value (2.1 µg/kgBW/day). However, As content in rice cooked in rainwater was significantly reduced, therefore, it is considered to be a safer and more sustainable option for this region.

Field observation of sequestration of uranium in iron-rich sediments under sequential reduction-oxidation conditions at the DOE Oak Ridge IFRC

At the U.S. DOE Oak Ridge Integrated Field Research Challenge (ORIFRC) site, the iron content of shallow subsurface materials (i.e. weathered saprolite) is relatively high (up to 5-6% as w/w), and therefore, the forms of the iron species present plays a critical role in the long-term sequestration of uranium. A long term pilot-scale study of the bioreduction and reoxidation of uranium conducted at the ORIFRC area 3 site, adjacent to the former S-3 disposal ponds (source zone), has provided us with the opportunity to study the impact of iron species on the sequestration of U(VI). The aqueous U(VI) concentrations at the site were decreased to below the EPA MCL through the intermittent injection of ethanol as the electron donor. Previous field tests indicated that both oxygen and nitrate could oxidize the bioreduced U(IV) and cause a short-term rebound of aqueous phase uranium concentration after the oxidative agents were delivered directly to the bioreduced zone.

A field test has been conducted to examine the long-term effect of exposure of bioreduced sediments to nitrate in contaminated groundwater for more than 1,380 days at the Area 3 site. Contaminated groundwater was allowed to invade the previously bioreduced zone via the natural groundwater gradient after an extended period in which reducing conditions were maintained and the bioreduced zone was protected from the influx of upgradient contaminated groundwater. The geochemical response to the invasion of contaminated groundwater was dependent on whether the monitoring location is in the middle or the fringe of the previously bioreduced zone. In general, the nitrate concentrations in the previously bioreduced area, increased gradually from near zero to ~50-300 mM within 200 days and then stabilized. The pH declined from bioreduced levels of 6.2-6.7 to below 5.0. Uranium concentrations rebounded in all monitoring wells but at different rates. At most locations U concentrations rebounded, declined and then rebounded again. Methane gas disappeared while a significant level (20,000 to 44,000 ppmv) N2O was found in the groundwater of monitoring wells after three years of reoxidization.

The U(IV) in sediments was mainly reoxidized to U(VI) species. Based on XANES analysis, the predominate uranium in all samples after re-oxidation was similar to a uranyl nitrate form. But the U content in the sediment remained as high as that determined after bioreduction activates were completed, indicating that much of the U is still sequestrated in situ. SEM observations of surged fine sediments revealed that clusters of colloidal-sized (200-500nm) U-containing precipitates appeared to have formed in situ, regardless from sample of FW106 in non-bioactivity control area or of pre-bioreduced FW101-2 and FW102-3. Additionally, SEM-EDS and microprobe analysis, showed that the U-containing precipitates (~1% U) in FW106 are notably higher in Fe, compared to the precipitates (~1-2.5% U) from FW101-2 and FW102-3. However, XRF analysis indicated that the U content was remained as high as 2180 and 1810 mg/kg with U/Fe ratio at 0.077 and 0.055 vs 0.037 g/g, respectively in pre-bioreduced FW101-2 and FW102-3, suggesting more U sequestrated by Fe in pre-bioreduced sediments.

Evaluation of in Situ DGT Measurements for Predicting the Concentration of Cd in Chinese Field-Cultivated Rice: Impact of Soil Cd:Zn Ratios

DGT (diffusive gradients in thin-films) has been proposed as a tool for predicting Cd concentrations in rice grain, but there is a lack of authenticating data. To further explore the relationship between DGT measured Cd and concentrations in rice cultivated in challenging, metal degraded, field locations with different heavy metal pollutant sources, 77 paired soil and grain samples were collected in Southern China from industrial zones, a "cancer village" impacted by mining waste and an organic farm. In situ deployments of DGT in flooded paddy rice rhizospheres were compared with a laboratory DGT assay on dried and rewetted soil. Total soil concentrations were a very poor predictor of plant uptake. Laboratory and field deployed DGT assays and porewater measurements were linearly related to grain concentrations in all but the most contaminated samples where plant toxicity occurred. The laboratory DGT assay was the best predictor of grain Cd concentrations, accommodating differences in soil Cd, pollutant source, and Cd:Zn ratios. Field DGT measurements showed that Zn availability in the flooded rice rhizospheres was greatly diminished compared to that of Cd, resulting in very high Cd:Zn ratios (0.1) compared to commonly observed values (0.005). These results demonstrate the potential of the DGT technique to predict Cd concentrations in field cultivated rice and demonstrate its robustness in a range of environments. Although, field deployments provided important details about in situ element stoichiometry, due to the inherent heterogeneity of the rice rhizosphere soils, deployment of DGT in dried and homogenized soils offers the best possibility of a soil screening tool.

Arsenic uptake and speciation in the rootless duckweed Wolffia globosa

Duckweeds are a common macrophyte in paddy and aquatic environments. Here, we investigated arsenic (As) accumulation, speciation and tolerance of the rootless duckweed Wolffia globosa and its potential for As phytofiltration.

When grown with 1 mu M arsenate, W. globosa accumulated two to 10 times more As than four other duckweed or Azolla species tested. W. globosa was able to accumulate > 1000 mg As kg(-1) in frond dry weight (DW), and tolerate up to 400 mg As kg-1 DW. At the low concentration range, uptake rate was similar for arsenate and arsenite, but at the high concentration range, arsenite was taken up at a faster rate.

Arsenite was the predominant As species (c. 90% of the total extractable As) in both arsenate-and arsenite-exposed duckweed. W. globosa was more resistant to external arsenate than arsenite, but showed a similar degree of tolerance internally. W. globosa decreased arsenate in solution rapidly, but also effluxed arsenite.

Wolffia globosa is a strong As accumulator and an interesting model plant to study As uptake and metabolism because of the lack of a root-to-frond translocation

Inorganic arsenic in Chinese food and its cancer risk

Even moderate arsenic exposure may lead to health problems, and thus quantifying inorganic arsenic (iAs) exposure from food for different population groups in China is essential. By analyzing the data from the China National Nutrition and Health Survey (CNNHS) and collecting reported values of iAs in major food groups, we developed a framework of calculating average iAs daily intake for different regions of China. Based on this framework, cancer risks from As in food was deterministically and probabilistically quantified. The article presents estimates for health risk due to the ingestion of food products contaminated with arsenic. Both per individual and for total population estimates were obtained. For the total population, daily iAs intake is around 42 mu g day(-1), and rice is the largest contributor of total iAs intake accounting for about 60%. Incremental lifetime cancer risk from food iAs intake is 106 per 100,000 for adult individuals and the median population cancer risk is 177 per 100,000 varying between regions. Population in the Southern region has a higher cancer risk than that in the Northern region and the total population. Sensitive analysis indicated that cancer slope factor, ingestion rates of rice, aquatic products and iAs concentration in rice were the most relevant variables in the model, as indicated by their higher contribution to variance of the incremental lifetime cancer risk. We conclude that rice may be the largest contributor of iAs through food route for the Chinese people. The population from the South has greater cancer risk than that from the North and the whole population. (C) 2011 Elsevier Ltd. All rights reserved.

Development and Validation of a PulseNet Standardized Protocol for Subtyping Isolates of Cronobacter Species

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

Paralytic shellfish poisoning (PSP) is a potentially fatal human health condition caused by the consumption of shellfish containing high levels of PSP toxins. Toxin extraction from shellfish and from algal cultures for use as standards and analysis by alternative analytical monitoring methods to the mouse bioassay is extensive and laborious. This study investigated whether a selected MAb antibody could be coupled to a novel form of magnetic microsphere (hollow glass magnetic microspheres, brand name Ferrospheres-N) and whether these coated microspheres could be utilized in the extraction of low concentrations of the PSP toxin, STX, from potential extraction buffers and spiked mussel extracts. The feasibility of utilizing a mass of 25 mg of Ferrospheres-N, as a simple extraction procedure for STX from spiked sodium acetate buffer, spiked PBS buffer and spiked mussel extracts was determined. The effects of a range of toxin concentrations (20-300 ng/mL), incubation times and temperature on the capability of the immuno-capture of the STX from the spiked mussel extracts were investigated. Finally, the coated microspheres were tested to determine their efficiency at extracting PSP toxins from naturally contaminated mussel samples. Toxin recovery after each experiment was determined by HPLC analysis. This study on using a highly novel immunoaffinity based extraction procedure, using STX as a model, has indicated that it could be a convenient alternative to conventional extraction procedures used in toxin purification prior to sample analysis.

Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation

Background: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes.

Results: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element.

Conclusions: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity

AimsThe main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus.

Methods and ResultsThe effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 075% O-2. 3 log(10) (999%) reductions in MS2 viability were achieved after 3min of exposure to the plasma source operated in a helium/oxygen (9925%:075%) gas mixture, with >7 log(10) reduction after 9min exposure.

ConclusionsAtmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications.

Significance and Impact of StudyThe atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.

Dietary exposure to aflatoxin and fumonisin among Tanzanian children as determined using biomarkers of exposure

SCOPE: The study aims to evaluate the status of dietary exposure to aflatoxin and fumonisin in young Tanzanian children, using previously validated biomarkers of exposure. METHODS AND RESULTS: A total of 148 children aged 12-22 months, were recruited from three geographically distant villages in Tanzania; Nyabula, Kigwa, and Kikelelwa. Plasma aflatoxin-albumin adducts (AF-alb) and urinary fumonisin B1 (UFB1) were measured by ELISA and LC-MS, respectively. AF-alb was detectable in 84% of children, was highest in fully weaned children (p <0.01) with higher levels being associated with higher maize intake (p <0.05). AF-alb geometric mean (95% CI) was 43.2 (28.7-65.0), 19.9 (13.5-29.2), and 3.6 (2.8-4.7) pg/mg albumin in children from Kigwa, Nyabula, and Kikelelwa, respectively. UFB1 was detectable in 96% of children and the level was highest in children who had been fully weaned (p <0.01). The geometric UFB1 mean (95% CI) was 327.2 (217.1-493.0), 211.7 (161.1-278.1), and 82.8 (58.3-117.7) pg/mL in Kigwa, Nyabula, and Kikelelwa, respectively. About 82% of all the children were exposed to both mycotoxins. CONCLUSION: Young children in Tanzania are chronically exposed to both aflatoxin and fumonisin through contaminated diet, although the level of exposure varies markedly between the three villages studied.