Detection of inorganic arsenic in rice using a field-deployable method with Cola extraction.

Rice is a staple food and known to accumulate inorganic arsenic (iAs), which is a class 1 carcinogen to humans. Arsenic field-deployable method kits, designed for water testing, are able to screen iAs in rice, to assure food safety and quick decision-making without the need for laboratory analysis. For the arsenic extraction within the field method, nitric acid is used. To make the field method on-site safer, cost-effective and easier to handle, the method was adapted using a Cola in the extraction process. The adapted field-deployable method was tested by screening a total of 30 rice and rice products from the Austrian market. To verify the results obtained by the Cola extraction field-deployable method, the obtained iAs concentration was compared to HPLC-ICP-MS results. The Cola extraction field method obtained an LOD of 39 µg iAs kg-1 rice, and with an average reproducibility of 14% RSD, the method was capable of recording no false-negative but 7% false-positive values at the 2023 updated European Commission (EC) limits for rice. All, but one, screened rice samples were within the EU limits for iAs in rice and rice products.

Arsenic, cadmium, and lead in rice and rice products on the Austrian market.

Fifty-one rice samples, i.e. 25 rice varieties, 8 rice products, and 18 rice containing baby foods from the Austrian market were surveyed for arsenic, cadmium, and lead. Inorganic arsenic (iAs) is most toxic to human health, and its mean concentrations in rice were 120 µg kg-1, 191 µg kg-1 in rice products, and 77 µg kg-1 in baby foods. The average dimethylarsinic acid and methylarsonic acid concentrations were 56 µg kg-1 and 2 µg kg-1, respectively. The highest iAs concentration was found in rice flakes (237 ± 15 µg kg-1), close to the Maximum Level (ML) set by the EU regulation for husked rice (250 µg kg-1). The levels of cadmium (12 to 182 µg kg-1) and lead (6 to 30 µg kg-1) in the majority of rice samples were below the European ML. Upland grown rice from Austria showed both, low inorganic arsenic (<19 µg kg-1) and cadmium (<38 µg kg-1) concentrations.

Multi trace element profiling in pathogenic and non-pathogenic fungi.

Maintaining appropriate levels of trace elements during infection of a host is essential for microbial pathogenicity. Here we compared the uptake of 10 trace elements from 3 commonly-used laboratory media by 3 pathogens, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and a model yeast, Saccharomyces cerevisiae. The trace element composition of the yeasts, C. albicans, C. neoformans and S. cerevisiae, grown in rich (YPD) medium, differed primarily in P, S, Fe, Zn and Co. Speciation analysis of the intracellular fraction, which indicates the size of the organic ligands with which trace elements are complexed, showed that the ligands for S were similar in the three fungi but there were significant differences in binding partners for Fe and Zn between C. neoformans and S.cerevisiae. The profile for Cu varied across the 3 yeast species. In a comparison of C. albicans and A. fumigatus hyphae, the former showed higher Fe, Cu, Zn and Mn, while A. fumigatus contained higher P, S Ca and Mo. Washing C. albicans cells with the cell-impermeable chelator, EGTA, depleted 50-90 % of cellular Ca, suggesting that a large proportion of this cation is stored in the cell wall. Treatment with the cell wall stressor, Calcofluor White (CFW), alone had little effect on the elemental profile whilst combined Ca + CFW stress resulted in high cellular Cu and very high Ca. Together our data enhance our understanding of trace element uptake by pathogenic fungi and provide evidence for the cell wall as an important storage organelle for Ca.

Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae.

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano-fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour-following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ∼ 8.7 µN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.

Enteric YaiW is a surface-exposed outer membrane lipoprotein that affects sensitivity to an antimicrobial peptide.

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.

Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen.

The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose-grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose- and lactate-grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections.

Biochemical characterization of Sinorhizobium meliloti mutants reveals gene products involved in the biosynthesis of the unusual lipid A very long-chain fatty acid.

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.

Internalization of a thiazole-modified peptide in Sinorhizobium meliloti occurs by BacA-dependent and -independent mechanisms.

BacA proteins play key roles in the chronic intracellular infections of Sinorhizobium meliloti, Brucella abortus and Mycobacterium tuberculosis within their respective hosts. S. meliloti, B. abortus and M. tuberculosis BacA-deficient mutants have increased resistance to the thiazole-modified peptide bleomycin. BacA has been previously hypothesized, but not experimentally verified, to be involved in bleomycin uptake. In this paper, we show that a BacA-dependent mechanism is the major route of bleomycin internalization in S. meliloti. We also determined that the B. abortus and S. meliloti BacA proteins are functional homologues and that the B. abortus BacA protein is involved in the uptake of both bleomycin and proline-rich peptides. Our findings also provide evidence that there is a second, BacA-independent minor mechanism for bleomycin internalization in S. meliloti. We determined that the BacA-dependent and -independent mechanisms of bleomycin uptake are energy-dependent, consistent with both mechanisms of bleomycin uptake involving transport systems.

The Sinorhizobium meliloti LpxXL and AcpXL proteins play important roles in bacteroid development within alfalfa.

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.

Investigation into antimony mobility in sewage sludge fermentation.

Antimony is distributed in the environment in inorganic and organic species with different solubility and mobility characters. Here we investigate the transformation of antimony in view of biomethylation during sewage sludge fermentation as a case study for an anaerobic environment. Our approach was to identify if antimony methylation follows the Challenger pathway by using isotopically enriched antimonite (123Sb(v)). The antimony source was subjected to methylation in sewage sludge, an anaerobic dominant methanogenic Archaea community. The antimony species were determined in the gas phase using cryotrapping (CT)-GC-ICP-MS, and in the medium (sewage slude) by hydride generation (HG) prior CT-GC-ICP-MS. The determined 123/121Sb isotope ratios in the volatile trimethylstibine and non-volatile methylantimony species indicated that the methylation follows the proposed methylation pathway. With this approach we were able to quantify 123Sb incorporation into monomethyl-, dimethyl- and trimethylantimony, respectively. The incorporation decreased with further methylation from 91% to 82% and 73%. Volatilisation as trimethystibine was generally lower than 0.1%, however, up to 0.8% of added antimony was found methylated to methylantimony species and mainly accumulated in the cell. Moreover, antimony biomethylation was enhanced by stimulation of the anaerobic communities of methanogenic Archaea and sulfate reducing bacteria (SRB), with the methanogens showing a higher activity.