Performance Characteristics of qPCR Assays Targeting Human- and Ruminant-Associated Bacteroidetes for Microbial Source Tracking across Sixteen Countries on Six Continents

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods

End-on And Side-on Peroxo Derivatives Of Non-heme Iron Complexes With Pentadentate Ligands: Models For Putative Intermediates In Biological Iron/dioxygen Chemistry

Mononuclear iron(III) species with end-on and side-on peroxide have been proposed or identified in the catalytic cycles of the antitumor drug bleomycin and a variety of enzymes, such as cytochrome P450 and Rieske dioxygenases. Only recently have biomimetic analogues of such reactive species been generated and characterized at low temperatures. We report the synthesis and characterization of a series of iron(II) complexes with pentadentate N5 ligands that react with H2O2 to generate transient low-spin Fe III -OOH intermediates. These intermediates have low-spin iron(III) centers exhibiting hydroperoxo-to-iron(III) charge-transfer bands in the 500-600-nm region. Their resonance Raman frequencies, vO-O, near 800 cm -1 are significantly lower than those observed for high-spin counterparts. The hydroperoxo-to-iron(III) charge-transfer transition blue-shifts and the vO-O of the Fe-OOH unit decreases as the N5 ligand becomes more electron donating. Thus, increasing electron density at the low-spin Fe(III) center weakens the O-O bond, in accord with conclusions drawn from published DFT calculations. The parent [(N4Py)Fe III (eta 1 -OOH)]2+ (1a) ion in this series (N4Py ) N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) can be converted to its conjugate base, which is demonstrated to be a high-spin iron(III) complex with a side-on peroxo ligand, [(N4Py)Fe III (eta 2 -O2)]+ (1b). A detailed analysis of 1a and 1b by EPR and Mo¨ ssbauer spectroscopy provides insights into their electronic properties. The orientation of the observed 57 Fe A-tensor of 1a can be explained with the frequently employed Griffith model provided the rhombic component of the ligand field, determined by the disposition of the hydroperoxo ligand, is 45° rotated relative to the octahedral field. EXAFS studies of 1a and 1b reveal the first metrical details of the iron-peroxo units in this family of complexes: [(N4Py)Fe III (eta 1 -OOH)]2+ has an Fe-O bond of 1.76 Å, while [(N4Py)Fe III (eta 2 -O2)]+ has two Fe-O bonds of 1.93 Å, values which are in very good agreement with results obtained from DFT calculations.