Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

After administration to livestock, a large fraction of antibiotics are excreted unchanged via excreta and can be transferred to agricultural land. For effective risk assessment a critical factor is to determine which antibiotics can be expected in the different environmental compartments. After excretion, the first relevant compartment is manure storage. In the current study, the fate of a broad scope of antibiotics (n = 46) during manure storage of different livestock animals (calves, pigs, broilers) was investigated. Manure samples were fortified with antibiotics and incubated during 24 days. Analysis was carried out by LC-MS. The dissipation of the antibiotics was modelled based on the recommendations of FOCUS working group. Sulphonamides relatively quickly dissipate in all manure types, with a DT90 of in general between 0.2 and 30 days. Tetracyclines (DT90 up to 422 days), quinolones (DT90 100-5800 days), macrolides (DT90 18-1000 days), lincosamides (DT90 135-1400 days) and pleuromutilins (DT90 of 49-1100 days) are in general much more persistent, but rates depend on the manure type. Specifically lincomycin, pirlimycin, tiamulin and most quinolones are very persistent in manure with more than 10% of the native compound remaining after a year in most manure types. For all compounds tested in the sub-set, except the macrolides, the dissipation was an abiotic process. Based on the persistence and current frequency of use, oxytetracycline, doxycycline, flumequine and tilmicosin can be expected to end up in environmental compartments. Ecotoxicological data should be used to further prioritize these compounds.

Prenylated flavonoids and isoflavonoids are phytochemicals with remarkable antibacterial activity. In this study, 30 prenylated (iso)flavonoids were tested against Listeria monocytogenes and Escherichia coli (the latter in combination with an efflux pump inhibitor). Minimum inhibitory concentrations of the most active compounds ranged between 6.3–15.0 µg/mL. Quantitative structure-activity relationships (QSAR) analysis was performed and linear regression models were proposed with R2 between 0.77–0.80, average R2m between 0.70–0.75, Q2LOO between 0.66–0.69, and relatively low amount of descriptors. Shape descriptors (related to flexibility and globularity), together with hydrophilic/hydrophobic volume and surface area descriptors, were identified as important molecular characteristics related to activity. A 3D pharmacophore model explaining the effect of the prenyl position on the activity of compounds was developed for each bacterium. These models predicted active compounds with an accuracy of 71–88%. With regard to the mode of action, good antibacterial prenylated (iso)flavonoids with low relative hydrophobic surface area caused remarkable membrane permeabilization, whereas those with higher relative hydrophobic surface area did not. Based on the QSAR and membrane permeabilization studies, the mode of action of antibacterial prenylated (iso)flavonoids was putatively rationalized.

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Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERβ were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.

In vitro liver metabolism of 11 prenylated flavonoids and isoflavonoids was investigated by determining their phase I glucuronyl and sulfate metabolites using pork liver preparations. One hundred metabolites were annotated using RP-UHPLC-ESI-MS(n). A mass spectrometry-based data interpretation guideline was proposed for the tentative annotation of the position of hydroxyl groups, considering its relevance for estrogenic activity. To relate structure to metabolism, compounds were classified on the basis of three criteria: backbone structure (isoflavene, isoflavan, or flavanone), number of prenyl groups (0, 1, or 2), and prenyl configuration (chain or pyran). Glucuronidation was most extensive for isoflavenes and for unprenylated compounds (yield of 90-100%). Pyran and chain prenylation gave more complex hydroxylation patterns with 4 or more than 6 hydroxyl isomers, respectively, as compared to unprenylated compounds (only 1 hydroxyl isomer). Moreover, the number of hydroxyl isomers also increased with the number of prenyl groups.

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