Default predicted no-effect target concentrations for antibiotics in the absence of data for the protection against antibiotic resistance and environmental toxicity.

The pharmaceutical manufacturing industry, via the AMR Industry Alliance, has developed and implemented steps to help minimize the potential impact of pharmaceutical manufacturing on the spread of antimicrobial resistance (AMR). One of these steps was to publish predicted no-effect concentrations (PNECs) to serve as targets for antibiotic manufacturing wastewater effluent risk assessments aimed to help protect environmental receptors and to mitigate against the spread of antibiotic resistance. Concentrations below which adverse effects in the environment are not expected to occur (PNECs) were first published in 2018 and are updated annually. The current list now stands at 125 antibiotics; however, it is recognized that this list does not encompass all manufactured antibiotics. Therefore, a statistical evaluation of currently available data was conducted and a default PNEC of 0.05 µg/L for antibiotics in the absence of other data was derived. Integr Environ Assess Manag 2022;18:863-867. © 2022 Merck, Sanofi, Johnson & Johnson Services, Inc, F.Hoffmann-La Roche Ltd, Teva Pharmaceuticals, GlaxoSmithKline, Novartis Pharma AG, and Pfizer lnc. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Resorcinol Hydroxylase of Azoarcus anaerobius: Molybdenum Dependence, Activity, and Heterologous Expression.

The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg-1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg-1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.

Science-based Targets for Antibiotics in Receiving Waters from Pharmaceutical Manufacturing Operations.

In 2016, the United Nations declared the need for urgent action to combat the global threat of antimicrobial resistance (AMR). In support of this effort, the pharmaceutical industry has committed to measures aimed at improving the stewardship of antibiotics both within and outside the clinic. Notably, a group of companies collaborated to specifically address concerns related to antibiotic residues being discharged from manufacturing sites. In addition to developing a framework of minimum environmental expectations for antibiotic manufacturers, science-based receiving water targets were established for antibiotics discharged from manufacturing operations. This paper summarizes the holistic approach taken to derive these targets and includes previously unpublished, company-generated, environmental toxicity data.

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius.

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.

Anaerobic degradation of protocatechuate (3,4-dihydroxybenzoate) by Thauera aromatica strain AR-1.

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxybenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or benzoate readily degraded benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.