Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Optimising antimicrobial use in humans - review of current evidence and an interdisciplinary consensus on key priorities for research.

Addressing the silent pandemic of antimicrobial resistance (AMR) is a focus of the 2021 G7 meeting. A major driver of AMR and poor clinical outcomes is suboptimal antimicrobial use. Current research in AMR is inequitably focused on new drug development. To achieve antimicrobial security we need to balance AMR research efforts between development of new agents and strategies to preserve the efficacy and maximise effectiveness of existing agents. Combining a review of current evidence and multistage engagement with diverse international stakeholders (including those in healthcare, public health, research, patient advocacy and policy) we identified research priorities for optimising antimicrobial use in humans across four broad themes: policy and strategic planning; medicines management and prescribing systems; technology to optimise prescribing; and context, culture and behaviours. Sustainable progress depends on: developing economic and contextually appropriate interventions; facilitating better use of data and prescribing systems across healthcare settings; supporting appropriate and scalable technological innovation. Implementing this strategy for AMR research on the optimisation of antimicrobial use in humans could contribute to equitable global health security.

In vitro characterization of cationic copolymer-complexed recombinant human butyrylcholinesterase.

Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly-L-lysine (PLL; high MW, 30-70 kDa; medium MW, 15-30 kDa; low MW, 4-15 kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 °C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro. While these results are promising for further development, additional studies are needed to evaluate in vivo performance.

Functional xenobiotic metabolism and efflux transporters in trout hepatocyte spheroid cultures.

Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.

Enantioselective biotransformation of hexabromocyclododecane by in vitro rat and trout hepatic sub-cellular fractions.

α-, ß-, and γ-Hexabromocyclododecanes (HBCDs) were subjected to in vitro biotransformation experiments with rat and trout liver S9 fractions for different incubation times (10, 30, and 60 min) at 2 concentration levels (1 and 10 µM). The metabolic degradation of target HBCDs followed first order kinetics. Whereas ß-HBCD undergoes rapid biotransformation (t0.5 = 6.4 and 38.1 min in rat and trout, respectively), α-HBCD appears the most resistant to metabolic degradation (t0.5 = 17.1 and 134.9 min). The biotransformation rate in trout was slower than in rat. Investigation of HBCD degradation profiles revealed the presence of at least 3 pentabromocyclododecene (PBCD) and 2 tetrabromocyclododecadiene (TBCD) isomers indicating reductive debromination as a metabolic pathway for HBCDs. Both mono- and di- hydroxyl metabolites were identified for parent HBCDs, while only mono hydroxyl metabolites were detected for PBCDs and TBCDs. Interestingly, δ-HBCD was detected only in trout S9 fraction assays indicating metabolic interconversion of test HBCD diastereomers during biotransformation in trout. Finally, enantioselective analysis showed significant enrichment of the (-)-α-HBCD enantiomer (EF = 0.321 and 0.419 after 60 min incubation in rat and trout, respectively). The greater enrichment of (-)-α-HBCD in rat than in trout underlines the species-specific differences in HBCD metabolism and the need for caution when extending similar results from animal studies to humans.

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies.

1. In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000 g supernatants (S9) in environmental risk assessment. 2. The rates of ethoxyresorufin-O-deethylation (relative to S9 protein) measured under different conditions of culture of rainbow trout hepatocytes were significantly higher than those detected in S9, in the order of freshly isolated hepatocytes > 10-day spheroid cultures > primary hepatocytes in culture > S9. The percentage of conjugated metabolites was also similar between freshly isolated and spheroid cultured hepatocytes (9.9 and 13.5%). 3. The rate of oxidation was enhanced (1.7 fold) when S9 was supplemented with cofactors for phase II conjugation but this was only approximately one tenth of the rate in freshly isolated hepatocytes (7.1 pmol/min/mg S9 protein equivalent). 4. Hepatocytes also hydroxylated ibuprofen, producing two metabolites, in contrast to only one (identified as the 1-hydroxy derivative) using hepatic S9 fractions. 5. Since the bioaccumulation potential of chemicals is often based on un-supplemented S9 in incubations ≥ 1 h when activity declines, it is recommended that predictability would be greatly improved through the use of hepatocyte spheroids, due to their maintenance of activity and longevity.