Developing Surrogate Markers for Predicting Antibiotic Resistance "Hot Spots" in Rivers Where Limited Data Are Available.

Pinpointing environmental antibiotic resistance (AR) hot spots in low-and middle-income countries (LMICs) is hindered by a lack of available and comparable AR monitoring data relevant to such settings. Addressing this problem, we performed a comprehensive spatial and seasonal assessment of water quality and AR conditions in a Malaysian river catchment to identify potential "simple" surrogates that mirror elevated AR. We screened for resistant coliforms, 22 antibiotics, 287 AR genes and integrons, and routine water quality parameters, covering absolute concentrations and mass loadings. To understand relationships, we introduced standardized "effect sizes" (Cohen's D) for AR monitoring to improve comparability of field studies. Overall, water quality generally declined and environmental AR levels increased as one moved down the catchment without major seasonal variations, except total antibiotic concentrations that were higher in the dry season (Cohen's D > 0.8, P < 0.05). Among simple surrogates, dissolved oxygen (DO) most strongly correlated (inversely) with total AR gene concentrations (Spearman's ρ 0.81, P < 0.05). We suspect this results from minimally treated sewage inputs, which also contain AR bacteria and genes, depleting DO in the most impacted reaches. Thus, although DO is not a measure of AR, lower DO levels reflect wastewater inputs, flagging possible AR hot spots. DO measurement is inexpensive, already monitored in many catchments, and exists in many numerical water quality models (e.g., oxygen sag curves). Therefore, we propose combining DO data and prospective modeling to guide local interventions, especially in LMIC rivers with limited data.

Antimicrobial resistance in rural rivers: Comparative study of the Coquet (Northumberland) and Eden (Cumbria) River catchments.

Many studies have characterised resistomes in river microbial communities. However, few have compared resistomes in parallel rural catchments that have few point-source inputs of antimicrobial genes (ARGs) and organisms (i.e., AMR) - catchments where one can contrast more nebulous drivers of AMR in rural rivers. Here, we used quantitative microbial profiling (QMP) to compare resistomes and microbiomes in two rural river catchments in Northern England, the Coquet and Eden in Northumberland and Cumbria, respectively, with different hydrological and geographical conditions. The Eden has higher flow rates, higher annual surface runoff, and longer periods of soil saturation, whereas the Coquet is drier and has lower flowrates. QMP analysis showed the Eden contained significantly more abundant microbes associated with soil sources, animal faeces, and wastewater than the Coquet, which had microbiomes like less polluted rivers (Wilcoxon test, p < 0.01). The Eden also had greater ARG abundances and resistome diversity (Kruskal Wallis, p < 0.05), and higher levels of potentially clinically relevant ARGs. The Eden catchment had greater and flashier runoff and more extensive agricultural land use in its middle reach, which explains higher levels of AMR in the river. Hydrological and geographic factors drive AMR in rural rivers, which must be considered in environmental monitoring programmes.

Improved quantitative microbiome profiling for environmental antibiotic resistance surveillance.

BACKGROUND: Understanding environmental microbiomes and antibiotic resistance (AR) is hindered by over reliance on relative abundance data from next-generation sequencing. Relative data limits our ability to quantify changes in microbiomes and resistomes over space and time because sequencing depth is not considered and makes data less suitable for Quantitative Microbial Risk Assessments (QMRA), critical in quantifying environmental AR exposure and transmission risks. RESULTS: Here we combine quantitative microbiome profiling (QMP; parallelization of amplicon sequencing and 16S rRNA qPCR to estimate cell counts) and absolute resistome profiling (based on high-throughput qPCR) to quantify AR along an anthropogenically impacted river. We show QMP overcomes biases caused by relative taxa abundance data and show the benefits of using unified Hill number diversities to describe environmental microbial communities. Our approach overcomes weaknesses in previous methods and shows Hill numbers are better for QMP in diversity characterisation. CONCLUSIONS: Methods here can be adapted for any microbiome and resistome research question, but especially providing more quantitative data for QMRA and other environmental applications.

An orally available non-nucleotide STING agonist with antitumor activity.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-ß secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Occupational health and safety in cannabis production: an Australian perspective.

The legal Australian cannabis industry has been rapidly expanding due to increased awareness of the plant's therapeutic potential, as well its diverse range of applications including biofuel, textiles, building materials, food, nutritional supplement, and animal feed. The objective of this paper is to describe the current landscape of the commercial Australian cannabis industry, summarise occupational health and safety (OHS) hazards in cannabis-related working environments, and provide suggestions for safeguarding worker health and well-being in this emerging industry. A comprehensive search of peer-reviewed and grey literature published between 1900 and 2017 was undertaken to identify case studies and original epidemiological research on OHS hazards associated with the cannabis cultivation and the manufacture of cannabis-based products. The review found that the majority of OHS studies were undertaken in the hemp textile industry during the late twentieth century, with a small number of articles published from a variety of occupational environments including forensic laboratories and recreational marijuana farms. Cannabis harvesting and initial processing is labour intensive, and presents a physical hazard Depending on the operation, workers may also be exposed to a variety of biological, chemical, and physical hazards including: organic dusts, bioaerosols, pollen/allergens, volatile organic compounds, psychoactive substances (tetrahydrocannabinol [THC])), noise, and ultraviolet radiation. Little research has been undertaken on the exposure to inhalable organic dust and other bioaerosols during the commercial cultivation and manufacture of cannabis-based products. Furthermore, there is an absence of Australian-based research and OHS guidance materials to help professionals develop risk management strategies in this evolving industry. It is recommended that: Investigation into the toxicological properties of cannabis dusts, specifically in relation to potential occupational exposures during cultivation and manufacture, should be a priority. The interim adoption of the respirable cotton dust exposure standard of 0.2 mg/m3 for workplace exposure in hemp facilities until a cannabis workplace exposure standard is developed, and that exposure to medicinal cannabis containing THC are kept as low as reasonably practicable. An industry partnership be established for the development of an Australian health and safety guideline for the production of medicinal cannabis and hemp. A classification to meet the requirements of the Global Harmonization Scheme should be undertaken to ensure consistency in the use of safety and risk phrases in cannabis-related industries.

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5.

The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7, resulting in either loss-of-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5 as confirmed in electrophysiology.