Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

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.

Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types.

The emergence and spread of antibiotic resistance genes (ARGs) in soil due to animal excreta and organic waste is a major threat to human health and ecosystems, and global efforts are required to tackle the issue. However, there is limited knowledge of the variation in ARG prevalence and diversity resulting from different land-use patterns and underlying driving factors in soils. This study aimed to comprehensively characterize the profile of ARGs and mobile genetic elements and their drivers in soil samples collected from 11 provinces across China, representing three different land-use types, using high-throughput quantitative polymerase chain reaction and 16S rRNA amplicon sequencing. Our results showed that agricultural soil had the highest abundance and diversity of ARGs, followed by tea plantation and forest land. A total of 124 unique ARGs were detected in all samples, with shared subtypes among different land-use patterns indicating a common origin or high transmission frequency. Moreover, significant differences in ARG distribution were observed among different geographical regions, with the greatest enrichment of ARGs found in southern China. Biotic and abiotic factors, including soil properties, climatic factors, and bacterial diversity, were identified as the primary drivers associated with ARG abundance, explaining 71.8% of total ARG variation. The findings of our study demonstrate that different land-use patterns are associated with variations in ARG abundance in soil, with agricultural practices posing the greatest risk to human health and ecosystems regarding ARGs. Our identification of biotic and abiotic drivers of ARG abundance provides valuable insights into strategies for mitigating the spread of these genes. This study emphasizes the need for coordinated and integrated approaches to address the global antimicrobial resistance crisis.

Why don't the British eat locally harvested shellfish? The role of misconceptions and knowledge gaps.

Although the UK consumes a substantial amount of shellfish, most is imported (e.g. prawns), while locally harvested molluscs and crustaceans (e.g. mussels, crab) tend to be exported. This study aimed to investigate whether a low rate of local shellfish consumption in the UK is due to misunderstandings or knowledge gaps about the potential health and environmental risks and benefits of consumption. Following the Mental Models Approach, the present paper reveals: 1) qualitative results from 26 stakeholder/public interviews which identified 10 key misunderstandings and knowledge gaps, including incorrect beliefs about health risks and a lack of knowledge about the relative environmental benefits compared to other foods (key misunderstandings included some parts of a crab are poisonous if eaten, and the majority of UK shellfish is farmed), and 2) quantitative results from a survey (n = 1,433) that explored the degree to which these misunderstandings and knowledge gaps may influence consumption intentions in the wider UK population. Survey results suggested the number of misunderstandings and knowledge gaps significantly predicted shellfish consumption intentions even after controlling for demographics, food related values, and past consumption behaviour. Path analyses revealed their impact on intentions was partially mediated via Theory of Planned Behaviour variables. Results could inform information campaigns supporting consumers to make more informed decisions regarding a group of foods that are potentially both healthy and relatively environmentally friendly.

Wild small mammals as sentinels for the environmental transmission of antimicrobial resistance.

Antimicrobial resistance (AMR) represents a serious threat to human health worldwide. We have tested the use of free-living small mammals (mice, voles and shrews) as sentinels of variation in the distribution of AMR in the environment and the potential for transmission from the natural environment to animal hosts. Escherichia coli isolated from the faeces of small mammals trapped at paired coastal and inland sites were tested for resistance to four antibiotics: trimethoprim, ampicillin, ciprofloxacin and cefotaxime. Coastal individuals were over twice as likely to carry AMR E. coli than inland individuals (79% and 35% respectively), and both between-site and between-species variation was observed. Animals from coastal populations also excreted increased numbers of AMR E. coli and a greater diversity of E. coli phylotypes, including human-associated pathogenic strains. Small mammals appear to be useful bioindicators of fine-scale spatial variation in the distribution of AMR and, potentially, of the risks of AMR transmission to mammalian hosts, including humans.

Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-ß-Lactamase Identified from a Functional Metagenomic Study.

ß-Lactamase production increasingly threatens the effectiveness of ß-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known ß-lactamases and mobilization from environmental reservoirs. The spread of metallo-ß-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other ß-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant ß-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αß/ßα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for ß-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of ß-lactam resistance.

The scourge of antibiotic resistance: the important role of the environment.

Antibiotic resistance and associated genes are ubiquitous and ancient, with most genes that encode resistance in human pathogens having originated in bacteria from the natural environment (eg, ß-lactamases and fluoroquinolones resistance genes, such as qnr). The rapid evolution and spread of "new" antibiotic resistance genes has been enhanced by modern human activity and its influence on the environmental resistome. This highlights the importance of including the role of the environmental vectors, such as bacterial genetic diversity within soil and water, in resistance risk management. We need to take more steps to decrease the spread of resistance genes in environmental bacteria into human pathogens, to decrease the spread of resistant bacteria to people and animals via foodstuffs, wastes and water, and to minimize the levels of antibiotics and antibiotic-resistant bacteria introduced into the environment. Reducing this risk must include improved management of waste containing antibiotic residues and antibiotic-resistant microorganisms.

Influence of humans on evolution and mobilization of environmental antibiotic resistome.

The clinical failure of antimicrobial drugs that were previously effective in controlling infectious disease is a tragedy of increasing magnitude that gravely affects human health. This resistance by pathogens is often the endpoint of an evolutionary process that began billions of years ago in non-disease-causing microorganisms. This environmental resistome, its mobilization, and the conditions that facilitate its entry into human pathogens are at the heart of the current public health crisis in antibiotic resistance. Understanding the origins, evolution, and mechanisms of transfer of resistance elements is vital to our ability to adequately address this public health issue.

Functional metagenomic libraries generated from anthropogenically impacted environments reveal importance of metabolic genes in biocide and antibiotic resistance.

Anthropogenic activities result in the release of antimicrobial resistant bacteria and a cocktail of antimicrobial compounds into the environment that may directly select or indirectly co-select for antimicrobial resistance (AMR). Many studies use metagenome sequencing or qPCR-based approaches to study the environmental resistome but these methods are limited by a priori knowledge. In this study, a functional metagenomic approach was used to explore biocide resistance mechanisms in two contaminated environments and a pristine site, and to identify whether potentially novel genes conferring biocide resistance also conferred resistance or reduced susceptibility to antibiotics. Resistance was predominately mediated through novel mechanisms exclusive of the well-known qac efflux genes. UDP-galactose 4-epimerase (galE) -like genes were identified in both contaminated environments and were shown to confer cross-resistance to biocides and clinically important antibiotics for the first time (to our knowledge), compared to knockout mutants. GalE -like genes were also co-located with transposons, suggesting mobilisation potential. These results show that housekeeping genes may play a significant yet underappreciated role in AMR in environmental microbiomes.

Interspecific competition can drive plasmid loss from a focal species in a microbial community.

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.

Assessing the impact of recreational water use on carriage of antimicrobial resistant organisms.

Understanding the role of exposure to natural recreational waters in the acquisition and transmission of antimicrobial resistance (AMR) is an area of increasing interest. A point prevalence study was carried out in the island of Ireland to determine the prevalence of colonisation with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) in recreational water users (WU) and matched controls. A total of 411 adult participants (199 WU, 212 controls) submitted at least one faecal sample between September 2020 - October 2021. In total, 80 Enterobacterales were isolated from 73 participants. ESBL-PE were detected in 29 (7.1 %) participants (7 WU, 22 controls), and CRE were detected in nine (2.2 %) participants (4 WU, 5 controls). No carbapenemase-producing Enterobacterales (CPE) were detected. WU were significantly less likely to harbour ESBL-PE than controls (risk ratio = 0.34, 95 % CI 0.148 to 0.776, χ2 7.37, p = 0.007). This study demonstrates the occurrence of ESBL-PE and CRE in healthy participants in Ireland. Recreational exposure to bathing water in Ireland was associated with a decreased prevalence of colonisation with ESBL-PE and CRE.

Natural recreational waters and the risk that exposure to antibiotic resistant bacteria poses to human health.

Antimicrobial resistance (AMR) is widely recognised as a considerable threat to human health, wellbeing and prosperity. Many clinically important antibiotic resistance genes are understood to have originated in the natural environment. However, the complex interactions between humans, animals and the environment makes the health implications of environmental AMR difficult to quantify. This narrative review focuses on the current state of knowledge regarding antibiotic resistant bacteria (ARB) in natural bathing waters and implications for human health. It considers the latest research focusing on the transmission of ARB from bathing waters to humans. The limitations of existing evidence are discussed, as well as research priorities. The authors are of the opinion that future studies should include faecally contaminated bathing waters and people exposed to these environments to accurately parameterise environment-to-human transmission.

Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics.

Wastewater treatment plants have been highlighted as a potential hotspot for the development and spread of antibiotic resistance. Although antibiotic resistant bacteria in wastewater present a public health threat, it is also possible that these bacteria play an important role in the bioremediation through the metabolism of antibiotics before they reach the wider environment. Here we address this possibility with a particular emphasis on stereochemistry using a combination of microbiology and analytical chemistry tools including the use of supercritical-fluid chromatography coupled with mass spectrometry for chiral analysis and high-resolution mass spectrometry to investigate metabolites. Due to the complexities around chiral analysis the antibiotic chloramphenicol was used as a proof of concept to demonstrate stereoselective metabolism due to its relatively simple chemical structure and availability over the counter in the U.K. The results presented here demonstrate the chloramphenicol can be stereoselectively transformed by the chloramphenicol acetyltransferase enzyme with the orientation around the first stereocentre being key for this process, meaning that accumulation of two isomers may occur within the environment with potential impacts on ecotoxicity and emergence of bacterial antibiotic resistance within the environment.

Non-antibiotic pharmaceuticals promote conjugative plasmid transfer at a community-wide level.

BACKGROUND: Horizontal gene transfer (HGT) plays a critical role in the spread of antibiotic resistance and the evolutionary shaping of bacterial communities. Conjugation is the most well characterized pathway for the spread of antibiotic resistance, compared to transformation and transduction. While antibiotics have been found to induce HGT, it remains unknown whether non-antibiotic pharmaceuticals can facilitate conjugation at a microbial community-wide level. RESULTS: In this study, we demonstrate that several commonly consumed non-antibiotic pharmaceuticals (including carbamazepine, ibuprofen, naproxen and propranolol), at environmentally relevant concentrations (0.5 mg/L), can promote the conjugative transfer of IncP1-α plasmid-borne antibiotic resistance across entire microbial communities. The over-generation of reactive oxygen species in response to these non-antibiotic pharmaceuticals may contribute to the enhanced conjugation ratios. Cell sorting and 16S rRNA gene amplicon sequencing analyses indicated that non-antibiotic pharmaceuticals modulate transconjugant microbial communities at both phylum and genus levels. Moreover, microbial uptake ability of the IncP1-α plasmid was also upregulated under non-antibiotic pharmaceutical exposure. Several opportunistic pathogens, such as Acinetobacter and Legionella, were more likely to acquire the plasmid conferring multidrug resistance. CONCLUSIONS: Considering the high possibility of co-occurrence of pathogenic bacteria, conjugative IncP1-α plasmids and non-antibiotic pharmaceuticals in various environments (e.g., activated sludge systems), our findings illustrate the potential risk associated with increased dissemination of antibiotic resistance promoted by non-antibiotic pharmaceuticals in complex environmental settings. Video abstract.

Antifungal Exposure and Resistance Development: Defining Minimal Selective Antifungal Concentrations and Testing Methodologies.

This scoping review aims to summarise the current understanding of selection for antifungal resistance (AFR) and to compare and contrast this with selection for antibacterial resistance, which has received more research attention. AFR is an emerging global threat to human health, associated with high mortality rates, absence of effective surveillance systems and with few alternative treatment options available. Clinical AFR is well documented, with additional settings increasingly being recognised to play a role in the evolution and spread of AFR. The environment, for example, harbours diverse fungal communities that are regularly exposed to antifungal micropollutants, potentially increasing AFR selection risk. The direct application of effect concentrations of azole fungicides to agricultural crops and the incomplete removal of pharmaceutical antifungals in wastewater treatment systems are of particular concern. Currently, environmental risk assessment (ERA) guidelines do not require assessment of antifungal agents in terms of their ability to drive AFR development, and there are no established experimental tools to determine antifungal selective concentrations. Without data to interpret the selective risk of antifungals, our ability to effectively inform safe environmental thresholds is severely limited. In this review, potential methods to generate antifungal selective concentration data are proposed, informed by approaches used to determine antibacterial minimal selective concentrations. Such data can be considered in the development of regulatory guidelines that aim to reduce selection for AFR.

The Complex Interplay Between Antibiotic Resistance and Pharmaceutical and Personal Care Products in the Environment.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are important environmental contaminants. Nonetheless, what drives the evolution, spread, and transmission of antibiotic resistance dissemination is still poorly understood. The abundance of ARB and ARGs is often elevated in human-impacted areas, especially in environments receiving fecal wastes, or in the presence of complex mixtures of chemical contaminants, such as pharmaceuticals and personal care products. Self-replication, mutation, horizontal gene transfer, and adaptation to different environmental conditions contribute to the persistence and proliferation of ARB in habitats under strong anthropogenic influence. Our review discusses the interplay between chemical contaminants and ARB and their respective genes, specifically in reference to co-occurrence, potential biostimulation, and selective pressure effects, and gives an overview of mitigation by existing man-made and natural barriers. Evidence and strategies to improve the assessment of human health risks due to environmental antibiotic resistance are also discussed. Environ Toxicol Chem 2023;00:1-16. © 2022 SETAC.

Dawning of a new ERA: Environmental Risk Assessment of antibiotics and their potential to select for antimicrobial resistance.

Antibiotics and antimicrobials are used, misused and overused in human and veterinary medicine, animal husbandry and aquaculture. These compounds can persist in both human and animal waste and then enter the environment through a variety of mechanisms. Though generally measured environmental concentrations (MECs) of antibiotics in aquatic systems are significantly lower than point of therapeutic use concentrations, there is increasing evidence that suggests these concentrations may still enrich antimicrobial resistant bacteria. In light of this evidence, a rigorous and standardised novel methodology needs to be developed which can perform environmental risk assessment (ERA) of antimicrobials in terms of their selective potential as well as their environmental impact, to ensure that diffuse and point source discharges are safe. This review summarises and critically appraises the current methodological approaches that study selection at below point of therapeutic use, or sub-inhibitory, concentrations of antibiotics. We collate and compare selective concentration data generated to date. We recommend how these data can be interpreted in line with current ERA guidelines; outlining and describing novel concepts unique to risk assessment of AMR (such as direct selection of AMR or increased persistence of AMR). We consolidate terminology used thus far into a single framework that could be adopted moving forward, by proposing predicted no effect concentrations for resistance (PNECRs) and predicted no effect concentrations for persistence (PNECPs) be determined in AMR risk assessment. Such a framework will contribute to antibiotic stewardship and by extension, protection of human health, food security and the global economy.

Spatiotemporal profiling of antibiotics and resistance genes in a river catchment: Human population as the main driver of antibiotic and antibiotic resistance gene presence in the environment.

Studies to understand the role wastewater treatment plants (WWTPs) play in the dissemination of antibiotics (ABs), and in the emergence of antibiotic resistance (ABR), play an important role in tackling this global crisis. Here we describe the abundance and distribution of 16 ABs, and 4 corresponding antibiotic resistance genes (ARGs), sampled from the influent to five WWTPs within a single river catchment. We consider four classes of antibiotics: fluroquinolones, macrolides, sulfamethoxazole and chloramphenicol, as well the corresponding antibiotic resistance genes qnrS, ermB, sul1 and catA. All antibiotics, apart from four fluroquinolones (besifloxacin, lomefloxacin, ulifloxacin, prulifloxacin), were detected within all influent wastewater from the 5 cities (1 city = 1 WWTP), as were the corresponding antibiotic resistance genes (ARGs). Strong correlations were observed between the daily loads of ABs and ARGs versus the size of the population served by each WWTP, as well as between AB and ARG loads at a single site. The efficiency of ABs and ARGs removal by the WWTPs varied according to site (and treatment process utilized) and target, although strong correlations were maintained between the population size served by WWTPs and daily loads of discharged ABs and ARGs into the environment. We therefore conclude that population size is the main determinant of the magnitude of AB and ARG burden in the environment.

Stereoselective Bacterial Metabolism of Antibiotics in Environmental Bacteria - A Novel Biochemical Workflow.

Although molecular genetic approaches have greatly increased our understanding of the evolution and spread of antibiotic resistance genes, there are fewer studies on the dynamics of antibiotic - bacterial (A-B) interactions, especially with respect to stereochemistry. Addressing this knowledge gap requires an interdisciplinary synthesis, and the development of sensitive and selective analytical tools. Here we describe SAM (stereoselective antimicrobial metabolism) workflow, a novel interdisciplinary approach for assessing bacterial resistance mechanisms in the context of A-B interactions that utilise a combination of whole genome sequencing and mass spectrometry. Chloramphenicol was used to provide proof-of-concept to demonstrate the importance of stereoselective metabolism by resistant environmental bacteria. Our data shows that chloramphenicol can be stereoselectively transformed via microbial metabolism with R,R-(-)-CAP being subject to extensive metabolic transformation by an environmental bacterial strain. In contrast S,S-(+)-CAP is not metabolised by this bacterial strain, possibly due to the lack of previous exposure to this isomer in the absence of historical selective pressure to evolve metabolic capacity.