Combined effects of composting and antibiotic administration on cattle manure-borne antibiotic resistance genes

Background: Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here we carried out a comprehensive parallel examination of the effects of small-scale (>55⁰C ×3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally-heated to >55⁰C ×15 days. Results: Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at small scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally-heated. Sul1, intI1, beta lactam ARGs, and plasmid-associated genes increased in all finished composts as compared to the initial condition. External heating more effectively reduced certain clinically-relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotics condition and slower in the beef manure with antibiotics condition. Conclusions: This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems

Sanitation in urban areas may limit the spread of antimicrobial resistance via flies

Synanthropic filth flies are common where sanitation is poor and fecal wastes are accessible to them. These flies have been proposed as mechanical vectors for the localized transport of fecal microbes including antimicrobial resistant (AMR) organisms and associated antimicrobial resistance genes (ARGs), increasing exposure risks. We evaluated whether an onsite sanitation intervention in Maputo, Mozambique reduced the concentration of enteric bacteria and the frequency of detection of ARGs carried by flies collected in household compounds of low-income neighborhoods. Additionally, we assessed the phenotypic resistance profile of Enterobacteriaceae isolates recovered from flies during the pre-intervention phase. After fly enumeration at study compounds, quantitative polymerase chain reaction was used to quantify an enteric 16S rRNA gene (i.e., specific to a cluster of phylotypes corresponding to 5% of the human fecal microflora), 28 ARGs, and Kirby Bauer Disk Diffusion of Enterobacteriaceae isolates was utilized to assess resistance to eleven clinically relevant antibiotics. The intervention was associated with a 1.5 log10 reduction (95% confidence interval: -0.73, -2.3) in the concentration of the enteric 16S gene and a 31% reduction (adjusted prevalence ratio = 0.69, [0.52, 0.92]) in the mean number of ARGs per fly compared to a control group with poor sanitation. This protective effect was consistent across the six ARG classes that we detected. Enterobacteriaceae isolates–only from the pre-intervention phase–were resistant to a mean of 3.4 antibiotics out of the eleven assessed. Improving onsite sanitation infrastructure in low-income informal settlements may help reduce fly-mediated transmission of enteric bacteria and the ARGs carried by them

Metagenomic tracking of antibiotic resistance genes through a pre‐harvest vegetable production system: an integrated lab‐, microcosm‐ and greenhouse‐scale analysis

Summary: Prior research demonstrated the potential for agricultural production systems to contribute to the environmental spread of antibiotic resistance genes (ARGs). However, there is a need for integrated assessment of critical management points for minimizing this potential. Shotgun metagenomic sequencing data were analysed to comprehensively compare total ARG profiles characteristic of amendments (manure or compost) derived from either beef or dairy cattle (with and without dosing antibiotics according to conventional practice), soil (loamy sand or silty clay loam) and vegetable (lettuce or radish) samples collected across studies carried out at laboratory‐, microcosm‐ and greenhouse‐scale. Vegetables carried the greatest diversity of ARGs (n = 838) as well as the most ARG‐mobile genetic element co‐occurrences (n = 945). Radishes grown in manure‐ or compost‐amended soils harboured a higher relative abundance of total (0.91 and 0.91 ARGs/16S rRNA gene) and clinically relevant ARGs than vegetables from other experimental conditions (average: 0.36 ARGs/16S rRNA gene). Lettuce carried the highest relative abundance of pathogen gene markers among the metagenomes examined. Total ARG relative abundances were highest on vegetables grown in loamy sand receiving antibiotic‐treated beef amendments. The findings emphasize that additional barriers, such as post‐harvest processes, merit further study to minimize potential exposure to consumers

Integrated metagenomic assessment of multiple pre-harvest control points on lettuce resistomes at field-scale

An integrated understanding of factors influencing the occurrence, distribution, and fate of antibiotic resistance genes (ARGs) in vegetable production systems is needed to inform the design and development of strategies for mitigating the potential for antibiotic resistance propagation in the food chain. The goal of the present study was to holistically track antibiotic resistance and associated microbiomes at three distinct pre-harvest control points in an agroecosystem in order to identify the potential impacts of key agricultural management strategies. Samples were collected over the course of a single growing season (67 d) from field-scale plots amended with various organic and inorganic amendments at agronomic rates. Dairy-derived manure and compost amendment samples (n= 14), soil samples (n= 27), and lettuce samples (n= 12) were analyzed via shotgun metagenomics to assess multiple pre-harvest factors as hypothetical control points that shape lettuce resistomes. Pre-harvest factors of interest included manure collection during/post antibiotic use, manure composting, and soil amendment soil with organic (stockpiled manure/compost) versus chemical fertilizer. Microbial community resistome and taxonomic compositions were unique from amendment to soil to lettuce surface according to dissimilarity analysis. The highest resistome alpha diversity (i.e., unique ARGs, n=642) was detected in amendment samples prior to soil application, while the composted manure had the lowest total ARG relative abundance (i.e., 16S rRNA gene-normalized). Regardless of amendment type, soils acted as an apparent ecological buffer, i.e., soil resistome and taxonomic profiles returned to background conditions 120 d-post amendment application. Effects of amendment conditions surprisingly re-emerged in lettuce phyllosphere resistomes, with the highest total ARG relative abundances recovered on the surface of lettuce plants grown in organically-fertilized soils (i.e. compost- and manure-amended soils). Co-occurrence analysis identified 55 unique ARGs found both in the soil amendments and on lettuce surfaces. Among these, arnA and pmrF were the most abundant ARGs co-occurring with mobile genetic elements (MGE). Other prominent ARG-MGE co-occurrences throughout this pre-harvest lettuce production chain included: TetM to transposon (Clostridiodies difficile) in the manure amendment and TriC to plasmid (Ralstonia solanacearum) on the lettuce surfaces. This suggests that, even with imposing manure management and post-amendment wait periods in agricultural systems, ARGs originating from manure can still be found on crop surfaces. This study demonstrates a comprehensive approach to identifying key control points for the propagation of ARGs in vegetable production systems, identifying potential ARG-MGE combinations that could inform future surveillance. The findings suggest that additional pre-harvest and potentially post-harvest interventions may be warranted to minimize risk of propagating antibiotic resistance in the food chain

ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem

DataSheet2_ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization.PDF

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.</p

Structured Ethical Review for Wastewater-Based Testing

Wastewater-based testing (WBT) for SARS-CoV-2 has rapidly expanded over the past three years due to its ability to provide a comprehensive measurement of disease prevalence independent of clinical testing. The development and simultaneous application of the field blurred the boundary between measuring biomarkers for research activities and for pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process (or associated data management safeguards), introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary group developed a framework for a structured ethical review of WBT. The workshop employed a consensus approach to create this framework as a set of 11-questions derived from primarily public health guidance because of the common exemption of wastewater samples to human subject research considerations. This study retrospectively applied the set of questions to peer- reviewed published reports on SARS-CoV-2 monitoring campaigns covering the emergent phase of the pandemic from March 2020 to February 2022 (n=53). Overall, 43% of the responses to the questions were unable to be assessed because of lack of reported information. It is therefore hypothesized that a systematic framework would at a minimum improve the communication of key ethical considerations for the application of WBT. Consistent application of a standardized ethical review will also assist in developing an engaged practice of critically applying and updating approaches and techniques to reflect the concerns held by both those practicing and being monitored by WBT supported campaigns. Development of a structured ethical review facilitates retrospective analysis of published studies and drafted scenarios in the context of wastewater-based testing

DataSheet1_ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization.CSV

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.</p

Table1_ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization.XLSX

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.</p