Tracking antimicrobial resistance transmission in urban and rural communities in Bangladesh: a One Health study of genomic diversity of ESBL-producing and carbapenem-resistant Escherichia coli.

Antimicrobial resistance (AMR) poses a significant threat to global health and sustainable development goals, especially in low- and middle-income countries (LMICs). This study aimed to understand the transmission of AMR between poultry, humans, and the environment in Bangladesh using a One Health approach. We analyzed the whole genome sequences (WGS) of 117 extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-Ec) isolates, with 46 being carbapenem resistant. These isolates were obtained from human (n = 20) and poultry feces (n = 12), as well as proximal environments (wastewater) (n = 85) of three different study sites, including rural households (n = 48), rural poultry farms (n = 20), and urban wet markets (n = 49). The WGS of ESBL-Ec isolates were compared with 58 clinical isolates from global databases. No significant differences in antibiotic resistance genes (ARGs) were observed in ESBL-Ec isolated from humans with and without exposure to poultry. Environmental isolates showed higher ARG diversity than human and poultry isolates. No clonal transmission between poultry and human isolates was found, but wastewater was a reservoir for ESBL-Ec for both. Except for one human isolate, all ESBL-Ec isolates were distinct from clinical isolates. Most isolates (77.8%) carried at least one plasmid replicon type, with IncFII being the most prevalent. IncFIA was predominant in human isolates, while IncFII, Col(MG828), and p0111 were common in poultry. We observed putative sharing of ARG-carrying plasmids among isolates, mainly from wastewater. However, in most cases, bacterial isolates sharing plasmids were also clonally related, suggesting clonal spread was more probable than just plasmid transfer. IMPORTANCE: Our study underscores that wastewater discharged from households and wet markets carries antibiotic-resistant organisms from both human and animal sources. Thus, direct disposal of wastewater into the environment not only threatens human health but also endangers food safety by facilitating the spread of antimicrobial resistance (AMR) to surface water, crops, vegetables, and subsequently to food-producing animals. In regions with intensive poultry production heavily reliant on the prophylactic use of antibiotics, compounded by inadequate waste management systems, such as Bangladesh, the ramifications are particularly pronounced. Wastewater serves as a pivotal juncture for the dissemination of antibiotic-resistant organisms and functions as a pathway through which strains of human and animal origin can infiltrate the environment and potentially colonize new hosts. Further research is needed to thoroughly characterize wastewater isolates/populations and understand their potential impact on interconnected environments, communities, and wildlife.

Modelling the impact of wastewater flows and management practices on antimicrobial resistance in dairy farms.

Dairy slurry is a major source of environmental contamination with antimicrobial resistant genes and bacteria. We developed mathematical models and conducted on-farm research to explore the impact of wastewater flows and management practices on antimicrobial resistance (AMR) in slurry. Temporal fluctuations in cephalosporin-resistant Escherichia coli were observed and attributed to farm activities, specifically the disposal of spent copper and zinc footbath into the slurry system. Our model revealed that resistance should be more frequently observed with relevant determinants encoded chromosomally rather than on plasmids, which was supported by reanalysis of sequenced genomes from the farm. Additionally, lower resistance levels were predicted in conditions with lower growth and higher death rates. The use of muck heap effluent for washing dirty channels did not explain the fluctuations in cephalosporin resistance. These results highlight farm-specific opportunities to reduce AMR pollution, beyond antibiotic use reduction, including careful disposal or recycling of waste antimicrobial metals.

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies.

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid assembly approaches that combine long- and short-read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock bacteriophage community of previously sequenced phage genomes and sequenced them using Illumina, Nanopore and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies 41 and 157 % higher than found in Illumina only assemblies, respectively. While the best PacBio assemblies had SNPs at frequencies 12 and 78 % higher than found in Illumina-only assemblies, respectively. Despite high-read coverage, long-read-only assemblies recovered a maximum of one complete genome from any assembly, unless reads were down-sampled prior to assembly. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short-read-only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes.

Is it selfish to be filamentous in biofilms? Individual-based modeling links microbial growth strategies with morphology using the new and modular iDynoMiCS 2.0.

Microbial communities are found in all habitable environments and often occur in assemblages with self-organized spatial structures developing over time. This complexity can only be understood, predicted, and managed by combining experiments with mathematical modeling. Individual-based models are particularly suited if individual heterogeneity, local interactions, and adaptive behavior are of interest. Here we present the completely overhauled software platform, the individual-based Dynamics of Microbial Communities Simulator, iDynoMiCS 2.0, which enables researchers to specify a range of different models without having to program. Key new features and improvements are: (1) Substantially enhanced ease of use (graphical user interface, editor for model specification, unit conversions, data analysis and visualization and more). (2) Increased performance and scalability enabling simulations of up to 10 million agents in 3D biofilms. (3) Kinetics can be specified with any arithmetic function. (4) Agent properties can be assembled from orthogonal modules for pick and mix flexibility. (5) Force-based mechanical interaction framework enabling attractive forces and non-spherical agent morphologies as an alternative to the shoving algorithm. The new iDynoMiCS 2.0 has undergone intensive testing, from unit tests to a suite of increasingly complex numerical tests and the standard Benchmark 3 based on nitrifying biofilms. A second test case was based on the "biofilms promote altruism" study previously implemented in BacSim because competition outcomes are highly sensitive to the developing spatial structures due to positive feedback between cooperative individuals. We extended this case study by adding morphology to find that (i) filamentous bacteria outcompete spherical bacteria regardless of growth strategy and (ii) non-cooperating filaments outcompete cooperating filaments because filaments can escape the stronger competition between themselves. In conclusion, the new substantially improved iDynoMiCS 2.0 joins a growing number of platforms for individual-based modeling of microbial communities with specific advantages and disadvantages that we discuss, giving users a wider choice.

The need for One Health systems-thinking approaches to understand multiscale dissemination of antimicrobial resistance.

Although the effects of antimicrobial resistance (AMR) are most obvious at clinical treatment failure, AMR evolution, transmission, and dispersal happen largely in environmental settings, for example within farms, waterways, livestock, and wildlife. We argue that systems-thinking, One Health approaches are crucial for tackling AMR, by understanding and predicting how anthropogenic activities interact within environmental subsystems, to drive AMR emergence and transmission. Innovative computational methods integrating big data streams (eg, from clinical, agricultural, and environmental monitoring) will accelerate our understanding of AMR, supporting decision making. There are challenges to accessing, integrating, synthesising, and interpreting such complex, multidimensional, heterogeneous datasets, including the lack of specific metrics to quantify anthropogenic AMR. Moreover, data confidentiality, geopolitical and cultural variation, surveillance gaps, and science funding cause biases, uncertainty, and gaps in AMR data and metadata. Combining systems-thinking with modelling will allow exploration, scaling-up, and extrapolation of existing data. This combination will provide vital understanding of the dynamic movement and transmission of AMR within and among environmental subsystems, and its effects across the greater system. Consequently, strategies for slowing down AMR dissemination can be modelled and compared for efficacy and cost-effectiveness.

The role of multisystemic resilience in fostering critical agency: UK adolescents during the COVID-19 Pandemic.

Critical agency (CA) refers to an individual's feeling of power in relation to social inequalities. Research has demonstrated that high CA is associated with positive adolescent outcomes, however, less is known about what supports are important for its development. Moreover, a large majority of the literature is based on studies from the US and various countries in Africa; although the UK is saturated with inequalities there is little research within a UK context. In this paper we examine (a) the validity of using an existing measure of CA with a sample of UK adolescents and (b) the extent to which resilience supports account for variance in CA. Our analysis identified two distinct factors of CA: justice-oriented and community-oriented. High CA in both factors was explained by resilience supports associated with peer relationships (p < 0.01). Our findings push us towards new relational, ecological ways of understanding adolescent CA. We close by instantiating a translational framework for those devising policies in support of youth resilience and CA. Supplementary Information: The online version contains supplementary material available at 10.1007/s12144-023-04578-1.

Predicting bioactivity of antibiotic metabolites by molecular docking and dynamics.

Antibiotics enter the environment through waste streams, where they can exert selective pressure for antimicrobial resistance in bacteria. However, many antibiotics are excreted as partly metabolized forms, or can be subject to partial breakdown in wastewater treatment, soil, or through natural processes in the environment. If a metabolite is bioactive, even at sub-lethal levels, and also stable in the environment, then it could provide selection pressure for resistance. (5S)-penicilloic acid of piperacillin has previously been found complexed to the binding pocket of penicillin binding protein 3 (PBP3) of Pseudomonas aeruginosa. Here, we predicted the affinities of all potentially relevant antibiotic metabolites of ten different penicillins to that target protein, using molecular docking and molecular dynamics simulations. Docking predicts that, in addition to penicilloic acid, pseudopenicillin derivatives of these penicillins, as well as 6-aminopenicillanic acid (6APA), could also bind to this target. MD simulations further confirmed that (5R)-pseudopenicillin and 6APA bind the target protein, in addition to (5S)-penicilloic acid. Thus, it is possible that these metabolites are bioactive, and, if stable in the environment, could be contaminants selective for antibiotic resistance. This could have considerable significance for environmental surveillance for antibiotics as a means to reduce antimicrobial resistance, because targeted mass spectrometry could be required for relevant metabolites as well as the native antibiotics.

Multidrug-Resistant ESBL-Producing E. coli in Clinical Samples from the UK.

Globally, cephalosporin therapy failure is a serious problem for infection control. One causative agent of cephalosporin-resistant infections is multidrug-resistant (MDR) E. coli producing extended-spectrum ß-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) ß-lactamases. We evaluated the occurrence of ESBL/pAmpC genetic determinants in phenotypically MDR E. coli isolated from clinical samples of blood, faeces, ear effusion, urine and sputum from a UK hospital. Phenotypic resistance profiling for 18 antibiotics (from seven classes) showed that 32/35 isolates were MDR, with resistance to 4-16 of the tested antibiotics. Of the isolates, 97.1% showed resistance to ampicillin, 71.4% showed resistance to co-amoxiclav, cefotaxime, ceftazidime and ceftiofur, and 68.5% showed resistance to cefquinome. blaCTX-M, blaTEM and blaOXA-1 genes were detected in 23, 13 and 12 strains, respectively, and Intl1 was detected in 17 isolates. The most common subtypes among the definite sequence types were CTX-M-15 (40%) and TEM-1 (75%). No E. coli isolates carried pAmpC genes. Significant correlations were seen between CTX-M carriage and cefotaxime, ceftiofur, aztreonam, ceftazidime and cefquinome resistance; between blaCTX-M, blaTEM and blaOXA-1 carriage and ciprofloxacin resistance; and between Intl1 carriage and trimethoprim/sulfamethoxazole resistance. Thus, MDR phenotypes may be conferred by a relatively small number of genes. The level and pattern of antibiotic resistance highlight the need for better antibiotic therapy guidelines, including reduced use and improved surveillance.

Antimicrobial resistance in dairy slurry tanks: A critical point for measurement and control.

Waste from dairy production is one of the largest sources of contamination from antimicrobial resistant bacteria (ARB) and genes (ARGs) in many parts of the world. However, studies to date do not provide necessary evidence to inform antimicrobial resistance (AMR) countermeasures. We undertook a detailed, interdisciplinary, longitudinal analysis of dairy slurry waste. The slurry contained a population of ARB and ARGs, with resistances to current, historical and never-used on-farm antibiotics; resistances were associated with Gram-negative and Gram-positive bacteria and mobile elements (ISEcp1, Tn916, Tn21-family transposons). Modelling and experimental work suggested that these populations are in dynamic equilibrium, with microbial death balanced by fresh input. Consequently, storing slurry without further waste input for at least 60 days was predicted to reduce ARB spread onto land, with > 99 % reduction in cephalosporin resistant Escherichia coli. The model also indicated that for farms with low antibiotic use, further reductions are unlikely to reduce AMR further. We conclude that the slurry tank is a critical point for measurement and control of AMR, and that actions to limit the spread of AMR from dairy waste should combine responsible antibiotic use, including low total quantity, avoidance of human critical antibiotics, and choosing antibiotics with shorter half-lives, coupled with appropriate slurry storage.

EMBRACE-WATERS statement: Recommendations for reporting of studies on antimicrobial resistance in wastewater and related aquatic environments.

BACKGROUND: A One Health approach requires integrative research to elucidate antimicrobial resistance (AMR) in the environment and the risks it poses to human health. Research on this topic involves experts from diverse backgrounds and professions. Shortcomings exist in terms of consistent, complete, and transparent reporting in many environmental studies. Standardized reporting will improve the quality of scientific papers, enable meta-analyses and enhance the communication among different experts. In this study, we aimed to generate a consensus of reporting standards for AMR research in wastewater and related aquatic environments. METHODS: Based on a risk of bias assessment of the literature in a systematic review, we proposed a set of study quality indicators. We then used a multistep modified Delphi consensus to develop the EMBRACE-WATERS statement (rEporting antiMicroBial ResistAnCE in WATERS), a checklist of recommendations for reporting in studies of AMR in wastewater and related aquatic environments. FINDINGS: Consensus was achieved among a multidisciplinary panel of twenty-one experts in three steps. The developed EMBRACE-WATERS statement incorporates 21 items. Each item contains essential elements of high-quality reporting and is followed by an explanation of their rationale and a reporting-example. The EMBRACE-WATERS statement is primarily intended to be used by investigators to ensure transparent and comprehensive reporting of their studies. It can also guide peer-reviewers and editors in evaluation of manuscripts on AMR in the aquatic environment. This statement is not intended to be used to guide investigators on the methodology of their research. INTERPRETATION: We are hopeful that this statement will improve the reporting quality of future studies of AMR in wastewater and related aquatic environments. Its uptake would generate a common language to be used among researchers from different disciplines, thus advancing the One Health approach towards understanding AMR spread across aquatic environments. Similar initiatives are needed in other areas of One Health research.

Laboratory Stock Variants of the Archetype Silver Resistance Plasmid pMG101 Demonstrate Plasmid Fusion, Loss of Transmissibility, and Transposition of Tn7/pco/sil Into the Host Chromosome.

Salmonella Typhimurium carrying the multidrug resistance (MDR) plasmid pMG101 was isolated from three burns patients in Boston United States in 1973. pMG101 was transferrable into other Salmonella spp. and Escherichia coli hosts and carried what was a novel and unusual combination of AMR genes and silver resistance. Previously published short-read DNA sequence of pMG101 showed that it was a 183.5Kb IncHI plasmid, where a Tn7-mediated transposition of pco/sil resistance genes into the chromosome of the E. coli K-12 J53 host strain had occurred. We noticed differences in streptomycin resistance and plasmid size between two stocks of E. coli K-12 J53 pMG101 we possessed, which had been obtained from two different laboratories (pMG101-A and pMG101-B). Long-read sequencing (PacBio) of the two strains unexpectedly revealed plasmid and chromosomal rearrangements in both. pMG101-A is a non-transmissible 383Kb closed-circular plasmid consisting of an IncHI2 plasmid sequence fused to an IncFI/FIIA plasmid. pMG101-B is a mobile closed-circular 154 Kb IncFI/FIIA plasmid. Sequence identity of pMG101-B with the fused IncFI/IncFIIA region of pMG101-A was >99%. Assembled host sequence reads of pMG101-B showed Tn7-mediated transposition of pco/sil into the E. coli J53 chromosome between yhiM and yhiN. Long read sequence data in combination with laboratory experiments have demonstrated large scale changes in pMG101. Loss of conjugation function and movement of resistance genes into the chromosome suggest that even under long-term laboratory storage, mobile genetic elements such as transposons and insertion sequences can drive the evolution of plasmids and host. This study emphasises the importance of utilising long read sequencing technologies of plasmids and host strains at the earliest opportunity.

A Model to Investigate the Impact of Farm Practice on Antimicrobial Resistance in UK Dairy Farms.

The ecological and human health impact of antibiotic use and the related antimicrobial resistance (AMR) in animal husbandry is poorly understood. In many countries, there has been considerable pressure to reduce overall antibiotic use in agriculture or to cease or minimise use of human critical antibiotics. However, a more nuanced approach would consider the differential impact of use of different antibiotic classes; for example, it is not known whether reduced use of bacteriostatic or bacteriolytic classes of antibiotics would be of greater value. We have developed an ordinary differential equation model to investigate the effects of farm practice on the spread and persistence of AMR in the dairy slurry tank environment. We model the chemical fate of bacteriolytic and bacteriostatic antibiotics within the slurry and their effect on a population of bacteria, which are capable of resistance to both types of antibiotic. Through our analysis, we find that changing the rate at which a slurry tank is emptied may delay the proliferation of multidrug-resistant bacteria by up to five years depending on conditions. This finding has implications for farming practice and the policies that influence waste management practices. We also find that, within our model, the development of multidrug resistance is particularly sensitive to the use of bacteriolytic antibiotics, rather than bacteriostatic antibiotics, and this may be cause for controlling the usage of bacteriolytic antibiotics in agriculture.

Hybrid assembly of an agricultural slurry virome reveals a diverse and stable community with the potential to alter the metabolism and virulence of veterinary pathogens.

BACKGROUND: Viruses are the most abundant biological entities on Earth, known to be crucial components of microbial ecosystems. However, there is little information on the viral community within agricultural waste. There are currently ~ 2.7 million dairy cattle in the UK producing 7-8% of their own bodyweight in manure daily, and 28 million tonnes annually. To avoid pollution of UK freshwaters, manure must be stored and spread in accordance with guidelines set by DEFRA. Manures are used as fertiliser, and widely spread over crop fields, yet little is known about their microbial composition. We analysed the virome of agricultural slurry over a 5-month period using short and long-read sequencing. RESULTS: Hybrid sequencing uncovered more high-quality viral genomes than long or short-reads alone; yielding 7682 vOTUs, 174 of which were complete viral genomes. The slurry virome was highly diverse and dominated by lytic bacteriophage, the majority of which represent novel genera (~ 98%). Despite constant influx and efflux of slurry, the composition and diversity of the slurry virome was extremely stable over time, with 55% of vOTUs detected in all samples over a 5-month period. Functional annotation revealed a diverse and abundant range of auxiliary metabolic genes and novel features present in the community, including the agriculturally relevant virulence factor VapE, which was widely distributed across different phage genera that were predicted to infect several hosts. Furthermore, we identified an abundance of phage-encoded diversity-generating retroelements, which were previously thought to be rare on lytic viral genomes. Additionally, we identified a group of crAssphages, including lineages that were previously thought only to be found in the human gut. CONCLUSIONS: The cattle slurry virome is complex, diverse and dominated by novel genera, many of which are not recovered using long or short-reads alone. Phages were found to encode a wide range of AMGs that are not constrained to particular groups or predicted hosts, including virulence determinants and putative ARGs. The application of agricultural slurry to land may therefore be a driver of bacterial virulence and antimicrobial resistance in the environment. Video abstract.

Towards a general model for predicting minimal metal concentrations co-selecting for antibiotic resistance plasmids.

Many antibiotic resistance genes co-occur with resistance genes for transition metals, such as copper, zinc, or mercury. In some environments, a positive correlation between high metal concentration and high abundance of antibiotic resistance genes has been observed, suggesting co-selection due to metal presence. Of particular concern is the use of copper and zinc in animal husbandry, leading to potential co-selection for antibiotic resistance in animal gut microbiomes, slurry, manure, or amended soils. For antibiotics, predicted no effect concentrations have been derived from laboratory measured minimum inhibitory concentrations and some minimal selective concentrations have been investigated in environmental settings. However, minimal co-selection concentrations for metals are difficult to identify. Here, we use mathematical modelling to provide a general mechanistic framework to predict minimal co-selective concentrations for metals, given knowledge of their toxicity at different concentrations. We apply the method to copper (Cu), zinc (Zn), mercury (Hg), lead (Pb) and silver (Ag), predicting their minimum co-selective concentrations in mg/L (Cu: 5.5, Zn: 1.6, Hg: 0.0156, Pb: 21.5, Ag: 0.152). To exemplify use of these thresholds, we consider metal concentrations from slurry and slurry-amended soil from a UK dairy farm that uses copper and zinc as additives for feed and antimicrobial footbath: the slurry is predicted to be co-selective, but not the slurry-amended soil. This modelling framework could be used as the basis for defining standards to mitigate risks of antimicrobial resistance applicable to a wide range of environments, including manure, slurry and other waste streams.

INfrastructure for a PHAge REference Database: Identification of Large-Scale Biases in the Current Collection of Cultured Phage Genomes.

Background: With advances in sequencing technology and decreasing costs, the number of phage genomes that have been sequenced has increased markedly in the past decade. Materials and Methods: We developed an automated retrieval and analysis system for phage genomes (https://github.com/RyanCook94/inphared) to produce the INfrastructure for a PHAge REference Database (INPHARED) of phage genomes and associated metadata. Results: As of January 2021, 14,244 complete phage genomes have been sequenced. The INPHARED data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated on only 30 bacterial genera. There is further bias, with significantly more lytic phage genomes (∼70%) than temperate (∼30%) within our database. Collectively, this results in ∼54% of temperate phage genomes originating from just three host genera. With much debate on the carriage of antibiotic resistance genes and their potential safety in phage therapy, we searched for putative antibiotic resistance genes. Frequency of antibiotic resistance gene carriage was found to be higher in temperate phages than in lytic phages and again varied with host. Conclusions: Given the bias of currently sequenced phage genomes, we suggest to fully understand phage diversity, efforts should be made to isolate and sequence a larger number of phages, in particular temperate phages, from a greater diversity of hosts.

A generalised model for generalised transduction: the importance of co-evolution and stochasticity in phage mediated antimicrobial resistance transfer.

Antimicrobial resistance is a major global challenge. Of particular concern are mobilizable elements that can transfer resistance genes between bacteria, leading to pathogens with new combinations of resistance. To date, mathematical models have largely focussed on transfer of resistance by plasmids, with fewer studies on transfer by bacteriophages. We aim to understand how best to model transfer of resistance by transduction by lytic phages. We show that models of lytic bacteriophage infection with empirically derived realistic phage parameters lead to low numbers of bacteria, which, in low population or localised environments, lead to extinction of bacteria and phage. Models that include antagonistic co-evolution of phage and bacteria produce more realistic results. Furthermore, because of these low numbers, stochastic dynamics are shown to be important, especially to spread of resistance. When resistance is introduced, resistance can sometimes be fixed, and at other times die out, with the probability of each outcome sensitive to bacterial and phage parameters. Specifically, that outcome most strongly depends on the baseline death rate of bacteria, with phage-mediated spread favoured in benign environments with low mortality over more hostile environments. We conclude that larger-scale models should consider spatial compartmentalisation and heterogeneous microenviroments, while encompassing stochasticity and co-evolution.

DirtyGenes: testing for significant changes in gene or bacterial population compositions from a small number of samples.

High throughput genomics technologies are applied widely to microbiomes in humans, animals, soil and water, to detect changes in bacterial communities or the genes they carry, between different environments or treatments. We describe a method to test the statistical significance of differences in bacterial population or gene composition, applicable to metagenomic or quantitative polymerase chain reaction data. Our method goes beyond previous published work in being universally most powerful, thus better able to detect statistically significant differences, and through being more reliable for smaller sample sizes. It can also be used for experimental design, to estimate how many samples to use in future experiments, again with the advantage of being universally most powerful. We present three example analyses in the area of antimicrobial resistance. The first is to published data on bacterial communities and antimicrobial resistance genes (ARGs) in the environment; we show that there are significant changes in both ARG and community composition. The second is to new data on seasonality in bacterial communities and ARGs in hooves from four sheep. While the observed differences are not significant, we show that a minimum group size of eight sheep would provide sufficient power to observe significance of similar changes in further experiments. The third is to published data on bacterial communities surrounding rice crops. This is a much larger data set and is used to verify the new method. Our method has broad uses for statistical testing and experimental design in research on changing microbiomes, including studies on antimicrobial resistance.

Removal of copper from cattle footbath wastewater with layered double hydroxide adsorbents as a route to antimicrobial resistance mitigation on dairy farms.

Copper and zinc are routinely used in livestock antimicrobial footbaths in commercial farming. The footbath mix is a cost to farmers, and the disposal of spent footbath into slurry tanks leads to soil contamination, as well as the potential for antimicrobial metal resistance and co-selection. This study assesses the potential to mitigate a source of antimicrobial metal resistance in slurry tanks while recovering copper and zinc from spent cattle footbaths. This is the first study in literature to investigate the potential of recovering copper from cattle footbath solutions via any method. The sorbent, Ca2Al-EDTA Layered Double Hydroxides (LDH), were used to remove Cu2+ from a Cu2SO4·5H20 solution at different temperatures. The maximum Cu2+ uptake from the Cu2SO4·5H20 solution was 568 ±â€¯88 mg g-1. Faster and higher equilibrium uptake was achieved by increasing the temperature of the solution. The sorbent was found to be effective in removing copper and zinc from a commercially available cattle footbath solution (filtered footbath solution Cu2+ uptake 283 ±â€¯11.05 mg g-1, Zn2+ uptake 60 ±â€¯0.05 mg g-1). Thus, this study demonstrates the opportunity for a completely novel and potentially economically beneficial method of mitigating antimicrobial resistance in agriculture and the environment, while also providing a new valuable copper and zinc waste stream for secondary metal production.

Investigating the Association Between the Caecal Microbiomes of Broilers and Campylobacter Burden.

One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonization in its natural niche, and the effect of the local microbiome on colonization. Previous studies have shown that the microbiome differed between Campylobacter colonized and non-colonized chicken intestinal samples. To characterize the microbiome of Campylobacter-colonized broilers, caecal samples of 100 randomly selected birds from four farms were analyzed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7-63.5 and 30.2-59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes, or Tenericutes levels in the 16S rRNA operational taxonomic unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (>9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonization. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety.

Regulatory feedback response mechanisms to phosphate starvation in rice.

Phosphorus is a growth-limiting nutrient for plants. The growing scarcity of phosphate stocks threatens global food security. Phosphate-uptake regulation is so complex and incompletely known that attempts to improve phosphorus use efficiency have had extremely limited success. This study improves our understanding of the molecular mechanisms underlying phosphate uptake by investigating the transcriptional dynamics of two regulators: the Ubiquitin ligase PHO2 and the long non-coding RNA IPS1. Temporal measurements of RNA levels have been integrated into mechanistic mathematical models using advanced statistical techniques. Models based solely on current knowledge could not adequately explain the temporal expression profiles. Further modeling and bioinformatics analysis have led to the prediction of three regulatory features: the PHO2 protein mediates the degradation of its own transcriptional activator to maintain constant PHO2 mRNA levels; the binding affinity of the transcriptional activator of PHO2 is impaired by a phosphate-sensitive transcriptional repressor/inhibitor; and the extremely high levels of IPS1 and its rapid disappearance upon Pi re-supply are best explained by Pi-sensitive RNA protection. This work offers both new opportunities for plant phosphate research that will be essential for informing the development of phosphate efficient crop varieties, and a foundation for the development of models integrating phosphate with other stress responses.