Opportunities and challenges with transitioning to non-lethal sampling of wild fish for microbiome research.

The microbial communities of fish are considered an integral part of maintaining the overall health and fitness of their host. Research has shown that resident microbes reside on various mucosal surfaces, such as the gills, skin, and gastrointestinal tract, and play a key role in various host functions, including digestion, immunity, and disease resistance. A second, more transient group of microbes reside in the digesta, or feces, and are primarily influenced by environmental factors such as the host diet. The vast majority of fish microbiome research currently uses lethal sampling to analyse any one of these mucosal and/or digesta microbial communities. The present paper discusses the various opportunities that non-lethal microbiome sampling offers, as well as some inherent challenges, with the ultimate goal of creating a sound argument for future researchers to transition to non-lethal sampling of wild fish in microbiome research. Doing so will reduce animal welfare and population impacts on fish while creating novel opportunities to link host microbial communities to an individual's behavior and survival across space and time (e.g., life-stages, seasons). Current lethal sampling efforts constrain our ability to understand the mechanistic ecological consequences of variation in microbiome communities in the wild. Transitioning to non-lethal sampling will open new frontiers in ecological and microbial research.

Impact of wastewater treatment upgrade and nitrogen removal on bacterial communities and their interactions in eutrophic prairie streams.

Eutrophication can impact bacteria by altering fluxes and processing of nutrients and organic matter. However, relatively little is known of how bacterial communities, diversity, and interactions with phytoplankton might respond to nutrient management. We used 16S rRNA amplicon sequencing to compare bacterial assemblages in the water column upstream (control) and downstream (impact) of a wastewater treatment plant (WWTP) located on a eutrophic prairie stream. Sampling occurred before (2012) and after (2018) the 2016 biological nutrient removal (BNR) upgrade that removed >90% of nitrogen (N, mainly NH4+). Multivariate ordination suggested that effluent-impacted bacterial communities were associated mainly with elevated NH4+ concentrations before the upgrade, whereas those after BNR were characteristic of reference systems (low NO3-, diverse regulation). Genera such as Betaproteobacteria and Rhodocyclacea were abundant at impacted sites in 2012, whereas Flavobacterium and a potential pathogen (Legionella) were common at impacted sites in 2018. Nitrifier bacteria (Nitrospira and Nitrosomonas) were present but rare at all sites in 2012, but recorded only downstream of the WWTP in 2018. Generalized additive models showed that BNR reduced bacterial diversity, with ∼70% of the deviance in diversity explained by hydrology, pH, nutrients, and phytoplankton abundance. Overall, NH4+ removal reduced symptoms of cultural eutrophication in microbe assemblages.

Complete genome sequences of bacteria isolated from cockroaches collected in a Bangladeshi hospital.

We report the complete genome sequences of four bacterial strains that were isolated from Blattella germanica (German cockroaches) that were found in three wards of the Rajshahi Medical College Hospital. Multiple antibiotic resistance genes were identified in each genome, with one genome containing multiple plasmid-encoded resistance genes.

Phylogenomic analysis of the genus Delftia reveals distinct major lineages with ecological specializations.

Delftia is a diverse betaproteobacterial genus with many strains having agricultural and industrial relevance, including plant-growth promotion, bioremediation of hydrocarbon-contaminated soils, and heavy metal immobilization. Delftia spp. are broadly distributed in the environment, and have been isolated from plant hosts as well as healthy and diseased animal hosts, yet the genetic basis of this ecological versatility has not been characterized. Here, we present a phylogenomic comparison of published Delftia genomes and show that the genus is divided into two well-supported clades: one 'Delftia acidovorans' clade with isolates from soils and plant rhizospheres, and a second 'Delftia lacustris and Delftia tsuruhatensis' clade with isolates from humans and sludge. The pan-genome inferred from 61 Delftia genomes contained over 28 000 genes, of which only 884 were found in all genomes. Analysis of industrially relevant functions highlighted the ecological versatility of Delftia and supported their role as generalists.

Proceedings of the Dual Use Research of Concern Panel Discussion: challenges and perspectives.

To address real and perceived emerging risks originating from the ever-accelerating breakthroughs in life science research, the Dual Use Research of Concern (DURC) Panel Discussion, organized by Synbio Canada and the Alberta RNA Research and Training Institute (ARRTI), took place on June 23rd, 2021. It brought together six stakeholders from different levels of academic research, administration, governance, and science publishing to explore the current and future challenges in addressing DURC. Technological advancements within the life sciences, especially within the field of omics technology, make it difficult to apply a simple checklist for dual-use assessment and require continuous and integrated effort. Bottom-up approaches from within the scientific community are suggested by all stakeholders to enable efficient governance and address the true risks resulting from DURC, not just the alleged risks. To address such alleged risks, open and broadscale communication of DURC and its oversight policies may be required. At the same time, any form of open communication also contains the risk of information hazards, defined as potentially creating public fear or informing malicious actors. Here, an overview of the DURC panel and its outcomes is provided.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element.

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

The ISOTILT software for discovering cooperative rigid-unit rotations in networks of interconnected rigid units.

A user-friendly web-based software tool called 'ISOTILT' is introduced for detecting cooperative rigid-unit modes (RUMs) in networks of interconnected rigid units (e.g. molecules, clusters or polyhedral units). This tool implements a recently described algorithm in which symmetry-mode patterns of pivot-atom rotation and displacement vectors are used to construct a linear system of equations whose null space consists entirely of RUMs. The symmetry modes are first separated into independent symmetry-mode blocks and the set of equations for each block is solved separately by singular value decomposition. ISOTILT is the newest member of the ISOTROPY Software Suite. Here, it is shown how to prepare structural and symmetry-mode information for use in ISOTILT, how to use each of ISOTILT's input fields and options, and how to use and interpret ISOTILT output.

Metagenomic and metatranscriptomic analysis reveals enrichment for xenobiotic-degrading bacterial specialists and xenobiotic-degrading genes in a Canadian Prairie two-cell biobed system.

Biobeds are agriculture-based bioremediation tools used to safely contain and microbially degrade on-farm pesticide waste and rinsate, thereby reducing the negative environmental impacts associated with pesticide use. While these engineered ecosystems demonstrate efficient pesticide removal, the microbiomes in these environments remain largely understudied both taxonomically and functionally. This study used metagenomic and metatranscriptomic techniques to characterize the microbial community in a two-cell Canadian biobed system before and after a field season of pesticide application. These culture-independent approaches identified an enrichment of xenobiotic-degrading bacteria, such as Afipia, Sphingopyxis and Pseudomonas, and enrichment and transcription of xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; we were able to directly link the transcription of these genes to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa.

Comparative genomic analyses of ß-lactamase (blaCMY-42)-encoding plasmids isolated from wastewater treatment plants in Canada.

Wastewater treatment plants (WWTPs) are useful environments for investigating the occurrence, diversity, and evolution of plasmids encoding clinically relevant antibiotic resistance genes (ARGs). Our objective was to isolate and sequence plasmids encoding meropenem resistance from bacterial hosts within Canadian WWTPs. We used two enrichment culture approaches for primary plasmid isolation, followed by screening for antibiotic resistance, conjugative mobility, and stability in enteric bacteria. Isolated plasmids were sequenced using Illumina MiSeq and Sanger sequencing methods. Bioinformatics analyses resolved a multi-resistance IncF/MOBF12 plasmid, pFEMG (209 357 bp), harbouring resistance genes to ß-lactam (blaCMY-42, blaTEM-1ß, and blaNDM-5), macrolide (mphA-mrx-mphR), tetracycline (tetR-tetB-tetC-tetD), trimethoprim (dfrA12), aminoglycoside (aadA2), and sulfonamide (sul1) antibiotic classes. We also isolated an IncI1/MOBP12 plasmid pPIMR (172 280 bp) carrying similar ß-lactamase and a small multi-drug efflux resistance gene cluster (blaCMY-42-blc-sugE) to pFEMG. The co-occurrence of different ARGs within a single 24 552 bp cluster in pFEMG - interspersed with transposons, insertion sequence elements, and a class 1 integron - may be of significant interest to human and veterinary medicine. Additionally, the presence of conjugative and plasmid maintenance genes in the studied plasmids corresponded to observed high conjugative transfer frequencies and stable maintenance. Extensive investigation is required to further understand the fitness trade-offs of plasmids with different types of conjugative transfer and maintenance modules.

Complete Genome Sequence of a Pseudomonas simiae Strain with Biocontrol Potential against Aphanomyces Root Rot.

Aphanomyces euteiches is a soilborne plant pathogen. It causes severe root rot in leguminous crop species. We report the complete genome sequence of a biocontrol strain, Pseudomonas simiae K-Hf-L9. The strain inhibited Aphanomyces euteiches mycelia and zoospores and suppressed root rot in field peas grown under controlled growth chamber conditions.

The effects of oral anticancer parity laws on out-of-pocket spending and adherence among commercially insured patients with chronic myeloid leukemia.

BACKGROUND: Over the past 12 years, 43 states and Washington DC have implemented oral anticancer medication parity laws in response to the burden of pharmacy cost sharing. Parity laws are designed to provide equal coverage and cost sharing between orally and parenterally administered anticancer medications for patients in commercial, fully insured health plans (FIHPs). However, there is considerable state-level variation in the requirements to achieve compliance with parity laws, and the clinical and economic effectiveness of parity is not fully known. OBJECTIVES: To (a) understand the impact of parity laws on out-of-pocket (OOP) spending and adherence to tyrosine kinase inhibitors (TKI) among commercially insured patients with chronic myeloid leukemia (CML) and (b) compare these effects across states with and without per prescription or per 30-day OOP spending limits as part of their parity laws. METHODS: Patients aged 18-64 years with CML, at least 1 pharmacy claim for a TKI, and residence in a state that implemented oral anticancer parity legislation between January 1, 2007, and January 1, 2017, were identified from the IBM MarketScan Commercial Claims and Encounters database. A propensity score-weighted difference-in-difference approach was used to measure the impact of parity on OOP spending and adherence in the 6 months after the first pharmacy claim for a TKI (index date) for patients enrolled in FIHPs (subject to parity) and self-funded health plans (SFHPs; exempt from parity). OOP spending was standardized to a 30-day equivalent amount and adjusted to 2017 US dollars. Adherence was assessed using the proportion of days covered (PDC), and patients were categorized as adherent with PDC ≥ 0.80. RESULTS: Of 1,887 patients initiating a TKI before or after their state's parity law, 678 (35.9%) were enrolled in FIHPs (480 before vs 198 after parity), and 1,209 (64.1%) were enrolled in SFHPs (688 before vs 521 after parity). Implementation of parity laws was not associated with any changes in mean OOP spending; however, it was associated with a reduced likelihood of paying $0 per 30 days across all states (adjusted difference-in-difference [aDD] OR = 0.662; 95% CI = 0.535-0.820) and states without OOP spending limits (aDD OR = 0.654; 95% CI = 0.508-0.848), but not in states with limits. Nonsignificant but directionally opposite changes at each end of the OOP spending distribution were observed for states with and without OOP spending limits, with increased spending observed at the 75th, 90th, and 95th percentiles in states without limits. Mean PDC and adherence showed a nonsignificant increase among FIHP and SFHP patients across all states, states with limits, and states without limits. CONCLUSIONS: Oral anticancer parity laws are not associated with reduced OOP spending or improved adherence in a commercially insured sample of patients with CML. These findings were consistent for states that included OOP spending limits as a component of their parity laws. DISCLOSURES: This study did not receive any external funding. Spargo, Yost, Raju, and Schroader are or were employees of Xcenda, which receives contracts from various industry partners unrelated to this work. There are no other conflicts of interest to disclose.

Fate and distribution of determinants of antimicrobial resistance in lateral flow sand filters used for treatment of domestic wastewater.

Residuals of antimicrobial products from anthropogenic uses can create a selective environment in domestic wastewater treatment systems and receiving environments and contribute to the spread of antimicrobial resistance (AMR). On-site wastewater treatment systems are widely used for domestic wastewater management in rural and remote regions, but the fate of determinants of AMR in these types of environments has received little attention. In this study, the mechanisms responsible for the attenuation of determinants of AMR in lateral flow sand filters were explored using a combination of lab, field and modeling investigations. The degradation kinetics and adsorption potential in the sand filter medium of three antibiotic resistance genes (ARGs; sul1, tetO, and ermB) and culturable bacteria resistant to sulfamethoxazole, tetracycline, and erythromycin were measured using lab experiments. The spatial distribution of ARGs and antibiotic resistant bacteria were also assessed in field scale sand filters, and mechanistic modeling was conducted to characterize filtration processes. The results indicated that the primary mechanisms responsible for AMR attenuation within the sand filters were degradation and filtration. The spatial distribution of AMR determinants illustrated that attenuation was occurring along the entire length of each filter. This study provides new insights on primary mechanisms of AMR attenuation in on-site wastewater treatment systems and supports the use of conservative design guidelines and separation distances for reducing AMR transmission.

A bacteriophage infecting Mesorhizobium species has a prolate capsid and shows similarities to a family of Caulobacter crescentus phages.

Mesorhizobium phage vB_MloS_Cp1R7A-A1 was isolated from soil planted with chickpea in Saskatchewan. It is dissimilar in sequence and morphology to previously described rhizobiophages. It is a B3 morphotype virus with a distinct prolate capsid and belongs to the tailed phage family Siphoviridae. Its genome has a GC content of 60.3% and 238 predicted genes. Putative functions were predicted for 57 genes, which include 27 tRNA genes with anticodons corresponding to 18 amino acids. This represents the highest number of tRNA genes reported yet in a rhizobiophage. The gene arrangement shows a partially modular organization. Most of the structural genes are found in one module, whereas tRNA genes are in another. Genes for replication, recombination, and nucleotide metabolism form the third module. The arrangement of the replication module resembles the replication module of Enterobacteria phage T5, raising the possibility that it uses a recombination-based replication mechanism, but there is also a suggestion that a T7-like replication mechanism could be used. Phage termini appear to be long direct repeats of just over 12 kb in length. Phylogenetic analysis revealed that Cp1R7A-A1 is more closely related to PhiCbK-like Caulobacter phages and other B3 morphotype phages than to other rhizobiophages sequenced thus far.

A Meta-Analysis to Determine the State of Biological Control of Aphanomyces Root Rot.

The increasing incidence and prevalence of the pathogen Aphanomyces euteiches in various pulse-growing regions worldwide necessitates the development of effective management strategies, including biological control agents. Numerous labs have undertaken research examining biological control methods to evaluate aphanomyces root rot suppression in multistep processes that include isolation of inhibitory organisms, lab assays, growth chamber assays, and field trials. Given the emergence of various biocontrol agents and the need to mitigate aphanomyces yield losses, we have undertaken a meta-analysis approach to analyze the effectiveness of biocontrol agents in relation to application method, biocontrol agent richness, biocontrol agent type, the type of study, and reporting system-oriented moderator variables. An effect size, calculated as a natural log response ratio, resulted in a summary weighted mean of -0.411, suggesting the overall effectiveness of biocontrol agents (p < .001). Aphanomyces root rot suppression using biological treatments showed significant heterogeneity for all moderator variables, confirming that the studies do not share a common effect size and the use of a random effect model was appropriate. Across studies, meta-analyses revealed that soil amendments, biocontrol agent application as a seed coating and suspension, bacterial and fungal biocontrol agents, mixed applications, growth chamber and field studies, and qualitative and quantitative reporting systems were all associated with significantly positive outcomes for aphanomyces root rot suppression. Our findings suggest that there is potential promise for biological control of aphanomyces root rot, and more field trials need to be conducted to demonstrate the efficacy level observed under growth chamber conditions. Moreover, we identified a lack of detailed understanding of the mechanism(s) of biological control of aphanomyces root rot as a research priority.

Alternative, environmentally conscious approaches for removing antibiotics from wastewater treatment systems.

Prevalence of antibiotic resistance in the environment is of critical concern from a public health perspective, with many human impacted environments showing increased incidence of antibiotic resistant bacteria. Wastewater treatment environments are of particular interest due to their high levels of antibiotic residuals, which can select for antibiotic resistance genes in bacteria. However, wastewater treatment plants are generally not designed to remove antibiotics from collected waste, and many of the currently proposed methods are unsafe for environmental use. This has prompted researchers to identify alternative environmentally safe methods for removing antibiotics from wastewater to be used in parallel with conventional wastewater treatment, as it is a potential strategy towards the mitigation of environmental antibiotic resistance selection. This paper reviews several methods developed to absorb and/or degrade antibiotics from aqueous solutions and wastewater biosolids, which includes ligninolytic fungi and ligninolytic enzymes, algae-driven photobioreactors and algae-activated sludge, and organically-sourced biochars.

Transcriptomics reveal core activities of the plant growth-promoting bacterium Delftia acidovorans RAY209 during interaction with canola and soybean roots.

The plant growth-promoting rhizobacterium Delftia acidovorans RAY209 is capable of establishing strong root attachment during early plant development at 7 days post-inoculation. The transcriptional response of RAY209 was measured using RNA-seq during early (day 2) and sustained (day 7) root colonization of canola plants, capturing RAY209 differentiation from a medium-suspended cell state to a strongly root-attached cell state. Transcriptomic data was collected in an identical manner during RAY209 interaction with soybean roots to explore the putative root colonization response to this globally relevant crop. Analysis indicated there is an increased number of significantly differentially expressed genes between medium-suspended and root-attached cells during early soybean root colonization relative to sustained colonization, while the opposite temporal pattern was observed for canola root colonization. Regardless of the plant host, root-attached RAY209 cells exhibited the least amount of differential gene expression between early and sustained root colonization. Root-attached cells of either canola or soybean roots expressed high levels of a fasciclin gene homolog encoding an adhesion protein, as well as genes encoding hydrolases, multiple biosynthetic processes, and membrane transport. Notably, while RAY209 ABC transporter genes of similar function were transcribed during attachment to either canola or soybean roots, several transporter genes were uniquely differentially expressed during colonization of the respective plant hosts. In turn, both canola and soybean plants expressed genes encoding pectin lyase and hydrolases - enzymes with purported function in remodelling extracellular matrices in response to RAY209 colonization. RAY209 exhibited both a core regulatory response and a planthost-specific regulatory response to root colonization, indicating that RAY209 specifically adjusts its cellular activities to adapt to the canola and soybean root environments. This transcriptomic data defines the basic RAY209 response as both a canola and soybean commercial crop and seed inoculant.

Genome Sequences of vB_RleM_RL38JI and vB_RleM_RL2RES, Two Virulent Rhizobium leguminosarum Transducing Phages.

Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in Rhizobium leguminosarum The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.

Isolation and Characterization of vB_PagP-SK1, a T7-Like Phage Infecting Pantoea agglomerans.

Background: Pantoea is a genus within the Enterobacterales whose members encompass free-living and host-associated lifestyles. Despite our growing understanding of the role of mobile genetic elements in the biology, ecology, and evolution of this bacterial group, few Pantoea bacteriophages have been identified and characterized. Materials and Methods: A bacteriophage that could infect Pantoea agglomerans was isolated from barnyard soil. We used electron microscopy and complete genome sequencing to identify the viral family, and evaluated its host range across 10 different Pantoea species groups using both bacterial lawn and phage lawn assays. The latter assays were carried out using a scalable microplate assay to increase throughput and enable spectrophotometric quantitation. We also performed a phylogenetic analysis to determine the closest relatives of our phage. Results: Phage vB_PagP-SK1 belongs to the genus Teseptimavirus of the Podoviridae family in the order Caudovirales. The 39,938 bp genome has a modular structure with early, middle, and late genes, along with the characteristic direct terminal repeats of 172 bp. Genome composition and synteny were similar to that of the Erwinia amylovora phage, vB_EamP-L1, with the exception of a few loci that are most similar to genes of phage infecting other members of the Enterobacteriaceae. A total of 94 Pantoea strains were surveyed and vB_PagP-SK1 was found to infect 15 Pantoea strains across three species, predominantly P. agglomerans, along with one Erwinia billingiae strain. Conclusions: vB_PagP-SK1 belongs to the Teseptimavirus genus and has a host range that spans multiple species groups, and is most closely related to the E. amylovora phage, vB_EamP-L1. The presence of xenologous genes in its genome indicates that the genome is a mosaic of multiple Teseptimavirus phages that infect members of the Enterobacteriaceae.

Lateral flow sand filters are effective for removal of antibiotic resistance genes from domestic wastewater.

The ability of lateral flow sand filters, used as on-site wastewater treatment systems (OWTS), to remove antibiotic resistance genes (ARGs), antibiotic resistant bacteria (ARB), and other relevant genetic markers (HF183, 16S rRNA, and int1) was assessed. Municipal wastewater was settled in a septic tank prior to loading into six pilot-scale lateral flow sand filters comprised of three different sand media types, at 5 and 30% slopes. The sand filters were sampled bi-weekly for: 9 ARGs and 3 other complimentary gene markers (sul1, sul2, qnrS, tetO, ermB, blaTEM, blaCTX-M, mecA, vanA, int1, HF183, 16S rRNA), and conventional microbial and water quality indicators, from July to November in 2017, and four times in the summer of 2018. The sand filters were observed to attenuate 7 of the ARGs to mostly below 2 log gene copies per mL. Log reductions ranging from 2.9 to 5.4 log were observed for the removal of absolute abundances of ARGs from septic tank effluent in 5 of the 6 sand filters. The fine-grained filter on the 5% slope did not perform as well for ARG attenuation due to hydraulic failure. The apportionment of cell-associated versus cell-free DNA was determined for the gene markers and this indicated that the genes were primarily carried intracellularly. Average log reductions of ARB with resistance to either sulfamethoxazole, erythromycin, or tetracycline were approximately 2.3 log CFU per mL within the filters compared to the septic tank effluent. This field study provides in-depth insights into the attenuation of ARB, ARGs, and their genetic compartmentalization in variably saturated sand OWTS. Overall, this type of OWTS was found to pose little risk of antimicrobial resistance contamination spread into surrounding environments when proper hydraulic function was maintained.