Non-target analysis of Danish wastewater treatment plant effluent: Statistical analysis of chemical fingerprinting as a step toward a future monitoring tool.

In an attempt to discover and characterize the plethora of xenobiotic substances, this study investigates chemical compounds released into the environment with wastewater effluents. A novel non-targeted screening methodology based on ultra-high resolution Orbitrap mass spectrometry and nanoflow ultra-high performance liquid chromatography together with a newly optimized data-processing pipeline were applied to effluent samples from two state-of-the-art and one small wastewater treatment facility. In total, 785 molecular structures were obtained, of which 38 were identified as single compounds, while 480 structures were identified at a putative level. Most of these substances were therapeutics and drugs, present as parent compounds and metabolites. Using R packages Phyloseq and MetacodeR, originally developed for bioinformatics, significant differences in xenobiotic presence in the wastewater effluents between the three sites were demonstrated.

Insights into the circular: The cryptic plasmidome and its derived antibiotic resistome in the urban water systems.

Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes (ARGs) in different compartments of urban water systems (UWSs) in three European countries representing different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified 9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as conjugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs' plasmidome was dominated by small plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs compartments, these results should alarm and call for attention.

Cessation of grazing causes biodiversity loss and homogenization of soil food webs.

There is widespread concern that cessation of grazing in historically grazed ecosystems is causing biotic homogenization and biodiversity loss. We used 12 montane grassland sites along an 800 km north-south gradient across the UK, to test whether cessation of grazing affects local α- and ß-diversity of below-ground food webs. We show cessation of grazing leads to strongly decreased α-diversity of most groups of soil microbes and fauna, particularly of relatively rare taxa. By contrast, the ß-diversity varied between groups of soil organisms. While most soil microbial communities exhibited increased homogenization after cessation of grazing, we observed decreased homogenization for soil fauna after cessation of grazing. Overall, our results indicate that exclusion of domesticated herbivores from historically grazed montane grasslands has far-ranging negative consequences for diversity of below-ground food webs. This underscores the importance of grazers for maintaining the diversity of below-ground communities, which play a central role in ecosystem functioning.

Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria.

Environmental bacteria host an enormous number of prophages, but their diversity and natural functions remain largely elusive. Here, we investigate prophage activity and diversity in 63 Erwinia and Pseudomonas strains isolated from flag leaves of wheat grown in a single field. Introducing and validating Virion Induction Profiling Sequencing (VIP-Seq), we identify and quantify the activity of 120 spontaneously induced prophages, discovering that some phyllosphere bacteria produce more than 108 virions/mL in overnight cultures, with significant induction also observed in planta. Sequence analyses and plaque assays reveal E. aphidicola prophages contribute a majority of intraspecies genetic diversity and divide their bacterial hosts into antagonistic factions engaged in widespread microbial warfare, revealing the importance of prophage-mediated microdiversity. When comparing spontaneously active prophages with predicted prophages we also find insertion sequences are strongly correlated with non-active prophages. In conclusion, we discover widespread and largely unknown prophage diversity and function in phyllosphere bacteria.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Fecal virome transfer improves proliferation of commensal gut Akkermansia muciniphila and unexpectedly enhances the fertility rate in laboratory mice.

Probiotics are intended to improve gastrointestinal health when consumed. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from mice with high relative abundance of A. muciniphila. All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly (p < 0.05) increased the abundance of naturally occurring A. muciniphila compared to the control. This highlights the potential of propagating the existing commensal "probiotics" that have already permanently colonized the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly (p < 0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating interactions between the gut microbiome and fertility.

Pruning Wound Protection Products Induce Alterations in the Wood Mycobiome Profile of Grapevines.

Fungal pathogens involved in grapevine trunk diseases (GTDs) may infect grapevines throughout their lifetime, from nursery to vineyard, via open wounds in stems, canes or roots. In vineyards, pruning wound protection products (PWPPs) offer the best means to reduce the chance of infection by GTD fungi. However, PWPPs may affect non-target microorganisms that comprise the natural endophytic mycobiome residing in treated canes, disrupting microbial homeostasis and indirectly influencing grapevine health. Using DNA metabarcoding, we characterized the endophytic mycobiome of one-year-old canes of cultivars Cabernet Sauvignon and Syrah in two vineyards in Portugal and Italy and assessed the impact of established and novel PWPPs on the fungal communities of treated canes. Our results reveal a large fungal diversity (176 taxa), and we report multiple genera never detected before in grapevine wood (e.g., Symmetrospora and Akenomyces). We found differences in mycobiome beta diversity when comparing vineyards (p = 0.01) but not cultivars (p > 0.05). When examining PWPP-treated canes, we detected cultivar- and vineyard-dependent alterations in both alpha and beta diversity. In addition, numerous fungal taxa were over- or under-represented when compared to control canes. Among them, Epicoccum sp., a beneficial genus with biological control potential, was negatively affected by selected PWPPs. This study demonstrates that PWPPs induce alterations in the fungal communities of grapevines, requiring an urgent evaluation of their direct and indirect effects on plants health with consideration of factors such as climatic conditions and yearly variations, in order to better advise viticulturists and policy makers.

Four novel Curtobacterium phages isolated from environmental samples.

Despite Curtobacterium spp. often being associated with the plant phyllosphere, i.e., the areal region of different plant species, only one phage targeting a member of the genus Curtobacterium has been isolated so far. In this study, we isolated four novel plaque-forming Curtobacterium phages, Reje, Penoan, Parvaparticeps, and Pize, with two novel Curtobacterium strains as propagation hosts. Based on the low nucleotide intergenomic similarity (<32.4%) between these four phages and any phage with a genome sequence in the NCBI database, we propose the establishment of the four genera, "Rejevirus", "Pizevirus", "Penoanvirus", and "Parvaparticepsvirus", all in the class of Caudoviricetes.

Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Replication Protein Rep Provides Selective Advantage to Viruses in the Presence of CRISPR-Cas Immunity.

Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δgp16) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

Detection of nucleotide modifications in bacteria and bacteriophages: Strengths and limitations of current technologies and software.

RNA and DNA modifications occur in eukaryotes and prokaryotes, as well as in their viruses, and serve a wide range of functions, from gene regulation to nucleic acid protection. Although the first nucleotide modification was discovered almost 100 years ago, new and unusual modifications are still being described. Nucleotide modifications have also received more attention lately because of their increased significance, but also because new sequencing approaches have eased their detection. Chiefly, third generation sequencing platforms PacBio and Nanopore offer direct detection of modified bases by measuring deviations of the signals. These unusual modifications are especially prevalent in bacteriophage genomes, the viruses of bacteria, where they mostly appear to protect DNA against degradation from host nucleases. In this Opinion article, we highlight and discuss current approaches to detect nucleotide modifications, including hardwares and softwares, and look onward to future applications, especially for studying unusual, rare, or complex genome modifications in bacteriophages. The ability to distinguish between several types of nucleotide modifications may even shed new light on metagenomic studies.

Antibiotic resistance genes are differentially mobilized according to resistance mechanism.

BACKGROUND: Screening for antibiotic resistance genes (ARGs) in especially environmental samples with (meta)genomic sequencing is associated with false-positive predictions of phenotypic resistance. This stems from the fact that most acquired ARGs require being overexpressed before conferring resistance, which is often caused by decontextualization of putative ARGs by mobile genetic elements (MGEs). Consequent overexpression of ARGs can be caused by strong promoters often present in insertion sequence (IS) elements and integrons and the copy number effect of plasmids, which may contribute to high expression of accessory genes. RESULTS: Here, we screen all complete bacterial RefSeq genomes for ARGs. The genetic contexts of detected ARGs are investigated for IS elements, integrons, plasmids, and phylogenetic dispersion. The ARG-MOB scale is proposed, which indicates how mobilized detected ARGs are in bacterial genomes. It is concluded that antibiotic efflux genes are rarely mobilized and even 80% of ß-lactamases have never, or very rarely, been mobilized in the 15,790 studied genomes. However, some ARGs are indeed mobilized and co-occur with IS elements, plasmids, and integrons. CONCLUSIONS: In this study, ARGs in all complete bacterial genomes are classified by their association with MGEs, using the proposed ARG-MOB scale. These results have consequences for the design and interpretation of studies screening for resistance determinants, as mobilized ARGs pose a more concrete risk to human health. An interactive table of all results is provided for future studies targeting highly mobilized ARGs.

Salmonella phage akira, infecting selected Salmonella enterica Enteritidis and Typhimurium strains, represents a new lineage of bacteriophages.

Some serovars of Salmonella can cause life-threatening diarrhoeal diseases and bacteriemia. The emergence of multidrug-resistant strains has led to a need for alternative treatments such as phage therapy, which requires available, well-described, diverse, and suitable phages. Phage akira was found to lyse 19 out of 32 Salmonella enterica serovars and farm isolates tested, although plaque formation was observed with only two S. Enteritidis and one S. Typhimurium strain. Phage akira encodes anti-defence genes against type 1 R-M systems, is distinct (<65% nucleotide sequence identity) from related phages and has siphovirus morphology. We propose that akira represents a new genus in the class Caudoviricetes.

Critical Assessment of Metagenome Interpretation: the second round of challenges.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

The Bacillus cereus Strain EC9 Primes the Plant Immune System for Superior Biocontrol of Fusarium oxysporum.

Antibiosis is a key feature widely exploited to develop biofungicides based on the ability of biological control agents (BCAs) to produce fungitoxic compounds. A less recognised attribute of plant-associated beneficial microorganisms is their ability to stimulate the plant immune system, which may provide long-term, systemic self-protection against different types of pathogens. By using conventional antifungal in vitro screening coupled with in planta assays, we found antifungal and non-antifungal Bacillus strains that protected the ornamental plant Kalanchoe against the soil-borne pathogen Fusarium oxysporum in experimental and commercial production settings. Further examination of one antifungal and one non-antifungal strain indicated that high protection efficacy in planta did not correlate with antifungal activity in vitro. Whole-genome sequencing showed that the non-antifungal strain EC9 lacked the biosynthetic gene clusters associated with typical antimicrobial compounds. Instead, this bacterium triggers the expression of marker genes for the jasmonic and salicylic acid defence pathways, but only after pathogen challenge, indicating that this strain may protect Kalanchoe plants by priming immunity. We suggest that the stimulation of the plant immune system is a promising mode of action of BCAs for the development of novel biological crop protection products.

Combining reverse osmosis and microbial degradation for remediation of drinking water contaminated with recalcitrant pesticide residue.

Groundwater contamination by recalcitrant organic micropollutants such as pesticide residues poses a great threat to the quality of drinking water. One way to remediate drinking water containing micropollutants is to bioaugment with specific pollutant degrading bacteria. Previous attempts to augment sand filters with the 2,6-dichlorobenzamide (BAM) degrading bacterium Aminobacter niigataensis MSH1 to remediate BAM-polluted drinking water initially worked well, but the efficiency rapidly decreased due to loss of degrader bacteria. Here, we use pilot-scale augmented sand filters to treat retentate of reverse osmosis treatment, thus increasing residence time in the biofilters and potentially nutrient availability. In a first pilot-scale experiment, BAM and most of the measured nutrients were concentrated 5-10 times in the retentate. This did not adversely affect the abundances of inoculated bacteria and the general prokaryotic community of the sand filter presented only minor differences. On the other hand, the high degradation activity was not prolonged compared to the filter receiving non-concentrated water at the same residence time. Using laboratory columns, it was shown that efficient BAM degradation could be achieved for >100 days by increasing the residence time in the sand filter. A slower flow may have practical implications for the treatment of large volumes of water, however this can be circumvented when treating only the retentate water equalling 10-15% of the volume of inlet water. We therefore conducted a second pilot-scale experiment with two inoculated sand filters receiving membrane retentate operated with different residence times (22 versus 133 min) for 65 days. While the number of MSH1 in the biofilters was not affected, the effect on degradation was significant. In the filter with short residence time, BAM degradation decreased from 86% to a stable level of 10-30% degradation within the first two weeks. The filter with the long residence time initially showed >97% BAM degradation, which only slightly decreased with time (88% at day 65). Our study demonstrates the advantage of combining membrane filtration with bioaugmented filters in cases where flow rate is of high importance.

Muskoxen homogenise soil microbial communities and affect the abundance of methanogens and methanotrophs.

Grazing herbivores may affect soil microbial communities indirectly by impacting soil structure and vegetation composition. In high arctic environments, this impact is poorly elucidated, while having potentially wide-reaching effects on the ecosystem. This study examines how a key arctic herbivore, the muskox Ovibos moschatus, affects the soil microbial community in a high arctic fen. Environmental DNA was extracted from soil samples taken from grazed control plots and from muskox exclosures established 5 years prior. We sequenced amplicons of the 16S rRNA gene to provide insight into the microbial communities. We found that in the grazed control plots, microbial communities exhibited high evenness and displayed highly similar overall diversity. In plots where muskoxen had been excluded, microbial diversity was significantly reduced, and had more uneven intra-sample populations and overall lower ecological richness and evenness. We observed that the composition of microbial communities in grazed soils were significantly affected by the presence of muskoxen, as seen by elevated relative abundances of Bacteroides and Firmicutes, two major phyla found in muskox faeces. Furthermore, an increase in relative abundance of bacteria involved in degradation of recalcitrant carbohydrates and cycling of nitrogen was observed in grazed soil. Ungrazed soils displayed increased abundances of bacteria potentially involved in anaerobic oxidation of methane, whereas some methanogens were more abundant in grazed soils. This corroborates a previous finding that methane emissions are higher in arctic fens under muskox grazing. Our results show that the presence of large herbivores stimulates soil microbial diversity and has a homogenizing influence on the inter-species dynamics in soil microbial communities. The findings of this study, thus, improve our understanding of the effect of herbivore grazing on arctic ecosystems and the derived methane cycling.

Bacteriophages Roam the Wheat Phyllosphere.

The phyllosphere microbiome plays an important role in plant fitness. Recently, bacteriophages have been shown to play a role in shaping the bacterial community composition of the phyllosphere. However, no studies on the diversity and abundance of phyllosphere bacteriophage communities have been carried out until now. In this study, we extracted, sequenced, and characterized the dsDNA and ssDNA viral community from a phyllosphere for the first time. We sampled leaves from winter wheat (Triticum aestivum), where we identified a total of 876 virus operational taxonomic units (vOTUs), mostly predicted to be bacteriophages with a lytic lifestyle. Remarkably, 848 of these vOTUs corresponded to new viral species, and we estimated a minimum of 2.0 × 106 viral particles per leaf. These results suggest that the wheat phyllosphere harbors a large and active community of novel bacterial viruses. Phylloviruses have potential applications as biocontrol agents against phytopathogenic bacteria or as microbiome modulators to increase plant growth-promoting bacteria.

Long-Term Safety Following Faecal Microbiota Transplantation as a Treatment for Recurrent Clostridioides difficile Infection Compared with Patients Treated with a Fixed Bacterial Mixture: Results from a Retrospective Cohort Study.

Faecal microbiota transplantation (FMT) is the recommended treatment for recurrent C. difficile infection (rCDI) following a second recurrence. FMT is considered safe in the short term when procedures for the screening of donors and transferred material are followed. However, the long-term safety profile of FMT treatment is largely unknown. In a retrospective cohort study, we assessed the long-term safety of patients treated for rCDI with FMT or a fixed bacterial mixture, rectal bacteriotherapy (RBT). The overall survival, risk of hospital admission, onset of certain pre-specified diseases (cancer, diabetes mellitus, hypertension and inflammatory bowel disease) and risk of being diagnosed with a multidrug-resistant organism were assessed by undertaking a review of the treated patients' medical records for up to five years following treatment. A total of 280 patients were treated for rCDI with FMT (n = 145) or RBT (n = 135) between 2016 and 2020. In the five years following treatment, there were no differences in survival (adjusted hazard ratio (aHR) 1.03; 95% CI 0.68-1.56), p = 0.89), risk of hospital admission ((aHR 0.92; 95% CI 0.72-1.18), p = 0.5) or onset of any of the analysed diseases. In conclusion, FMT was not associated with increased mortality, risk of hospital admission or onset of disease following treatment when compared with RBT.