Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.09.08.750087

Single-time-point shotgun metagenomics of four Irish Integrated Constructed Wetlands reveals microbial dynamics in wastewater treatment.

Tumeo, A.·Streparola, G.·Harrington, C.·Carty, A.·Leonard, F.·Burgess, C. M.·Morris, D.·Miliotis, G.
View original source ↗Export metadata ↓

Abstract

Over the past 20 years, the Integrated Constructed Wetland (ICW) concept has been applied in Ireland for wastewater treatment, offering a nature-based solution for reducing pollutants and antimicrobial resistance genes (ARGs) in effluents and receiving environments. However, wastewater microbial communities remain inadequately characterized and their dynamics across treatment largely unexplored. Here, we present a culture-independent investigation of four Irish ICWs, aiming to advance our understanding of microbial dynamics in wastewater treatment. Shotgun metagenomic sequencing was conducted on influent and effluent samples collected in biological triplicates from four ICWs treating agricultural, companion animal, industrial, and municipal wastewater, alongside eight positive and ten negative controls for filtering and sequencing. End-to-end metagenomic analysis was performed with SqueezeMeta. Taxonomic placement of high-quality metagenome-assembled-genomes (HQ-MAGs) was confirmed with GTDB-Tk. Decontamination and statistical analyses were conducted in R. Coassembled contigs were screened for ARGs, virulence- and plasmid-associated sequences. Results revealed statistically significant shifts in taxonomic and functional profiles across treatment. Effluent populations exhibited generally higher richness than corresponding influents, yet were more similar to one another across locations. Multi-log-scale increases were observed in the relative abundance of environmental genera (Legionella, Methylotenera, Thiothrix), alongside reductions in common faecal/human indicators (Bacteroides, Lactococcus, Prevotella), and up to 80% ARGs removal. Collectively, our findings provide insights into ICW efficacy, showing that they can drive marked shifts in wastewater microbiome and ARG reduction. Our culture-independent approach retrieved 34 HQ-MAGs, including 19 potentially novel taxa, uncovering previously uncharacterized microbial diversity that is potentially unique to the Irish environment and warrants further investigation.

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Tumeo, A., Streparola, G., Harrington, C., Carty, A., Leonard, F., Burgess, C. M., Morris, D., Miliotis, G.. 2026-09-09. Single-time-point shotgun metagenomics of four Irish Integrated Constructed Wetlands reveals microbial dynamics in wastewater treatment.. https://doi.org/10.64898/2026.09.08.750087

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗
bioRxiv2026

The iron-binding siderophore enterobactin is required for the response of multi-drug resistant Klebsiella pneumoniae to zinc limitation

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

microbiology↗
bioRxiv2026

A microbiota-derived protease links phage susceptibility to host epithelial responses

Bacteriophages are major ecological drivers of gut microbial ecology, yet whether bacterial mechanisms that determine phage susceptibility have consequences for the mammalian host remains poorly understood. Here, we identify dipeptidyl peptidase 11 (Dpp11a), the predominant active serine protease of the prevalent gut commensal Phocaeicola vulgatus, as an unexpected bacterial defence factor. Dpp11a protects against environmental proteases and confers resistance to bacteriophage infection. Metatranscriptomic analyses further reveal increased expression of both dpp11a and P. vulgatus-associated phage transcripts in ulcerative colitis stool samples, indicating that both components of this interaction are transcriptionally active in disease-associated human microbiomes. Using the microfluidic gut-on-a-chip co-culture model HuMiX, we show that the absence of Dpp11 is accompanied by altered epithelial tight-junction remodelling during phage-bacterial infection. Together, our findings reveal that the consequences of bacterial phage defence can extend beyond phage-bacterium interactions to the mammalian epithelium.

microbiology↗