ISME communications最新文献

{"title":"Targeted genomic analysis of a predominant uncultured marine pelagiphage-host model via microfluidics and semipermeable capsule technology.","authors":"Manuel Martinez-Garcia, Monica Lluesma-Gomez, Laura Perez-Martin, Esther Rubio-Portillo, Ana Belen Martin-Cuadrado, Francisco Nadal-Molero, Aitana Escolano-Vico, Fernando Santos Sanchez, Victoria Orphan, Josefa Antón","doi":"10.1093/ismeco/ycaf123","DOIUrl":"10.1093/ismeco/ycaf123","url":null,"abstract":"<p><p>Microbes and their viruses drive central biogeochemical cycles on a global scale. Understanding the biology and ecology of virus-host interactions and their impact on ecosystems depends on our ability to develop tools that enable high-throughput screening of ecologically relevant, uncultured virus-host pairs. Viruses infecting Pelagibacterales, the predominant bacteria in surface oceans, have been studied through computational analyses and cultivation efforts. Here, we employ an accessible microfluidics and semi-permeable capsule (SPC) technology to investigate the uncultured pelagiphage vSAG 37-F6-host interactions since it is one of the most abundant and ubiquitous viruses in the marine virosphere. First, we validated this technology using cultured virus-host pairs. Then, marine single cells were microfluidically encapsulated in SPCs, lysed, whole-genome amplified, and screened using fluorescent polymerase chain reaction (PCR) for the presence of a hallmark gene of vSAG 37-F6. Data indicate that ~30% of the targeted cell population (cell fraction ≤0.45 μm) contained the virus vSAG 37-F6-like. A total of ~500 putatively infected cells were sorted, combined, and sequenced. Data showed that most reads (~60%) and assembled genome fragments (~85%) were identified as viral, indicating that the sorted host cells were likely in the final stages of infection. Two major viral clusters were detected: one corresponding to vSAG 37-F6 and another mixed viral cluster consisting of cyanophages, pelagiphages, and vibriophages. A significant proportion of total reads (~20%) were assigned to <i>Pelagibacter</i> spp. TMED287, a bacterium reported to be abundant in the Mediterranean Sea. This flexible microfluidic-SPC technology holds enormous potential for exploring uncultured microbial and viral communities across various perspectives and microbiology fields.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf123"},"PeriodicalIF":6.1,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12404659/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144994502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ecological resilience in ulcerative colitis: microbial dynamics of donor and resident species in a longitudinal fecal microbiota transplantation study.","authors":"Susanne Pinto, Elisa Benincà, Sam Nooij, Elisabeth M Terveer, Josbert J Keller, Andrea E van der Meulen-de Jong, Ewout W Steyerberg, Johannes A Bogaards","doi":"10.1093/ismeco/ycaf119","DOIUrl":"10.1093/ismeco/ycaf119","url":null,"abstract":"<p><p>Fecal microbiota transplantation (FMT) is a promising treatment for the chronic immune-mediated disease ulcerative colitis (UC). However, the microbial dynamics underlying clinical remission remain poorly understood. To investigate these dynamics, we analysed data from 22 UC patients treated with four rounds of FMT donated by two healthy donors. Microbiota samples from patients were collected at nine timepoints before, during, and after treatment, covering a period of 14 weeks. Additionally, 27 donor samples were analysed. Species in the recipients' gut microbiota were categorised into ecological categories based on their origin and temporal dynamics: species already present in the recipient pre-FMT, species derived from the donor, or novel species, i.e. absent before FMT in both recipient and donor but detected during or after treatment. Overdispersed Poisson regression models were employed to model the number of species within each category over time. Furthermore, we investigated the change in relative abundance for recipient, colonising, and novel species. The results revealed that recipient species with higher relative abundances prior to FMT were more likely to persist following FMT. Notably, patients who achieved combined clinical and endoscopic remission at week 14 retained a higher number of recipient species compared to non-responders. In contrast, non-responders initially exhibited colonisation of more donor species than responders, but colonisation rate decreased over time in non-responders whereas colonisation rate remained stable in responders. These findings suggest that clinical remission following FMT is associated with controlled incorporation of donor species without replacement of resident species, which may reflect a resilient recipient gut community.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf119"},"PeriodicalIF":6.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12378841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144981530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Salinity-driven niche differentiation within the aquatic Luna-1 subcluster.","authors":"Annie G West, Jian Sheng Boey, Hwee Sze Tee, Kim M Handley","doi":"10.1093/ismeco/ycaf122","DOIUrl":"10.1093/ismeco/ycaf122","url":null,"abstract":"<p><p>Salinity imposes a major barrier to microbial dispersal and colonization due to the requirement for osmoadaptations to maintain cell turgor and protein stability. Estuaries may facilitate infrequent evolutionary transitions between freshwater and marine habitats, which are characterized by differences in both salinity and resource availability. Here we illustrate niche differentiation of the <i>Actinomycetota</i> Luna-1 subcluster sister lineages within an estuarine system: freshwater-adapted <i>Rhodoluna</i> and saltwater-adapted <i>Aquiluna</i>. Comparative genomic and transcriptomic analyses highlighted key differences in osmoregulation, photoheterotrophy, and nutrient acquisition. Both genera are differentiated by mechanisms for osmoregulation, phosphate and iron uptake, and carbohydrate utilization, and by their rhodopsin preference (actinorhodopsin or heliorhodopsin). To clarify which traits are habitat versus lineage specific, we investigated the global distribution of Luna-1 subcluster taxa. The two constituent genera are both more commonly known from freshwater sources, although there are reports of <i>Aquiluna</i> isolated from saltwater. Results here confirm that <i>Rhodoluna</i> is almost exclusively freshwater-derived. <i>Aquiluna</i> instead comprises distinct clades of predominantly freshwater- or saltwater-derived taxa, with approximately half of <i>Aquiluna</i> representing slight halophiles from brackish and marine waters. Consistent with observations from the estuary, traits associated with osmoregulation and photoheterotrophy (rhodopsin preference and carbohydrate utilization) differentiated saltwater <i>Aquiluna</i> and freshwater members of the global dataset (both <i>Aquiluna</i> and <i>Rhodoluna</i>), and are therefore likely to be habitat rather than lineage-specific traits. Together, findings demonstrate various genomic characteristics enabling habitat-based niche differentiation between and within lineages of the Luna-1 subcluster, providing insights into microbial adaptation across salinity gradients.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf122"},"PeriodicalIF":6.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12422013/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145042392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Pseudosulfitobacter pseudonitzschiae</i> hitchhikes on gliding colonies of <i>Cellulophaga lytica</i>.","authors":"Asimenia Gavriilidou, Maria Murace, Marina Portoghese, Sanne Schouten, Raditijo Hamidjaja, Álvaro Escobar Doncel, Sjef Boeren, Marcel Giesbers, Jérémie Capoulade, Silvia Vignolini, Hauke Smidt, Colin J Ingham","doi":"10.1093/ismeco/ycaf118","DOIUrl":"10.1093/ismeco/ycaf118","url":null,"abstract":"<p><p>Interspecies interactions shape microbial communities; this is central for microbial ecology. <i>Cellulophaga lytica</i> PlyA2 is a marine flavobacterium, which glides over surfaces and forms ordered, structurally coloured colonies, which display angle-dependent reflection of light. <i>Pseudosulfitobacter pseudonitzschiae</i> SW is an apparently nonmotile, nonstructurally coloured marine bacterium. Here, we aim to understand the interaction of both strains at cellular, genomic, optical, and proteomic levels. Cocultivation on agar showed that <i>P. pseudonitzschiae</i> uses gliding <i>C. lytica</i> to spread by microbial hitchhiking in which <i>Pseudosulfitobacter</i> appears to \"surf\" on basal layers of motile <i>Cellulophaga</i>. This dispersal mechanism was found to be often beneficial for <i>P. pseudonitzschiae,</i> which could maximally expand its population up to 350-fold relative to monoculture. Coculture was often of limited benefit for <i>C. lytica</i>, only in extended cultivation on rich medium was the presence of <i>P. pseudonitzschiae</i> detrimental to its viability. The proteome of <i>P. pseudonitzschiae</i> was strongly impacted by the association with <i>C. lytica</i>. Quorum-sensing signalling, potential exchange of amino acids, vitamins, and other metabolites are likely mediating this hitchhiking interaction. In contrast, <i>C. lytica</i> made minimal adjustments to its proteome composition in coculture. Supported by optical analysis, <i>P. pseudonitzschiae</i> patterned <i>C. lytica</i> by changing how groups of the latter organised to reflect light. Our results underscore the unusual, dynamic interplay between two bacterial species and provide insights on the mechanisms underlying this relationship.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf118"},"PeriodicalIF":6.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12376047/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144981445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Greater host influence and promiscuity: how an invasive seaweed host has advantages over co-occurring natives.","authors":"Marjan Ghotbi, Guido Bonthond, Mitra Ghotbi, Sven Künzel, David M Needham, Florian Weinberger","doi":"10.1093/ismeco/ycaf120","DOIUrl":"10.1093/ismeco/ycaf120","url":null,"abstract":"<p><p>The surface microbiome of seaweed hosts is a multi-domain biofilm regulated by host-microbe and microbe-microbe interactions. The extent to which hosts influence these interactions, and potentially affect their resilience and invasion success, remains unclear. We experimentally tested whether hosts with invasion history exert greater influence over their biofilms than native hosts. Biofilm formation on proxy surfaces adjacent to one invasive (<i>Gracilaria vermiculophylla</i>) and two native (<i>Fucus serratus, Fucus vesiculosus</i>) co-occurring hosts was monitored and compared to mature epiphytic biofilms of the same hosts. Only <i>Gracilaria's</i> Proxy Biofilms (PBs) were significantly different in community composition compared to control surfaces. <i>Gracilaria</i>'s PBs also showed the highest similarity to their adjacent algae sharing certain bacterial taxa that were absent in control treatments, indicating that colonization of the proxy surface was influenced by the host. <i>Gracilaria</i> and its proxy biofilms showed highest similarity in microbial network variables, suggesting a higher ability of the invader to influence connectivity and microbial associations within its biofilm. Meanwhile <i>Gracilaria</i>'s mature biofilm also showed higher variability in its prokaryotic composition over experiments, which was also reflected in a less robust microbial network in both <i>Gracilaria</i> and its proxy biofilms. This suggests that in addition to stronger influence in the invasive host, it was also more promiscuous towards potential symbionts from the environment. Ultimately, through examining microbial interactions, in line with previous research we found that host influence and promiscuity may play an important role in seaweed hosts to acclimate to different environmental condition and successfully thrive in new ecosystems.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf120"},"PeriodicalIF":6.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12449580/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145115145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput cultivation and isolation of environmental anaerobes using selectively permeable hydrogel capsules.","authors":"Hugo Sallet, Marion Calvo, Matteo Titus, Nicolas Jacquemin, Karin Lederballe Meibom, Rizlan Bernier-Latmani","doi":"10.1093/ismeco/ycaf117","DOIUrl":"10.1093/ismeco/ycaf117","url":null,"abstract":"<p><p>Over the past two decades, metagenomics has greatly expanded our understanding of microbial phylogenetic and metabolic diversity. However, most microbial taxa remain uncultured, hindering research and biotechnological applications. Isolating environmental anaerobes using traditional methods is particularly cumbersome and low throughput. Here, we present a novel, high-throughput approach for the cultivation and isolation of anaerobes, which involves trapping and growing single microbes within selectively permeable hydrogel capsules followed by fluorescence-activated cell sorting to distribute compartmentalized isolates into liquid medium for further growth. We show that diverse anaerobes can grow within capsules and that slower-growing ones (e.g. methanogens) can be enriched with this platform. We also applied this approach to isolate anaerobes from soil, including strains of the sulfate-reducing bacteria <i>Desulfovibrio desulfuricans</i> and <i>Nitratidesulfovibrio vulgaris</i>. Overall, this work introduces a robust, high-throughput alternative to traditional techniques for isolating environmental anaerobes and expands the emerging set of microfluidics-based tools for the cultivation of novel taxa.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf117"},"PeriodicalIF":6.1,"publicationDate":"2025-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12319321/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144786131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Expanding the use of circulating microbiome in fish: contrast between the gut and blood microbiome of <i>Sebastes fasciatus</i>.","authors":"Fanny Fronton, Arthur Gandin, David Deslauriers, Daniel Small, Dominique Robert, Yves St-Pierre","doi":"10.1093/ismeco/ycaf116","DOIUrl":"10.1093/ismeco/ycaf116","url":null,"abstract":"<p><p>The study of microbiomes in fish populations offers vital insights for ecological and fisheries management, particularly in responses to environmental changes. Although traditional studies have concentrated on the gut microbiome, the emerging concept of a circulating blood microbiome suggests it may act as an early indicator of dysbiosis and various health conditions by reflecting transient bacterial DNA presence. In this study, we examined the gut and blood microbiomes of <i>Sebastes fasciatus</i> (Storer, 1854), a species of redfish of significant economic and ecological importance in the Gulf of St. Lawrence, to obtain critical information for health monitoring, pathogen detection, and ecological management in fisheries. Our results revealed that the gut and blood microbiomes of <i>S. fasciatus</i> have distinct bacterial DNA signatures, with significant differences in microbial diversity. Notably, although both microbiomes exhibited similar dominant genera, specific amplicon sequence variants varied significantly. Through a controlled experimental design, we found that the dietary impacts on microbiome composition were statistically significant yet minimal, suggesting that environmental factors play a more substantial role in shaping microbial communities. Finally, we report the presence of potential pathogens and opportunistic bacteria found exclusively in the blood microbiome. Our results highlight the blood microbiome's value as a sensitive health and environmental stress indicator, essential for sustainable fish population management. Integrating microbiome indicators can improve fisheries management and ecosystem sustainability, offering a model applicable to various marine species and environments.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf116"},"PeriodicalIF":6.1,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12342392/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Use of sequence barcodes for tracking horizontal gene transfer of antimicrobial resistance genes in a microbial community.","authors":"Veera Partanen, Svjetlana Dekić Rozman, Antti Karkman, Johanna Muurinen, Teppo Hiltunen, Marko Virta","doi":"10.1093/ismeco/ycaf113","DOIUrl":"10.1093/ismeco/ycaf113","url":null,"abstract":"<p><p>One of the most important knowledge gaps in the antimicrobial resistance crisis is the lack of understanding regarding how genes spread from their environmental origins to bacteria pathogenic to humans. In this study our aim was to create a system that allows the conduction of experiments in laboratory settings that mimic the complexity of natural communities with multiple resistance genes and mobile genetic elements circulating at the same time. Here we report a new sequence-based barcode system that allows simultaneous tracking of the spread of antimicrobial resistance genes from multiple genetic origins. We tested this concept with an experiment in which we added an antimicrobial resistance gene to different genetic environments in alive and dead donors and let the gene spread naturally in an artificial microbial community under different environmental conditions to provide examples of factors that can be investigated. We used emulsion, paired-isolation, and concatenation polymerase chain reaction to detect the new gene carriers and metagenomic analysis to see changes in the genetic environment. We observed the genes moving and were able to recognise the barcode from the gene sequences, thus validating the idea of barcode use. We also saw that temperature and gene origin had effects on the number of new host species. Our results confirmed that our system worked and can be further developed for more complicated experiments.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf113"},"PeriodicalIF":6.1,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12343072/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Water mass mixing controls methane cycling and emission in highly hydrodynamic regions of the open ocean.","authors":"Xiao-Jun Li, Jinyan Wang, Hao-Nan Wang, Shuang Li, Zhen Zhou, Zhao-Hui Chen, Jiarui Liu, Gui-Ling Zhang, Hong-Hai Zhang, Gui-Peng Yang, Jonathan D Todd, Guang-Chao Zhuang","doi":"10.1093/ismeco/ycaf114","DOIUrl":"10.1093/ismeco/ycaf114","url":null,"abstract":"<p><p>Ocean circulations and water mass exchange can exert significant influences on seawater biogeochemistry, microbial communities, and carbon cycling in marine systems. However, the detailed mechanisms of the impacts of physical processes in the open ocean on the cycle of greenhouse gases, particularly methane, remain poorly understood. In this study, we integrated high-resolution underway observations, experimental incubations, radioisotope labelling, and molecular analysis to constrain the controls of methanogenic pathways, methanotrophic activity, and emission fluxes in the highly hydrodynamic Kuroshio and Oyashio Extension (KOE) region of the Northwest Pacific. The mixing of high-temperature, nutrient-rich Kuroshio waters with methane-rich Oyashio currents significantly affected not only methane abundance, but also methane production pathways and oxidation rates. Water mass mixing caused changes in the dominance of phytoplankton communities to <i>Bacillariophyta</i>, with less production of the methane precursor dimethylsulphoniopropionate, thus reducing dimethylsulphoniopropionate-dependent methanogenesis. The alteration of nutrient levels due to mixing of Kuroshio and Oyashio at KOE is also likely to affect microbial utilization of dissolved organic phosphorus, thus influencing methane production from the C-P cleavage of methylphosphonate. Furthermore, the abundances of methanotrophs, such as <i>Methylocystis</i> and <i>Methylosinus</i>, were much higher at the KOE sites than those observed at the Oyashio Extension, which contributed to elevated methane oxidation rates in the mixing region. Microbial oxidation as a biological sink of methane accounted for ~43.7% ± 28.8% of the total methane loss, which reduced methane emissions to the atmosphere. These data highlight the physical controls on biogeochemical methane cycling, indicating that intensive mixing of water masses may regulate methane emissions from the open oceans.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf114"},"PeriodicalIF":6.1,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12342928/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples.","authors":"Veronika V Koziaeva, Katja Engel, Josh D Neufeld","doi":"10.1093/ismeco/ycaf115","DOIUrl":"10.1093/ismeco/ycaf115","url":null,"abstract":"<p><p>Digital polymerase chain reaction (dPCR) is a DNA quantification technology that offers absolute quantification of DNA templates. In this study, we optimized and validated a chip-based dPCR EvaGreen assay with commonly used 16S rRNA gene primer pairs and compared its performance to quantitative real-time PCR (qPCR). We compared measurements of low amounts of template DNA using a newly designed synthetic DNA standard to assess precision, accuracy, and sensitivity. Optimization approaches were tested to minimize partitions with intermediate fluorescence levels between true positive and true negative partitions (so-called \"rain\") for dPCR. Both dPCR and qPCR demonstrated similar quantification performance, with variability in accuracy increasing for samples containing fewer than 30 copies μl^-1 template concentrations. Both tested 16S rRNA gene primer sets amplified non-target template contaminants within both qPCR and dPCR mixtures, which could not be eliminated by ultraviolet light or DNAse treatment and negatively affected the apparent sensitivity of both PCR assays. Digital PCR was less susceptible to common PCR inhibitors, such as ethanol and humic acids, but was more susceptible to tannic acid inhibition than qPCR. These findings demonstrate the suitability of dPCR for 16S rRNA gene quantification of low biomass environmental samples.</p>","PeriodicalId":73516,"journal":{"name":"ISME communications","volume":"5 1","pages":"ycaf115"},"PeriodicalIF":6.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12342375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}