Because ducks live in direct contact with their litter, a fecal-oral transmission route is established that raises the risk of enteritis, viral hepatitis, and parasitic disease. Standard practice calls for thorough litter removal and replacement after each flock, followed by stringent washing, disinfection, and resting of the housing. But rising costs and limited availability of litter materials mean many farmers simply sun-dry and till the existing litter between batches, a shortcut that produces frequent disease outbreaks, heavier antibiotic use, and reduced farming efficiency, particularly in southern China. Against this backdrop, a team of researchers from Fujian Agriculture and Forestry University and the Fujian Academy of Agricultural Sciences set out to test whether a fermentation technology could attack the problem at its biological source, and their findings, published in Frontiers in Microbiomes, offer a detailed portrait of what happens to the viral and resistance-gene landscape of duck litter when high-temperature fermentation is applied.

The technology under investigation, known as compound microbial intelligent membrane high-temperature fermentation, integrates three components: a compound microbial agent, functional nano- and molecular membranes that cover the compost pile, and an intelligent control system that regulates the fermentation process. The microbial agent used in the study combined Bacillus subtilis, Bacillus licheniformis, Lactobactillus plantarum, and Saccharomyces cerevisiae at defined concentrations, applied at one kilogram per ton of litter material. The functional molecular membrane allows water vapor to evaporate while containing malodorous gases including ammonia and hydrogen sulfide, and the enclosed process prevents the leachate contamination of soil and groundwater that traditional open composting can cause through rainwater runoff. Bioheat generated by microbial fermentation maintains pile temperatures within a range of 80 to 85 degrees Celsius, hot enough to inactivate pathogens and weed seeds, and the entire process requires no turning or dedicated factory buildings, allowing one operator to manage multiple piles simultaneously.

To evaluate the technology’s impact, the researchers collected litter samples from four commercial duck farms in Fujian Province, five samples per farm for a total of twenty. Farm 1 employed deep-layer fermentation without prophylactic antibiotics, designated DL-D, with fermentation piles constructed to a height of 2.0 to 2.5 meters. Farm 2 used shallow-layer fermentation without prophylactic antibiotics, DL-S-NAF, with piles 0.8 to 1.2 meters high. Farm 3 was a conventional operation without fermentation or prophylactic antibiotics, DL-S, and Farm 4 was a conventional farm using prophylactic antibiotics without fermentation, DL-S-ANF. Piles were covered with the functional intelligent membrane and fermented for 15 to 18 days, with the intelligent control system maintaining aeration at 0.3 to 0.5 cubic meters per minute per ton of material and temperature monitored daily at three depths. Initial moisture content was adjusted to 55 to 65 percent.

The analytical approach, called metaviromics, involved extracting both DNA and RNA from the litter samples, enriching viral particles through sequential filtration and polyethylene glycol precipitation, and subjecting the resulting nucleic acids to high-throughput sequencing. Approximately 1.01 billion raw paired-end reads were generated from the twenty DNA virome samples, totaling roughly 151.5 gigabases. After quality filtering, host genome subtraction against the duck reference genome, and de novo assembly using MEGAHIT, the team screened contigs of at least 2,000 base pairs using DeepVirFinder, VirSorter2, VIBRANT, and the IMG/VR database, identifying 57,032 candidate viral sequences. Quality assessment with CheckV and removal of sequences classified as Not-determined left 23,130 viral sequences, which clustered at 95 percent average nucleotide identity over 85 percent alignment coverage into 18,321 viral operational taxonomic units, or vOTUs, with a combined length of 95.33 megabases.

The results revealed a clear structural pattern in the litter virome. DNA viral communities were dominated by the phyla Uroviricota and Nucleocytoviricota, with Caudoviricetes and Megaviricetes as the predominant classes, and their relative abundances remained remarkably stable across all four treatment groups, with Uroviricota accounting for roughly 94 to 95 percent of DNA viral reads in every group. RNA viral communities told a different story. Pisuviricota and Lenarviricota emerged as the dominant phyla, and their proportions fluctuated markedly among treatments: Pisuviricota reached 85.27 percent in the DL-S-NAF group but only 68.79 percent in the unfermented DL-S group, while Lenarviricota ranged from 13.75 percent in DL-S-NAF to 28.07 percent in DL-S. At the class level, Pisoniviricetes and Leviviricetes dominated the RNA viral assemblage, with Pisoniviricetes peaking at 82.17 percent in the shallow-layer fermentation group.

Crucially, the fermentation treatments significantly reduced RNA viral abundance, with a corresponding decreasing trend for DNA viruses. Alpha diversity analysis showed no significant differences in DNA viral richness or diversity among groups, suggesting high structural stability of the bacteriophage-dominated DNA communities, but the Shannon and Simpson indices for RNA viruses revealed significant differences between the unfermented DL-S group and both fermentation groups. Beta diversity analysis using non-metric multidimensional scaling and principal coordinates analysis confirmed significant differences in viral community structure across the experimental groups, with the first two principal coordinates accounting for 63.32 percent of DNA viral community variation and 55.39 percent of RNA viral community variation. The heightened sensitivity of RNA viruses is mechanistically attributable to the chemical lability of RNA under the alkaline pH generated by ammonia release during fermentation, coupled with lipolytic disruption of viral envelopes at elevated temperatures.

The resistome findings were equally striking. Total antibiotic resistance gene abundance followed a clear hierarchical trend of DL-D less than DL-S-NAF less than DL-S less than DL-S-ANF, indicating that antibiotic usage was the principal driver of ARG enrichment while fermentation was an effective method for their reduction. Fermentation treatments markedly decreased the loads of specific ARGs including sul1, sul2, tetB(P), and qacEdelta1, which confer resistance to sulfonamides and tetracyclines. The abundances of floR, aadA, qacEdelta1, tet(X), and cfr(A) in the three antibiotic-free groups were significantly lower than in the antibiotic-using farm group. Metal resistance genes including arsC, arsM, merA, copR, and corR showed a parallel reduction, with the overall MRG trend being DL-D less than DL-S-NAF less than DL-S-ANF less than DL-S. Biocide resistance genes including mdeA, actP, smdB, cpxA, and galE, and bacterial virulence factors including groEL2 and lirB, were similarly attenuated by the fermentation process.

A counterintuitive finding was the relatively high abundance of efflux pump mechanism genes such as TaeA, conferring pleuromutilin and tiamulin resistance, in the deep-layer fermentation group. The researchers suggest this may reflect adaptive mechanisms activated by microorganisms in response to metabolic stress such as organic acid accumulation within the fermentation environment, or alternatively that these efflux pumps serve dual physiological functions including metabolite export and pH homeostasis rather than mediating antibiotic resistance per se. The substrate promiscuity of MFS and RND-family efflux transporters, fulfilling ancestral roles in metabolite export under acidic fermentation conditions, offers a plausible explanation for this apparent outlier. Similarly, the opposing MRG trends between unfermented and fermented groups likely reflect redox-driven metal speciation shifts: oxidative dissolution in aerated shallow litter mobilizes bioavailable metals that select for MRG maintenance, whereas sulfidogenic conditions during thermophilic fermentation promote metal precipitation, alleviating this selective pressure.

An exploratory single-sample in-depth metavirome analysis, conducted on a representative DL-D sample because it exhibited the most distinct viral community profile and the lowest ARG abundance, identified 34 viral operational taxonomic units and 343 viral genes, of which 210 received functional annotation in at least one database. The viral community was dominated by Uroviricota members of the families Winoviridae, Salasmaviridae, Mesyanzhinovviridae, and Orlajensenviridae, with Nucleocytoviricota members including Mimiviridae also well-represented. KEGG pathway enrichment showed viral genes were predominantly associated with metabolism and genetic information processing, with translation-related functions constituting the largest category. The resistome analysis identified ARG subtypes including TaeA, Bado_rpoB_RIF, and aminocoumarin resistance genes, MRGs conferring resistance to arsenic, mercury, chromium, copper and zinc, and nickel and cobalt, BRGs associated with quaternary ammonium compounds, triclosan, hydrogen peroxide, and hydrochloric acid, and virulence factors involved in effector delivery, immune modulation, biofilm formation, exoenzyme production, and stress survival.

The co-enrichment of biocide resistance genes and virulence factors within the antibiotic-using farm group provides evidence for the prevalent co-occurrence of disinfectant resistance, antibiotic resistance, and virulence determinants in duck farming environments, driven by physical linkage of these determinants on class 1 integrons and conjugative plasmids that enable antibiotic-mediated co-selection to amplify resistance and virulence traits through genetic hitchhiking. This finding aligns with prior work on Riemerella anatipestifer, a major duck pathogen, in which isolates from Guizhou province harbored quaternary ammonium compound resistance genes while demonstrating severe multidrug resistance, and whole-genome studies reporting that over 90 percent of R. anatipestifer isolates carried tet(X). The detection of Riemerella phage vB_RanS_CRP19 in the litter virome suggests the viral community may harbor phages with potential biocontrol applications against this pathogen.

The study’s authors conclude that RNA viruses, particularly Pisuviricota, are sensitive bioindicators for ecological health assessment in litter environments, and that the antibiotic-free fermentation system represents a promising strategy for improving duck health and welfare while curbing the dissemination of antimicrobial resistance at its source in poultry production. The phylum Pisuviricota encompasses significant duck pathogens including Duck Tembusu virus, and its abundance patterns may reflect the interplay of sustained high temperatures and acidic conditions during fermentation that inhibit certain bacteria while providing competitive advantages to thermotolerant or acidophilic organisms and their associated viruses. The notable enrichment of Negarnaviricota, which includes influenza viruses, in the deep-layer fermentation group indicates that deep anaerobic environments may be more conducive to the persistence of such viruses, suggesting that fermentation design parameters matter.

Several limitations temper the conclusions. The sample size of five per group was relatively modest, potentially limiting statistical power to detect subtle differences in viral community composition. The cross-sectional design provides only a snapshot of the virome and resistome, and longitudinal studies are needed to understand temporal dynamics during fermentation. Functional validation through culture-based or in vitro experiments is warranted to confirm the biological significance of the identified resistance genes and virulence factors, and the study was conducted in a single geographic region, so generalizability to other climatic zones and farming systems requires further investigation. The mechanistic interpretations advanced, including thermal inactivation kinetics, host-community turnover, and redox-driven metal speciation, remain hypotheses warranting validation through time-course studies integrating metaviromic, metatranscriptomic, and metabolomic measurements.

Nevertheless, the work provides the first comprehensive metaviromic characterization of duck litter treated with compound microbial intelligent membrane fermentation, and its central message is clear: this technology effectively reduces viral abundance, particularly that of RNA viruses, and significantly decreases the loads of antibiotic resistance genes, metal resistance genes, biocide resistance genes, and virulence factors. In an era when antimicrobial resistance is recognized as one of the foremost threats to global health, interventions that curb the dissemination of resistance genes at their agricultural source carry significance well beyond the duck house. The finding that RNA viruses respond more sensitively to treatment than their DNA counterparts offers a practical monitoring tool, and the demonstration that an antibiotic-free fermentation system can attenuate the co-enrichment of resistance and virulence determinants points toward farming practices that protect both animal welfare and public health.

Subject of Research: Compound microbial intelligent membrane fermentation reshapes the virome and reduces various resistance genes in duck litter

Article Title: Compound microbial intelligent membrane fermentation reshapes the virome and reduces various resistance genes in duck litter

Article References: Sun, T., Mei, G., Mo, J., Li, M., Ma, Y., Zhao, M., Peng, S., Lin, F., Chen, J., Ma, Y., & Li, Z. (2026). Compound microbial intelligent membrane fermentation reshapes the virome and reduces various resistance genes in duck litter. Frontiers in Microbiomes, 5, Article 1897965. https://doi.org/10.3389/frmbi.2026.1897965

Image Credits: AI Generated

DOI: 10.3389/frmbi.2026.1897965

Keywords: Compound, microbial, intelligent, membrane, fermentation, reshapes, virome, reduces, various, resistance, genes, duck