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Phage-mediated resistome dynamics in global aquifers

Nature Water volume 4, pages 78–90 (2026) Cite this article

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Abstract

While mobile genetic elements (MGEs) critically influence antibiotic resistance gene (ARG) dissemination, the regulatory role of bacteriophages as unique MGEs remains enigmatic in natural ecosystems. Through a global-scale phage-resistome interrogation spanning 840 groundwater metagenomes, we established a large aquifer resistome repository and uncovered three paradigm-shifting discoveries. First, phages harboured markedly fewer ARGs compared to plasmids and integrative elements, but their bacterial hosts paradoxically maintained the highest anti-phage defence gene inventories, showing an evolutionary equilibrium where investment in phage defence constrains ARG acquisition. Second, lytic phages demonstrated dual functionality characterized with directly suppressing ARG transmission through host lysis while indirectly enriching defence genes that inhibit horizontal gene transfer. Third, vertical inheritance sustained ARGs in 11.2% of MGE-free groundwater microbes. We further extended linkages between ARG profiles, phage defences and biogeochemical genes, revealing phage-mediated co-occurrence of ARGs and denitrification genes in shared hosts. These findings pioneer a phage-centric framework for resistome evolution, guiding phage-based ARG mitigation in groundwater ecosystems.

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Fig. 1: A global atlas of groundwater resistomes reveals expansive ARG landscapes.
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Fig. 2: MGE-mediated ARG mobilization patterns in global groundwater.
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Fig. 3: Interplay between phage infection, host defence systems and ARG dissemination.
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Fig. 4: Vertical gene transfer sustains ARG persistence in aquifer microbiomes.
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Fig. 5: Phage–host dynamics link microbial resistance, defence and aquifer ecosystem functions.
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Data availability

Domestic groundwater data generated for this study have been deposited in the NCBI Sequence Read Archive under accession code PRJNA858913. Publicly available groundwater metagenomes are listed with their BioProject accession numbers in Supplementary Table 2. Source data are provided with the paper.

Code availability

The R scripts used are publicly available via Zenodo at https://doi.org/10.5281/zenodo.17540538 (ref. 76).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant numbers U2240205 and 51721006 to J.R.N.).

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  1. Jinren Ni

    Present address: College of Environmental Sciences and Engineering, Peking University, Beijing, P. R. China

Authors and Affiliations

  1. Eco-environment and Resource Efficiency Research Laboratory, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen, People’s Republic of China

    Huaiyu Cao, Songfeng Liu, Jiawen Wang & Jinren Ni

  2. Environmental Microbiome and Innovative Genomics Laboratory, College of Environmental Sciences and Engineering, Peking University, Beijing, People’s Republic of China

    Huaiyu Cao, Pinggui Cai & Pengwei Li

  3. Environmental Protection Key Laboratory of All Material Fluxes in River Ecosystems, Ministry of Ecology and Environment, Beijing, People’s Republic of China

    Pengwei Li & Jinren Ni

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Contributions

J.R.N. designed the research. H.Y.C., S.F.L. and P.G.C. conducted the statistical analysis with help of P.W.L. and J.W.W. H.Y.C. and J.R.N. wrote the paper. All the authors read and approved the final paper.

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Correspondence to Jinren Ni.

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Nature Water thanks Liping Ma, Yanni Sun and Pingfeng Yu for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Composition and geographic distribution of antibiotic resistance genes (ARGs) across global aquifer metagenomes.

a. Total abundance and prevalence of ARGs detected in all metagenomes. The majority of ARGs (6,935 of 9,681) are sparsely distributed, appearing in < 10% of samples (peripheral ARGs); whereas a core set of 392 ARGs occurs in > 75% of samples (core ARGs). b. Relative abundance of ARG types based on read-level profiling; MLS: macrolide-lincosamide-streptogramin. c. Upset plot showing ARG subtypes uniquely detected in specific continental combinations. d. Composition of ARGs by resistance mechanisms based on MAG-level analysis, reflecting host-associated ARG preferences.

Source data

Extended Data Fig. 2 Characterization of mobile genetic elements (MGEs) associated with transferable ARGs in groundwater.

a. Functional composition of annotated MGEs linked to ARGs in groundwater metagenomes. b. Distribution of transferable ARGs across different MGE types. Transferable ARGs were categorized as MGE-single if they were associated with only one type of MGE, and as MGE-multi if detected in association with more than one type of MGE. c. MGE repertoire associated with the most pervasive transferable ARGs (detected in > 100 MGE sequences). For each ARG, both the number and diversity of linked MGEs are shown.

Source data

Extended Data Fig. 3 Distribution and characteristics of bacterial defence systems in groundwater microbiomes.

a. Composition of defence gene families identified across all groundwater MAGs. b. Distribution of defence-encoding genomes across the ten most represented bacterial phyla. Bars indicate the proportion of genomes carrying defence systems, with the total number of unique DGs per phylum shown alongside. c. Defence gene (DG) counts in MAGs predicted to be phage-susceptible (P-phage, n = 1,458) versus those without phage-linked contigs (NP-phage, n = 1,452). Each point represents the number of DGs in an individual host genome. Statistical significance was evaluated using a two-sided Wilcoxon rank-sum test (p < 2 × 10−16). d. DG counts in L-phage (n = 908) versus NL-phage (n = 376) hosts. Each point represents an individual host genome. Box plots indicate the median (center line and red point), interquartile range (box), and 1.5 × the interquartile range (whiskers). Statistical significance was determined using a two-sided Wilcoxon rank-sum test. e. Number of ARG types associated with NP-phage, NL-phage, and L-phage hosts.

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Extended Data Fig. 4 Defence system distribution between P-phage and NP-phage MAGs across aquatic ecosystems (freshwater, marine, wastewater).

P-phage MAGs consistently encoded more defence systems (DSs) than NP-phage MAGs, with significant differences detected across all ecosystems (two-sided Wilcoxon rank-sum test; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). The magnitude of phage–host antagonism decreased along the gradient from freshwater to marine and wastewater environments. Each point represents the number of DSs in an individual host genome. Box plots indicate the median (center line and red point), interquartile range (box), and 1.5 × the interquartile range (whiskers). Sample sizes and exact p values were as follows: Freshwater: NP-phage (n = 432) and P-phage (n = 249), p = 3.12 × 10⁻11; Marine: NP-phage (n = 252) and P-phage (n = 379), p = 6.82 × 10⁻4; Wastewater: NP-phage (n = 203) and P-phage (n = 210), p = 8.26 × 10⁻3.

Source data

Extended Data Fig. 5 Vertical gene transfer sustains ARG persistence in aquifer microbiomes.

Comparison between the MAG-based phylogenetic tree and the phylogeny of the rsmA resistance gene within two bacterial genera: 202FULL6113 (a) and JADFDG01 (b). Topological similarity between the two phylogenies was assessed using Robinson–Foulds (RF) distance.

Source data

Extended Data Fig. 6 Assessment of vertical and horizontal contributions to ARG dissemination in groundwater microbial communities.

Procrustes analysis at the phylum (a) and genus (b) levels compares microbial community composition (read-based) with ARG subtype profiles. Higher Procrustes m2 values indicate greater deviation from vertical inheritance, suggesting stronger influence of HGT on ARG distribution. Statistical significance was evaluated using a two-sided Procrustes permutation test (999 permutations; p < 0.001).

Source data

Extended Data Fig. 7 Phage–host interactions shape microbial nitrogen cycling potential in aquifer ecosystems.

a. Module completeness of functional gene markers associated with eight nitrogen cycling processes in P-phage (n = 1,458) and NP-phage (n = 1,452) genomes. Each bar represents the mean metabolic completeness for the corresponding nitrogen transformation process within each group, and error bars indicate the standard error of the mean (SEM). Statistical differences between groups were evaluated using a two-sided Wilcoxon rank-sum test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Exact p-values for A–H are: A, p = 1.36 × 10−2; B, p = 0.19; C, p = 8.37 × 10−7; D, p = 5.24 × 10−3; E, p = 2.39 × 10−3; F, p = 0.30; G, p = 2.73 × 10−12; H, p = 1.97 × 10−6. b. Contribution of lytic phages to nitrogen cycling in all phage-infected microbial hosts, illustrated by a metabolic pathway map highlighting their role in aquifer nitrogen transformations.

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Cao, H., Liu, S., Cai, P. et al. Phage-mediated resistome dynamics in global aquifers. Nat Water 4, 78–90 (2026). https://doi.org/10.1038/s44221-025-00558-w

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