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. 2025 Apr 24;13(5):972.
doi: 10.3390/microorganisms13050972.

Multi-Metagenome Analysis Unravels Community Collapse After Sampling and Hints the Cultivation Strategy of CPR Bacteria in Groundwater

Kai Jiang  1   2 , Lijia Ye  1 , Chunling Cao  3 , Gen Che  1 , Yanxing Wang  1 , Yu Hong  1   2
Affiliations

Multi-Metagenome Analysis Unravels Community Collapse After Sampling and Hints the Cultivation Strategy of CPR Bacteria in Groundwater

Kai Jiang et al. Microorganisms. .

Abstract

Groundwater harbors phylogenetically diverse Candidate Phyla Radiation (CPR) bacteria, representing an ideal ecosystem for studying this microbial dark matter. However, no CPR strains have been successfully isolated from groundwater, severely limiting further research. This study employed a multi-metagenome approach, integrating time-resolved sampling, antibiotic/nutrient interventions, and microbial correlation networks to unravel CPR ecological roles in groundwater and provide insights into their subsequent cultivation. Through 36 metagenomes from a groundwater system containing at least 68 CPR phyla, we revealed the time-sensitive collapse of CPR communities: total abundance plummeted from 7.9% to 0.15% within 48 h post-sampling, driven by competition with rapidly dividing non-CPR bacteria, such as members of Pseudomonadota. Ampicillin (100 mg/L) stabilized CPR communities by suppressing competitors, whereas low-nutrient conditions paradoxically reversed this effect. Long-term enrichment (14 months) recovered 63 CPR phyla (0.35% abundance), revealing their survival resilience despite nutrient deprivation. Correlation networks prioritized Actinomyces, a novel Acidimicrobiaceae genus, Aestuariivirga, Baekduia and Caedimonadaceae as potential CPR partners, providing actionable targets for co-culture trials. Here, we propose actionable recommendations spanning groundwater sampling, activation status, identification of CPR symbiotic partners, and optimization of culture conditions, which bypass traditional blind cultivation and are critical for future efforts to cultivate CPR bacterial strains from groundwater. Cultivating CPR bacteria will contribute to clarifying their diversity, ecological roles, evolutionary mechanisms, metabolic pathways, and genetic potential.

Keywords: Candidate Phyla Radiation (CPR); cultivation strategy; groundwater ecosystem; metagenomics; microbial community interactions.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Sample correlations based on gene number. (a) Effects of post-sampling duration. (b) Impact of antibiotics and nutrients supplementation. Darker colors and larger circles indicate higher absolute correlation coefficients.
Figure 2
Figure 2
Effects of post-sampling duration on CPR and DPANN. (a) Relative abundance changes in CPR bacterial phyla. (b) Relative abundance changes in DPANN archaeal phyla. (c) Overall relative abundance trends in CPR bacteria and DPANN archaea. ** denotes ANOVA statistics with p ≤ 0.01, *** denotes ANOVA statistics with p ≤ 0.001.
Figure 3
Figure 3
Impact of antibiotics and nutrients on CPR and DPANN. (a) Relative abundance changes in CPR bacterial phyla. (b) Relative abundance changes in DPANN archaeal phyla. (c) Overall relative abundance trends in CPR bacteria and DPANN archaea. **** denotes ANOVA statistics with p ≤ 0.0001.
Figure 4
Figure 4
Co-occurrence networks. (a) Network based on post-sampling duration experiment. (b) Network based on antibiotic/nutrient experiment. Purple lines: negative correlations; orange lines: positive correlations. Node colors: red (CPR phyla), blue (DPANN phyla), green (family-level microbes positively correlated with CPR in both networks), black (other family-level microbes).
Figure 5
Figure 5
Carbon metabolic potential in groundwater. Filled symbols represent presence, while open symbols indicate absence.
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