Mario Romero-Rivera1,2,3†Miguel D. Fernández-de-Bobadilla4†María Beltrán5
Rosa del Campo1,6,7*
José Avendaño-Ortiz1,6*
Cristina Herencias1,6*- 1Departamento de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria IRYCIS, Madrid, Spain
- 2Department of Microbiology, Universidad Complutense de Madrid, Madrid, Spain
- 3Facultad de Química y Farmacia, Universidad de El Salvador, San Salvador, El Salvador
- 4Instituto de Biología Funcional y Genómica, CSIC, Salamanca, Spain
- 5Universidad Autónoma de Madrid, Madrid, Spain
- 6CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid, Spain
- 7Facultad de Ciencias de la Salud, Universidad de La Rioja, Logroño, Spain
Introduction: Predatory bacteria of the Bdellovibrio and like organisms (BALOs) have long been postulated as living antimicrobials, yet their occurrence and ecological roles within human-associated microbiota have remained uncertain due to the absence of culturable human-derived isolates. Here, we report the first successful isolation and comprehensive characterization of viable Bdellovibrio bacteriovorus from human fecal samples.
Methods: Targeted enrichment was applied to five pooled fecal samples to facilitate predator recovery. We performed whole-genome sequencing on the isolates and conducted comparative genomics across 162 publicly available Bdellovibrio genomes. Additionally, pangenome analysis of 22 high-quality genomes and phenotypic assays against clinical pathogens were conducted to assess genomic diversity, prey specificity, and biosafety profiles.
Results: Despite extremely low natural abundance, targeted enrichment recovered predators in two of five pooled samples, which produced characteristic lytic plaques. Sequencing revealed >99% average nucleotide identity to reference strain HD100 with only 26 core single-nucleotide polymorphisms across both isolates, indicating minimal divergence between human-associated and environmental lineages. Comparative genomics showed that only 10.4% of public genomes fulfill criteria for B. bacteriovorus sensu stricto. Pangenome analysis revealed a stable, highly conserved core (~2,500–2,650 genes) and an expanding accessory genome. Phenotypically, the human-derived isolates displayed narrower prey ranges concentrated on Pseudomonas spp., including multidrug-resistant clinical strains, and no acquired virulence factors were detected.
Discussion: Collectively, these findings suggest predation in the human gut and that viable Bdellovibrio could be natural, genomically conserved members of the intestinal ecosystem. This work advances a testable keystone-predator framework for human microbiome ecology and opens an ecologically informed therapeutic pathway in which human-associated Bdellovibrio may help control multidrug-resistant pathogens while supporting microbiota homeostasis.
Introduction
Predatory bacteria belonging to the Bdellovibrio and Like Organisms (BALOs) group have garnered significant attention as potential “living antibiotics” to counter multidrug-resistant pathogens (Cavallo et al., 2021; Zhang et al., 2024; Rai et al., 2025). These small and motile Gram-negative bacteria exhibit a unique obligate predatory lifestyle, reproducing by invading and killing other Gram-negative bacteria through a sophisticated biphasic life cycle (Sockett and Lambert, 2004; Cavallo et al., 2021; Caulton et al., 2024; Herencias et al., 2024). In natural ecosystems, including soil, freshwater, and marine environments, predatory bacteria function as keystone species that regulate bacterial populations, maintain microbial diversity, and prevent competitive dominance (mechanisms analogous to classical predator–prey dynamics that structure ecological communities) (Johnke et al., 2014; Cavallo et al., 2021; Hungate et al., 2021). During their attack phase, BALOs locate and attach to susceptible prey cells, penetrate the outer membrane, and enter the periplasmic space where they form a structure known as a bdelloplast. Within this protected environment, the predator consumes the host cell’s contents, replicates, and ultimately lyses the prey to release progeny that continue the predatory cycle (Rendulic et al., 2004; Caulton et al., 2024; Maher et al., 2025). This obligate predatory strategy represents a fundamentally different mechanism of bacterial control than conventional antibiotics, which target specific cellular processes through chemical inhibition (Sockett, 2009; Pasternak et al., 2013; Herencias et al., 2024).
Bdellovibrio bacteriovorus, the most extensively studied member of this group in natural environments, demonstrates remarkable therapeutic potential owing to its broad prey spectrum and ability to target clinical isolates regardless of their antibiotic resistance profiles (Saralegui et al., 2022; Ajao et al., 2025; Rai et al., 2025). Unlike conventional antibiotics that target specific cellular processes, BALOs physically invade and destroy their prey, making them particularly effective against biofilms and persister cells that often evade traditional antimicrobial treatments (Kadouri et al., 2013; Tang et al., 2025). Recent studies have demonstrated the safety and efficacy of B. bacteriovorus in in vivo models, showing successful reduction of pathogen loads without adverse effects on host health or immune response, including groundbreaking work in wound healing and biofilm eradication (Shanks et al., 2013; Shatzkes et al., 2015; Willis et al., 2016; Maraş et al., 2023).
The known ecological distribution of BALOs extends across diverse natural environments, including soil, freshwater, marine ecosystems, and wastewater treatment facilities, where they serve as important regulators of bacterial populations (Sockett and Lambert, 2004; Chen and Williams, 2012; Pasternak et al., 2013; Zhang et al., 2024). Interestingly, molecular detection studies have occasionally identified BALOs in human-associated environments, including duodenal biopsies from healthy individuals and respiratory samples from cystic fibrosis patients (Iebba et al., 2013; de Dios Caballero et al., 2017). These findings suggest a potential natural association between predatory bacteria and the human microbiome, although the extent and functional significance of this relationship remains largely unexplored.
The human gut microbiome represents a complex ecosystem where bacterial interactions, including predation, play crucial roles in maintaining microbial diversity and preventing pathogen overgrowth (Lozupone et al., 2012; Afzaal et al., 2022). Theoretical frameworks suggest that predatory bacteria could function as ecological regulators within the intestinal environment, similar to their role in natural ecosystems (Johnke et al., 2014; Hungate et al., 2021). However, despite compelling evidence for their presence using molecular methods, viable BALOs have never been successfully isolated from human samples, representing a significant knowledge gap in understanding their natural ecology and therapeutic potential. We cannot exclude the possibility that predatory activity in the intestine is performed by bacteria in which this behavior has not been previously reported. Additionally, the detection of classic predators, such as bacteriolytic obligate predators (BALOs), may reflect transient colonization, likely resulting from dietary intake.
The isolation and characterization of viable predatory bacteria from human sources would provide critical insights into their adaptation to host-associated environments, prey specificity patterns, and safety profiles for potential clinical applications (Cavallo et al., 2021; Rai et al., 2025). Such findings would also advance our understanding of the complex predator–prey dynamics within the human microbiome and their implications for maintaining intestinal homeostasis and preventing dysbiosis-related diseases (Iebba et al., 2016; Schoultz et al., 2025). Furthermore, genomic characterization of human-associated BALOs could reveal adaptive features that distinguish them from environmental isolates, potentially identifying novel genetic determinants associated with host colonization, prey recognition, or survival in the intestinal environment (Hobley et al., 2012; Pasternak et al., 2013; Maher et al., 2025).
Herein, we report the successful isolation of viable B. bacteriovorus strains from human microbiome. Through comprehensive 16S rDNA amplicon metagenomic profiling of donor microbiota, whole-genome sequencing of isolates, pangenomic analysis across 162 publicly available Bdellovibrio genomes, phenotypic characterization, and predation assays against clinically relevant pathogens, this work provides novel insights into the natural occurrence, ecological context, and genetic features of human-associated predatory bacteria. These findings represent a significant advancement in our understanding of bacterial predation within the human microbiome and establish the foundation for future BALOs research as novel antimicrobial agents in the fight against antibiotic-resistant infections.
Materials and methods
Ethical consideration and human fecal samples
This study was conducted in accordance with the ethical standards of Hospital Universitario Ramón y Cajal. Fifty anonymized, non-diarrheal stool samples previously submitted to the Microbiology Department for routine parasitological screening were collected without identifiable patient information. Samples were pooled into five groups of ten individual specimens each to facilitate BALO enrichment and detection.
Bacterial strains and culture conditions
B. bacteriovorus HD100 (ATCC 15356) was used as control predator strain and Pseudomonas putida KT2440 as the reference prey (Martínez et al., 2016). Additional prey strains included P. aeruginosa PAO1 (ATCC 47085) and Escherichia coli (ATCC 25922), and clinical isolates of P. aeruginosa obtained from cystic fibrosis patients (Díez-Aguilar et al., 2021), and Klebsiella pneumoniae isolates from urinary tract infections (Avendaño-Ortiz et al., 2023). Clinical strains were selected based on their antibiotic resistance profiles and previously documented lipopolysaccharide modifications. The strains used in this study are listed in Supplementary Table S1.
The prey strains were cultured from glycerol stock in blood agar (CAN-I-BD) medium at 37 °C and were cultivated in liquid with shaking at 180 rpm in LB medium. All predation co-cultures were performed in diluted nutrient broth (DNB, 1:10 dilution of standard nutrient broth) supplemented with 2 mM CaCl₂ and 1 mM MgSO₄ (Herencias et al., 2017).
BALO enrichment from fecal samples
BALO enrichment was performed following an adaptation of established protocols (Iebba et al., 2013; Herencias et al., 2017). Ten grams of pooled fecal material were suspended in 50 mL of sterile saline solution (0.9% NaCl) and vortexed vigorously for 5 min to homogenize. To this suspension, 5 mL of DNB containing 108 CFU/mL of P. putida KT2440 was added as prey. The enrichment culture was incubated at 30 °C with shaking at 150 rpm for 48 h to allow the growth of predatory bacteria.
Following incubation, cultures were filtered through 0.45 μm cellulose acetate membrane filters (Millipore) to remove most intact prey cells while allowing small predatory bacteria to pass. One milliliter of filtrate was transferred to tubes containing 500 μL of each potential prey strain adjusted to 108 CFU/mL and co-incubated for 24 h at 30 °C and shaking 150 rpm.
Double-layer agar overlay assay for BALO enumeration
BALO viability and enumeration were determined using the double-layer agar overlay method (Bolyen et al., 2019). Briefly, 1.5 mL of co-culture was added to 4 mL of melted DNB soft agar (0.7% w/v), maintained at 50 °C, supplemented with 2 mM CaCl₂ and 1 mM MgSO₄. The mixture was gently vortexed and immediately poured onto plates containing DNB agar (1.5% w/v). Plates were allowed to solidify at room temperature for 30 min before incubation at 30 °C for 48–96 h. Clear lytic plaques as indicative of predatory activity were counted and expressed as plaque-forming units per milliliter (PFU/mL).
DNA extraction and molecular confirmation of BALOs
Genomic DNA was extracted from fecal pools and purified lytic plaques using the QIAamp DNA Stool Mini Kit (Qiagen, Germany) following the manufacturer’s protocol. For fecal samples, we used an aliquot of 3 mL, centrifuged and proceeded with the extraction protocol. For lytic plaque samples (Pool 1, 2, 4, 5), plaques were excised from soft agar, resuspended in 500 μL of sterile phosphate-buffered saline, and centrifuged at 10,000 × g for 10 min to pellet cells before proceeding with the extraction protocol. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and DNA integrity was evaluated by agarose gel electrophoresis. Extracted DNA was stored at −20 °C until further analysis.
To confirm the presence of predatory bacteria, PCR amplification targeting the Bdellovibrionaceae family was performed using specific primers Bd347F (5′-ATAAGGGATGACGACGACGGAGG-3′) and Bd549R (5′-GCTAG GATCCCTCGTCTTACC-3′), following the conditions previously established (Iebba et al., 2013).
16S rDNA gene amplicon sequencing
To characterize the bacterial composition of fecal pools and lytic plaques, 16S rDNA gene amplicon sequencing targeting the V3-V4 hypervariable region was performed. PCR amplification was conducted using universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) with Illumina adapter sequences. Amplicons were sequenced on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 (2 × 300 bp) for 600 cycles.
Raw sequencing reads were processed using the QIIME 2 bioinformatics platform (Bolyen et al., 2019). Quality filtering, denoising, and ASV identification were performed using the DADA2 (Callahan et al., 2016). Forward and reverse reads were trimmed to remove primers and low-quality bases, and reads were merged with a minimum overlap of 12 bp. Chimeric sequences were identified and removed using the consensus method in DADA2. Taxonomic classification of ASVs was performed using the SILVA 138 database (Quast et al., 2013) with the classify-sklearn naive Bayes classifier. ASVs with total abundances below 10 reads across all samples were removed to control for spurious sequences and sequencing errors.
Whole-genome sequencing and assembly
Due to the low abundance of predatory bacteria in co-cultures with prey, DNA enrichment was necessary prior to whole-genome sequencing. Isolated BALOs from pools 1 and 2 were subjected to sequential enrichment by co-culturing with increasing volumes of P. putida prey. Specifically, predators were incubated with 1 mL of prey (10⁸ CFU/mL) in 8 mL HEPES buffer (25 mM, pH 7.8) at 30 °C with shaking at 170 rpm for 72 h. Cultures were centrifuged at 15,000 × g for 5 min at room temperature, supernatants were discarded, and pellets were resuspended in double the previous prey volume with fresh HEPES buffer. This process was repeated four times until sufficient biomass was obtained, as evidenced by visible pellet formation.
Total genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). For short-read sequencing, DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina) and sequenced on an Illumina MiSeq platform with the MiSeq Reagent Kit v2 Nano (2 × 150 bp) for 300 cycles, generating 250,000–500,000 reads per sample. Genomic DNA were sequenced with Oxford Nanopore technology at Plasmidsaurus (Eugene, United States1) using the V10 chemistry library prep kit with the R10.4.1 flow cells. Sequences were annotated using BAKTA (Schwengers et al., 2021).
Genomic analysis, taxonomic typing and core genome
The 162 deposited genomes were initially annotated using Bakta 1.7.0 (Schwengers et al., 2021). Subsequently, core and accessory genomes were defined using the PATO tool (Fernández-de-Bobadilla et al., 2021). This allowed for an analysis of the population structure, the annotation of adaptive features, and the creation of gene networks. Specifically, we utilized the PATO functions “core_plots” to determine the size of the pangenome, core, and accessory genome, and “similarity_tree” to generate pseudo-phylogenetic trees.
We assigned the species to each isolate using the function “classifier” of PATO (Fernández-de-Bobadilla et al., 2021). “Classifier” assigns each query genome to the closest reference genome from the NCBI2 by calculating the ANI of each genome to the reference ones. It assigns a reference species if the ANI is over 95% of identity. The heatmap from Supplementary Figure S3 that included 162 sequenced genomes of Bdellovibrio from the NCBI was created using the function “similarity_tree” and the values from the function “mash” from PATO. Clades were defined based on minimum within-node pairwise ANI scores using the unsupervised clustering MClust (Fraley and Raftery, 2007).
Statistical analysis
Predatory capacity (PFU counts) data were analyzed using a negative binomial generalized linear model (GLM) to account for overdispersion and zero-inflation inherent in count data. The model included the predator strain and prey type as fixed effects. Pairwise comparisons between isolates (BD_H1, BD_H2) and the reference strain (HD100) were performed using Tukey’s Honestly Significant Difference (HSD) post-hoc tests. Statistical significance was set at p < 0.05. All analyses were conducted using R software (v.4.3.1).
Data availability
Assembled genome sequences for isolates BD_H1 and BD_H2 have been submitted to the EBI database under the project accession number PRJEB102169.
Results
Detection and isolation of viable predatory bacteria associated to human microbiomes
Four out of the five pooled fecal samples analyzed (pools 1, 2, 4, and 5) yielded lytic plaques following enrichment with P. putida KT2440 as prey bacteria. These plaques displayed characteristic features of BALO predation: round morphology with sharp boundaries, progressive size increase over 3–5 days, and absence of bacterial colonies at their centers (Figure 1A; Supplementary Figure S1A). Viable predatory bacteria were quantified using the double-layer agar overlay method.
Figure 1. Isolation of BALOs from human fecal samples. (A) After incubation for 72–96 h of incubation on double-layer agar culture containing P. putida, human associated predators formed circular plaques (arrows). (B) Taxonomic composition at the family level of bacterial DNA extracted from lytic plaques formed by predatory bacteria isolated from human fecal pools 1, 2, 4, 5, and the positive control B. bacteriovorus HD100. Relative abundances were determined by 16S rDNA gene amplicon sequencing (V3–V4 region) analyzed using QIIME 2 and DADA2. The Bdellovibrionaceae family is highlighted, representing the predatory bacteria isolated in this study.
16S rDNA gene amplicon sequencing of DNA extracted from lytic plaques revealed variable relative abundances of Bdellovibrionaceae across plaque-derived samples: 28% in pool 1, 40% in pool 2, 5% in pool 4, 38% in pool 5, and 50% in the B. bacteriovorus HD100 positive control (Figure 1B; Supplementary Figure S1B). The remaining bacterial composition consisted primarily of prey organisms (Pseudomonas spp.) and commensal bacteria from the fecal matrix. Notably, despite comprehensive 16S rDNA gene sequencing of the five original fecal pools prior to enrichment, no Bdellovibrionaceae sequences were detected above the detection threshold (>10 reads per amplicon sequence variant, ASV), indicating extremely low natural abundance in the gut microbiota (Supplementary Figure S2). This finding is consistent with previous molecular detection studies showing transient or rare presence of BALOs in human-associated environments (Iebba et al., 2013; de Dios Caballero et al., 2017). Finally, family-specific PCR amplification using primers Bd347F/Bd549R confirmed the presence of Bdellovibrionaceae DNA in Pool1 and Pool2 plaque-positive pools, yielding the expected 202 bp amplicon (Iebba et al., 2013).
Taxonomic classification of human-associated predatory bacteria
After successful enrichment in liquid medium and repeated and robust formation of lytic plaques, two of the predator isolates were selected and sequenced (from pool 1 and pool 2, hereafter BD_H1 and BD_H2, respectively). To establish taxonomic identity and phylogenetic relationships, we employed a comprehensive comparative genomic approach using the PATO pipeline (Fernández-de-Bobadilla et al., 2021), which integrates multiple analytical modules for bacterial genome characterization. Taxonomic assignment was performed using the PATO “classifier” function, which determines species identity by calculating average nucleotide identity (ANI) values against reference genomes in the NCBI database. The classifier assigns a reference species when ANI exceeds 95% identity. Both isolates were confirmed as B. bacteriovorus, with ANI values of 99.10% (BD_H1) and 99.09% (BD_H2) relative to the reference strain HD100 (Supplementary Table S2). These high ANI values, well above the 95% species delineation threshold, confirm conspecific status while indicating strain-level genetic variation.
The BD_H1 genome was assembled into a single circular chromosome of 3,782,514 bp with a GC content of 50.65%. The BD_H2 genome comprised three contigs, identified through hybrid assembly using Illumina and Oxford Nanopore sequencing technologies. The main chromosome (Contig_2, 3,782,519 bp) has a GC content of 50.67%, consistent with BD_H1 and typical of B. bacteriovorus. The additional contigs (Contig_1, 5,354 bp; Contig_3, 5,667 bp) harbor ribosomal RNA operons and transfer RNA genes with elevated GC content (overall assembly: 56.69%), likely representing duplicated chromosomal regions or horizontally transferred elements rather than separate replicons. Detailed analysis of the smaller contigs revealed important structural and functional features. Both Contig_1 and Contig_3 harbor ribosomal RNA (rDNA) operons and transfer RNA (tRNA) genes (Supplementary Table S3), which are typically chromosomally encoded in bacteria.
Comparative alignment showed that Contig_1 shares 100% nucleotide identity with a specific region of the main chromosome (Contig_2) (Supplementary Table S3), strongly suggesting it represents a duplicated chromosomal segment or assembly artifact arising from repetitive rDNA operons rather than an independent replicon. In contrast, Contig_3 exhibited 99.75% nucleotide identity with Stenotrophomonas maltophilia genomic sequences (Supplementary Table S3). The presence of complete rDNA and tRNA genes on this contig, combined with its foreign origin, suggests either: (i) acquisition of a mobile genetic element carrying accessory metabolic functions during gut colonization or (ii) a genuine integrative element that has mobilized between distantly related species. Despite the presence of these additional contigs, the core genome of BD_H2 (Contig_2) exhibited typical B. bacteriovorus architecture and completeness metrics.
Phylogenetic analysis of human-associated predatory bacteria
To position our human-derived isolates within the broader diversity of predatory bacteria, we retrieved 162 publicly available genomes annotated as Bdellovibrio spp. from the NCBI RefSeq and GenBank databases (Supplementary Table S4). Given the limited number of Bdellovibrio genomes historically available in public databases and the recent taxonomic reclassification of predatory bacteria within the phylum Bdellovibrionota (Waite et al., 2020; Davis et al., 2024), we performed comprehensive genome-wide validation to confirm that deposited sequences truly represent authentic members of the genus B. bacteriovorus. To accomplish this, we employed the PATO “classifier” function, which establishes species boundaries using average nucleotide identity (ANI) calculations against rigorously validated reference genomes.
Pairwise ANI calculations between Bdellovibrio genomes (including our isolates BD_H1, BD_H2) revealed striking phylogenetic heterogeneity within publicly available sequences (Supplementary Figure S3). Most of the deposited genomes showed low ANI values (<90%), indicating they represent distinct species or even genera within the Bdellovibrionaceae family. This finding highlights the substantial taxonomic diversity that remains poorly characterized within this predatory bacterial group and underscores the need for systematic genomic-based reclassification of historical isolates.
Applying the widely accepted >94% ANI threshold for bacterial species delineation (Jain et al., 2018), we identified only 16 genomes (10.4% of total) that qualified as B. bacteriovorus sensu stricto. To examine fine-scale genomic variation within this species complex, we performed a single nucleotide polymorphism (SNP) analysis across the core genome and calculated SNP density normalized per megabase (SNPs/Mb) for all pairwise comparisons (Figure 2A; Supplementary Table S5). SNP-based heatmap analysis revealed distinct patterns of genomic divergence within the B. bacteriovorus clade. BD_H1 and BD_H2 exhibited remarkably low SNP densities when compared to each other and to the reference strain HD100 (Supplementary Table S2). These extremely low SNP densities indicate a recent common ancestry and minimal evolutionary divergence between human-derived isolates and well-characterized environmental strains.
Figure 2. Genomic characterization and comparative analyses of Bdellovibrio strains. (A) Heatmap showing pairwise core genome SNP counts per megabase calculated across 16 genomes with an ANI ≥ 94%. The accession numbers are listed on the axes. (B) Maximum likelihood phylogenetic tree based on core genome SNPs (n = 22 genomes). The tree topology recapitulates the ANI-based clustering, with distinct branching patterns separating B. bacteriovorus sensu stricto (short internal branches, indicating recent divergence) from the more distantly related 90–94% ANI group (longer branches). The tree demonstrates that both phylogenetic (SNP-based) and genomic similarity (ANI-based) metrics converge on the same population structure, validating the taxonomic classification. Accession numbers of each strain are listed in Supplementary Table S5.
To further resolve the relationships within the B. bacteriovorus sensu stricto and its closest relatives, we selected the 16 genomes with ANI ≥ 95% and an additional 24 genomes with ANI of 90–94%, for a total of 22 high-quality genomes (Supplementary Figure S3). We then performed a core genome analysis and constructed a matrix of pairwise average nucleotide identity (ANI) values, alongside single nucleotide polymorphism (SNP)-based phylogenetic analysis of the core annotated genome (Supplementary Figures S4, S5).
Complementing the ANI-based clustering, the SNP-based maximum likelihood phylogenetic tree of the core genome (Figure 2B) provides additional resolution of relationships among these 22 genomes. The human-derived isolates clustered together with HD100 and closely related reference strains, supporting both their classification and their close evolutionary relationship, while other clades showed longer branches, indicative of additional divergence.
We then analyzed the pangenome composition and evolution across the 22 Bdellovibrio genomes, stratified by core genome, accessory genome, and total pangenomes (Figure 3). The core genome (green line), representing genes present in ≥95% of the genomes, remained remarkably stable and nearly constant across all 22 analyzed strains, plateauing at approximately 2,500–2,650 orthologous gene families. This genomic invariance, even across geographically and ecologically diverse isolates (including human-derived, environmental aquatic, soil, and wastewater strains), indicates strong functional constraints on the predatory lifestyle. The obligate predatory strategy, which requires specialized systems for prey sensing, attachment, invasion, and intracellular lysis, appears to impose a well-defined minimal genomic blueprint that cannot be substantially altered without compromising fitness. In contrast, the accessory genome (red line) exhibited substantial variation and consistent expansion as more genomes were sequenced, reaching approximately 4,100–4,200 gene families by the 22 genomes. The steep trajectory of the red curve indicates that each newly added genome contributes an average of ~60–100 novel genes not found in the previously sequenced strains. Finally, the total pangenome (blue line) demonstrated logarithmic growth, increasing from ~4,000 genes in the first 10 genomes to ~6,500–7,000 genes at n = 22, approaching saturation but not yet fully converging. This incomplete saturation suggests that additional Bdellovibrio diversity remains undescribed, consistent with our finding that only 10.4% of deposited Bdellovibrio-labeled genomes are truly B. bacteriovorus sensu stricto. The continued accumulation of novel accessory genes underscores the substantial hidden diversity within this genus and supports the need for systematic taxonomy-guided exploration and characterization of predatory bacterial isolates from undersampled ecological niches.
Figure 3. Pangenome composition and scaling dynamics. Plot showing the accumulation of core genes (green line), accessory genes (red line), and total pangenome size (blue line) as a function of sequenced genomes (n = 22). Error bars represent 95% confidence intervals from 100 rarefaction replicates.
The remaining 122 genomes (74.8%) displayed ANI values below 90%, confirming their assignment to divergent lineages within the broader Bdellovibrionaceae family. This distribution reveals that current NCBI annotations significantly oversimplify the taxonomic complexity of predatory bacteria, with many genomes labeled as “Bdellovibrio sp.” actually representing phylogenetically distant taxa. Our analysis demonstrated that the genus Bdellovibrio encompasses far greater genomic diversity than previously recognized, with multiple cryptic species awaiting formal description.
Predatory activity and prey specificity of human-associated BALOs
To assess prey range and specificity, both human-derived BD_H1 and BD_H2 predators and the reference strain B. bacteriovorus HD100 were challenged against a panel of clinically relevant prey organisms in three to five independent replicate predation assays (Table 1; Supplementary Table S6).
Table 1. Predatory capability of B. bacteriovorus HD100 and BALOs isolates from BD_H1 and BD_H2.
Predation assays revealed marked strain-specific prey preferences among the three predators tested. The reference strain HD100 demonstrated the broadest prey spectrum, efficiently preying on P. aeruginosa, P. putida, E. coli, and select K. pneumoniae isolates. In contrast, BD_H1 and BD_H2 predators exhibited narrower prey ranges, with their activity predominantly restricted to Pseudomonas spp. Neither BD_H1 nor BD_H2 predators showed detectable lytic activity against K. pneumoniae or E. coli isolates tested.
To investigate potential genomic correlates of these phenotypic differences, we performed BLAST analyses of predation-associated genes (MAT adhesins, MIDAS, etc.) against assemblies from both BD_H1 and BD_H2 pools (Supplementary Table S7). All 12 core predation genes showed identical hits (100% identity/coverage) across both contigs, indicating no sequence variation in known predatory machinery. Core genome SNP analysis (Figure 2A) further confirmed minimal genomic divergence between isolates. These findings suggest that observed prey specificity differences likely arise from regulatory mechanisms, expression timing, or yet-unidentified genomic factors rather than variation in predation-associated coding sequences.
Variability between replicate experiments was observed for certain predator–prey combinations, likely reflecting the dynamic nature of predatory interactions and potential heterogeneity in prey surface structures. This variability underscores the importance of standardized assay conditions and replicate testing when evaluating predatory phenotypes (Caulton et al., 2024; Tyson et al., 2024).
Discussion
The successful isolation of viable B. bacteriovorus from human fecal samples bridges the gap between molecular detection studies and actual cultivation of these organisms from human sources. While previous molecular surveys detected Bdellovibrio DNA in human intestinal samples with higher prevalence in healthy controls versus patients with inflammatory bowel disease (Iebba et al., 2013; de Dios Caballero et al., 2017), our work now demonstrates that these predators are not merely transient DNA signatures but viable, metabolically active organisms capable of maintaining their predatory activity under laboratory conditions (Table 1; Supplementary Table S6).
Our findings demonstrate that despite extremely low natural abundance in the intestinal environment (where Bdellovibrionaceae were undetectable in the original samples before enrichment, as shown in Supplementary Figure S2), viable BALOs are represented in the human microbiome and may play ecological roles in microbial community regulation. Nature provides evidence that, across all scales (from macroscopic to microscopic) the long-term viability and stability of ecosystems rely on mechanisms that control predator populations. This finding aligns with recent advances in culturomics showing that approximately 70–80% of the human gut microbiota can be cultured using diverse enrichment approaches, enabling recovery of numerically rare taxa that exert disproportionate functional impacts on community structure and ecosystem function (Lagier et al., 2018; Armetta et al., 2025). The necessity of enrichment protocols reflects that predatory bacteria, like many ecological specialists, occupy specific microniches and may exist in density-dependent equilibrium with prey populations through frequency-dependent predator–prey dynamics rather than constituting dominant community members (Thakur and Geisen, 2019; Cohen et al., 2021).
The isolation of BD_H1 and BD_H2 with genomic signatures nearly identical to well-characterized environmental strains (ANI > 99% to HD100, with minimal SNP densities of 26 total polymorphisms across both isolates) is particularly significant for biosafety considerations, as it indicates that human-associated predators have not acquired novel virulence factors or substantially diverged from laboratory, reference, or environmental strains. We cannot rule out the possibility that the detected predators were transient microorganisms; however, previous findings of their molecular detection (Iebba et al., 2013; de Dios Caballero et al., 2017) suggest that they are naturally represented in the human microbiota. Our comparative genomic analysis of 162 Bdellovibrio-labeled sequences revealed striking taxonomic heterogeneity within public databases, with only 10.4% qualifying as true B. bacteriovorus sensu stricto based on the 94% ANI species delineation threshold (Supplementary Figure S3). This finding underscores the substantial cryptic diversity within predatory bacteria that remains poorly characterized and formally undescribed, highlighting the importance of rigorous genome-based taxonomy and systematic exploration of predatory isolates from diverse ecological niches. The pangenome analysis across 22 genomes demonstrated a paradoxical pattern: a highly conserved core genome (~2,500–2,650 genes) reflecting stringent functional constraints imposed by the obligate predatory strategy, combined with substantial expansion of the accessory genome. This pattern suggests that while the fundamental predatory machinery is tightly constrained, individual strains acquire diverse metabolic and regulatory capabilities, potentially reflecting adaptation to specific ecological niches or prey populations.
The observed strain-specific prey preferences constitute perhaps the most functionally significant finding, with BD_H1 and BD_H2 isolates demonstrating markedly narrower prey ranges restricted primarily to Pseudomonas spp., whereas reference strain HD100 efficiently predated Pseudomonas, Escherichia, and Klebsiella spp. This specialization may reflect ecological adaptation to the human gut environment or selection during laboratory enrichment of P. putida prey and suggests that prey range determination is multifactorial and potentially influenced by lipopolysaccharide structure, particularly lipid A composition and modifications. Critically, variable predation efficiencies against isogenic pairs of clinical isolates differed in documented LPS modifications (Díez-Aguilar et al., 2021; Avendaño-Ortiz et al., 2023). Particularly P. aeruginosa strains with murepavadin resistance conferred by lipid A acylation changes (Díez-Aguilar et al., 2021), K. pneumoniae isolates from urinary tract infections, and K. pneumoniae strains with colistin resistance due to 4-amino-arabinose addition to lipid A phosphate groups (Avendaño-Ortiz et al., 2023), suggesting that antimicrobial resistance-associated surface modifications can mechanistically influence predator attachment efficiency and invasion kinetics. Bacteria can acquire antibiotic resistance through outer membrane remodeling paradoxically create or abolish predator epitopes, establishing an ecological link between antibiotic resistance evolution and predation susceptibility with potentially significant implications for sequential evolution of multiple defense mechanisms. The therapeutic implications of narrow prey specificity present potential advantage over broad-spectrum activity for targeted applications. This approach could be directed against specific ESKAPE pathogens, particularly P. aeruginosa, a leading cause of healthcare-associated infections and the dominant pathogen in cystic fibrosis lung disease (Rossi et al., 2020).
Notably, genomic analysis identified a contig of 99.75% nucleotide identity to S. maltophilia genomic sequences in isolate BD_H2, containing complete rDNA and tRNA operons. While this finding could represent contamination or assembly artifacts, an alternative hypothesis warrants consideration. These sequences may be derived from prey nucleotides incorporated during Bdellovibrio DNA replication within the bdelloplasts, through horizontal gene transfer or other DNA uptake mechanisms. Classic radiolabeling experiments using (14C)uracil-labeled E. coli demonstrated that approximately 50% of prey RNA radioactivity is incorporated into Bdellovibrio RNA with identical specific activity, and prey DNA contributed substantially to predator DNA synthesis, with 73% of 3H-thymidine from labeled prey incorporated into Bdellovibrio DNA (Matin and Rittenberg, 1972; Hespell and Odelson, 1978). Given that BD_H2 was enriched through serial predation on Pseudomonas spp., the detected S. maltophilia sequences (if representing salvaged prey nucleotides) would suggest natural predation on this organism in the original human microbiota prior to isolation. This interpretation gains ecological significance when contextualized within documented presence of S. maltophilia in human fecal microbiota: 10.9% carriage in diarrheal patients and 33% in hematologic malignancies (Denton and Kerr, 1998; Apisarnthanarak et al., 2003), recent metagenomic detection in colorectal cancer intestinal samples, and increased respiratory colonization in cystic fibrosis (3–15%).
Our study provides novelty insights into the natural ecology of predatory bacteria in the human microbiome and establishes a foundation for evaluating their therapeutic potential. Critically, the isolation of human-derived predators represents a new avenue in predatory bacteria research, although we cannot exclude the possibility that other microorganisms act as predators, this aspect should therefore be investigated in low-abundance species To date, essentially all laboratory and therapeutic development studies investigating B. bacteriovorus have employed isolates derived from environmental sources (primarily soil and freshwater ecosystems) or reference strains maintained through decades of laboratory cultivation (Shatzkes et al., 2015; Willis et al., 2016; Herencias et al., 2020). These environmentally-sourced or laboratory-adapted strains, while demonstrating impressive antimicrobial activity in vitro and efficacy in animal infection models, were never naturally selected for persistence or predatory activity within human-associated microbiota. The isolation of BD_H1 and BD_H2 directly from human fecal samples, coupled with their demonstration of viability, functional predatory capacity, genomic integrity (>99% ANI to reference strains), and activity against clinically-relevant multidrug-resistant pathogens, opens a novel opportunity: the possibility of developing Bdellovibrio-based therapeutics using naturally human-associated strains that may be inherently optimized for survival, predatory activity, and immunological compatibility within human biological systems (Alexakis et al., 2024; Rai et al., 2025). The transition from environmental isolates to human-derived predatory bacteria as therapeutic agents could substantially enhance clinical translatability while simultaneously advancing our understanding of predatory bacteria ecology, prey–predator coevolution, and the role of top-down regulation in human microbiome homeostasis, ultimately positioning predatory bacteria as a revolutionary biocontrol strategy for the global antimicrobial resistance crisis or dysbiotic states and contributing to microbiome-mediated therapeutic strategies.
Conclusion
We reported the detection of viable Bdellovibrio isolates recovered from human fecal microbiota. Quantitative analysis reveals differential prey specificity among isolates, with P. putida emerging as the most susceptible prey. Genomic characterization identifies BD_H1 and BD_H2 as novel Bdellovibrio species with distinct genomic architectures, including potential horizontal gene transfer events. These findings expand our understanding of predatory bacteria in the human microbiota and provide a foundation for investigating their ecological roles and therapeutic potential. Future work should employ longitudinal sampling and quantitative metagenomic profiling to assess in vivo activity and ecological stability of these organisms within human gut communities.
Data availability statement
Assembled genome sequences for isolates BD_H1 and BD_H2 have been submitted to the EBI database under the project accession number PRJEB102169.
Ethics statement
The studies involving humans were approved by Comisión de investigación. Instituto Ramón y Cajal de Investigación Sanitaria. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from gifted from another research group. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.
Author contributions
MR-R: Data curation, Methodology, Writing – original draft, Investigation. MD-F: Investigation, Software, Visualization, Methodology, Writing – original draft, Data curation. MB: Writing – review & editing, Investigation, Methodology. RC: Writing – review & editing, Project administration, Writing – original draft, Resources, Investigation, Supervision, Conceptualization, Funding acquisition. JA-O: Investigation, Resources, Funding acquisition, Writing – review & editing, Project administration, Methodology, Supervision. CH: Investigation, Methodology, Writing – review & editing, Funding acquisition, Supervision, Writing – original draft, Data curation, Visualization, Formal analysis, Resources, Validation.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Work in the Metagenomics lab is supported by PRECICOLON P2022/BMD-7212 from Comunidad de Madrid, METOXISAN project from Fundación Mutua Madrileña, END-RAM PLEC2024011123 project by Agencia Estatal de Investigación and MePRAM PMP22/00092 funded by Instituto de Salud Carlos III. Work of MR-R was supported by Fundación Carolina (C2022). During implementation of this study, MD-F was supported by the Instituto de Salud Carlos III (pFIS F19/00366). CH is supported by the European Union (ERC, HorizonGT, 101077809), PI23/01945 by the Carlos III Health Institute (ISCIII), and FERP-2024-182 (2024/0357) funded by Fundación Eugenio Rodríguez Pascual.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2026.1752098/full#supplementary-material
Footnotes
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Keywords: bacterial predation, Bdellovibrio bacteriovorus, cultivation, human gut microbiome, microbiome ecology, pangenomics, predatory bacteria, prey specificity
Citation: Romero-Rivera M, D. Fernández-de-Bobadilla M, Beltrán M, del Campo R, Avendaño-Ortiz J and Herencias C (2026) Genome assembly and functional predation analysis of novel Bdellovibrio isolates from human gut microbiota. Front. Microbiol. 17:1752098. doi: 10.3389/fmicb.2026.1752098
Edited by:
Mohammad Tahir Siddiqui, Indian Institute of Technology Delhi, IndiaReviewed by:
S. Adeeb Mujtaba Ali, Deccan College of Medical Sciences, IndiaYin Wang, University of Georgia, United States
Nihan Altinsoy Marun, Sigma BioVet Sciences LLC, United States
Copyright © 2026 Romero-Rivera, D. Fernández-de-Bobadilla, Beltrán, del Campo, Avendaño-Ortiz and Herencias. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Rosa del Campo, cm9zYWNhbXBvQHlhaG9vLmNvbQ==; José Avendaño-Ortiz am9zZWF2ZW5vcnRAZ21haWwuY29t; Cristina Herencias, Y2hlcm9kckBnbWFpbC5jb20=
†These authors have contributed equally to this work and share first authorship