Metagenomic analysis of the human gut virome reveals functional signatures and viral stability across hospitalized and non-hospitalized diarrheal and non-diarrheal individuals

The human gut virome, defined as the collection of viruses inhabiting the gastrointestinal tract, represents a highly abundant yet underexplored fraction of the microbiome, with estimates suggesting up to 10 viruses for every bacterial cell [1, 2]. Within this community, bacteriophages dominate, followed by eukaryotic viruses and dietary-derived viruses [2, 3]. Phages play an essential ecological role by modulating bacterial composition through lytic cycles or lysogenic cycles. These interactions not only shape microbial community structure but also facilitate horizontal gene transfer, through which bacteriophages can introduce new genetic material into bacterial hosts [4]. This process can confer adaptive advantages such as antibiotic resistance, increased stress tolerance, and enhanced metabolic capabilities, traits that not only promote bacterial survival but may also have significant clinical implications, particularly in the context of microbial resistance to antibiotics [5].

Despite their functional relevance, characterizing the gut virome remains challenging due to the lack of universal viral marker genes and the experimental difficulty of recovering viral particles. As a result, viral sequences, especially from Latin American populations, are underrepresented in public databases, limiting both taxonomic and functional classification [6]. Nevertheless, recent studies have proposed the existence of a “core virome” which are recurrently found across individuals regardless of health status or geographic location. This core is believed to be largely composed of temperate phages [6, 7], whose composition may shift in disease states, including inflammatory bowel diseases [8, 9], HIV infection [10], and diabetes mellitus [11].

Some researchers have even proposed that members of the core virome could be used therapeutically to restore intestinal eubiosis, the balanced state of the intestinal microbiota [12, 13, 20]. For example, Ott et al. showed that sterile fecal filtrates (FFTs) containing viral particles could reverse Clostridioides difficile-associated dysbiosis, likely by transferring phages that modulate bacterial communities [14] In this context, one emerging focus in virome research is the identification of vAMGs, viral genes that modulate bacterial metabolism to favor host adaptation and enhance viral replication [15]. While AMGs are well studied in marine and environmental contexts [16], their presence and role in the human gut is still incipient, and their clinical relevance remains underestimated.

Among the clinical conditions that alter the gut microbiome, hospital-acquired diarrhea is a frequent event, particularly in patients exposed to antibiotics [17] or healthcare-associated infections. This condition is often marked by reduced bacterial richness and diversity, disrupting intestinal ecosystem stability [18]. While fecal microbiota transplantation (FMT) has proven effective in restoring bacterial communities [12], therapeutic interventions remain almost exclusively bacteria-focused. In contrast, the impact and role of the virome during diarrheal episodes, especially in hospitalized patients, remain largely unexplored [19]. Given that (i) viruses are critical components of the intestinal microbiota, (ii) the virome represents a potential therapeutic target to restore balance after gut dysbiosis and (iii) little is known about its composition during diarrhea, this study aimed to characterize the gut virome using a metagenomic approach in three groups: ICU, Non-ICU, and Non-diarrheic individuals, providing insights into the structure and potential role of the virome in hospital-acquired diarrhea, an area largely understudied, particularly in Latin American populations.

Through a hybrid sequencing approach combining Illumina short reads and Nanopore long reads, we analyzed the virome of 37 human stool samples. This strategy facilitated the recovery of extended viral contigs, improving the reconstruction of vOTUs and enabling detailed taxonomic and functional characterization of the viral communities. Additionally, it allowed the robust identification of viral functions and AMGs, providing a comprehensive view of viral communities within hospital environments.

In this study, we observed that bacteriophages constituted the most abundant viral group, regardless of hospitalization status or the presence of diarrhea (Fig. 1), a pattern consistent with previous research [27‐30] reinforcing their central role as functional regulators of the microbial ecosystem. Notably, the majority of the vOTUs were assigned to Caudoviricetes (Fig. 1A), a morphologically diverse group of tailed viruses that includes both virulent and temperate phages [28]. The absence of statistically significant differences in their abundance across the three groups, suggests that Caudoviricetes represent a stable component of the intestinal virome.

This stability is particularly relevant in the clinical context of diarrhea, a frequent clinical manifestation of intestinal imbalance [18, 31], often associated with disrupted intestinal homeostasis due to antibiotic use, nosocomial infections such as those caused by Clostridioides difficile, or inflammatory diseases [32]. While these conditions are typically associated with reduced bacterial diversity and loss of key taxa like Faecalibacterium, Roseburia, and Bacteroides [33], their impact on the virome remains less explored. Our results show that, despite potential bacterial dysbiosis, certain viral groups maintain a stable presence, suggesting greater resilience.

A similar pattern was observed for the family Intestiviridae, which was consistently identified in all samples analyzed (Fig. 1A). This family has been proposed as one of the main representatives of the human core virome [34, 35]. The ubiquitous presence of Intestiviridae supports its role as a stable component of the gut ecosystem. This family includes crAssphages, prevalent viruses that infect Bacteroides species such as B. intestinalis and B. xylanisolvens [35, 36]. These bacteria are key to intestinal metabolism through polysaccharide degradation and short-chain fatty acid production, which may explain the persistence of their phages even during dysbiosis.

In line with this hypothesis, our phylogenetic analyses revealed that the vOTUs assigned to Intestiviridae, Caudoviricetes, and Peduoviridae consistently clustered by viral family rather than clinical condition (Fig. 3). The genetic similarity of vOTUs between hospital and community samples may reflect active horizontal transmission between populations or the long-term conservation of shared viral lineages. We hypothesize that viral communities are continually transmitted between hospitalized and non-hospitalized populations, which may help explain the absence of marked differentiation between them.

Upon further examination at the taxonomic level, just 22% of viral sequences could be assigned to the genus level. Among these, Triavirus stood out, representing approximately 30% of all vOTUs assigned at the genus level. Of the 24 Triavirus occurrences, 13 were observed in the Non-diarrheic group, while the remaining 11 were in diarrheic subjects (6 in Non-ICU and 5 in ICU; Sup Fig. 1). This pattern aligns with findings reported by Wu et. al. [37], who described a significant reduction in Triavirus in patients with multiple primary malignancies, including colorectal cancer, compared to healthy individuals. Although functional evidence regarding the role of this genus remains limited, these findings suggest that Triavirus may represent a viral component associated with an eubiotic microbiome. Future studies should explore Triavirus dynamics through longitudinal sampling, assessing its persistence in health and shifts during dysbiosis. Correlating its abundance with markers of eubiosis, like key taxa such as Faecalibacterium, could clarify its role in gut homeostasis.

To infer the functional potential of the virome, we performed gene annotation of the identified vOTUs. Most of the detected functions were associated with infection cycles, including genome replication, capsid assembly, and host infection (Fig. 4A). This functions aligns with previous findings [38] and are consistent with the Kill-the-Winner (KtW) ecological model, which suggests that phages apply selective pressure on the most abundant bacteria to prevent their overgrowth and maintain microbial balance [39].

In clinical contexts, the predominance of lytic cycles may be particularly relevant. In such scenarios, lytic viral activity could contribute to the control of opportunistic strains but may also, paradoxically, exacerbate ecological instability by destroying commensal bacteria and potentially releasing resistance genes or virulence factors [40]. The dominance of lytic-cycle functions across all clinical groups (Fig. 4A) suggests that phage-driven bacterial modulation is a core ecological process in the gut. Rather than responding solely to dysbiosis, lytic activity may help maintain microbial balance by limiting overgrowth and supporting dynamic equilibrium.

Beyond the basic functions related to viral replication, one of the most striking findings was the detection of vAMGs. A total of 309 vAMGs were identified, spanning functions related to energy metabolism, genetic information processing, cellular processes, and environmental signaling (Fig. 4B). Notably, the non-diarrheic group exhibited the highest number of vAMGs (N = 176), which is particularly interesting given that this same group displayed the lowest viral diversity (Shannon index; Fig. 2A).

Consistently, AMG-based alpha diversity analyses revealed significantly higher functional richness and diversity in non-diarrheic samples compared to ICU and Non-ICU groups. This indicates that increased functional potential is not necessarily linked to a more taxonomically diverse virome. In line with this, AMG composition showed substantial overlap among clinical groups, supporting the existence of a conserved viral functional core, with differences likely reflecting quantitative shifts in AMG abundance rather than major functional turnover.

Importantly, when ICU and non-ICU samples were pooled into a single diarrheic group, the same pattern was retained, with diarrheic individuals showing significantly reduced AMG diversity and increased inter-individual variability compared to non-diarrheic subjects (Suppl. Figure 3). This consistency across analytical frameworks reinforces the notion that diarrhea status, rather than hospitalization setting, represents a major driver of viral functional destabilization.

This inverse relationship between functional richness and viral taxonomic diversity suggests a more active lysogenic dynamic, possibly associated with a more stable and less disrupted microbial ecosystem. This hypothesis aligns with the ecological Piggyback-the-Winner (PtW) model [41], which proposes that in environments with high bacterial density but low viral diversity, phages tend to adopt lysogenic strategies to maximize persistence. To experimentally assess this hypothesis, phage-infected bacterial cultures could be exposed to antibiotic-induced. The prevalence of lysogeny-associated markers, such as reporter phages or integration genes, could provide evidence of whether phages adopt lysogenic strategies under unstable microbiome conditions.

Of particular interest was the detection of genes encoding ATP-dependent bacitracin transporters, which were predominantly observed in the hospitalized groups. These proteins are part of active efflux systems for toxic compounds, and their presence suggests a viral mechanism associated with antimicrobial resistance [42] in clinical settings with high selective pressure. Previous studies have shown that certain phages can carry efflux pump genes [42, 43], enabling their bacterial hosts to expel antibiotics from the cytoplasm [44]. The identification of these genes in the intestinal virome of hospitalized patients reinforces the idea that phages function not only as ecological regulators but also as vehicles for adaptive genetic resistance.

Additionally, we identified the gene encoding the enzyme Zinc D-Ala-D-Ala carboxypeptidase, which is involved in the modification of bacterial peptidoglycan by hydrolyzing terminal D-Ala-D-Ala residues. This activity prevents the binding of antibiotics such as vancomycin, thereby conferring resistance to glycopeptides [45]. Unlike efflux pump genes, Zinc D-Ala-D-Ala carboxypeptidase was detected not only in the hospitalized groups but also in the non-diarrheic group, suggesting that it may represent a broadly conserved mechanism within the intestinal virome.

Our findings highlight the need to incorporate the virome as a key component of the intestinal resistome, challenging the traditionally bacteria-centered perspective. This is particularly relevant considering the ability of viruses to act as mobile reservoirs of resistance genes [46] within the human microbiome, as evidenced by studies reporting the presence of genes associated with β-lactams, vancomycin, tetracyclines, and macrolides in viral metagenomes derived from saliva, sputum, and skin, many of which were located on phage-associated sequences [47, 48]. In the future, understanding the mechanisms that promote viral gene mobilization will be crucial for designing effective strategies for surveillance and mitigation in the face of rising antimicrobial resistance.

Despite the strengths of our hybrid sequencing approach, this study has some limitations. First, the use of a bulk metagenomic strategy likely reduced the relative representation of viral sequences, potentially limiting the detection of low-abundance viruses. The inclusion of exogenous standards to assess viral recovery efficiency would help address this limitation, particularly for low-abundance viral populations. Additionally, bacterial reads were filtered using the SILVA_138.1 database, which targets 16 S rRNA sequences; complementary filtering against complete bacterial genome databases could further improve the removal of bacterial contamination.

Second, functional annotation relied primarily on sequence-based approaches and existing reference databases, which may underestimate the functional diversity of viral genes. Emerging strategies such as protein structure prediction combined with FoldSeek-based clustering offer promising opportunities to uncover conserved viral functions that are not detectable through sequence similarity alone.

Third, experimental validation, including cloning and heterologous expression of key genes such as Zinc D-Ala-D-Ala carboxypeptidase and bacitracin transporters, would strengthen functional inferences regarding their role in antimicrobial resistance. Moreover, the inability to distinguish between community and hospital-acquired diarrhea limits clinical interpretation; incorporating this variable, along with metadata on diarrhea severity or duration, would improve the resolution of virus–host associations.

Finally, although we attempted to overcome the small sample size by pooling ICU and Non-ICU samples into a single diarrheic group, future studies with larger cohorts will be essential to more comprehensively capture fine-scale shifts in virus–host interactions in health and disease. Future studies with clinical phenotyping and longitudinal sampling will be required to directly assess the relationships between the virome and diarrhea characteristics.

In summary, our findings expand current knowledge of the human gut virome by revealing its taxonomic stability across clinical conditions and its active functional role in microbial regulation. We demonstrate a high taxonomic stability dominated by Caudoviricetes, Intestiviridae and Triavirus phages, alongside a core set of viral functions involved in host infection. Notably, the non-diarrheic group exhibited a higher number of viral auxiliary metabolic genes (vAMGs), particularly those related to transport and metabolism, suggesting a more functionally diverse and stable virome in the absence of gastrointestinal symptoms. Furthermore, the widespread detection of genes associated with antimicrobial resistance highlights the potential role of viruses in the gut resistome, opening new avenues for future research into virus–host interactions in human health and disease. Together, these findings emphasize the functional relevance of the gut virome and its implications for microbial ecology and host health.