A roadmap of isolating and investigating bacteriophage infecting human gut anaerobes

Bacteriophages are highly abundant and diverse within the human gastrointestinal tract (GIT), playing a crucial role in shaping the structure and stability of the gut microbiota. They hold promising therapeutic potential for chronic disease caused by imbalances in human bacteria. Previously, several comprehensive human gut virome databases have been published [1–4], each containing hundreds of thousands of non-redundant virus genomes [5]. Compared with the vast amount of human gut virome data, research on phage isolates specifically targeting human gut bacteria, especially those that target the predominant obligate anaerobes in the gut, remains sparse—this is not due to a lack of interest, but rather the difficulty in obtaining such phages. Nevertheless, combined with the advanced metagenomics and culturomics technologies, several phages infecting different human gut bacterial taxa have been isolated by separate studies successively [6–16], providing a multitude of representative strategies for phage isolation. Despite these advances, our understanding of phages infecting gut bacteria—particularly those targeting obligate anaerobes—remains limited, and the current strategies have yet to fully overcome the barriers to isolation [1,17]. This knowledge gap has led to a limited number of culturable phages that infect human gut obligate anaerobic bacteria, constraining the full exploration of their potential. To provide a ‘Roadmap’ for research on phages targeting human gut bacteria, we briefly review phage isolates, particularly those targeting human gut obligate anaerobes, summarize available data on the diversity, taxonomy, and basic characteristics of these phages, suggest an approach for phage isolation, and explore their implications. By discussing their value and future perspectives, we hope to illuminate the ‘dark matter’ of the gut microbiota and contribute to the understanding of the virus.

To comprehensively review phages targeting human gut bacteria, we first cataloged these bacteria from four databases established based on extensive metagenome data sets or isolated biobanks: the Unified Human Gastrointestinal Genome (UHGG) [18], the Culturable Genome Reference (CGR) [19], the Human Gastrointestinal Bacteria Culture Collection (HBC) [20], and human Gut Microbial Biobank (hGMB) [21]. Almeida et al. compiled and analyzed 204,938 genomes and 170,602,708 genes from human gut microbiome data sets to generate the UHGG [18]. Zou et al. presented a reference catalog of genomes of cultivated human gut bacteria (CGR) by collecting 1,520 non-redundant, high-quality draft genomes from over 6,000 bacteria cultivated from fecal samples of healthy humans [19]. Forster et al. presented the HBC, including a comprehensive set of 737 whole-genome-sequenced bacterial isolates, representing 273 species from 31 families [20]. Liu et al. established the hGMB, which contains 1,170 strains and represents 400 human gut microbial species [21]. These four studies comprehensively demonstrate the diversity of human gut bacteria, serving as sources of information for our search to identify host bacteria for targeted phages.

To summarize the proportions of obligate anaerobes and aerobes or facultative anaerobes in the human gut microbiota, as well as the corresponding proportions of isolated phages, we organized the data through the following steps: (1) We conducted a comprehensive review of four human gut bacterial data sets mentioned above, removing redundancies to retain only those with clearly defined taxonomic classifications. This resulted in a dataset of human gut bacteria with clearly defined species classifications, including obligate anaerobes and aerobe or facultative anaerobes. (2) We retrieved information on phage isolates from the NCBI database (updated on April 8, 2024), which included details on the species of targeted host bacteria, isolation sources. (3) We integrated and compared the data from the previous two steps. By cross-referencing the phage isolates’ host bacteria information with our compiled lists of obligate anaerobes and aerobe or facultative anaerobes, we identified the phage isolates that specifically target these two categories of bacteria.

Based on these criteria, we compiled a total of 315 species of human gut bacteria with clearly defined classifications. Among these, the proportion of obligate anaerobes ranged from 61.98% to 93.31%, while the remaining 6.69% to 37.93% were aerobes or facultative anaerobes, indicating that obligate anaerobes pre-dominated in the gut bacterial community (Table 1). Regarding phages, we identified 8,583 phage isolates targeting the aforementioned 315 species of human gut bacteria from the NCBI data base, with the number of phages targeting aerobic or facultative anaerobes (95.50%) significantly exceeding that targeting obligate anaerobes (4.50%) (Table 1). While preliminary, this finding suggests that despite the predominance of obligate anaerobic bacteria among the human gut bacteria community, phage isolates specific to these bacteria are remarkably scarce and underrepresented in research. This may be due to (1) existing isolation methods may not be entirely suitable for isolating phages that infect gut obligate anaerobes, (2) the biological characteristics of these phages remain unclear, and (3) the interaction mechanisms between the phage and their host bacteria have not been elucidated, further complicating the isolation process. Therefore, reviewing phage isolates targeting obligate anaerobes in the human gut is urgent, as it will provide valuable insights into their unique biological properties and interactions with other biological entities. A deeper understanding will advance the knowledge of these phages and improve their isolation, thereby facilitating their application.

To delve into phages targeting human gut obligate anaerobes, we selected existing phages that target these anaerobes based on the literature (Table 2). These phages must have corresponding literature references to allow for traceability and review, thereby providing insights into their biological and genomic characteristics and ensuring that their host bacteria are obligate anaerobes isolated from the human gut (Table 2).

Through this process, we collected a total of 212 phage isolates, of which 137 infect Bacteroides, 23 infect Parabacteroides, 22 infect Clostridium, and 11 infect Bifidobacterium, while the remaining isolates infect bacterial genera with fewer than 10 isolates (Figure 1A). Notably, highly abundant bacterial genera in the gut, which host over 10 phage isolates, are well-represented across three biobanks, indicating that the richness of the host bacterial genus is essential for isolating phages. However, some high-abundance bacteria have few associated phages. For example, Ruminococcus and Alistipes, accounting for 4.08% and 3.80% of the UHGG database, respectively, have only 6 and 1 phage isolates. This discrepancy may be attributed to the unique life cycle of such phages or distinct defense mechanisms carried by these obligate anaerobes against phage infection. Interestingly, despite the high viral diversity targeting Prevotella/Segatella in the viral database [1], no phages infecting these bacteria have been isolated to date [22]. These phages are coined ‘Lak megaphages’ due to their large genome size [17], which may be one of the reasons for their difficulty in isolation, as larger phages are challenging to isolate through plaque assays [17,23]. Furthermore, most phages originate from sewage (195 isolates), while notably, only 17 phages infecting human gut obligate anaerobes have been isolated from feces, indicating a significant gap in exploring and isolating these phages from feces (Figure 1B). The limited recovery from feces could be due to the complexity of the gut environment or the presence of phages that are not easily cultivable under standard laboratory conditions.

Furthermore, based on relevant literature (Table 2), we reviewed the basic characteristics of these phages, including their genome sizes and taxonomic classifications. These summaries are based on information provided in the literature. The genome sizes of these phages range from 10,891 bp to 179,283 bp, with an average size of 49,477 bp (Figure 1C). Among them, except for one genome lacking classification information, all others belong to the class Caudoviricetes, including 28 strains in the family Steigviridae, 1 in Intestiviridae, and 182 unassigned genomes (Figure 1D). This indicates the diversity of phages targeting obligate anaerobic bacteria and highlights the presence of ‘dark matter’ [24]. Additionally, existing publications on these phages have primarily focused on their biological characteristics, the interaction between phage and host bacteria, and their applications (Figure 1E). Among these topics, ‘phage applications’ have been the least studied. By continuously accumulating resources of phages that infect human gut obligate anaerobic bacteria and expanding our understanding of their interactions with host bacteria and humans, the potential applications of these phages are expected to be significantly enhanced.

Since the first bacteriophage targeting obligate anaerobic gut bacteria was isolated in 2018, a slight increase was observed in 2020. Subsequently, approximately five new phage isolates were discovered each year from 2020 to 2023, with a particularly rapid increase in numbers observed in 2023 (Figure 1F). This trend indicates a growing interest and progress in the study of phages targeting obligate anaerobes, highlighting their importance in gut microbiome research and potential applications. Nonetheless, further exploration is necessary to fully understand the diversity, functionality, and therapeutic potential of bacteriophages targeting obligate anaerobes.

The procedures employed for phage isolation in publications are diverse. In summary, these methods can be categorized into three principal approaches: the classical method, the enrichment method, and the fermentation method. Each approach typically involves three steps: sample pretreatment, enrichment co-culture, and phage screening/isolation.

The classical method, widely utilized for isolating phages targeting ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens or lactic acid bacteria (LAB) [25,26], is particularly suitable for processing large-volume environmental samples. This method involves two main steps: pretreatment and phage isolation (Figure 2A). First, in the pretreatment step, the original source samples should be centrifuged and filtered to obtain virus-like particles (VLPs) [27]. The VLPs are then co-cultured with the targeted bacterial strains. After propagating for a few hours, the co-culture needs to be centrifuged, filtered, and concentrated to increase the titer of phages [16]. Finally, a plaque assay will be used in the isolation step.

The enrichment method involves all three steps. This method shares similarities with the classical method in the sample pretreatment step but diverges in subsequent steps, notably in the enrichment assay(Figure 2B). There is variability in the enrichment assays across different studies as well, for instance, some studies opt for a single round of enrichment [8], while others employ repetitive enrichment to achieve a higher concentration of the target phages [28,29]. These two methods are not only applicable to isolating phages that infect gut obligate anaerobic bacteria, but also extend to isolating other types of phages that are typically challenging to obtain.

The fermentation method exhibits unique applicability in isolating phages from fecal samples [7,30] (Figure 2C). During fermentation, anoxic conditions are employed, supplemented by antibiotics to suppress the growth of non-targeted bacteria while promoting the proliferation of targeted bacteria. This approach enhances the chance of phage encountering and infecting their host bacteria, facilitating their in situ replication. Simultaneously, the bacteria used for phage isolation are derived from the same fecal sample used for fermentation, contributing to the specific targeting of phages. Additionally, the following enrichment co-culture assay is needed to increase the abundance of targeted phages. The phage screening/isolation step utilizes either a traditional plaque assay or q-PCR detection, especially for phages that do not form plaques, such as the crAss002 phage [7]. This method maximizes the preservation of target phages and their host bacteria within the samples, thus enhancing the efficiency of isolating highly specific phages. Concurrently, conducting q-PCR detection on phages that cannot form plaques expands the opportunities for screening phages.

These three established methods isolate phages directly from the samples without prior identification of the microbiome composition, resulting in a blind isolation process and yielding random outcomes. Recently, Ramos-Barbero et al. [29] employed qPCR assays to detect the presence of specific phages with known genome data, such as crAss-like phages, in resource samples. This strategy enables the selection of optimal samples for subsequent phage screening [29], thereby enhancing isolation efficiency. However, it may not be suitable for the discovery of previously unknown phages. With the advancement of sequencing technologies and the decreasing costs associated, researchers have attempted to conduct metagenomic sequencing before phage isolation, using the analysis of the metagenome data to identify phage sequences and predict their bacterial hosts [6]. It provides insight that this approach could be applied to the isolation of phages targeting human gut bacteria. Particularly, as an increasing number of human cohorts have been established, resulting in a growing number of fecal samples with metagenomic data. For these fecal samples containing metagenomic data, we suggest a method that integrates bioinformatics analysis with selected strategies from the aforementioned three methods to achieve the precise isolation of phage targeting specific host bacteria from these samples (Figure 2D). In this method, (1) the host bacteria of viral gene fragments in the samples are predicted through metagenomic data analysis [6]. (2) The relative abundance of phages targeting the specific host bacteria in different samples is analyzed, and the ‘best sample’ with the highest relative abundance of phages targeting the desired host bacteria is identified. (3) These ‘best samples’ are co-cultured with the target host bacterial strains [28], and (4) plaque assays or growth curve analysis and q-PCR techniques can be utilized to validate the success of phage isolation [7,30]. This method effectively achieves precise screening of phages while significantly saving labor and sample resources.

Isolation of phages targeting human gut bacteria provides valuable insights into their characteristics, dynamic interplay between the microbiota and humans, bacterial inhibition, and the potential for disease intervention. Among these phages, crAss-like phages are the most abundant family in the gut and have been studied for their biological properties after isolation [31,32]. By obtaining isolates, researchers can verify their morphology and host specificity [7,28]. Furthermore, the interactions between crAss-like phages and their hosts have been explored, shedding light on the potential mechanisms underlying their long-term persistence in the gut alongside host bacteria [14]. For instance, gut bacteria may use invertible promoters to mediate rapid phase variation of alternate capsular polysaccharides, thereby maintaining a dynamic equilibrium between phage sensitivity and resistance [8,14,33]. However, our current understanding of various elements in crAss-like phages and their interactions with hosts is limited to a small subset of phages. Thus, further screening and exploration of phage isolates are warranted to expand our knowledge in this field.

Phages play a crucial role not only in shaping the human gut microbiota but also in bypassing mammalian physical barriers, influencing human health by directly or indirectly interacting with human cells and the immune system [34,35]. Through predation, phages affect the bacterial host and also affect non-host bacteria through cascading effects mediated by interbacterial interactions, thereby contributing to the architecture of the gut microbiota and influencing the composition of gut metabolites [36]. Moreover, phages can embed themselves within the mucus layer to regulate invasive bacteria, maintaining the integrity of the intestinal barrier [37]. Furthermore, phages can be internalized by epithelial cells and transported to the opposite side, where they release active phages [38]. They also stimulate immune responses by triggering the production of interferon-gamma (IFN-γ) via Toll-like receptor 9 (TLR9), potentially exacerbating intestinal inflammation and colitis [39]. Phages have also been found to impact bacterial phase variation and regulate the levels of regulatory T cells (Tregs), highlighting that phage and host inflammation can drive the dynamic bacterial phase variations [40]. Understanding the dynamic interactions between bacteriophages and gut bacteria offers novel insights into the manipulation of the gut microbiota and the development of targeted therapies.

Additionally, due to their inhibitory properties and specific targeting, phages have the potential to intervene in human chronic disease. Recently, phages of anaerobes, such as Ruminococcus phages, have been utilized to treat age-related cognitive dysfunction and have shown potential for treating inflammatory bowel disease (IBD) [13,27]. Furthermore, researchers have successfully modified bacteriophages to carry antimicrobial factors or specific medications. By capitalizing on the targeted capabilities of bacteriophages, these phages can precisely reach the lesions of colorectal cancer (CRC), effectively intervening in the disease progression. It is noteworthy that the modified bacteriophages, carrying drugs, not only deliver the medications directly to the tumor site but also employ the bacteriophage’s antimicrobial properties to reduce the population of Fusobacterium nucleatum, a bacterium associated with CRC. As a result, this innovative approach significantly enhances the efficacy of chemotherapy treatments for CRC [12,41].

In conclusion, we reviewed the isolation, characteristics, and applications of phage isolates infecting human gut obligate anaerobes and suggested a strategy for phage isolation. It is important to note that the isolation of these phages is merely the first step in phage applications. As the number of phage isolates continues to increase, they will accumulate and supplement the genomic information of gut virome, enhancing the accuracy of viral sequence prediction tools. This will enable us to predict the various phage isolates present in different samples and selectively target and screen phage isolates, thereby effectively improving the efficiency of phage isolation. Furthermore, the phage isolates will increase the genomic diversity of phages and impart additional biological characteristics to the phage, thus elucidating the viral ‘dark matter’, promoting further exploration of the interactions between phages and bacteria in vitro and in vivo, and confirming the causal relationships between gut bacteria and diseases. Additionally, to expand the applications of phages infecting gut bacteria, we can engineer phages to possess tunable host ranges, design and synthesize novel phages and express phage-derived functional proteins for purposes of host recognition, binding, or lysis. These insights are essential for developing targeted therapeutic approaches and interventions, and they will also expand the boundaries of the phage applications.

• Bacteriophages play a crucial role in maintaining the balance of the gut microbiome and have significant implications for human health and disease.

• Despite extensive virome data in the gut, the limited number of culturable phages, especially those targeting human gut obligate anaerobes, constrains a comprehensive exploration of their potential functions.

• We reviewed the phage isolates infecting human gut obligate anaerobes, summarized their characteristics and applications, discussed phage isolation methods, and suggested an approach based on the existing methods to improve isolation efficiency.

• The isolation and research of phages targeting human gut obligate anaerobic bacteria contribute to elucidating their interactions with host bacteria, providing new perspectives for disease intervention.

The authors declare that there are no competing interests associated with the manuscript.

This work is supported by the National Natural Science Foundation of China (NSFC) [grant number 82250901].

C.L. wrote the manuscript. C.L. and B.X. prepared the figures. B.X. and Z.L. prepared the table. M.X. and J.L. offered critical insights. All authors edited, corrected, and approved the manuscript.

CGR

Culturable Genome Reference

CRC

colorectal cancer

ESKAPE

Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

GIT

gastrointestinal tract

HBC

Human Gastrointestinal Bacteria Culture Collection

hGMB

human Gut Microbial Biobank

IBD

inflammatory bowel disease

IFN-γ

interferon-gamma

LAB

lactic acid bacteria

TLR9

Toll-like receptor 9

UHGG

Unified Human Gastrointestinal Genome

VLP

virus-like particle