Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease over decades, which, however, remains the leading cause of morbidity and mortality in preterm infants.[1] Although multiple factors, including gastrointestinal tract bacterial colonization, immaturity, and formula feeding, are reported to be involved in the pathogenesis of NEC, the precise mechanisms are not well understood. Owing to the advances in metagenomic sequencing, ample evidences have illustrated the presence of gut dysbacteriosis in infants with NEC, which is characterized by an elevated abundance of Proteobacteria, reduced abundance of Firmicutes phyla, and an overall decrease in bacterial diversity, providing novel insights into NEC progression.[1,2] Additionally, the bacterial replication rates, particularly those of Enterobacteriaceae, rapidly increase two days prior to NEC diagnosis.[2] Moreover, aberrant changes of the gut bacteriome are associated with NEC severity. During NEC, the occurrence of pneumatosis intestinalis and the presence of air in the gastrointestinal wall suggest impaired mucosal regeneration and bacterial invasion.[3] From this point of view, invading bacteria or their toxins can induce the secretion of a cascade of inflammatory mediator, which is postulated to facilitate the onset of NEC. However, it has to be mentioned that although the specific bacterial signatures precede NEC onset, the causal relationship between gut dysbacteriosis and NEC onset needs to be investigated, and further questions of to what extent the aberrant gut bacteriome is attributable to the progression of NEC should be the area of further investigation.

In addition to the gut bacteriome, the gut virome (known as the viral “dark matter”) is rapidly recognized to constitute remarkably the gut microbial ecosystem, which is an important modulator of the gut bacterial structure. The gut virome consists of a diverse set of bacteriophages (hereafter referred to as phages), eukaryotic DNA and RNA viruses.[4] Compared to eukaryotic DNA and RNA viruses, phages are the predominant component of the gut virome, and function as natural killers of bacteria, which thus can potentially prevent bacteria-related diseases, such as inflammatory bowel diseases (IBD). A previous keystone study reported an abnormal gut virome with increased overall viral richness and relative abundance of Caudovirales in patients with Crohn’s disease and ulcerative colitis.[5] The core virome with a prevalence of over 50% in healthy subjects is absent in patients with IBD, and most of the increased Siphoviridae viral clusters in IBD were classified as temperate with Firmicutes as the predicted host.[6]Klebsiella pneumoniae (K. pneumoniae), a multidrug-resistant species, has been suggested to link with the flare-up state of IBD in multicenter IBD cohorts.[7] To specifically eliminate the IBD-associated pathobionts without disturbing the gut microbial homeostasis and stimulating the emergence of antibiotic-resistant strains, Federici et al[7] recently demonstrated that a combination of five lytic phages could effectively suppress K. pneumoniae in colitis-prone mice, thereby ameliorating the gastrointestinal inflammation and disease severity. Interestingly, K. pneumoniae-targeted phages were tolerated by and stably persisted in the human gastrointestinal tract, warranting further studies of their clinical implications.[7]

In contrast to the diverse gut virome in adults, the early-life gut virome is initially absent in the neonatal gut of infants before delivery, but rapidly colonizes during the first week after birth. The diversity and richness of the phage-dominated virome smoothly develop toward an adult-like viral profile until 2–3 years of age, which highly relies on external exposures. The initial phage colonizers mainly originate from induced prophages of pioneering gut bacteria,[4] which are primarily acquired from sources such as maternal microbiota (including the gut and breastmilk).[8] Duranti et al[8] observed that a number of prophage-harboring Bifidobacterium spp. in breast milk could be vertically transmitted to the gut of infants, which may attribute to the beneficial effects of breastmilk feeding for infants. A recent study has identified specific gut viral signatures, including reduced viral beta diversity and 137 viral contigs in preterm infants over 10 days before the onset of NEC using longitudinal fecal sampling.[9] Of these NEC-associated contigs that had at least five open reading frames, the majority (68%) were predicted to be lytic and the others were predicted to be temperate. During NEC, some viruses, such as enterovirus and human bocavirus, are specifically present in infants with NEC, and the presence of adenovirus and Epstein–Barr virus has been observed to be associated with NEC severity.[10] To restore the balance of the gut bacteriome and reduce the severity of NEC using the gut virome, Brunse et al[11] used a piglet model and showed that orogastric administration of fecal filtrate (containing pure and virus-like particles but not bacteria) could completely prevent NEC, with an increase in the diversity of virome in both mucosal and luminal compartments as well as the relative abundance of Streptococcus genus. Although this study could not rule out the potential influence of the microbial metabolites in the fecal filtrate, this evidence highlights the potential intermediate role of the gut virome between NEC and gut bacteriome, and further investigation is necessary for more insight in this regard.

Moreover, transkingdom interactions between the gut virome and bacteriome emphasize the need to disclose the contribution of the virome or viral linkage in the context of the balance between NEC and host immunity. In normal conditions, the symbiotic relationship between the gut virome and bacteriome contributes to immune homeostasis and is essential to maintain the host’s health.[12] Abnormal gut virome, which is associated with disease progression as discussed above, may cause dysfunctions in the host immune system. In addition to modulating the bacterial host communities to indirectly influence the human host, phages directly affect the host’s immunity.[12] Some phages can inhabit the intestinal mucosal layer and bind to mucin glycoproteins via their variable immunoglobin (Ig)-like domains on capsids, which thus establishes an antimicrobial layer to reduce the attachment and colonization of bacterial pathogens in the mucus.[13] Moreover, some phages have been reported with capacities to cross through the intestinal epithelium and enter the human circulation to communicate with the host immune system and induce pro- or anti-inflammatory responses by regulating the release of specific cytokines or the activities of T and B cells.[12] For instance, feeding Escherichia coli phages or T4 phages to germ-free mice can increase the proportion and number of interferon gamma (IFN-γ)-producing T cells in the intestinal mucosal layer, including CD4+ and CD8+ T cells.[14] However, the specific roles of the gut virome in the development of the host immune system in infants with NEC remain unknown. With the development of metagenomic sequencing technologies and explicit experimental designs with the human or animal models,[15] the complex dynamic virome–bacteriome relationships regarding their coexistence, coevolution, and interactions, as well as vital roles of the gut virome in diseases, such as NEC, will be disclosed.

In conclusion, the mechanisms underlying the pathogenesis of NEC in preterm infants are still under investigation. The roles of gut bacteriome in NEC have been extensively studied with consistent dynamic patterns disclosed, whereas thus far the composition, changes and functions of the gut virome in NEC remain poorly understood. Given the major role of the gut virome in shaping bacterial homeostasis directly or indirectly, and the aberrant gut bacteriome involved in the progression of NEC, future studies should emphasize the roles of the gut virome and its interactions with gut bacteriome to develop effective strategies for the prediction and prevention of NEC onset in early life. Additionally, the gut virome affects the intestinal barrier integrity and immune response of the host, which may be related to the etiology of NEC in preterm infants. Therefore, we hypothesize that the gut virome is the next frontier in the investigation of NEC, which will deepen our appreciation of its pathology and facilitate the development of effective clinical therapies.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 82100590 and 82241036) and the Department of Science and Technology of Sichuan Province (No. 2023NSFSC0579).

Conflicts of interest

None.

References 1. Hackam DJ, Sodhi CP. Bench to bedside—New insights into the pathogenesis of necrotizing enterocolitis. Nat Rev Gastroenterol Hepatol 2022;19:468–479. doi: 10.1038/s41575-022-00594-x. 2. Olm MR, Bhattacharya N, Crits-Christoph A, Firek BA, Baker R, Song YS, et al. Necrotizing enterocolitis is preceded by increased gut bacterial replication, Klebsiella, and fimbriae-encoding bacteria. Sci Adv 2019;5:eaax5727. doi: 10.1126/sciadv.aax5727. 3. Vieten D, Corfield A, Carroll D, Ramani P, Spicer R. Impaired mucosal regeneration in neonatal necrotising enterocolitis. Pediatr Surg Int 2005;21:153–160. doi: 10.1007/s00383-004-1312-6. 4. Liang G, Bushman FD. The human virome: Assembly, composition and host interactions. Nat Rev Microbiol 2021;19:514–527. doi: 10.1038/s41579-021-00536-5. 5. Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 2015;160:447–460. doi: 10.1016/j.cell.2015.01.002. 6. Clooney AG, Sutton TDS, Shkoporov AN, Holohan RK, Daly KM, O’Regan O, et al. Whole-virome analysis sheds light on viral dark matter in inflammatory bowel disease. Cell Host Microbe 2019;26:764–778.e5. doi: 10.1016/j.chom.2019.10.009. 7. Federici S, Kredo-Russo S, Valdés-Mas R, Kviatcovsky D, Weinstock E, Matiuhin Y, et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation. Cell 2022;185:2879–2898.e24. doi: 10.1016/j.cell.2022.07.003. 8. Duranti S, Lugli GA, Mancabelli L, Armanini F, Turroni F, James K, et al. Maternal inheritance of bifidobacterial communities and bifidophages in infants through vertical transmission. Microbiome 2017;5:66. doi: 10.1186/s40168-017-0282-6. 9. Kaelin EA, Rodriguez C, Hall-Moore C, Hoffmann JA, Linneman LA, Ndao IM, et al. Longitudinal gut virome analysis identifies specific viral signatures that precede necrotizing enterocolitis onset in preterm infants. Nat Microbiol 2022;7:653–662. doi: 10.1038/s41564-022-01096-x. 10. Cheng C, He Y, Xiao S, Ai Q, Yu J. The association between enteric viruses and necrotizing enterocolitis. Eur J Pediatr 2021;180:225–232. doi: 10.1007/s00431-020-03746-w. 11. Brunse A, Deng L, Pan X, Hui Y, Castro-Mejía JL, Kot W, et al. Fecal filtrate transplantation protects against necrotizing enterocolitis. ISME J 2022;16:686–694. doi: 10.1038/s41396-021-01107-5. 12. Cao Z, Sugimura N, Burgermeister E, Ebert MP, Zuo T, Lan P. The gut virome: A new microbiome component in health and disease. EBioMedicine 2022;81:104113. doi: 10.1016/j.ebiom.2022.104113. 13. Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, et al. Bacteriophage adhering to mucus provide a non-host-derived immunity. Proc Natl Acad Sci U S A 2013;110:10771–10776. doi: 10.1073/pnas.1305923110. 14. Gogokhia L, Buhrke K, Bell R, Hoffman B, Brown DG, Hanke-Gogokhia C, et al. Expansion of bacteriophages is linked to aggravated intestinal inflammation and colitis. Cell Host Microbe 2019;25:285–299.e8. doi: 10.1016/j.chom.2019.01.008. 15. Qian XB, Chen T, Xu YP, Chen L, Sun FX, Lu MP, et al. A guide to human microbiome research: Study design, sample collection, and bioinformatics analysis. Chin Med J 2020;133:1844–1855. doi: 10.1097/CM9.0000000000000871.