PEPHI: €1.5 Million ERC Project to Investigate Why Some Phages Persist Without Killing Their Hosts
A new European Research Council project will investigate an unusual bacteriophage lifestyle that challenges one of the simplest assumptions surrounding lytic phages: successful viral replication does not necessarily mean that the infected bacterium will immediately be destroyed. The five-year PEPHI project, short for Permanent Phage Infections, has received €1,499,982 in European funding to determine how certain nucleus-forming phages can apparently coexist with dividing bacteria over multiple generations and whether this persistent state can be deliberately disrupted.
The project will be hosted by the Helmholtz Centre for Infection Research in Germany. According to the European Commission's CORDIS database, the grant agreement was signed on October 2, 2026, with the project scheduled to begin on March 1, 2027 and continue until February 29, 2032. The public record was updated on October 7 and identifies the project under the 2026 European Research Council Starting Grant programme. The entire listed project cost of €1,499,982 is covered by the European Union contribution.
PEPHI is built around a biological problem that could have important implications for both fundamental phage biology and the development of bacteriophages as antibacterials. Virulent phages are conventionally described as viruses that infect a bacterial cell, replicate inside it and eventually lyse the bacterium, releasing newly produced viral particles. Temperate phages, by contrast, can establish longer-term relationships with their hosts, most characteristically by integrating their genomes into bacterial DNA or otherwise maintaining a latent state before potentially returning to productive replication.
Recent research has shown that this traditional separation is incomplete. Some phages classified as virulent appear capable of persisting alongside viable bacterial populations without behaving like conventional temperate phages. PEPHI intends to investigate one particularly striking version of this phenomenon involving nucleus-forming jumbo phages and what the project calls a “permanent phage replication cycle.”
These phages are biologically unusual even before persistence is considered. Members of nucleus-forming groups such as the Chimalliviridae can construct a proteinaceous compartment inside an infected bacterial cell that resembles a nucleus in its spatial organization. The structure is not a true eukaryotic nucleus, but it creates a physically separated environment in which the phage genome can be replicated and protected while other stages of viral production take place elsewhere in the bacterial cytoplasm.
The shell of this phage nucleus is built from viral proteins, including the major shell protein chimallin in well-characterized nucleus-forming phages. Compartmentalization can protect the replicating viral genome from several bacterial defence mechanisms while forcing the virus to organize transport between the phage compartment and the surrounding bacterial cytoplasm. This unusually sophisticated intracellular organization has made nucleus-forming jumbo phages important models for studying viral manipulation of bacterial cells.
PEPHI will investigate evidence that these phages can use such intracellular organization to maintain replication without immediately destroying their bacterial host. The project description states that recent observations show nucleus-forming phages replicating in synchrony with their hosts across generations without lysis. If confirmed mechanistically and shown to be reproducible across defined conditions, this would expand the recognized spectrum of behaviours available to phages normally described as virulent.
The idea has gained support from a 2026 study in Nature Microbiology examining persistent virulent phages across bacterial isolates. Researchers analysed more than 267,000 publicly available Escherichia genome assemblies and identified 373 virulent phage genomes unexpectedly associated with bacterial assemblies. Persistent phages were not distributed randomly across viral diversity, with very large jumbo phages and particularly the nucleus-forming family Chimalliviridae strongly represented.
The researchers also identified 285 related phage genomes in bacterial taxa beyond Escherichia, suggesting that persistent associations between supposedly virulent phages and viable bacterial populations are not restricted to a single host species or isolated laboratory phenomenon. Culture experiments and genomic analyses supported the interpretation that at least some of these sequences represented genuine persistent infections rather than simple contamination or sequencing artefacts.
Among the experimental systems investigated was the nucleus-forming Escherichia phage Goslar. The published work examined Goslar infection of E. coli cultures and detected phage persistence alongside surviving bacterial populations. Electron microscopy revealed several stages of infection, including early infection compartments, phage nuclei and intracellular viral particles. Milan Gerovac of the Helmholtz Centre for Infection Research contributed to the study and performed the Goslar phage culture experiments, linking the published observations closely to ongoing research on complex phage–host interactions at HZI.
Research presented at the International SPP 2330 Symposium in Berlin in October 2026 has provided additional context for the questions PEPHI intends to address. Gerovac presented observations in which nucleus-forming phages were detected replicating in dividing bacterial cells, alongside extensive remodelling of host protein complexes associated with stress responses, translation and central metabolism. The researchers interpreted these findings as evidence for an infection state in which nucleus-forming phages can maintain prolonged replication rather than following an exclusively rapid lytic programme.
At the same meeting, HZI researcher Milena Dziomba presented work specifically examining the potential for a permanent replication cycle in Chimalliviridae. The work proposed that adverse environmental or physiological conditions may favour a state of long-term coexistence between nucleus-forming phages and their bacterial hosts. This idea is central to PEPHI because bacterial physiology changes dramatically under stress, and the conditions experienced by bacteria in persistent infections are often very different from those used in standard laboratory plaque assays.
Traditional plaque assays are optimized to reveal phages that efficiently kill bacteria. A phage is added to a susceptible bacterial lawn, and repeated cycles of infection and lysis generate a visible zone of clearing. A virus capable of entering bacterial cells and replicating while allowing a substantial proportion of the population to remain viable could therefore be underestimated or behave unexpectedly in this type of assay.
CORDIS explicitly identifies this as one of the methodological problems PEPHI intends to address, describing permanent replication as a strategy that can remain invisible to classical plaque assays. The project consequently aims not only to understand the mechanism but also to establish experimental criteria and tools capable of detecting this alternative infection state.
The first major objective will be to define and quantitatively measure permanent phage replication. Establishing a rigorous definition is essential because several very different processes can produce apparently persistent phage–bacterium associations. Temperate lysogeny, pseudolysogeny, carrier states, incomplete infection, lysis inhibition and stable population-level coexistence can all produce conditions in which bacteria and phages are detected simultaneously. PEPHI is specifically concerned with productive replication of nucleus-forming phages across host generations without the expected rapid collapse of the bacterial population.
The project will then attempt to map the molecular mechanisms that allow this state to develop and persist. Its central hypothesis is that nucleus-forming phages combine their specialized intracellular compartments with host-takeover factors to maintain viral replication under stressful conditions. Identifying these factors will require determining how viral proteins alter bacterial physiology, how the phage nucleus behaves as the host divides, how viral production is coordinated with bacterial growth and which molecular switches determine whether infection proceeds toward rapid lysis or persistent replication.
The experimental programme is expected to combine quantitative virology with time-resolved single-cell measurements, fluorescence imaging and electron microscopy. These techniques will allow researchers to follow individual infected cells rather than averaging the behaviour of an entire bacterial population, which is particularly important if only a subset of cells enters a persistent infection state.
The project will use an E. coli–Goslarvirus system as an experimentally validated model. Goslar is especially suitable because it forms a phage nucleus and has already provided evidence for unusual persistence in bacterial cultures. Following the infection microscopically over time should allow researchers to determine whether the viral compartment is maintained through bacterial growth and division and how viral replication changes as environmental or cellular stress increases.
PEPHI also intends to identify the molecular determinants responsible for permanency rather than stopping at descriptive microscopy. The researchers plan to characterize the factors that establish or maintain the state and determine whether modifying those factors can shift the outcome of infection toward efficient bacterial killing.
This final objective gives the project its most direct connection with phage therapy. The CORDIS description proposes engineering phages that bypass permanency and describes the intended result as the generation of “hyper-virulent” phages capable of progressing more efficiently toward bacterial clearance. The term in this context refers to increased effectiveness of phage-mediated killing rather than enhanced bacterial pathogenicity.
For therapeutic phage development, the underlying problem is important because susceptibility cannot be reduced to whether a bacteriophage can enter a bacterial cell. A phage may adsorb to the bacterium, inject its genome and even replicate while still failing to eliminate the bacterial population efficiently. If permanent replication occurs under physiological stresses resembling those found during difficult infections, conventional laboratory screening could overestimate the ability of some phages to produce bacterial clearance.
Chronic bacterial infections are particularly relevant because bacteria within biofilms, nutrient-limited environments or inflamed tissues can exhibit physiological states that differ greatly from rapidly growing laboratory cultures. Metabolism can slow, stress responses can change and heterogeneous subpopulations can emerge. These factors are already known to influence phage infection, adsorption and replication, but PEPHI proposes an additional possibility: under some conditions, the phage itself may adopt a replication strategy that allows continued coexistence with its host instead of rapidly eliminating it.
The therapeutic implications remain hypothetical at this stage. PEPHI has not demonstrated that permanent replication is responsible for clinical failure of phage therapy, nor has it shown that engineered phages avoiding this state improve outcomes in patients. The project will only begin in 2027, and its proposed connection with chronic infection should therefore be understood as a research hypothesis to be tested rather than an established clinical mechanism.
That distinction is important because persistent bacterial infections are influenced by many factors, including biofilm architecture, limited antibiotic penetration, altered metabolism, immune responses, intracellular bacterial reservoirs and antimicrobial resistance. Permanent phage replication could eventually prove to be one component of this problem without being a universal explanation for why chronic infections are difficult to eradicate.
The project's broader importance lies in questioning the binary language traditionally used to describe phage lifestyles. The distinction between lytic and temperate phages remains extremely useful, particularly for selecting therapeutic candidates because phages capable of lysogeny are generally avoided. However, the discovery of virulent phages persisting in bacterial isolates suggests that productive viral lifestyles occupy a wider biological spectrum than the simple model implies.
Large genomic studies published around the same period reinforce this conclusion. A separate Nature Microbiology analysis examined 3.6 million bacterial genome assemblies representing 1,226 species and identified 119,510 sequences corresponding to apparently lytic phages embedded within bacterial assembly datasets. The authors described these as bacterial assembly-associated phage sequences and found previously underappreciated viral diversity, including phages related to Goslarvirus. Such findings indicate that bacterial genome databases may contain extensive evidence of phage–host associations that conventional assumptions about strictly lytic viruses had caused researchers to overlook.
PEPHI is designed to move from those large-scale observations toward mechanism. Detecting a virulent phage genome in a bacterial assembly establishes that something unusual may be occurring, but it does not explain how viral replication can coexist with continued bacterial proliferation. Direct measurements of individual infected cells, intracellular phage compartments, viral production and host physiology will be necessary to establish what the permanent replication state actually is.
The Helmholtz Centre for Infection Research provides a particularly relevant environment for this work. Its Complexes in Phage-infected Cells research group, led by Milan Gerovac, studies jumbo phages and the molecular mechanisms through which they take control of bacterial cells. The group combines multi-omics, molecular microbiology, biochemical approaches and structural techniques to investigate interactions between viral factors and the bacterial machinery, with the longer-term aim of translating mechanistic discoveries into antimicrobial applications.
If PEPHI succeeds, its contribution could therefore operate at several levels. It could establish a mechanistic definition of permanent phage replication, provide methods for detecting this state, clarify why nucleus-forming phages are disproportionately associated with persistent infections and identify viral or host factors controlling the transition between coexistence and bacterial destruction.
For phage therapy, the most consequential outcome would be a better ability to distinguish phages that merely replicate from phages that reliably clear their bacterial targets under clinically relevant physiological conditions. Screening programmes could eventually incorporate assays specifically designed to detect persistent replication, while phage engineering might be used to remove mechanisms that promote coexistence or reinforce mechanisms driving productive lysis.
The project also provides a useful reminder that the phrase “lytic phage” should not automatically be interpreted as a guarantee of rapid bacterial elimination under every condition. Infection outcome emerges from an interaction between the phage, the bacterial genotype, the physiological state of the host and the surrounding environment. PEPHI will spend five years investigating one of the more unexpected outcomes of that interaction: a supposedly virulent virus replicating inside a bacterial population without immediately destroying it.
By determining how that state is established and whether it can be broken, the €1.5 million project could reshape both the fundamental classification of phage lifestyles and the way therapeutic phages are evaluated for persistent infections. Whether the eventual findings translate into more effective phage medicines remains to be demonstrated, but the underlying question is already significant: successful phage infection and successful bacterial killing are not necessarily the same biological event.
Sources :
European Commission CORDIS — Permanent Phage Infections (PEPHI), Grant Agreement 101303516. CORDIS confirms the €1,499,982 EU contribution, October 2, 2026 signature date, March 1, 2027 start, February 29, 2032 end date, ERC-2026-STG funding scheme, HZI as beneficiary, the E. coli–Goslarvirus model and the project's mechanistic and therapeutic objectives. CORDIS
https://cordis.europa.eu/project/id/101303516
Dougherty PE, Bernard C, Carstens AB et al. — “Persistent virulent phages exist across bacterial isolates.” Nature Microbiology 11, 31–41 (2026). The study analysed more than 267,000 Escherichia assemblies, identified 373 virulent phage genomes and provided experimental evidence for persistent virulent phages, particularly among jumbo and nucleus-forming phages. Nature
https://doi.org/10.1038/s41564-025-02207-0
Perfilyev A, Gæde A, Hooton S et al. — “Large-scale analysis of bacterial genomes reveals thousands of lytic phages.” Nature Microbiology 11, 42–52 (2026). The study analysed 3.6 million bacterial assemblies from 1,226 species and identified 119,510 bacterial assembly-associated lytic phage sequences. DOI
https://doi.org/10.1038/s41564-025-02203-4
Helmholtz Centre for Infection Research — Complexes in Phage-infected Cells. HZI describes Milan Gerovac's group, its focus on jumbo and nucleus-forming phages, host takeover, phage intracellular compartments and translation of mechanistic knowledge toward antimicrobial applications. helmholtz-hzi.de
https://www.helmholtz-hzi.de/en/research/research-groups/details/complexes-in-phage-infected-cells/
International SPP 2330 Symposium, Berlin, October 5–7, 2026 — The Phage Nucleus shapes the Virulence Spectrum in Chimalliviridae and The permanent replication cycle potential in Chimalliviridae. The HZI presentations reported phage replication in dividing cells and proposed persistent coexistence of nucleus-forming phages under adverse conditions. evis.events
https://evis.events/event/691/timetable/
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