Inside METAMIC3: Laura Marsal and Mariagrazia Di Luca Explore Phages, Antimicrobial Peptides and the Biofilm Barrier

Some of the most difficult bacterial infections are not defined solely by antibiotic resistance. Their persistence often depends on biofilms, highly organized microbial communities in which bacteria become embedded within an extracellular matrix and adopt physiological states that can make conventional antimicrobial treatment considerably less effective. Understanding whether bacteriophages and antimicrobial peptides can overcome this barrier is therefore becoming an important direction in the search for new strategies against antimicrobial-resistant infections.

This question lies at the centre of the doctoral research of Laura Marsal Martinez at the University of Pisa. Marsal is part of METAMIC3, a European Marie Skłodowska-Curie Doctoral Network focused on microbiomes, metaproteomics and microbial effectors. Her project investigates the activity of bacteriophages and antimicrobial peptides against bacteria growing either freely in planktonic cultures or embedded within biofilms. The work is supervised by Mariagrazia Di Luca, Associate Professor of Microbiology at the University of Pisa and Group Leader of the Antimicrobial Resistance laboratory at the International Centre for Genetic Engineering and Biotechnology in Trieste.

METAMIC3, formally titled Metaproteome-based Leveraged Microbiome Management in the Context of One Health, began in October 2025 and is expected to continue until September 2029. Supported by approximately €4.4 million through the European Union's Marie Skłodowska-Curie Actions, the network brings together fifteen doctoral research projects and institutions across Europe. Its central idea is that understanding a microbiome requires more than determining which microorganisms are present. Researchers also need to understand which proteins these organisms are producing, which biological pathways are active and how microbial communities respond when their environment is perturbed.

This functional perspective is particularly relevant to phage research. A bacteriophage may appear extremely effective when tested against rapidly growing bacteria in liquid culture but behave very differently against the same bacterial strain inside a mature biofilm. Within these structures, gradients of oxygen, nutrients and metabolites create distinct bacterial physiological states over microscopic distances. Some cells continue to divide, while others grow slowly or enter tolerant and persister-like states. The surrounding extracellular matrix, composed of polysaccharides, proteins, extracellular DNA and other molecules, can further alter how antimicrobial agents reach their targets.

This helps explain why biofilm-associated infections can persist despite apparently appropriate antimicrobial therapy. Chronic wounds, implant-associated infections, respiratory infections and several other difficult clinical conditions may involve bacterial populations protected not only by conventional genetic resistance but also by spatial organization, altered metabolism and phenotypic tolerance. An antimicrobial capable of eliminating planktonic bacteria therefore cannot automatically be expected to eradicate a mature biofilm.

Marsal's project directly addresses this difference. By comparing the performance of bacteriophages and antimicrobial peptides against planktonic and biofilm-embedded bacteria, the work can help identify which antimicrobial effectors retain activity under conditions that more closely resemble persistent infections. It also creates an opportunity to investigate whether combinations of biologically distinct agents can complement one another.

Phages and antimicrobial peptides provide an especially interesting pairing because they act through very different mechanisms. Bacteriophages depend on recognition of specific bacterial receptors and successful replication within susceptible cells. Many antimicrobial peptides, in contrast, interact directly with bacterial membranes or other cellular targets. Their biological differences raise the possibility that one agent could modify the bacterial environment in a way that improves the effectiveness of another.

The outcome cannot simply be assumed to be synergistic. If an antimicrobial peptide rapidly damages or suppresses bacterial cells, for example, it could theoretically reduce the metabolic activity necessary for efficient phage replication. Conversely, partial disruption of a biofilm matrix or bacterial membrane could potentially improve phage access to previously protected bacterial populations. Dose, timing and treatment sequence may therefore be as important as the identity of the antimicrobials themselves.

Mariagrazia Di Luca's previous research provides a strong scientific foundation for this type of work. Her research has long focused on biofilms, antimicrobial resistance and alternative approaches to bacterial infection. In a study involving the Staphylococcus aureus bacteriophage Sb-1, Di Luca and colleagues found that the phage could reduce components of the extracellular biofilm matrix and act against persister populations. Selected phage-antibiotic combinations subsequently produced greater antibiofilm activity than some individual treatments.

Such observations have helped move phage research beyond the simple question of whether a virus can form plaques against a bacterial strain. Therapeutic performance depends on the physiological environment in which bacteria are growing. Biofilm architecture, bacterial growth state, receptor expression, antimicrobial exposure and even components of host biological fluids can alter the outcome of phage infection.

Some bacteriophages possess enzymes such as depolymerases capable of degrading specific polysaccharides associated with bacterial capsules or biofilms. These properties may improve penetration or expose bacterial cells that would otherwise remain protected. However, such activities are highly phage- and strain-dependent, meaning that identifying a bacteriophage with strong activity against planktonic cells remains only the beginning of therapeutic characterization.

Antimicrobial peptides introduce another layer of possibility. Many are naturally occurring molecules involved in host defence and can interact rapidly with bacterial membranes because of electrostatic differences between microbial and mammalian cell surfaces. Di Luca's earlier research involving the human peptide hepcidin 20 showed that antimicrobial peptides can also influence biofilm architecture and extracellular matrix organization rather than acting simply as bacterial killing agents.

The possibility of combining phages with peptides therefore deserves particular attention. Instead of searching for a single molecule capable of replacing antibiotics, researchers may increasingly develop combinations in which distinct biological mechanisms attack different components of bacterial persistence. Such approaches could be particularly valuable against mature biofilms, where bacterial heterogeneity makes complete eradication by a single antimicrobial mechanism difficult.

METAMIC3 places these experiments within a broader metaproteomic framework. Metaproteomics examines proteins expressed collectively by microbial communities, providing information about biological activity that cannot be obtained from DNA sequencing alone. In phage-related experiments, this could eventually help researchers examine how bacterial metabolism, defence pathways, resistance mechanisms and stress responses change following exposure to different antimicrobial effectors.

Instead of measuring only whether bacterial numbers decrease, researchers can begin asking why particular populations survive, which metabolic pathways are altered, whether resistance mechanisms are activated and how microbial communities reorganize following treatment. These questions are especially important for biofilms because surviving cells can rebuild an apparently disrupted community once antimicrobial pressure disappears.

The project also fits naturally within METAMIC3's One Health perspective. Antimicrobial resistance does not circulate exclusively within hospitals. Resistant bacteria, bacteriophages and resistance determinants move between humans, animals, food systems and environmental reservoirs. Understanding how microbial communities can be manipulated using highly specific biological effectors may therefore have implications extending beyond individual clinical infections.

For Marsal, the doctoral project sits at the intersection of microbiology, antimicrobial resistance, phage biology and microbiome research. For Di Luca, it extends a research programme that already connects fundamental biofilm biology with translational phage therapy and alternative antimicrobial strategies.

This type of work reflects a broader maturation of the phage field. The question is no longer simply whether bacteriophages can kill bacteria. They clearly can. The challenge is determining when they remain effective inside the complex biological environments where difficult infections actually persist, how resistance will develop and which complementary agents can make phage treatment more robust.

Biofilms represent one of the most demanding environments in which to answer those questions. Their spatial organization, physiological heterogeneity and ability to harbour tolerant bacterial populations expose the limitations of antimicrobial strategies developed exclusively in simplified laboratory cultures.

By studying phages and antimicrobial peptides across planktonic and biofilm conditions, Laura Marsal and Mariagrazia Di Luca are addressing precisely this translational gap. Their work within METAMIC3 may help identify which antimicrobial combinations remain effective when bacterial populations behave less like laboratory cultures and more like the persistent microbial communities encountered in real infections.

For phage therapy, learning how to cross the biofilm barrier could ultimately prove just as important as discovering the phages themselves.

Sources:

METAMIC3 official website and doctoral projects https://metamic3.isas.de/

https://metamic3.isas.de/research/projects

European Commission CORDIS, METAMIC3, Grant Agreement 101225682
https://cordis.europa.eu/project/id/101225682

International Centre for Genetic Engineering and Biotechnology, Mariagrazia Di Luca
https://www.icgeb.org/mariagrazia-di-luca/

University of Pisa, Laura Marsal Martinez
https://www.unipi.it/ateneo/organizzazione/persone/laura-marsal-martinez-218922/

Tkhilaishvili T, Lombardi L, Klatt AB, Trampuz A, Di Luca M. Bacteriophage Sb-1 enhances antibiotic activity against biofilm, degrades exopolysaccharide matrix and targets persisters of Staphylococcus aureus. International Journal of Antimicrobial Agents. 2018;52:842–853.
https://pubmed.ncbi.nlm.nih.gov/30236955/

Di Luca M et al. The antimicrobial peptide hepcidin 20 affects Staphylococcus epidermidis biofilm formation and matrix organization.
https://pubmed.ncbi.nlm.nih.gov/24645694/