The study, published in Applied Microbiology and Biotechnology, focused on two of the most clinically relevant and drug-resistant organisms found in chronic wounds: Staphylococcus aureus and Pseudomonas aeruginosa. These species are frequent co-inhabitants of non-healing wounds, where they cooperate within mixed biofilms that are far more tolerant to treatment than free-floating planktonic bacteria. Rather than reaching for another antibiotic, the Italian team, led by Silvia Di Lodovico and Mara Di Giulio, tested a combination protected by Italian patent N. 102022000024408, a composition designed for the treatment of skin lesion infections. The approach is deliberately multi-target, aiming to stress pathogens through several mechanisms at once rather than relying on a single lethal hit that bacteria can easily evolve around.

Methylglyoxal, the small molecule at the heart of the combination, is a reactive dicarbonyl compound best known as the major antibacterial principle of manuka honey. It works by glycating bacterial proteins and damaging cells through chemical modification, a mode of action that is difficult for microbes to neutralize. In the new experiments, the researchers first determined the minimum inhibitory concentration of methylglyoxal against clinical isolates of antimicrobial-resistant S. aureus and P. aeruginosa. The MIC values ranged from 32 to 128 milligrams per liter, confirming that the strains were susceptible at concentrations that are considered achievable and non-toxic in wound care contexts.

Graphene oxide, the second component, is a two-dimensional carbon nanomaterial decorated with oxygen-containing functional groups. Its antibacterial reputation rests on physical and chemical actions: sharp nanosheet edges can contact and stress bacterial membranes, while its surface chemistry can promote oxidative stress and disrupt cellular integrity. Crucially, graphene oxide can also serve as a delivery platform, adsorbing small molecules onto its expansive surface and presenting them to microbial cells at high local concentrations. The researchers hypothesized that combining the nanomaterial with methylglyoxal would produce more than the sum of its parts, and they set out to quantify exactly how the two agents interact.

To measure the interaction, the team used the checkerboard test, a standard microbiological method that exposes bacteria to a grid of two-dimensional dilutions of both agents and calculates whether the outcome is synergistic, additive, or antagonistic. The results revealed a striking species-specific pattern. Against S. aureus, graphene oxide and methylglyoxal acted synergistically, meaning the combination was significantly more effective than either agent alone at the same doses. Against P. aeruginosa, the interaction was additive: the two agents still worked better together than separately, but their effects simply stacked rather than multiplying. This distinction matters, because it shows the combination is broadly useful while hinting that the two pathogens respond differently to the treatment’s mechanisms.

The optimal formulation identified in the study combined 6.25 milligrams per liter of graphene oxide with 64 milligrams per liter of methylglyoxal. When the researchers probed what this combination was doing to the bacteria, one finding stood out: the treatment increased bacterial membrane fluidity. Membrane fluidity is a sensitive indicator of cellular stress, and a shift in the physical state of the lipid bilayer can impair transport, energy generation, and envelope integrity. This observation suggests that the GO plus MGO combination destabilizes the bacterial cell envelope, potentially making cells more vulnerable to the glycation damage inflicted by methylglyoxal and to the physical stress imposed by the nanosheets.

The most demanding test came in the Lubbock Chronic Wound Biofilm model, a recognized in vitro system that recreates the polymicrobial biofilms characteristic of real chronic wounds. The researchers applied the GO plus MGO combination both to biofilms that were still forming, described as informing biofilms, and to mature, established biofilms. In both cases, the treatment reduced viable bacterial counts by 60 to 80 percent, measured as colony-forming units per milligram of biofilm. Dismantling a mature polymicrobial biofilm is notoriously difficult, and a reduction of this magnitude using non-toxic, non-antibiotic concentrations is a notable result for a field where biofilm tolerance routinely defeats standard therapies.

The combination also struck a blow against Pseudomonas aeruginosa’s motility, specifically its twitching motility, the surface-crawling movement powered by type IV pili that helps the bacterium colonize tissue and spread across wound surfaces. Inhibiting this motility could slow the expansion of infection within a wound bed and reduce the pathogen’s ability to reach and colonize new territory. Taken together with the membrane effects and the biofilm reductions, the picture that emerges is one of multi-target action: the combination attacks the envelope, the biofilm structure, and the behavioral machinery of the pathogens simultaneously, leaving fewer escape routes for resistance to develop.

An important practical aspect of the study is that the effective concentrations fall within ranges recognized as non-toxic, which is essential for any topical wound treatment. The authors describe the GO plus MGO combination as a valid and innovative non-antibiotic solution for wound management, one that acts on polymicrobial chronic wound biofilms and on P. aeruginosa motility without contributing to the antibiotic resistance crisis. Because neither component is a conventional antibiotic, the selective pressure that drives classic resistance mechanisms is reduced, an increasingly urgent consideration as antimicrobial resistance continues to climb worldwide.

The work, which was published open access on 24 September 2026 and carried out at the Department of Pharmacy and the Department of Medical, Oral and Biotechnological Sciences in Chieti, adds to a growing body of research exploring nanomaterial-honey compound partnerships for infection control. The authors note that the article was shared early to provide faster access to peer-reviewed, accepted research, with a final version of record to follow. If the laboratory findings translate into clinical settings, the graphene oxide and methylglyoxal pairing could offer clinicians a much-needed tool for wounds that have exhausted every antibiotic option, turning a two-pronged chemical and physical assault into a practical strategy for healing that has, until now, remained out of reach.

Subject of Research: A graphene oxide and methylglyoxal combination as a non-antibiotic treatment for polymicrobial chronic wound biofilms

Article Title: Graphene Oxide and Methylglyoxal: a combined strategy against chronic wound pathogens

Article References: Di Lodovico, S., Fontana, A., Di Fermo, P., Diban, F., Di Campli, E., Pilato, S., D’Ercole, S., Cellini, L., & Di Giulio, M. (2026). Graphene Oxide and Methylglyoxal: a combined strategy against chronic wound pathogens. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14050-2

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14050-2

Keywords: graphene oxide, methylglyoxal, chronic wounds, biofilms, Staphylococcus aureus, Pseudomonas aeruginosa, antimicrobial resistance, wound healing, nanomaterials, Lubbock Chronic Wound Biofilm model, membrane fluidity, non-antibiotic therapy