The urgency behind the search is hard to overstate. MRSA and the other so-called ESKAPE pathogens, including Enterococcus faecium, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and drug-resistant Enterobacter species, sit at the center of a worsening global crisis. MRSA strains carry the mecA gene, which encodes a penicillin-binding protein with low affinity for beta-lactam antibiotics, allowing the bacteria to keep building their cell walls in the presence of drugs that once destroyed them. With multidrug-resistant strains spreading faster than new antibiotics are being developed, scientists have turned increasingly to underexplored ecological niches, and endophytic fungi, the microorganisms that live within plant tissues without harming their hosts, have proven to be especially rich sources of structurally diverse bioactive metabolites.

What makes the new study particularly striking is how little was known about its star organism. Only about 25 publications on the genus Neocucurbitaria existed prior to this work, most focusing on fungal ecology, and just three had examined secondary metabolites. Those earlier studies, conducted on the marine fungus Neocucurbitaria unguis-hominis, yielded 17 diterpenoids, most of which showed little antibacterial or cytotoxic activity. The Groningen team, led by Kristina Haslinger and Jan Maarten van Dijl with first author Xiao Li, took a different route: they had previously built an in-house library of endophytic fungi from Vinca minor, and when they screened crude ethyl acetate extracts for antibacterial activity, isolate VM-36 consistently showed one of the strongest inhibitory effects against the indicator bacterium Bacillus subtilis.

To understand what the fungus was capable of, the researchers sequenced its entire genome using long-read Oxford Nanopore technology, generating 870,296 raw reads that assembled into a 32.8-megabase genome across 19 contigs, with a completeness score of 97.2 percent. The assembly predicted 11,817 protein-coding genes, along with 120 transfer RNA genes and 29 ribosomal RNA genes. Phylogenetic analysis using multiple barcode loci placed the fungus close to Neocucurbitaria salicis-albae, a species previously isolated from a willow twig in Germany. More importantly, genome mining with the antiSMASH pipeline revealed 34 putative biosynthetic gene clusters, a number comparable to the average of 36 found in related Cucurbitariaceae fungi, spanning polyketide synthases, non-ribosomal peptide synthetases, ribosomally synthesized peptide clusters, terpene clusters and hybrids.

Four of those gene clusters stood out because they appear to be unique to VM-36, with similarities to clusters known to produce the antifungal agent griseofulvin, the antibacterial compounds usnic acid and phomasetin, and the anticancer agent hypothemycin. The researchers also found evidence of a large carbohydrate-active enzyme arsenal, with 571 related genes, hinting at the fungus’s ecological role in decomposing plant biomass inside its host. On the metabolomic side, untargeted liquid chromatography-tandem mass spectrometry and molecular networking on the GNPS platform produced a network of 1,521 nodes and 1,896 edges, from which 19 compounds were annotated by library matching and 7 more were putatively assigned. Among the most abundant constituents were analogues of CJ-21058, a known fungal SecA inhibitor, belonging to the decalin-containing tetramic acid family.

The team then scaled up fermentation, growing the fungus in 13 liters of dextrose peptone yeast medium for 30 days and purifying the dominant compound, designated compound 1, to 114 milligrams. Using nuclear magnetic resonance spectroscopy, mass spectrometry, Marfey’s analysis of the acid hydrolysate, and electronic circular dichroism supported by time-dependent density functional theory calculations, they established that compound 1 is a decalin-containing tetramic acid with the molecular formula C23H33NO4, closely related to the known compounds CJ-21058 and hyalodendrin A. The serine-derived tetramic acid moiety was determined to have the L-configuration, and the absolute configuration was tentatively assigned as (2R, 3S, 6R, 8S, 11S, 5’S). Intriguingly, the compound appears to exist as a mixture of two tautomeric forms differing in where the keto and enol groups sit within the tetramic acid ring.

The antibacterial results are what elevate the compound to headline status. Against a panel of ESKAPE(E) pathogens, compound 1 showed strong activity against Gram-positive bacteria, including Enterococcus faecalis and multiple Staphylococcus aureus strains, but was entirely inactive against Gram-negative species such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, all with minimum inhibitory concentrations above 64 micrograms per milliliter. Against MRSA and methicillin-sensitive S. aureus alike, the compound achieved MIC values of 0.5 to 1 microgram per milliliter, matching or approaching the clinical antibiotics vancomycin and daptomycin used as positive controls. With minimum bactericidal concentrations of 2 to 4 micrograms per milliliter and MBC-to-MIC ratios at or below 4, compound 1 is clearly bactericidal, not merely bacteriostatic.

Kill kinetics reinforced that picture. When exponentially growing cultures of S. aureus strains USA300, D15, D17 and ATCC 29213 were exposed to compound 1 at 4 and 8 micrograms per milliliter, viable counts dropped by up to three orders of magnitude, sometimes to zero detectable colonies, outperforming both vancomycin and daptomycin at those concentrations. The optical density of the cultures declined after treatment, suggesting the compound may induce bacterial lysis, though the authors caution that membrane permeability assays and leakage experiments will be needed to confirm a bacteriolytic mechanism. In checkerboard combination assays, no antagonism was observed across 35 drug combinations, and compound 1 combined additively with daptomycin against the clinical MRSA isolate D17, with a fractional inhibitory concentration index of 0.625, a finding that suggests the molecule could complement existing therapies without undermining them.

Safety data, however, temper the excitement. Compound 1 caused 50 percent hemolysis of human red blood cells at 34.54 micrograms per milliliter, roughly four times its bactericidal concentration, indicating moderate hemolytic activity at effective doses. More concerning, the compound inhibited the metabolic activity of human bronchial epithelial cells with an IC50 of 2.39 micrograms per milliliter, well below its antibacterial MBC, whereas vancomycin and daptomycin showed IC50 values of 111.25 and above 200 micrograms per milliliter, respectively. That narrow selectivity window means compound 1 is far from ready for systemic use, and the authors emphasize that structural optimization, along with studies of stability, solubility, pharmacokinetics, resistance development and in vivo efficacy, will be essential before it can be considered a genuine drug lead.

Even so, the study reshapes expectations for an entire fungal genus. By connecting a candidate hybrid polyketide synthase-non-ribosomal peptide synthetase gene cluster to compound 1 production, noting that the core enzyme NeoA shares 72 percent amino acid identity with the phomasetin biosynthesis enzyme Phm1, and proposing a Diels-Alderase step catalyzed by NeoC, the researchers have laid a genomic framework for genome-guided discovery of related antibiotics. They also propose a possible biosynthetic origin for a cyclic siderophore detected in the metabolomic network. For a field racing to find new scaffolds against drug-resistant bacteria, the message is clear: some of the most promising chemical arsenals may be hidden not in exotic deep-sea sediments but in the quiet symbiosis between a common garden plant and the fungus living inside its leaves.

Subject of Research: Antibacterial metabolites from the endophytic fungus Neocucurbitaria sp. VM-36 active against MRSA

Article Title: Genomics and metabolomics analyses reveal the endophytic fungus Neocucurbitaria sp. VM-36 as a potential source of bioactive metabolites against methicillin-resistant Staphylococcus aureus

Article References: Li, X., Flores-Vallejo, R. D. C., He, T., van Dijl, J. M., & Haslinger, K. (2026). Genomics and metabolomics analyses reveal the endophytic fungus Neocucurbitaria sp. VM-36 as a potential source of bioactive metabolites against methicillin-resistant Staphylococcus aureus. Frontiers in Microbiology, 17, Article 1929829. https://doi.org/10.3389/fmicb.2026.1929829

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1929829

Keywords: MRSA, antibiotic resistance, endophytic fungi, Neocucurbitaria, Vinca minor, decalin-containing tetramic acids, natural products, genome mining, metabolomics, Staphylococcus aureus, biosynthetic gene clusters, daptomycin