The urgency behind the search is difficult to overstate. According to recent estimates from the World Health Organization, bacterial antimicrobial resistance was associated with approximately 1.14 million deaths in 2021 alone. Between 1990 and 2021, deaths linked to antimicrobial resistance among adults aged 70 and older rose by more than 80 percent, and projections suggest that by 2050 resistance could be directly responsible for roughly 1.91 million deaths annually, with a further 8.22 million deaths associated with resistant infections. Gram-negative bacteria such as E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Shigella are among the worst offenders, wielding structurally dynamic cell envelopes, antibiotic-inactivating enzymes, remodeled porins, efflux pumps and altered drug targets that render many existing drugs ineffective.

What makes the new study particularly compelling is its focus on biofilms, the dominant form of microbial growth in nature. Biofilms are structured communities of bacteria encased in a self-produced extracellular matrix of polysaccharides, proteins, nucleic acids and lipids. This matrix shields the inhabitants from antibiotics and immune attack, restricts drug penetration, and supports cell-to-cell communication, making biofilm-associated infections notoriously difficult to eradicate. Both E. coli and Shigella form robust biofilms that dramatically amplify their resistance. Most previous screens of natural products have tested only free-floating, planktonic bacteria, leaving antibiofilm activity largely unexplored. The Indian research team set out to close that gap by examining not just whether their fungal extract killed bacteria, but whether it could prevent biofilms from forming and dismantle those already established.

The journey began with leaves collected from healthy A. officinalis trees. After thorough washing and surface sterilization with ethanol and sodium hypochlorite, leaf segments were placed on potato dextrose agar supplemented with antibiotics to suppress bacterial contaminants. Fungal hyphae emerged only from the cut edges of the sterilized segments, confirming that the fungus truly lived inside the plant tissue rather than on its surface. Morphological examination under light and field-emission scanning electron microscopes revealed dense, septate, branched hyphae and a gray to grayish-green, velvety colony. Molecular identification followed: the researchers amplified the internal transcribed spacer region of the fungal genome with the universal primers ITS1 and ITS4, sequenced the product, and compared it against the Unite 9.0 database. The result was a 100 percent match with Daldinia sp., confirmed by a maximum-likelihood phylogenetic analysis built from the closest neighbors in the NCBI database.

To coax the fungus into releasing its chemical defenses, the team grew it in potato dextrose broth for 21 days in the dark, then extracted the culture filtrate with ethyl acetate and methanol. The ethyl acetate fraction, designated AO-EA, proved the more potent of the two and became the focus of all subsequent experiments. In agar well diffusion assays, the crude extract produced clear zones of inhibition ranging from 13.67 to 17.33 millimeters against E. coli and Shigella. Those zones are smaller than the 30 to 33 millimeters produced by the fluoroquinolone antibiotic ciprofloxacin, but the comparison is not straightforward. Ciprofloxacin is a purified single compound with a well-characterized target, whereas AO-EA is a crude mixture in which any individual active molecule is present at relatively low concentration, diluted among inactive constituents. Purification and enrichment of the active principles could substantially improve potency.

Quantitative testing reinforced the promise. Broth microdilution following Clinical and Laboratory Standards Institute guidelines yielded minimum inhibitory concentrations of 625 micrograms per milliliter for E. coli and 312.5 micrograms per milliliter for Shigella, with minimum bactericidal concentrations of 625 micrograms per milliliter for both organisms, indicating that the extract does not merely stall growth but kills the cells outright. Time-kill kinetics over 24 hours showed a significant, progressive decline in viable counts of both pathogens at the MIC, while untreated controls grew normally. Field-emission scanning electron microscopy then revealed what the killing looked like at the cellular level: after three hours of exposure, treated cells appeared deformed, wrinkled and broken, with altered shape and size, in stark contrast to the smooth, intact rods of the untreated controls.

The mechanism of action came into sharper focus through two complementary techniques. Flow cytometry with propidium iodide, a red fluorescent dye that enters only cells with compromised membranes, showed that after three hours at twice the MIC, 84.04 percent of E. coli cells and 62.15 percent of Shigella cells had lost membrane integrity, a rightward shift in fluorescence that signals catastrophic permeability failure. Confocal laser scanning microscopy confirmed the same pattern visually, with treated cells glowing red in the propidium iodide channel. The confocal experiments also used the dye DCFDA to probe for reactive oxygen species, and the results were striking: treated cells displayed a surge in fluorescence comparable to that produced by hydrogen peroxide, indicating that the extract floods the bacteria with oxidative stress. Membrane disruption and ROS-mediated damage together appear to form a two-pronged killing strategy.

The antibiofilm results may be the study’s most impressive contribution. In crystal violet staining assays, AO-EA at its MIC prevented new biofilm formation by 93.03 percent for E. coli and 89.38 percent for Shigella. More remarkably, when the researchers allowed mature 24-hour biofilms to establish first and then added the extract, biomass fell by 86.20 percent for E. coli and 83.02 percent for Shigella. Published criteria consider inhibition above 50 percent to be good antibiofilm activity, and the extract not only cleared that bar decisively but approached the performance of ciprofloxacin, which achieved just over 90 percent inhibition. An MTT assay, which measures the metabolic activity of living cells through a colorimetric reaction, showed that only around 10 to 15 percent of biofilm cells remained viable and metabolically active after treatment. Scanning electron micrographs of treated biofilms revealed scattered, loosely associated cells instead of the dense, layered architecture of untreated communities, pointing to impaired adhesion and destabilized cell-to-surface interactions.

Finally, high-resolution liquid chromatography mass spectrometry on an Orbitrap platform identified 15 distinct metabolites in the extract, spanning antimicrobial peptides, carboxylic acids, alkaloids, polyketides and phenols. Among the notable constituents were citrinin, a polyketide recently reported to show antimicrobial activity from other mangrove-derived fungi; radicinin, known to inhibit the grapevine pathogen Xylella fastidiosa; homovanillic acid, azelaic acid, xanthine, 5-hydroxyindole-3-acetic acid, gluconic acid and anthranilic acid, all of which have documented antibacterial credentials in other fungal systems; and peptide compounds including glycyl-L-leucine and two cyclic dipeptides of the octahydropyrrolo[1,2-a]pyrazine-dione family. Several of these molecules had not previously been reported from the genus Daldinia, although related phenols and carboxylic acids from Daldinia eschscholtzii have shown antibacterial and antibiofilm effects elsewhere. The extreme conditions of mangrove ecosystems, with their fluctuating salinity, tides and oxygen levels, are thought to drive these fungi toward chemically novel metabolism, and the competition among microorganisms packed into plant tissues likely selects for broad-spectrum defensive chemistry.

The authors caution that much work remains before any clinical application. The active compounds must be isolated and purified, their individual contributions disentangled, their mechanisms confirmed at the molecular level, and their safety and efficacy tested far beyond the culture dish. Nevertheless, the study demonstrates that a single fungus plucked from the leaves of an ecologically important, traditionally medicinal mangrove can simultaneously kill planktonic Gram-negative pathogens, shatter their membranes, provoke lethal oxidative stress, block nearly all new biofilm formation and dismantle the majority of established biofilms. As resistance continues to erode the value of existing antibiotics, such findings suggest that the world’s mangrove forests, among the most productive and least explored ecosystems on Earth, may hold a substantial share of the next generation of anti-infective drugs, provided they are studied before they disappear.

Subject of Research: Antimicrobial and antibiofilm activity of secondary metabolites from the mangrove endophytic fungus Daldinia sp. isolated from Avicennia officinalis

Article Title: Antimicrobial and Antibiofilm Activity of an Endophytic Fungus, Daldinia sp. Isolated From Avicennia officinalis

Article References: Dutta, N., Kumari, N., Lather, A., & Kumar, K. (2026). Antimicrobial and Antibiofilm Activity of an Endophytic Fungus, Daldinia sp. Isolated From Avicennia officinalis. MicrobiologyOpen, 15(5), Article e70422. https://doi.org/10.1002/mbo3.70422

Image Credits: AI Generated

DOI: 10.1002/mbo3.70422

Keywords: endophytic fungus, Daldinia, mangrove, Avicennia officinalis, antimicrobial resistance, antibiofilm, Escherichia coli, Shigella, secondary metabolites, LC-HRMS, membrane disruption, reactive oxygen species