Celery (Apium graveolens L.), coriander (Coriandrum sativum L.) and parsley (Petroselinum crispum) were purchased from a commercial market in Alexandria, Egypt, and their aerial parts were extracted separately with methanol. Each methanolic extract was then partitioned into a methylene chloride fraction and an aqueous mother liquor. The biological tests used 20 uropathogenic E. coli isolates obtained from private laboratories in Alexandria. Susceptibility testing showed that every isolate was resistant to amoxicillin-clavulanic acid, doxycycline and nalidixic acid, while all were susceptible to ertapenem, imipenem and meropenem. Twelve isolates were classified as strong biofilm formers, four as intermediate formers and four as non-biofilm formers.

The antibacterial results varied markedly among the herbs and fractions. Coriander’s total methanolic extract and aqueous mother liquor each produced a minimum inhibitory concentration of 3.75 micrograms per millilitre, the lowest value reported for the plant preparations. Its methylene chloride fraction required 100 micrograms per millilitre. Celery was less active: the total methanolic extract had a minimum inhibitory concentration of 50 micrograms per millilitre, while its aqueous mother liquor reached 25 micrograms per millilitre and its methylene chloride fraction showed no detectable activity under the tested conditions. Parsley’s total extract and aqueous mother liquor likewise had no detectable activity, although its methylene chloride fraction inhibited growth at 100 micrograms per millilitre. Ciprofloxacin and norfloxacin had minimum inhibitory concentrations between 0.9 and 1.9 micrograms per millilitre alone. When combined with coriander extract, the effects were described as additive, with no evidence of synergism or antagonism in the tested combinations.

Untargeted metabolomics produced markedly different profiles for the three herbs. Analyses were run in positive and negative electrospray ionization modes and incorporated retention time, accurate mass, predicted molecular formulas and tandem mass-spectrometry fragmentation patterns. Compound detection and processing initially yielded 4,861 features in negative mode and 3,155 in positive mode; after peak detection, deisotoping, chromatographic alignment and filtering, 580 and 512 aligned features, respectively, remained for chemometric analysis. The final annotation included flavonoids and glycosides, isoflavones, phenolic acids, coumarins, amino-acid derivatives, fatty acids and lipids, chlorins and a lignan. All 167 annotations were assigned level 2 confidence, meaning they were tentative identifications supported by library or literature spectral matches rather than confirmed with authentic standards.

Coriander had the richest and most diverse flavonoid and phenolic profile in this analysis. Its extracts contained quercetin, multiple quercetin conjugates, apigenin glucuronide, luteolin-7-O-glucoside, kaempferol-3-O-glucorhamnoside, several caffeoylquinic-acid derivatives, ferulic acid and assorted coumarins. Celery was distinguished by compounds including apiin, luteolin and apigenin conjugates, 4,5-dicaffeoylquinic acid and the coumarin marmesin. Parsley contained numerous furanocoumarins, including cnidilin, 5-hydroxyxanthotoxin, pinnatin, bergaptol and isoimperatorin, as well as rhamnetin and isorhamnetin conjugates. The researchers also detected chlorin-related molecules in all three herbs and the lignan eucommicin A in celery and coriander.

Principal component analysis, hierarchical cluster analysis and partial least squares discriminant analysis separated the three samples according to their metabolite profiles. In negative-ion mode, the first two principal components accounted for 95% of the observed variability, and the samples formed three distinct clusters. Coriander was associated with high levels of quercetin 3-O-rutinoside, eucommicin A and several hydroxylated C18 fatty acids, including three trihydroxy-octadecaenoic-acid features and hydroxy-octadecatrienoic- and hydroxy-octadecadienoic-acid features. Celery was associated with apiin, luteolin 7-O-apiosylglucoside, pyroglutamic acid, lunularic acid and a glycerophosphoinositol. Parsley was distinguished by fructose-phenylalanine and features including bergaptol. Supervised models separating celery from parsley reported R² and Q² values of 0.99 in both ionization modes, although the limited set of triplicate samples means these internal performance values should not be confused with independent validation.

Partial least squares regression then related metabolite abundance to the measured minimum inhibitory concentrations. The negative-ion model had cumulative R² of 0.92 and Q² of 0.87, while the positive-ion model had R² of 0.94 and Q² of 0.83. In both models, coriander’s metabolite pattern was inversely positioned relative to minimum inhibitory concentration, whereas parsley lay closest to the values associated with inactivity. Prominent variables associated with antibacterial activity included quercetin 3-O-rutinoside, trihydroxy-octadecaenoic-acid isomers, hydroxy-octadecatrienoic acid, hydroxy-octadecadienoic acid and eucommicin A. Positive-ion analysis additionally highlighted furanocoumarin hexosides, a methoxy-ethoxycarbonyl coumarin and a chlorin-related compound, 132-hydroxypheophorbide-A methyl ester. These are statistical associations within extract-level data, not demonstrations that any individual metabolite caused the observed inhibition.

All tested plant preparations also inhibited biofilm formation, whether used alone or in combinations with ciprofloxacin or norfloxacin. Biofilm mass was measured after 24 hours by fixing the attached biomass with methanol, staining it with crystal violet, releasing the dye with acetic acid and measuring absorbance at 630 nanometres. The study reported significant reductions in biofilm mass across the extract and antibiotic-combination conditions, including tests with sub-minimum-inhibitory-concentration levels of the plant extracts. The researchers discussed several possible contributors, including quercetin derivatives, phenolic compounds and coumarins that have activity against cell membranes, quorum sensing, extracellular polymeric substances and other biofilm-maintenance processes in earlier studies. Those mechanisms remain hypotheses for the herb extracts tested here, which were not separated into constituent compounds during the assays.

The bacterial collection also displayed several virulence-associated genes, adding context to the laboratory model. The fimH gene was present in 80% of the isolates, afa genes in 90%, papC in 76% and hly genes in 76%; the cytotoxic necrotizing factor gene occurred in 35%. These findings characterize the tested isolates but do not show that the herbal preparations affect the corresponding virulence traits. Moreover, the experiments were performed in vitro with commercial plant material, methanol-derived fractions and an artificial biofilm assay. They did not examine pharmacokinetics, host toxicity, urinary concentrations, treatment outcomes or the possibility of interactions with conventional drugs in patients.

For that reason, the results are best understood as a chemical and activity map for future research. The strongest candidates identified for isolation include quercetin 3-O-rutinoside, trihydroxy-octadecaenoic acid, hydroxy-octadecatrienoic acid, hydroxy-octadecadienoic acid and eucommicin A, together with selected coumarins and chlorin-related metabolites. The authors propose purifying these compounds, confirming their structures with authentic standards and complementary analytical methods, and testing them individually and in combinations against multidrug-resistant uropathogenic E. coli. Mechanistic work, cytotoxicity and selectivity assessments, and testing in appropriate in-vivo urinary tract infection models would still be required before any therapeutic or preventive application could be considered.

Subject of Research: Metabolite profiles associated with antibacterial and antibiofilm activity of celery, coriander and parsley against multidrug-resistant uropathogenic E. coli

Article Title: Integrative metabolomics and chemometric analysis reveal metabolites associated with antibacterial and antibiofilm activities of celery, Coriander, and parsley against multidrug-resistant uropathogenic Escherichia coli

Article References: Abdel-dayem, S. I. A., El Gedaily, R. A., Eltanany, B. M., Seiffein, N. L., Hani, G. A., Afifi, S. M., & Ali, D. E. (2026). Integrative metabolomics and chemometric analysis reveal metabolites associated with antibacterial and antibiofilm activities of celery, Coriander, and parsley against multidrug-resistant uropathogenic Escherichia coli. BMC Complementary Medicine and Therapies, 26(1), Article 313. https://doi.org/10.1186/s12906-026-05610-0

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05610-0

Keywords: Uropathogenic E. coli, Multidrug resistance, Antibiofilm activity, Coriander, Metabolomics, Plant extracts, Quercetin, Fatty acids, Urinary tract infections, Integrative, chemometric, analysis

The phage was recovered from sewage water collected in Karabük, Türkiye, using the standard laboratory strain E. coli ATCC 25922 as the indicator and propagation host. After enrichment and four consecutive rounds of single-plaque purification, the researchers obtained a homogeneous phage preparation producing circular, clear plaques with well-defined edges. Transmission electron microscopy of negatively stained particles revealed a polyhedral head roughly 45 nanometers in diameter and a long, flexible, non-contractile tail of about 176 nanometers, giving a total particle length of approximately 220 nanometers. The absence of a contractile sheath places KBU_SV1 firmly in the siphovirus morphotype within the class Caudoviricetes, the broad group of tailed double-stranded DNA phages that dominates the bacterial virosphere.

Host-range testing against a panel of 106 bacterial strains revealed a pattern of selective rather than broad lytic activity. Spot assays produced visible clearance on 16 strains, corresponding to a host range index of 0.15. Notably, clearance was detected on 7 of 20 carbapenem-resistant E. coli clinical isolates and 4 of 20 carbapenem-susceptible E. coli isolates, meaning that lysis was actually more frequent among the resistant strains. Four of 40 carbapenem-resistant Klebsiella pneumoniae isolates also showed spot clearance, while none of the 20 Enterococcus faecalis clinical isolates or the other reference strains tested were affected. The researchers caution that spot clearance alone does not prove productive infection, since clearing can arise through mechanisms that do not generate progeny phages, such as lysis from without, and efficiency-of-plating measurements will be needed to confirm true susceptibility, particularly for K. pneumoniae.

In liquid culture, KBU_SV1 suppressed the growth of E. coli ATCC 25922 across the entire range of multiplicities of infection tested, from 100 down to 0.01. During the first 14 hours of incubation, optical density in all phage-treated cultures remained below 0.10, a reduction of at least 93 percent relative to the untreated control, which reached an optical density of 1.70 by 24 hours. Regrowth was eventually observed under all conditions, a common feature of phage-bacterium systems that is often attributed to the emergence of phage-resistant subpopulations. Interestingly, the late growth response showed an apparent non-monotonic relationship with the initial phage dose: cultures treated at multiplicities of 0.1 and 0.01 ended up with lower optical densities at 24 hours than those treated at 10 and 1. The authors emphasize that the mechanism behind this pattern remains unresolved and that it should not be taken as evidence that lower doses are generally superior.

Physicochemical stability is a key practical consideration for any phage envisaged as a therapeutic candidate, and here KBU_SV1 performed well. The phage retained infectivity after one hour of incubation across a pH range from 4 to 12, with only modest declines of roughly 0.25 log units at the extremes of that interval compared with the neutral pH reference. No infectivity was detected at pH 2, however, a point worth noting given the acidic environment of the stomach. Temperature testing showed stable titers at 4, 23 and 37 degrees Celsius, but exposure to 65 degrees Celsius eliminated detectable infectivity. Taken together, these results suggest that KBU_SV1 is robust under moderate conditions relevant to storage, formulation and physiological temperature, although long-term storage stability was not evaluated.

Biofilms, structured bacterial communities embedded in an extracellular polymeric matrix, are a major driver of treatment failure because they dramatically reduce susceptibility to both antibiotics and immune defenses. KBU_SV1 demonstrated meaningful antibiofilm activity in crystal violet assays. Treatment of established biofilms of E. coli ATCC 25922 reduced biomass by 84.5 to 86.0 percent across all tested multiplicities of infection, while inhibition of biofilm formation reached 78.0 to 83.7 percent. Against the carbapenem-resistant clinical isolate Ec59, the effects were smaller but still substantial, with biomass reductions of 22.3 to 54.2 percent and inhibition of formation ranging from 24.6 to 57.5 percent, with the strongest effects at the highest multiplicity. The authors note that crystal violet staining measures total attached biomass rather than viable cells, so these findings reflect reductions in biofilm mass rather than proof of bacterial killing or complete eradication.

The in vivo experiments used the greater wax moth Galleria mellonella, a widely used invertebrate infection model. Larvae infected with a lethal dose of E. coli ATCC 25922 died rapidly, with 40 percent mortality at 12 hours and complete mortality by 18 hours. Larvae that received phage treatment one hour after infection survived significantly longer: mortality was distributed across the 48-hour observation period, and 40 percent of treated larvae were still alive at 24 hours. Kaplan-Meier survival analysis confirmed a statistically significant delay in mortality compared with the infection control, with a log-rank test yielding a p-value of 1.2 × 10⁻⁷. Importantly, no deaths occurred in larvae receiving phage alone or mock injections, indicating no detectable toxicity of the preparation under the tested conditions. The authors stress, however, that the phage delayed rather than prevented death, and that G. mellonella does not fully reproduce mammalian physiology, so these results should be viewed as preliminary evidence supporting further investigation rather than demonstration of therapeutic efficacy.

Genomic sequencing provided the most reassuring safety-relevant findings. The KBU_SV1 genome is a 45,190-base-pair double-stranded DNA molecule with a G plus C content of 53.56 percent, confirmed as complete by CheckV with 100 percent estimated completeness, zero contamination and a high-confidence 272-base-pair direct terminal repeat at both ends. Sequencing depth averaged nearly 24,000-fold with complete positional coverage. The genome encodes 71 putative open reading frames organized into five functional modules covering DNA and RNA replication, DNA metabolism, structural proteins, assembly and lysis, and contains no tRNA genes. Critically, screening with ResFinder, VirulenceFinder, the Comprehensive Antibiotic Resistance Database and the Virulence Factor Database detected no known antimicrobial resistance genes, no virulence determinants and no lysogeny-associated genes such as integrases, excisionases or phage repressors. Machine-learning-based lifestyle prediction tools PhageAI and PhageLeads both classified the phage as virulent, meaning strictly lytic, a property generally considered desirable for therapeutic candidates because it rules out the temperate lifestyle that could mediate horizontal gene transfer.

Comparative genomics clarified where KBU_SV1 fits in the phage taxonomy. BLASTn searches revealed 99.12 percent nucleotide identity with Escherichia phage EPIMAM03, and VIRIDIC intergenomic similarity calculations showed values above the 95 percent species-level threshold with several related genomes, including Dhillonvirus LM40P1 at 98.7 percent. Independent taxonomic analysis with taxMyPhage assigned KBU_SV1 to the genus Dhillonvirus within the class Caudoviricetes, and phylogenetic trees based on the large terminase subunit and whole-proteome comparisons were congruent with this placement. The complete genome has been deposited in GenBank under accession number PZ050807.1. Rather than representing an entirely new species, KBU_SV1 belongs to an existing species-level genomic cluster, which the authors note is consistent with contemporary genome-based approaches to phage classification.

The study has limitations that the authors themselves enumerate candidly. Efficiency-of-plating measurements were not performed on the spot-test-positive isolates, one-step growth experiments to determine adsorption kinetics, latent period and burst size were not carried out, the mechanism of bacterial regrowth was not investigated, and the DNA packaging mechanism implied by the direct terminal repeat was not experimentally resolved. The growth-reduction and in vivo experiments used the reference strain rather than carbapenem-resistant clinical isolates, and endotoxin levels in the phage preparation were not quantified. Nevertheless, the overall profile of KBU_SV1, combining lytic activity against carbapenem-resistant E. coli, broad pH and temperature stability, antibiofilm effects, delayed mortality in an infection model and a genome apparently free of resistance, virulence and lysogeny genes, marks it as a promising candidate for further development, whether deployed alone or as a component of phage cocktails. Before any clinical application, the authors conclude, future work must address productive host range, replication parameters, resistance development, optimized dosing and efficacy in mammalian infection models.

Subject of Research: Isolation and genomic characterization of the bacteriophage KBU_SV1 as a candidate therapeutic against carbapenem-resistant Escherichia coli

Article Title: Biological and genomic characterization of Escherichia phage KBU_SV1 active against carbapenem-resistant Escherichia coli

Article References: Öztaş, S., Karaynir, A., Ozkan, R. G., Bozdogan, B., & Dündar, D. (2026). Biological and genomic characterization of Escherichia phage KBU_SV1 active against carbapenem-resistant Escherichia coli. Frontiers in Microbiology, 17, Article 1927801. https://doi.org/10.3389/fmicb.2026.1927801

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1927801

Keywords: bacteriophage, phage therapy, carbapenem-resistant E. coli, antimicrobial resistance, Dhillonvirus, biofilm, Galleria mellonella, Caudoviricetes, siphovirus, phage genome, antibiofilm activity, wastewater

The logic of the approach rests on a simple contrast. Conventional antibiotic pipelines are slow, expensive and increasingly outpaced by bacterial evolution, while chemical methods for manufacturing nanoparticles rely on toxic solvents and costly instrumentation. Biological synthesis, by contrast, is inexpensive, clean and operates at ambient conditions. The idea dates back to 1980, when Beveridge and Murray first showed that Bacillus subtilis could build nanosized gold. Since then, bacteria, plants, yeasts, fungi and actinomycetes have all been recruited as living nanofactories. Streptomyces species, famous for producing roughly half of all clinically used antibiotics, have a particular advantage: their rich secondary metabolism provides a natural toolkit of biomolecules that can reduce metal ions and cap the resulting particles, preventing them from clumping together.

In the new work, the team collected the cell-free supernatant of S. thinghirensis and mixed it in equal volume with a freshly prepared solution of copper sulfate pentahydrate at ten millimolar concentration. The mixture was incubated at 30 degrees Celsius in darkness for up to 120 hours. As the reaction proceeded, the solution changed color from light blue to greenish brown, the first visible hint that copper ions were being transformed into copper oxide nanoparticles. Ultraviolet-visible spectroscopy tracked the reaction kinetically: absorption in the 210 to 220 nanometer region rose steadily over the incubation period, reflecting progressive complexation of the bacterial secondary metabolites during the capping process, while the characteristic broad absorption shoulder of monoclinic copper oxide between 250 and 300 nanometers emerged clearly after 24 hours and peaked at 120 hours, which the researchers selected as the optimum reaction time.

A battery of analytical techniques then confirmed the identity, purity and dimensions of the product. X-ray diffraction revealed peaks at 38.04, 44.16, 62.05, 76.80 and 81.14 degrees, matching the (111), (200), (-202), (022) and (222) planes of monoclinic CuO and agreeing closely with the standard reference data, with no trace of metallic copper or cuprous oxide impurity phases. Fourier transform infrared spectroscopy identified the chemical fingerprints of the capping layer: strong bands near 490 and 563 per centimeter corresponding to copper-oxygen vibrations, alongside signals from amines, peptide carbonyls and hydroxyl groups that demonstrate how the bacterial metabolites wrap around the growing crystals. Energy-dispersive X-ray analysis showed the surface dominated by copper at roughly 48 percent and oxygen at about 28 percent, with smaller contributions of phosphorus, carbon, calcium and potassium inherited from the actinobacterial metabolites.

Transmission electron microscopy provided the most striking image of the material: well-dispersed, quasi-spherical particles averaging about 9.8 nanometers in diameter, with sizes ranging from 6 to 16 nanometers. That is considerably smaller than many copper oxide particles previously made with plant extracts, such as the 40 to 80 nanometer particles produced from papaya leaf extracts or the roughly 15 nanometer particles from Calotropis procera latex. Dynamic light scattering, which measures the hydrodynamic diameter of particles in solution rather than the dry inorganic core, gave a larger average of 54.2 nanometers with a bimodal distribution, a discrepancy the authors attribute to the organic capping ligands and hydration shell that DLS counts but TEM does not. A zeta potential of minus 13.5 millivolts indicated moderate colloidal stability, and selected-area electron diffraction confirmed crystallinity with lattice spacings matching the diffraction data.

With the material thoroughly characterized, the researchers turned to the microbes. They tested the particles, dubbed ST@CuONPs, against six strains: vancomycin-sensitive E. faecalis ATCC 29212, vancomycin-resistant E. faecium ATCC 700221 carrying the vanA gene, methicillin-susceptible S. aureus ATCC 29213, methicillin-resistant S. aureus NCTC 12493, ceftazidime-sensitive P. aeruginosa ATCC 27853 and ceftazidime-resistant P. aeruginosa ATCC BAA-2108. In disk diffusion assays, the lowest nanoparticle dose produced no inhibition, but activity rose steeply and dose-dependently at higher loads. At 13.5 micrograms per disk, the particles generated inhibition zones of 29 millimeters against E. faecalis, 30 millimeters against susceptible S. aureus and 23 millimeters against the resistant S. aureus strain, all statistically superior to the standard antibiotic disks used as comparators.

The most dramatic results came from the pathogens that no longer respond to conventional therapy. The vancomycin-resistant E. faecium strain, which showed a zero-millimeter zone against the reference antibiotic, was highly susceptible to the nanoparticles, yielding a 29.17-millimeter inhibition zone. The ceftazidime-resistant Pseudomonas strain also proved susceptible, with a 10-millimeter zone, although the resistant Gram-negative pathogen was clearly harder to kill than its Gram-positive counterparts. Broth microdilution confirmed this pattern quantitatively: minimum inhibitory concentrations were 12.5 micrograms per milliliter for the sensitive E. faecalis and S. aureus strains, 25 micrograms per milliliter for the resistant E. faecium and S. aureus, and 100 micrograms per milliliter for both Pseudomonas strains. The Gram-negative envelope, with its outer lipid-rich membrane, appears to blunt the copper attack, whereas copper’s affinity for proteins makes the thick peptidoglycan walls of Gram-positive bacteria, roughly 30 nanometers thick compared with 3 to 4 nanometers in Gram-negative species, especially vulnerable.

Perhaps the most clinically significant finding concerns biofilms, the slimy, surface-attached bacterial communities that shield pathogens from both antibiotics and immune cells and serve as hotspots for the exchange of resistance genes. Using crystal violet staining to quantify preformed biofilms, the team showed that the nanoparticles dismantled these structures in a concentration-dependent manner. Against the vancomycin-resistant E. faecium strain, biofilm mass fell by 64 percent at a quarter of the MIC, by 83 percent at half the MIC and by 89 percent at the full MIC. The susceptible E. faecalis strain was even more responsive, retaining only 8 percent of its biofilm at the full MIC, a 92 percent eradication. Even the recalcitrant Pseudomonas biofilms were significantly reduced, by 68 and 58 percent at full MIC for the sensitive and resistant strains respectively. The authors propose that the bactericidal mechanism operates on several fronts at once: generation of reactive oxygen species, disruption of bacterial cell membranes, DNA damage and inhibition of protein synthesis, a multipronged assault that is difficult for bacteria to counter with single-gene resistance mechanisms.

The study is not without caveats. The authors acknowledge that they lacked a concurrent, internal cell-free supernatant control within the same experimental runs, although their earlier work established only a modest baseline effect for the supernatant alone, 12 to 14 millimeter zones, far short of the much larger zones observed here with the nanoparticles. They also frame the antibacterial mechanism as a hypothetical framework drawn from prior literature rather than a fully resolved pathway, and the moderate zeta potential suggests the colloidal stability could be improved. Still, the eco-friendly synthesis, which avoids toxic reagents entirely and uses a cheap, abundant metal instead of gold, platinum or silver, makes the route economically attractive, particularly for developing countries where antimicrobial resistance exacts its heaviest toll.

For now, the particles remain a laboratory-scale proof of concept rather than a bedside therapy. Toxicity testing, pharmacokinetics, formulation and large-scale manufacturing all lie ahead. But the demonstration that a single soil-dwelling Streptomyces strain can fabricate near-ten-nanometer copper oxide particles capable of shrinking vancomycin-resistant biofilms by nearly 90 percent offers a tantalizing glimpse of where the fight against superbugs may go next: not toward yet another tweak on an old antibiotic molecule, but toward materials engineered, or rather, grown, by biology itself.

Subject of Research: Green synthesis of copper oxide nanoparticles by Streptomyces thinghirensis and their activity against multidrug-resistant bacteria

Article Title: Green synthesis of copper oxide nanoparticles ST@CuONPs by Streptomyces thinghirensis: current treatment path against multidrug-resistant bacteria

Article References: Loqman, A., Esmaeel, Q., Sayen, S., Remy, S., Renault, J.-H., Smani, Y., & Loqman, S. (2026). Green synthesis of copper oxide nanoparticles ST@CuONPs by Streptomyces thinghirensis: current treatment path against multidrug-resistant bacteria. Frontiers in Pharmacology, 17, Article 1904965. https://doi.org/10.3389/fphar.2026.1904965

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1904965

Keywords: copper oxide nanoparticles, green synthesis, Streptomyces thinghirensis, antimicrobial resistance, multidrug-resistant bacteria, biofilms, nanotechnology, vancomycin-resistant Enterococcus faecium, MRSA, Pseudomonas aeruginosa, actinomycetes, antibiofilm activity

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

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that thrives in the damaged tissue of severe burns, where the loss of the skin barrier and prolonged hospitalization create ideal conditions for colonization. The organism is intrinsically resistant to many antimicrobial classes and readily acquires further resistance, and its capacity to form biofilms—structured bacterial communities encased in a self-produced extracellular matrix—makes eradication exceptionally difficult. Biofilms shield cells from antibiotics and immune defenses, driving therapeutic failure and recurrence in burn units and intensive care settings worldwide.

To gauge the scale of the problem in their region, the team collected 75 non-duplicate clinical P. aeruginosa isolates from patients at educational hospitals in Hamedan, Iran, between October 2024 and March 2025. Forty-nine isolates came from burn wounds and 26 from respiratory samples. Using Kirby-Bauer disk diffusion testing interpreted against Clinical and Laboratory Standards Institute breakpoints, the researchers found resistance was highest for ceftazidime at 78.67 percent, imipenem at 72 percent, and gentamicin at 69.33 percent. Overall, 57 isolates, or 76 percent, met the internationally accepted definition of multidrug resistance, meaning non-susceptibility to at least one agent in three or more antimicrobial categories.

Biofilm formation proved nearly universal. Seventy-four of the 75 isolates, or 98.7 percent, produced biofilms in the crystal violet microtiter assay, and every multidrug-resistant isolate did so, with 37 of the 57 classified as strong producers. From the burn-wound collection, the investigators selected nine isolates for detailed analysis—seven multidrug-resistant and two non-multidrug-resistant—all of which showed strong or intermediate biofilm formation, alongside the reference strain PAO1 as a standardized comparator.

The compound under investigation, (5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)(pyridin-2-yl)methanol, is a heterocyclic scaffold bearing a 3-methoxyphenyl substituent and a pyridin-2-yl methanol moiety, synthesized and characterized previously with purity above 95 percent confirmed by NMR. Against the nine selected burn isolates, the derivative yielded geometric mean minimum inhibitory and bactericidal concentrations of 20.54 and 41.21 micrograms per milliliter for planktonic cells. For biofilm-associated cells, the minimum biofilm inhibitory and eradication concentrations were 52.56 and 105.11 micrograms per milliliter, respectively. The higher eradication value reflects the well-known tolerance of mature biofilms, whose extracellular matrix limits drug penetration and access to embedded cells.

Checkerboard microdilution assays then tested whether the oxadiazole could potentiate gentamicin, an aminoglycoside protein synthesis inhibitor, and imipenem, a broad-spectrum carbapenem. Against planktonic cells, the gentamicin combination produced geometric mean fractional inhibitory and bactericidal concentration indices of 0.54 and 0.50, while the imipenem combination yielded 0.72 and 0.62. Against biofilm cells, the corresponding fractional biofilm indices ranged from 0.55 to 0.62, indicating similar gains in antibiofilm activity. Full synergy, defined as an index below 0.5, was observed in three of the nine isolates, with most others showing partial synergy, and no antagonism was detected in any combination.

The most mechanistically revealing results came from gene expression analysis. When the researchers exposed PAO1 and two clinical isolates to sub-inhibitory concentrations of the oxadiazole, quantitative real-time PCR revealed significant, concentration-dependent downregulation of two biofilm-associated genes: lasR, the master transcriptional regulator of the quorum-sensing system that coordinates virulence factor production and biofilm maturation, and algD, which encodes GDP-mannose dehydrogenase, the key enzyme in alginate biosynthesis that determines biofilm matrix thickness and stability. Log2 fold changes reached as low as minus 4.12 for both genes, with regression analysis showing strong concentration-response relationships and coefficients of determination between 0.888 and 0.969.

Because these transcriptional changes occurred at concentrations below the biofilm-inhibitory endpoint, the authors interpret them as evidence that the compound interferes with the regulatory machinery of biofilm formation rather than simply killing cells. They caution, however, that reduced gene expression alone does not establish a definitive molecular mechanism, since changes in viability or broader transcriptional responses could contribute, and that exopolysaccharide production and quorum-sensing signal levels were not directly measured in this study. Plausible mechanisms proposed for oxadiazoles elsewhere include disruption of bacterial membrane integrity, inhibition of essential enzymatic pathways, interference with nucleic acid synthesis, and altered permeability that enhances antibiotic uptake.

The dose-reduction implications are clinically significant. In previous work by the same group, the compound showed no significant cytotoxicity at concentrations up to 15.62 micrograms per milliliter, with an estimated IC50 range of 15.62 to 31.25 micrograms per milliliter. Combining the oxadiazole with gentamicin or imipenem lowered the fractional concentrations needed for antibacterial and antibiofilm effects, potentially allowing the compound to operate within its non-cytotoxic range while restoring activity to antibiotics against which the isolates had substantial resistance. The authors emphasize that the findings support the derivative as a promising adjunctive or alternative strategy for biofilm-related multidrug-resistant P. aeruginosa infections in burn patients, but that translation requires pharmacokinetic profiling, stable formulations, direct cytotoxicity testing of the combinations, and rigorous in vivo efficacy and toxicity studies in animal burn-infection models before clinical use can be considered.

Subject of Research: Antibacterial and antibiofilm activity of a 1,3,4-oxadiazole derivative against multidrug-resistant Pseudomonas aeruginosa from burn wound infections

Article Title: Antibacterial and antibiofilm properties of 1,3,4-oxadiazoles against multidrug-resistant Pseudomonas aeruginosa isolated from burn infections – an in vitro study

Article References: Nazari, M., Majzoobi, M. M., Alikhani, M. Y., & Imani Fooladi, A. A. (2026). Antibacterial and antibiofilm properties of 1,3,4-oxadiazoles against multidrug-resistant Pseudomonas aeruginosa isolated from burn infections – an in vitro study. International Microbiology. https://doi.org/10.1007/s10123-026-00890-5

Image Credits: AI Generated

DOI: 10.1007/s10123-026-00890-5

Keywords: Pseudomonas aeruginosa, burn wound infection, multidrug resistance, biofilm, 1,3,4-oxadiazole, lasR, algD, quorum sensing, synergistic therapy, gentamicin, imipenem, antimicrobial resistance