Proteus mirabilis is a Gram-negative bacterium best known for causing complicated urinary tract infections, particularly in patients with indwelling urinary catheters. Its defining feature is the ability to swarm across surfaces and to form crystalline biofilms that encase the bacteria in a protective matrix, frequently mineralized with struvite stones that make eradication with conventional antibiotics extremely difficult. As multidrug resistance spreads among uropathogens, clinicians are running out of reliable options, which has renewed scientific interest in inexpensive, locally available plants whose antimicrobial reputations stretch back centuries. Moringa oleifera is prized for its antimicrobial, antioxidant, and nephroprotective properties, while Hygrophila auriculata carries a similar traditional pedigree, making both attractive candidates for systematic laboratory evaluation.

The research team, led by Ayuni Tiranya Hansalee with colleagues including corresponding author Ayomi Dilhari and senior author Neluka Fernando, was careful to control how the plant material was processed. Leaf extracts were prepared using four distinct methods: simple boiling, steam extraction, a classical Ayurvedic decoction, and 50% ethanolic Soxhlet extraction. This design matters because extraction technique dramatically changes which phytochemicals end up in the final preparation, and therefore how potent the extract is against bacteria. By comparing methods head to head, the researchers could identify not just whether the plants work, but which traditional and laboratory preparation styles yield the most active material.

Antibacterial activity was assessed using a battery of standard microbiological assays. Agar well diffusion provided an initial screen of growth inhibition, while minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays quantified how much extract was needed to stop bacterial growth outright and to kill the cells completely. To probe the biofilm dimension, the team used an MTT assay, a colorimetric technique that measures metabolic activity and therefore the viability of bacteria embedded within established biofilms, yielding minimum biofilm eradication concentration values (MBEC50). The results showed a clear hierarchy: individual Moringa extracts achieved MIC values ranging from 0.2441 to 0.9766 milligrams per milliliter, with MBC values of 0.4883 to 1.9531 milligrams per milliliter and MBEC50 values of 1.229 to 4.227 milligrams per milliliter. Hygrophila extracts alone were somewhat less potent, with MIC values of 0.4883 to 0.9766 milligrams per milliliter, MBC values of 0.9766 to 1.9531 milligrams per milliliter, and MBEC50 values of 1.029 to 3.322 milligrams per milliliter.

The headline finding emerged when the two plants were paired. The combined treatment of Hygrophila auriculata prepared as an Ayurvedic decoction with Moringa oleifera prepared by boiling exhibited enhanced antibacterial and antibiofilm efficacy across the board. MIC values for the combination dropped to a range of 0.1220 to 0.4883 milligrams per milliliter, MBC values fell to 0.2441 to 0.9766 milligrams per milliliter, and MBEC50 values ranged from 0.6098 to 2.161 milligrams per milliliter. In practical terms, the mixture needed roughly half the concentration of the weaker individual extracts to achieve the same inhibitory and bactericidal effects, and it also chipped away at biofilms more efficiently. This kind of synergistic interaction is exactly what researchers hunting for botanical alternatives to antibiotics hope to find, because lower effective doses reduce both cost and the risk of off-target effects.

Potency alone is not enough for any therapeutic candidate; safety must be evaluated in parallel. The team turned to the zebrafish embryo toxicity assay, a widely accepted whole-organism model in developmental toxicology. Zebrafish embryos are transparent, develop rapidly, and share many physiological pathways with vertebrates, making them a sensitive early warning system for embryotoxic compounds. Embryos were exposed to increasing concentrations of each extract and the combination, and mortality was tracked to calculate the lethal concentration for 50 percent of the population (LC50). All three treatments produced a dose-dependent toxic response, meaning higher concentrations killed more embryos, but the combination again came out ahead. The combined extract showed the highest LC50 value at 1812.74 micrograms per milliliter, compared with 1527.5 micrograms per milliliter for Moringa alone and 1274.28 micrograms per milliliter for Hygrophila alone. A higher LC50 means lower toxicity, so the mixture was the gentlest of the three on developing embryos.

To begin understanding the chemistry behind these effects, the researchers characterized the selected aqueous extracts using Fourier-transform infrared spectroscopy (FTIR), which identifies the major functional groups and chemical classes present, such as phenolics, flavonoids, and other plant secondary metabolites often implicated in antimicrobial action. In parallel, gas chromatography-mass spectrometry (GC-MS) was performed on the corresponding dried leaf materials to profile their volatile and semi-volatile constituents. The authors emphasize that these analyses provide preliminary chemical characterization rather than definitive identification of the active molecules, but they offer a starting map for future fractionation studies aimed at isolating the compounds responsible for the antibacterial and antibiofilm activity.

The study’s design also reflects a thoughtful approach to research ethics and provenance. Because the work involved only secondary analysis of previously collected, anonymized bacterial isolates held in the culture collection of the Department of Microbiology at the University of Sri Jayewardenepura, no new human participants were recruited and no identifiable patient information was accessed. The isolates originally came from a study of biofilm formation in patients with urinary catheters that had already received ethical approval and informed consent, and institutional guidelines deemed additional approval unnecessary. The research itself was funded by a Research Grant from the University of Sri Jayewardenepura (Grant No. ASP/01/RE/AHS/2022/90), and the authors declared no competing interests.

It is important to keep the scope of these findings in perspective. This was an in vitro study, conducted entirely in laboratory glassware and microplates, and the authors are explicit that the results outline promising potential rather than proven therapies. Concentrations that inhibit bacteria in a well do not automatically translate into safe, effective doses in a human urinary tract, where factors such as dilution by urine, protein binding, metabolism, and tissue penetration all intervene. The zebrafish embryo data, while reassuring in their relative terms, are a first-tier toxicity screen rather than a substitute for mammalian safety studies. The path from a promising decoction to a clinically validated treatment typically runs through compound isolation, mechanism-of-action studies, pharmacokinetic profiling, and eventually controlled trials, and this study represents an early but genuinely encouraging step on that path.

Even so, the implications are significant for a field urgently seeking new weapons against biofilm-forming uropathogens. The demonstration that a traditional Ayurvedic preparation of Hygrophila can synergize with simply boiled Moringa leaves, while simultaneously lowering embryotoxicity, suggests that the combination may contain complementary compounds whose effects add up in ways that single-plant extracts cannot replicate. For communities where these plants grow abundantly and antibiotics are expensive or increasingly ineffective, the findings provide a scientific foundation for further investigation of accessible, low-cost botanical candidates. The open access publication means researchers anywhere can scrutinize the data, replicate the assays, and build on the chemical characterization. As antimicrobial resistance continues to erode the utility of conventional drugs, studies like this one, which pair rigorous quantitative microbiology with developmental toxicity screening, offer a template for how traditional knowledge can be tested, refined, and potentially translated into modern therapeutic options against Proteus mirabilis and the infections it causes.

Subject of Research: Antibacterial and antibiofilm activity of Moringa oleifera and Hygrophila auriculata leaf extracts against Proteus mirabilis

Article Title: In vitro evaluation of antibacterial, antibiofilm, and embryotoxicity profiles of individual and combined Moringa oleifera and Hygrophila auriculata leaf extracts against Proteus mirabilis

Article References: Hansalee, A. T., Nissanka, M. C., Dilhari, A., Weerasekera, M. M., & Fernando, N. (2026). In vitro evaluation of antibacterial, antibiofilm, and embryotoxicity profiles of individual and combined Moringa oleifera and Hygrophila auriculata leaf extracts against Proteus mirabilis. BMC Complementary Medicine and Therapies. https://doi.org/10.1186/s12906-026-05617-7

Image Credits: AI Generated

DOI: 10.1186/s12906-026-05617-7

Keywords: Moringa oleifera, Hygrophila auriculata, Proteus mirabilis, antibacterial, antibiofilm, embryotoxicity, zebrafish embryo assay, MIC, MBC, urinary tract infections, medicinal plants, antimicrobial resistance

The team, led by Dounya Achwak Chemmam of the University of Jijel, grew the fungus for twenty-one days and extracted its metabolic products with ethyl acetate, a solvent chosen for its ability to pull moderately polar organic molecules out of fungal biomass. When that crude extract was tested against a panel of pathogenic microorganisms, the numbers were striking. The minimum inhibitory concentration, or MIC, the lowest dose that halts visible microbial growth, was just 7.5 micrograms per milliliter against Staphylococcus aureus ATCC 43300 and 15 micrograms per milliliter against Bacillus cereus ATCC 14579. For a raw, unfractionated extract, those values are competitive with many purified natural-product antibiotics and suggest that the active constituents are present at meaningful levels rather than as trace contaminants.

Antibacterial potency was only the beginning. The same extract showed an IC50 of 33.27 micrograms per milliliter in an anti-inflammatory assay, meaning that half of the measured inflammatory activity was suppressed at that concentration. In an antidiabetic screen targeting enzymes that break down dietary sugars, the extract achieved an IC50 of just 3.32 micrograms per milliliter, an exceptionally low figure that points to strong inhibition of carbohydrate-hydrolyzing enzymes. Inhibiting these enzymes is a validated strategy for managing blood glucose spikes after meals, and fungal metabolites have increasingly been flagged as alternatives to synthetic drugs such as acarbose. Finally, the extract exhibited antiproliferative activity with an IC50 of 124.16 micrograms per milliliter against the tested cell line, a more modest but still noteworthy effect that rounds out the pharmacological profile.

Because the activity of a fungal extract depends heavily on how the organism is grown, the researchers next turned to fermentation optimization using the one-factor-at-a-time method, systematically varying culture conditions to find the settings that maximize antimicrobial output. The tuned conditions produced dramatic gains. Against Escherichia coli, the optimized cultures generated a zone of inhibition of 19 millimeters; against Klebsiella pneumoniae, 23 millimeters; and against Bacillus cereus, an impressive 34 millimeters. That last figure is particularly significant because Bacillus cereus is a spore-forming foodborne pathogen, and activity of that magnitude against it suggests the extract could have applications well beyond the clinic, including food preservation and agricultural biocontrol.

What exactly is inside the extract? To answer that, the team deployed two complementary analytical techniques. Gas chromatography coupled to mass spectrometry, or GC-MS, separates volatile and semi-volatile compounds and fragments them into characteristic mass fingerprints that can be matched against spectral libraries. High-performance liquid chromatography, or HPLC, handles compounds that are less volatile or more thermally fragile. Together, the two methods revealed fourteen distinct metabolites produced by PB1, a chemically diverse roster that includes small organic acids, sugars, and more exotic structures.

The identified compounds read like a short course in fungal chemistry. Among them are 3-methylbutanoic acid and propanoic acid, short-chain fatty acids with documented antimicrobial effects against oral and other pathogens; ethylene glycol and 2,3-butanediol, small alcohols whose antibacterial properties have only recently attracted attention; and lactic acid alongside 2,3-dihydroxy-3-methylpropanoic acid, both hydroxy acids that can disrupt microbial membranes. The list also contains phosphorylated and siloxane-containing species, the sugars D-fructofuranose pentakis and D-psicofuranose pentakis, the plant-like glycoside erigeside B, D-galactonic acid, a bufadienolide-type steroid called bufa-20,22-dienolide, and the nucleoside uridine, a molecule that has inspired synthetic small-molecule inhibitors in medicinal chemistry.

No single one of these compounds may account for the full spectrum of activity on its own. More likely, the extract works through additive or synergistic interactions, a common feature of crude natural-product mixtures that pharmaceutical chemists are only beginning to exploit systematically. The presence of short-chain fatty acids could underpin much of the antibacterial effect, while the glycosides and the bufadienolide are plausible contributors to the cytotoxic and enzyme-inhibiting activities. Disentangling those contributions, through bioassay-guided fractionation, will be the essential next step before any clinical relevance can be claimed.

Context matters for this discovery. Penicillium citrinum is a species with a storied double reputation. On one hand, it is the original source of mevastatin, the first statin cholesterol-lowering drug, and its relatives have given medicine compounds ranging from antibiotics to immunosuppressants. On the other hand, the species is known for citrinin, a mycotoxin that contaminates grains and other foods, which means any future development of PB1-derived products would need careful toxicological scrutiny. The current study did not report citrinin among the fourteen detected metabolites, but the genus-level history is a reminder that bioactivity and toxicity often travel together in fungal chemistry and must be evaluated in parallel.

The choice of source organism is also part of the story. Almonds are a nutritionally celebrated food, and the fungi that colonize them occupy a niche that sits at the intersection of food safety and chemical innovation. Isolating a strain from a common pantry item and finding MIC values in the single-digit microgram range underscores how much pharmacological potential remains locked in ordinary environments. The Algerian team has previously described bioactive Penicillium strains from electronic waste and sea sand, and this new work extends that survey into the food chain, reinforcing the idea that productive fungi are distributed across wildly different habitats.

For now, the findings remain at the level of in vitro biochemistry: enzyme assays, microbial cultures, and cell lines, not patients. Translating a crude extract into a drug candidate typically requires years of fractionation, structural elucidation, mechanism-of-action studies, toxicity testing, and formulation work. But the comprehensive nature of this assessment, spanning antimicrobial, anti-inflammatory, antidiabetic, and antiproliferative activities alongside full chemical profiling and fermentation optimization, gives the almond fungus PB1 a unusually complete starting dossier. It is exactly the kind of well-characterized lead that natural-products researchers hope will seed the next generation of microbe-derived medicines, and it arrived, fittingly, from a handful of almonds.

Subject of Research: Bioactive secondary metabolites from an almond-derived Penicillium citrinum strain

Article Title: Unveiling the pharmacological potential of Penicillium citrinum PB1 isolated from almonds: a comprehensive bioactivity assessment

Article References: Unveiling the pharmacological potential of Penicillium citrinum PB1 isolated from almonds: a comprehensive bioactivity assessment. (n.d.). https://doi.org/10.1007/s13205-026-05066-6

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05066-6

Keywords: Penicillium citrinum, almonds, secondary metabolites, antimicrobial activity, MIC, anti-inflammatory, antidiabetic, alpha-glucosidase, GC-MS, HPLC, fermentation optimization, natural products

The urgency behind the research reflects a grim epidemiological reality. Antimicrobial resistance is escalating worldwide as antibiotics are overused in medicine and agriculture, and the pipeline for genuinely new drugs has thinned to a trickle. The ESKAPE pathogens, a group that includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, have become emblematic of the crisis because they evade nearly every class of conventional antibiotic through efflux pumps, destructive enzymes such as beta-lactamases, target modifications, and the horizontal acquisition of resistance genes. Pseudomonas aeruginosa, for instance, deploys an array of secreted toxins delivered through type II and III secretion systems, while Acinetobacter baumannii survives desiccation, forms stubborn biofilms on hospital surfaces, and has become notorious for carbapenem resistance. Staphylococcus aureus rounds out the trio with immune-evasive proteins, pore-forming toxins, and the now-ubiquitous methicillin-resistant lineage known as MRSA.

Nanoparticles offer a fundamentally different attack strategy. Because of their tiny size and enormous surface-area-to-volume ratio, metal nanoparticles can interact with bacterial cells in ways that conventional drugs cannot. Silver and gold nanoparticles are thought to disrupt bacterial membranes, generate reactive oxygen species that damage cellular machinery, interfere with vital metabolic processes, and even potentiate existing antibiotics. Crucially, these mechanisms operate in concert and are less likely to trigger the classical resistance pathways that neutralize small-molecule drugs. Nanoparticles can also penetrate biofilms, the dense microbial communities that shield chronic infections from both antibiotics and immune cells. The catch has always been synthesis: traditional chemical and physical production methods can be expensive, energy-intensive, and environmentally unfriendly, and the resulting particles may carry toxic residues.

That is where green synthesis enters the picture. Rather than using synthetic reducing and stabilizing agents, green synthesis harnesses plant phytochemicals such as flavonoids, alkaloids, phenols, and terpenoids to simultaneously reduce metal salts into nanoparticles and cap their surfaces, all in a single step. The Nonea lutea plant, a member of the Boraginaceae family with a long history in traditional medicine, proved an ideal candidate. Its aerial parts, collected in Mazandaran province in northern Iran and verified by botanists at the Sari School of Pharmacy, were dried, ground, and extracted with methanol before being combined with silver nitrate under optimized conditions of pH, temperature, and reaction time.

The resulting silver nanoparticles were extensively characterized before any biological testing. Ultraviolet-visible spectroscopy revealed a characteristic surface plasmon resonance band at roughly 425 nanometers, confirming the conversion of silver ions to metallic silver, a change that was visible to the naked eye as the reaction mixture shifted from pale yellow to dark brown. Electron microscopy showed predominantly spherical particles: field-emission scanning electron microscopy placed diameters between 22 and 37 nanometers, while transmission electron microscopy yielded an average size of about 13.5 nanometers. Notably, the images revealed a thin organic coating around each particle, the fingerprint of plant-derived biomolecules acting as capping agents. X-ray diffraction confirmed a cubic crystalline structure with an average crystallite size near 26.9 nanometers, energy-dispersive X-ray spectroscopy detected elemental silver along with carbon and oxygen from adsorbed biomolecules, and a zeta potential of minus 32.9 millivolts indicated strong electrostatic repulsion and good colloidal stability.

With the particles characterized, the team turned to the clinical battlefield. They collected 100 isolates each of P. aeruginosa, A. baumannii, and S. aureus from patients at five hospitals affiliated with Mazandaran University of Medical Sciences, spanning burn units, intensive care, surgery, and emergency wards. From these, 15 multidrug-resistant isolates of each species were selected for nanoparticle testing based on strict criteria: resistance to at least three antibiotic classes, carriage of major resistance and virulence genes, and diverse clinical origins. Molecular screening by polymerase chain reaction revealed an alarming genetic landscape. Every one of the 15 Pseudomonas isolates carried the exoU, exoA, bla CTX-M, bla TEM, and bla OXA-2 genes. All Acinetobacter isolates harbored the biofilm genes bap and csuE, and every Staphylococcus isolate carried the icaA biofilm gene, with most also bearing the methicillin resistance determinant mecA.

When the green-synthesized silver nanoparticles met these formidable organisms in broth microdilution assays, the results were remarkable. Minimum inhibitory concentrations ranged from 0.19 to 1.56 micrograms per milliliter against Pseudomonas aeruginosa, 0.19 to 0.78 against Acinetobacter baumannii, and just 0.19 to 0.39 against Staphylococcus aureus. Minimum bactericidal concentrations tracked closely behind, suggesting the particles do not merely stall growth but actively kill the bacteria. The statistics confirmed what the raw numbers implied: the green silver nanoparticles significantly outperformed the green gold nanoparticles, the chemically synthesized silver nanoparticles, and the chemically synthesized gold nanoparticles for every species tested, with p-values below 0.001 across comparisons. Activity was slightly weaker against Pseudomonas than against Staphylococcus, a difference the authors attribute to the thick peptidoglycan armor of Gram-positive cell walls potentially slowing nanoparticle penetration, though the precise mechanism remains to be verified experimentally.

The comparative failures proved as instructive as the successes. Green-synthesized gold nanoparticles from the same plant extract required concentrations of 100 to 200 micrograms per milliliter to inhibit growth, and chemically synthesized silver and gold nanoparticles of both types showed essentially no useful activity, with minimum inhibitory and bactericidal concentrations exceeding 500 micrograms per milliliter. The stark gap between chemically produced and plant-derived silver particles points to a tantalizing conclusion: the phytochemical coating itself may be central to antimicrobial performance. Nonea lutea is rich in flavonoids, alkaloids, saponins, tannins, and phenolic compounds with documented biological activity, and the researchers suggest these bioactive surface molecules may act synergistically with the metallic core. The superior intrinsic antimicrobial nature of silver ions compared with gold likely contributes as well.

The findings compare favorably with earlier green synthesis studies. Silver nanoparticles made from Feijoa sellowiana, for example, achieved similar inhibitory concentrations against standard laboratory strains, but the Nonea lutea particles matched that potency against genuine multidrug-resistant clinical isolates and delivered lower bactericidal values in the same experimental setting. The proximity of inhibitory and bactericidal concentrations hints at a bactericidal mode of action, consistent with proposed mechanisms of membrane disruption and reactive oxygen species generation described across the wider literature, though the authors are careful to note that these mechanisms remain hypotheses requiring direct confirmation through techniques such as electron microscopy, membrane permeability assays, and time-kill experiments. Encouragingly, no significant correlation was found between specific resistance gene carriage and nanoparticle susceptibility, suggesting the silver particles may sidestep the genetic determinants that defeat conventional antibiotics.

The road from laboratory dish to clinic is long, and the authors are measured in their claims. This was an in vitro proof-of-concept study; biofilm disruption, synergy with existing antibiotics, pharmacokinetics, toxicity, and in vivo efficacy all remain untested. Nonetheless, the study makes a compelling case that sustainable, plant-mediated nanotechnology can produce particles capable of confronting the worst pathogens hospitals have to offer. As antibiotic discovery falters and resistance genes spread through mobile genetic elements, the idea that a desert wildflower’s chemistry, combined with nothing more exotic than silver and careful engineering, could help fill the therapeutic void is a reminder that some answers to modern medicine’s hardest problems may be growing quietly in the hills of northern Iran.

Subject of Research: Green synthesis of plant-mediated silver and gold nanoparticles and their antibacterial activity against multidrug-resistant clinical pathogens

Article Title: Antibacterial efficacy of Nonea lutea-mediated silver and gold nanoparticles against multidrug-resistant clinical pathogens harboring virulence and resistance genes

Article References: Jasim, H. H., Gholami, M., Ahanjan, M., Ebrahimzadeh, M. A., & Goli, H. R. (2026). Antibacterial efficacy of Nonea lutea-mediated silver and gold nanoparticles against multidrug-resistant clinical pathogens harboring virulence and resistance genes. International Microbiology. https://doi.org/10.1007/s10123-026-00898-x

Image Credits: AI Generated

DOI: 10.1007/s10123-026-00898-x

Keywords: antimicrobial resistance, silver nanoparticles, green synthesis, Nonea lutea, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, MDR pathogens, nanomedicine, virulence genes, MIC, plant extract