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