Melia azedarach Phenolic and Terpenoid Leaf Extracts Impair Biofilm Production and Quorum Sensing in Clinical Escherichia coli Isolates

Escherichia coli can cause diseases such as urinary tract infections (UTIs), bloodstream infections (sepsis), middle ear infections, wound sepsis, newborn meningitis, peritonitis, and respiratory tract infections (Naqid et al., 2020; Gagaletsios et al., 2025). The most common causative agent of UTIs in humans is E. coli (Rahman et al., 2019; Naqid et al., 2020; Mayer et al., 2025). Biofilm formation on multiple surface types substantially contributes to the prominence of uropathogenic Escherichia coli (UPEC ) as a leading cause of healthcare-associated complications in humans (Mayer et al., 2025). Cases of multidrug -resistant (MDR) E. coli have increased notably, showing the highest prevalence observed in UTIs (Batubara et al., 2025). Globally, E. coli ranks as the second critical MAR pathogen that urgently requires new therapies, heavily driving an infection-related mortality (Mayer et al., 2025).

Numerous studies have shown that constituents extracted from various medicinal plants possess a range of biological activities, including antibacterial and antifungal properties (Touzout et al., 2023; Naveed et al., 2024). M. azedarach, a Meliaceae tree commonly known as chinaberry or Persian lilac, provides well-established medicinal benefits with documented antifungal and antibacterial effects (Touzout et al., 2023; Hemdan et al., 2023). M. azedarach contains numerous secondary metabolites, including glycosides, tannins, phenols, and flavonoids, which are responsible for its various biological activities (Touzout et al., 2023; Naveed et al., 2024; Hassan et al., 2025). The leaf extract of this plant contains phytochemicals with an antibacterial activity against Gram-positive and Gram-negative bacteria (Naveed et al., 2024; Batubara et al., 2025).

The capacity to form biofilms, swarming motility, exopolysaccharide production, and the inhibition of violacein production represent critical QS-dependent factors regulated by the QS mechanism (Alrashidi et al., 2022; Chaichana et al., 2024). Biofilms constitute a critical threat to healthcare systems because of their elevated tolerance to conventional interventions. This resistance stems from multifactorial mechanisms, including restricted antimicrobial diffusion across the biofilm matrix and the upregulation of resistance mechanisms within the colonizing E. coli cells (Moradi et al., 2020; Mayer et al., 2025). Therefore, it is necessary to investigate and explore new anti-biofilm compounds (Ismail et al., 2024; David et al., 2026).

Quorum sensing (QS) crucially regulates target gene expression, particularly virulence genes such as those involved in antibiotic resistance, motility, pigment production, biofilm formation, and other virulence factors (Dimitrova et al., 2023). In addition, several protein studies have shown that downregulation of the proteins associated with QS influences the virulence-associated phenotypes, such as motility and biofilm formation in E. coli (Chaichana et al., 2024; Mayer et al., 2025). The use of quorum-sensing inhibitors (QSIs) offers a key advantage of an anti-virulence strategy relative to the standard antibiotic therapies (Maura et al., 2016; Saeki et al., 2020; Santos et al., 2021). Emerging research indicated that phenolic-rich plant extracts possess potent QSI properties (Nazir et al., 2020).

Given the scarcity of studies on the application of ultrasound-assisted extraction, we selected this technique for the present study. To the best of our knowledge, no previous studies have examined the effect of this extraction technique on the antibacterial, anti-biofilm, and anti-QS activity against E. coli isolated from various clinical samples in Babylon City, Iraq. Hence, this study aimed to assess the phytochemical composition of the ethanol and hexane leaf extracts of M. azedarach using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), respectively. The nature of the anti-QS properties of M. azedarach remains unknown. Therefore, the anti-QS activity of sub-MICs of these extracts was investigated by assessing the inhibition of motility (swimming and swarming) in E. coli, as well as the inhibition of violacein pigment production by Chromobacterium violaceum, representing an initial baseline evaluation.

Approximately 300 clinical samples, including 100 from each of urine, wound, and sputum were collected from patients, which attended to a Hospital in Babylon, Iraq, between October 2025 and December 2025. The suspected bacterial isolates obtained from these clinical samples were streaked on eosin methylene blue (EMB) agar and MacConkey agar (Oxoid, Basingstoke, UK), and then incubated for 24 h at 37°C. After incubation, to identify the developed E. coli isolates, conventional biochemical assays were conducted, including indole, carbohydrate fermentation, catalase, oxidase, H2S production, Voges–Proskauer, methyl red test, Kligler’s Iron Agar (KIA), urea hydrolysis, and citrate utilization, following the methods described by Abdul Hamid and Khoshaba (2024); Getahun et al. (2025). The Vitek 2 system (bioMérieux, France) and the API 20E identification kit (bioMérieux, France) further verified the identity of the recovered E. coli isolates. For the QS inhibition assay, stocks of C. violaceum ATCC 12472 strain obtained from the Microbiology Department, University of Babylon, were used.

Assessment of biofilm-forming capacity

Escherichia coli biofilm formation was quantified using the 96-well microtiter plate method described by Fink (2023); David et al. (2026). Briefly, overnight bacterial cultures in Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, UK) were adjusted to 0.5 McFarland standard, and 200 µl aliquots were inoculated into BHI agar plates, followed by incubation at 37 °C for 48 h. Afterward, the grown cells were washed with phosphate-buffered saline and the developed biofilm cells were stained with 0.1% crystal violet for 15 min. The bound dye was solubilized using 96% ethanol, foiiowed by absorbance measurement at 600 nm. Uninoculated BHI broth served as the negative control. Biofilm formation was categorized according to the following criteria: strong biofilm producer = OD > 4 × ODcut; moderate biofilm producer = 2 × ODcut < OD ≤ 4 × ODcut; weak biofilm producer = ODcut < OD ≤ 2 × ODcut; and non-biofilm producer = OD ≤ ODcut, where ODcut = OD of the negative control (Alshammari et al., 2023).

In November 2025, M. azedarach leaves were collected from the Babylon Tourist Resort, Iraq. An expert in plant taxonomy at the University of Babylon authenticated the plant identity. Following a water wash, the leaves were dried in the dark at room temperature before being ground into a fine powder using an electric laboratory mill and stored at 4°C until required (Kadhim et al., 2021).

Bioactive compounds were extracted from the dried leaf powder via ultrasound-assisted extraction using an ultrasonic bath (Bandelin Electronic, Berlin, Germany) for 60 min at 40°C following the methodology described by Batubara et al. (2025). Specifically, hexane was employed as the solvent for terpenoid extraction, while 70% ethanol was utilized for phenolic extraction. Quantification of total phenolics was performed spectrophotometrically via the Folin–Ciocalteau method against a standard curve of gallic acid (Sigma-Aldrich, St. Louis, MO, USA) (Mykhailenko et al., 2025; Kadhim et al., 2026). Moreover, total terpenoids were determined spectrophotometrically as per the procedure described by Biswas et al. (2021).

In vitro antibacterial efficacy of M. azedarach extracts

Stock solutions of M. azedarach leaf terpenoids extract (MLTE) and M. azedarach leaf phenolic extract (MLPE) were prepared at concentrations of 10, 25, 50, and 75 mg/ml (Al-Aboudi and Al-Azawi, 2025). The disc diffusion technique was performed to determine the growth inhibition zones according to Touzout et al. (2023); Getahun et al. (2025). Briefly, bacterial suspensions adjusted to 10⁸ cfu/ml were inoculated onto Mueller-Hinton Agar (MHA) plates. Sterile 6 mm paper discs were loaded with 50 µl of each extract dilutions and placed aseptically onto the agar surface. Ciprofloxacin (5 µg/ml) and Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) served as the positive and negative controls, respectively. Following incubation at 37 °C for 24 h, the inhibitory activity of the extracts was determined by measuring the diameter of the zone of inhibition (mm) using a calibrated ruler (Touzout et al., 2023; Getahun et al., 2025). The MIC values were determined via the resazurin microtiter assay following the methodology described by Fink (2023). Briefly, 100 µl of each extract dilution were individually mixed with 100 µl of Nutrient Broth (NB) and 5 µl of E. coli inoculum (10⁶ cfu/mL) in 96-well microplates. Following incubation at 37°C for 24 h, each well received 10 µl of 0.015% resazurin solution. The plates were incubated further under identical conditions for 4 h to evaluate the colorimetric changes. The MIC represented the lowest extract concentration that prevented conversion of the blue dye to pink . The MBC was determined by subculturing a 10 µl aliquot from each microtiter well showing no visible turbidity onto MHA (Oxoid, Basingstoke, UK) plates, before maintaining them at 37°C for 24 h (Getahun et al., 2025).

The anti-biofilm activity of M. azedarach extracts was tested at the MIC, MIC/2, MIC/4, and MIC/8, where all of them represented the sub-MIC concentrations. The biofilm assay was performed as described above (Al-Aboudi and AL-Azawi, 2025).

The percentage of biofilm inhibition = [(OD control–OD extract)/ OD control] ×100 (David et al., 2026)

The chemical composition of the hexane extract (terpenoid extract, MLTE) was determined via GC-MS (Agilent Technologies, Santa Clara, CA, USA) system equipped with an HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness), using helium as a carrier gas at a flow rate of 1.0 ml/min. Aliquots of 100 µL of extract were analyzed with an inlet temperature of 300°C. The mass spectrometer operated in electron ionization mode, and identification of compound involved spectral comparision with library databases and authentic standards. Relative abundances were quantified from the peak areas in the total ion chromatograms (Naveed et al., 2024; Batubara et al., 2025). The chemical composition of the ethanol extract (phenolic extract, MLPE) was characterized using a HPLC system (Shimadzu, Kyoto, Japan). Analyses were performed according to Touzout et al. (2023); Mykhailenko et al. (2025). By using an Agilent Technologies 1100 series liquid chromatograph equipped with a Kinetex Evo C18 analytical column (250 × 4.6 mm, 3 µm) maintained at 40°C. The mobile phase consisted of 1% formic acid in water (A) and acetonitrile plus 1% formic acid (B), with the elution rate set to 0.8 ml /min. Sample aliquots of 20 µl were injected, and peak monitoring was achieved at 254 nm. Identification of phenolic compounds involved matching their retention times and UV spectra against authentic reference standards analyzed under matching conditions.

The broth microdilution technique in 96-well microtiter plates was employed to monitor the inhibitory influence of the extracts on E. coli growth kinetics. 100 µl of a bacterial suspension adjusted to 10⁶ cfu/ml were mixed with 100 µl of graded plant extract concentrations (i.e., MIC, MIC/2, MIC/4, and MIC/8). Wells containing inoculated broth without extracts served as the growth control. The plates were incubated at 37°C for 24 h, and optical density readings were recorded at 600 nm every 1 h using a microplate reader (BMG LabTech, Offenburg, Germany) to construct continuous kinetic curves. (Chaichana et al., 2024).

To ensure that the extracts did not affect the bacterial growth, two QS assays were conducted at sub-inhibitory concentrations.

Following minor optimizations to the established protocol, swarming migration was evaluated using semi-solid LB agar matrices (0.5%) supplemented with 0.5% filter-sterilized D (+)-glucose (Santos et al. 2021). To evaluate the anti-motility effects, the plant extracts were incorporated individually into the agar at graded concentrations, specifically ranging from MIC down to MIC/16, including MIC/2, MIC/4, and MIC/8 (corresponding to 50, 25, 12.5, 6.25, and 3.12 mg/ml for MLTE, and 30, 15, 7.5, 3.75, and 1.87 mg/ml for MLPE, respectively). Swimming translocation profiles were monitored using a semi-solid LB agar (Oxoid, Basingstoke, UK) medium (0.25% agar) containing the extracts at sub-MICs. Approximately 2 μl aliquot of a bacterial culture inoculum containing 10⁸ cfu/ml of E. coli were spotted at the center of the plate, followed by incubation at 37°C for 12 h (swarming motility) or 20 h (swimming motility). The control plate consisted of a semi-solid agar prepared without the extracts. Both types of motility were assessed by measuring the diameter of the circular growth spread around the inoculation point by using a calibrated ruler (Bose et al., 2020).

To evaluate the potential of the extracts to inhibit the QS system at sub-MICs, suppression of violacein pigment synthesis by C. violaceum ATCC 12472 was assessed. An overnight Luria-Bertani (LB) broth culture of C. violaceum ATCC 12472 strain was prepared. The assay was performed according to Moradi et al. (2020). Violacein production inhibition was quantified in 96-well microtiter plates using C. violaceum ATCC 12472 strain. Briefly, 100 µl of LB broth containing sub-MICs of extracts were inoculated with 10 µl of a bacterial suspension 10⁶ cfu/ml and incubated at 30°C for 24 h in a shaking incubator. Untreated C. violaceum cultures served as the positive control, while wells containing broth and extracts alone acted as the negative control. Following incubation, the density of the extracted violacein pigment was measured colorimetrically at 585 nm using a microplate reader. The absorbance of each well was measured at 585 nm and 600 nm to determine pigment yields and host bacterial proliferation, respectively. Violacein pigment inhibition (%) was calculated using the following equation (Alrashidi et al., 2022):

Inhibition (%)= [(OD585nm C − OD585nm T)/ OD585nm C] × 100

Where OD585nm C is the OD585nm of violacein production in control wells (without extracts) and OD585nm T is the OD585nm of violacein production in wells containing C. violaceum ATCC 12472 strain treated with the extracts.

All treatment data were obtained from three replicate, and results are expressed as mean ± standard deviation (±SD). Data were subjected to analysis of variance (ANOVA) by Tukey’s post-hoc test using SPSS software (version 16.0, SPSS Inc., Chicago, IL, USA) and the Chi-square test. A completely randomized design was used, with significance evaluated at p ≤ 0.05.

Identification of Escherichia coli

Escherichia coli accounted for 24% of all isolates recovered during the study period. Of the 300 samples, 72 isolates (24%) were identified as E. coli: 14 E. coli isolates were from wound samples (WEC), nine isolates from sputum samples of pneumonia patients (SEC), and 49 isolates from urine samples of UTI patients (UEC). Identification of the isolates was further confirmed using the standard analytical profile index (API 20E) identification kits and the Vitek 2 system.

Biofilm production and density were measured using the microtiter plate assay technique, with absorbance readings recorded at 600 nm. The results of biofilm production are presented in Figure 1. All UEC isolates produced biofilms, but with varying degrees: 71.4% were strong producers, 20.4% moderate, and 8.2% weak. Among the WEC isolates, 71.4% produced biofilms, with 57.1% being strong producers and 14.3% moderate producers, whereas 28.6% were non-producers. For SEC isolates, 66.6% produced biofilms; 33.3% were strong, 22.2% moderate, and 11.1% weak, and 33.3% were being non-producers. Across all the combined clinical sources (i.e. urine, wound, and sputum), strong biofilm expression reached 63.8%, followed by moderate (19.4%) and weak (6.9%) production, whereas 9.7% of the isolates were non-biofilm-producers.

The antibacterial activities of MLPE and MLTE were evaluated through an in vitro inhibition zone assay using E. coli isolates. Antibacterial activities were observed for both MLPE and MLTE with concentrations ranging from 10 to 75 mg/ml, whereby strong inhibition of microbial growth was observed compared to ciprofloxacin (positive control) and DMSO (negative control), respectively. Compared to the control, the microbial proliferation was inhibited by both extracts, demonstrating high antibacterial efficacy with relative inhibition percentages ranging from 21.4% to 75.0% (Table 1). The highest relative inhibition percentage (75.0%) was observed for MLPE (75 mg/ml) against SEC isolates. The highest percentage efficacy for the hexane extract (75 mg/ml) was recorded as 46.4% against SEC isolates. Based on the established MIC and MBC parameters, both tested extracts demonstrated clear bactericidal properties against the isolates. However, MLPE showed stronger bacteriostatic effects against the isolates. The MIC values for both extracts ranged from 25 mg/ml to 50 mg/ml. Both extracts demonstrated a remarkable antibacterial potency, with the lowest MIC concentration of 25 mg/ml recorded for the MLPE extract against SEC isolates. The MIC value of MLTE was higher for UEC isolates (50 mg/ml) than for MLPE. The MBC of both extracts ranged from 50 to 100 mg/ml. The lowest MBC (50 mg/ml) was observed for MLPE against SEC isolates. Compared to the control, these MIC obtained results indicated that MLPE was more effective than MLTE against the E. coli isolates. Both UEC and WEC isolates had MIC values of 30 mg/ml for MLPE, while SEC isolates had an MIC of 25 mg/ml for MLPE. The MBC values required to completely inhibit growth of the tested E. coli isolates were approximately 50–60 mg/ml for MLPE and 80–100 mg/ml for MLTE.

Anti-biofilm activity of extracts

To determine the anti-biofilm efficacy of both extracts, crystal violet staining biomass assays were conducted using E. coli isolates, which were incubated with sub-MIC levels (MIC, MIC/2, MIC/4, and MIC/8), as shown in Figure 2. Both tested extracts showed different effects on the development of the preformed biofilms. The analysis displayed that the MIC of both extracts substantially reduced biofilm formation in all isolates. SEC isolates demonstrated a greater sensitivity to MLPE, followed by UEC isolates, whereas WEC isolates showed a lower sensitivity to the extracts at MIC/2. In contrast, WEC isolates demonstrated a greater sensitivity to MLTE, followed by SEC isolates, whereas UEC isolates showed a lower sensitivity to MLTE at MIC/2. Maximal anti-biofilm activity was achieved with MLPE, which disrupted the SEC isolates biofilm matrices by up to 90.9% at MIC/2, while MLTE reduced WEC isolate’s biofilm by up to 66.7% at MIC/2. Our data confirmed a concentration-dependent effect of the extracts, with a peak biofilm reduction recorded at the highest tested extract concentrations.

Table 1: Antibacterial activity and its relative percentage for both Melia azedarach phenolic leaf extract (MLPE) and Melia azedarach terpenoids leaf extract (MLTE) against E. coli isolates.

Where: UEC: E. coli isolate from urine, SEC: E. coli isolate from sputum, WEC: E. coli isolate from wound, SD: standard deviation, DMSO: dimethyl sulfoxide, MIC: minimal inhibitory concertration, MBC: minimal bactericidal concertration. Data mean ± standard deviation (±SD) of triplicate independent experiments. p < 0.001 indicates a statistically significant difference among the bacterial isolates within the same concentration. NO. indicates the isolates number.

The GC-MS analysis of hexane extract (terpenoid extract)

Chemical profiling of the MLTE fraction by GC-MS analysis identified 11 compounds and their corresponding retention times (Table 2, Figure 3). The prominent terpenoids in the MLTE fraction were caryophyllene-β (Peak 2; RT: 11.48 min), germacrene D (Peak 4; RT: 16.25 min), and phytol (Peak 8; RT: 19.23 min). Furthermore, the MLTE extract comprised several alkanes, including tridecane, eicosane, and heptacosane.

Phenolics in the M. azedarach MLPE fraction were fingerprinted using HPLC. These bioactive phenolics were detected based on their retention times and elution sequence (Table 3, Figure 4). The first three peaks were identified as the phenolic acids: gallic, chlorogenic, and caffeic acids. Other peaks were observed to be flavonoids, including catechin, catechin-7-O-glycoside, hyperoside, rutin, isoquercetin, quercetin, isorhamnetin, and chrysin. Chlorogenic acid (93.48 µg/ml) and catechin (86.42 µg/ml) were the most abundant constituents, with rutin being identified as the predominant flavonol.

Effect of extracts on the growth curves of Escherichia coli

To examine the effect of M. azedarach leaf extracts on the QS system, the growth of E. coli isolates was determined in the presence of various concentrations of MLPE and MLTE. The MIC values varied among the bacterial isolates: MLTE showed values of 50 mg/ml for UEC isolates and 40 mg/ml for WEC and SEC isolates. Meanwhile, MLPE showed MIC values of 30 mg/ml for UEC and WEC isolates and 25 mg/ml for SEC isolates. As shown in Figures 5 and 6, the growth curve of the control E. coli isolates entered the

Table 2: Identification of biologically active compounds in the M. azedarach hexane leaf extract (terpenoids extract) detected by Gas Chromatography-Mass Spectrometry (GC-MS) analysis.

Table 3: Identification of biologically active compounds in the M. azedarach ethanol leaf extract (phenolic extract) detected by High-Performance Liquid Chromatography (HPLC) analysis.

exponential phase within 10–15 h of incubation and reached the stationary phase after 15 h. Both MLPE and MLTE completely inhibited E. coli growth at the MIC, whereas bacterial growth was slower at concentrations below the MIC, eventually reaching the logarithmic and stationary phases. Sub-MIC concentrations were determined through growth curve analysis. The MIC/8 concentration showed no inhibitory effect on E. coli growth, and all subsequent experiments were conducted at concentrations below sub-MICs.

The influence of both extracts on the motility of E. coli isolates was also evaluated, given the pivotal role that motility plays in biofilm formation by these bacteria. In this study, the anti-QS potential of both extracts at sub-MICs on the swarming and swimming motility of E. coli isolates was examined. The obtained results displayed that both extracts inhibited the swarming and swimming motility of E. coli isolates at various sub-MIC concentrations. At MIC/2, swarming was inhibited by 79.4% and 89.4% for MLTE and MLPE, respectively (Figure 7), with SEC isolates showing the highest percentage of inhibition. At MIC/2, swimming was inhibited by 53.3% and 65.3% for MLTE and MLPE, respectively (Figure 8), with UEC isolates demonstrating the highest percentage of inhibition. For MLPE-treated SEC isolates, the diameters of the swarming and swimming motility zones were decreased to 55.1% and 89.4%, respectively, compared to the control samples (Table 4). The motility of E. coli isolates decreased following exposure to both extracts at sub-MICs, in a concentration-dependent manner. At MIC/2, both extracts caused a considerably greater inhibition of swimming motility than at MIC/4. At MIC/2 for MLTE, the swimming motility of the UEC, SEC, and WEC E. coli isolates was reduced by 53.3%, 49.4%, and 50.1%, respectively. While at MIC/2 for MLPE, the motility was decreased by 65.3%, 55.1%, and 57.8%, respectively.

Table 4: Swimming and swarming motility zone diameter (mm) of E. coli isolates treated with sub- minimal inhibitory concertrations of the two extracts.

Where: UEC: E. coli isolate from urine, SEC: E. coli isolate from sputum, and WEC: E. coli isolate from wound, SD: standard deviation, MIC: minimal inhibitory concertration. Data mean ± standard deviation (±SD) of triplicate independent experiments. p < 0.001 indicates a statistically significant difference among the bacterial isolates within the same concentration.

Violacein inhibition assay in chromobacterium violaceum

Figure 9 illustrates the effect of both extracts on violacein production by C. violaceum ATCC 12472 strain. Pigment production was reduced by 85.19% following treatment with MLPE (MIC/2), while MLTE achieved 76.35% inhibition at the same tested concentration. Sub-MIC concentrations did not affect the growth of C. violaceum ATCC 12472. Inhibition of violacein production (measured at 585 nm) was increased with increasing the extract concentrations below the MIC, relative to the extract-free control plates. Cell counts (OD600nm = 1) obtained results confirmed that growth of C. violaceum ATCC 12472 was not inhibited by the MLTE and MLPE extract’s treatment, where the results were compared to the untreated control group. Statistical analysis confirmed that both extracts elicited a peak suppression of violacein pigment synthesis at MIC/2. At lower concentrations of both extracts, such as MIC/32, no anti-QS efficacy was observed.

Regarding the comparative epidemiological prevalence, nearly 90% of community-acquired UTIs and up to half of nosocomial UTIs are caused by UPEC (Soujanya and Banashankari, 2023; Gagaletsios et al., 2025).The most common causative agent of UTIs is E. coli (91%) (Rahman et al., 2019). Consistent with previous literature, E. coli was the predominant isolate in this study, aligning with prevalence rates ranging from 36.8% to 49.1%, which were reported by Bharti et al. (2016); Nazmeen et al. (2018); Rafalskiy et al. (2020). Al-Taie et al. (2014) revealed that the percentage of E. coli isolated from wounds was 15.73%. Gagaletsios et al. (2025) reported that E. coli isolates were recovered from 65 urine samples, one swab, and one sputum sample. E. coli isolates were most frequently obtained from urine samples (92.2%), followed by wound samples (3.9%) (Naqid et al., 2020). Another study demonstrated that 35% of E. coli were isolated from urine samples, followed by 5% and 17% from wound and sputum, respectively (Tewari et al., 2018).

In terms of the phytochemical distribution trend, several compounds have been currently identified from M. azedarach extracts, including unique products belonging to the terpenoids, specifically limonoids and tirucallane-type triterpenoids. The obtained results showed higher levels of flavonoids and phenolic compounds in more polar fractions, especially in the ethanol fraction. In general, the absorption spectra reflected a trend related to polarity, with hexane extract containing non-polar components and ethanol extract containing highly polar compounds, as previously demonstrated by Batubara et al. (2025).

Regarding the chromatographic and phytochemical profiling, GC-MS analysis revealed 11 compounds, including β-caryophyllene, neophytadiene, and a phytol isomer, as major constituents. These chemical profiles align with previous findings reported by Hassan et al. (2025), confirming the consistent presence of diverse bioactive terpenoids and other phenolic compounds in M. azedarach leaves. Similarly, Batubara et al. (2025) revealed the presence of extended-chain hydrocarbons in the leaf extracts of the same plant. The current notable abundance of both heptacosane and eicosane within leaf extracts further supported this finding. HPLC analysis was used to identify and quantify flavonoids and phenolic compounds in the ethanolic extract of M. azedarach, confirming the findings of Hemdan et al. (2023), who reported the presence of numerous phenolic compounds in M. azedarach. Catechin and chlorogenic acid were observed to be the major constituents of the phenolic extract.

Concerning the comparative antibacterial efficacy, the inhibition zones diameters measured in this study (with corresponding relative inhibition percentages detailed in the results) agree with the previous reported values of 15-13 mm recorded at 195 mg/ml of the M. azedarach methanolic leaf extract against E. coli (Getahun et al., 2025). Similarly, the methanolic leaf extract suppressed the proliferation of MDR E. coli K1-1, recording a 10.93 mm inhibition zone diameter, whereas hexane leaf extract generated a 7.4 mm zone diameter (Batubara et al., 2025). This finding is also consistent with the reported ranges of 12.0–16.7 mm (at 300 mg/ml) and 10.0–13.0 mm (at 150 mg/ml) for methanol leaf extract of M. azedarach against E. coli (Touzout et al., 2023). Likewise, the ethanolic extract of M. azedarach has been shown to exert bactericidal activity against E. coli, with a recorded inhibition zone diameter of 16 mm (Hemdan et al., 2023).

In terms of the minimum inhibitory parameters, the MIC of MLPE against E. coli ranged from 25 to 30 mg/ml, and the MBC values ranged from 50 to 60 mg/ml. Meanwhile, these recorded values were higher than those reported by Getahun et al. (2025) for M. azedarach methanolic leaf extract against E. coli, where the MIC and MBC were 6.09 mg/ml and 12.18 mg/ml, respectively. This discrepancy is directly related to the different extraction solvents and the configurations evaluated across the studies. This is consistent with the findings of Hemdan et al. (2023), who reported that M. azedarach extract was effective against E. coli at an MIC of 50 mg/ml. The methanol leaf extract exhibited a superior antibacterial efficacy against E. coli compared to the hexane extract, demonstrating lower MIC (1 vs. 20 mg/ml) and MBC (2 vs. 20 mg/ml) values (Batubara et al., 2025).

Regarding the underlying molecular mechanisms of action, M. azedarach contained abundant polyphenols, which may express an antibiotic activity. Its antibacterial activity was attributed to the disruption of the cell wall’s integrity and cohesion, leading to the leakage of cell contents and cell lysis (Hemdan et al., 2023). Gallic acid, a phenolic acid known for its antibacterial efficacy, triggers irreversible permeability changes, membrane rupture, and pore formation (Sorrentino et al., 2018; Silva et al., 2018; Santos et al., 2021). The phenolic compounds induce microbial death by blocking enzyme active sites and acting as hydrogen ion carriers to disrupt ATP energy production (Hemdan et al., 2023; Al-Aboudi and Al-Azawi, 2025). The antibacterial activity of the hexane extract of M. azedarach leaves was attributed to its high proportion of sesquiterpenes, most notably β-caryophyllene, which is known to penetrate the microbial cell membranes and inhibit its growth (Dahham et al., 2015). The terpenoids induce membrane damage in bacteria through direct contact with the cells, increasing membrane permeability, interfering with ion transport and membrane proteins, affecting cellular enzymes, and inhibiting DNA synthesis (Fink, 2023).

With respect to biofilm formation dynamics, biofilm formation was observed in 90.1% of the evaluated isolates. This finding corroborates that of Gagaletsios et al. (2025), who reported that approximately 87% of E. coli strains exhibited a pronounced ability to form biofilms. In a similar study performed by Gagaletsios et al. (2025), E. coli strains were categorized based on their biofilm capabilities, where 71 isolates exhibited a moderate production, 16 showed high production, and 13 displayed a weak biofilm production. Bacterial biofilms facilitate adherence to host cells (Al-Aboudi and Al-Azawi, 2025) and act as a protective barrier against the immune system, thereby increasing antibiotic resistance and complicating the bacterial treatment (Fink, 2023).

Regarding the specific anti-biofilm mechanisms, the M. azedarach methanol leaf extract exhibited a peak anti-biofilm efficacy, achieving up to 60.26% inhibition of E. coli biofilm formation (Touzout et al., 2023). Fink (2023) demonstrated that eugenol can remove up to 67% of E. coli biofilm. A similar trend was observed for limonene (46%), while terpineol removed up to 44% of E. coli biofilm. Flavonoid compounds have likewise demonstrated an anti-biofilm efficacy by inhibiting biofilm production (Al-Aboudi and Al-Azawi, 2025). Flavonoids possess potent antibacterial and anti-biofilm properties that enable them to combat a wide range of pathogenic bacteria (Al-Aboudi and Al-Azawi, 2024). The biological activity of these extracts was influenced by several parameters, including the type of extraction solvent, geographic origin, harvesting period, storage climate, and dehydration techniques (Getahun et al., 2025).

Regarding the inhibition of QS and bacterial motility, the obtained results showed that both extracts suppressed the swimming and swarming motility, along with biofilm formation, across all E. coli isolates at sub-MIC levels without affecting the bacterial growth. However, these concentrations had a lesser inhibitory effect on swimming motility. Swimming, unlike swarming, results from rotation of the bacterial flagellar rotor and is not a multicellular behavior (Ren et al., 2001). Our study revealed the anti-QS activity of both extracts against the tested strain C. violaceum ATCC 12472, which synthesizes violacein under the control of the QS system (Moradi et al., 2020). Inhibition of QS-dependent violacein formation by sub-MIC concentrations of both extracts yielded favorable results. Bacterial locomotion and various virulence factors are heavily dependent on QS mechanisms (Chaichana et al., 2024). Through motility, E. coli strains can migrate and colonize new territories, which ultimately promotes biofilm proliferation (Benyoussef et al., 2022).

Regarding the surface migration and swarming dynamics, the swarming motility represents a QS-dependent, coordinated surface migration that drives biofilm assembly and infection by adapting the bacteria to environmental challenges, virulence, and drug resistance (Rütschlin and Böttcher, 2019; Santos et al., 2021). Natural phytochemicals are extensively documented for their anti-motility properties, particularly in weakening the migratory capabilities of E. coli (Garcia-Heredia et al., 2016; Sheng et al., 2016; Chaichana et al., 2024). Disrupting the QS pathways using botanical agents represents a viable strategy against MDR strains (Moradi et al., 2020). The phenolic phytochemicals can competitively inhibit QS receptors by blocking N-acyl homoserine lactones (AHL) signaling molecules; thus, disrupting the swarming motility, representing a remarkable alternative for limiting or preventing biofilm-associated infections (Santos et al., 2021). Decreased production of extracellular polysaccharides may explain this inhibition, negatively affecting the migratory capabilities of the microbial pathogens (Zheng et al., 2020; Santos et al., 2021). Multiple studies have also reported an inhibition of bacterial movement by phenolic compounds, which completely blocked the swarming motility (O’May and Tufenkji, 2011; Santos et al., 2021).

The results of the present investigation indicated that Melia azedarach contains potential antibacterial components, which may be of considerable value to the pharmaceutical industry as a therapy against infectious diseases. Specifically, the experimental findings demonstrated the distinct effectiveness of the extracts. Notably, the maximum relative growth inhibition reached 75.0% for MLPE and 46.4% for the hexane extract at a concentration of 75 mg/ml. Additionally, peak biofilm inhibition reached 90.9% for MLPE and 66.7% for MLTE at their respective MIC/2 values. Furthermore, at the same concentration (MIC/2), swarming motility was suppressed by up to 89.4% for MLPE and 79.4% for MLTE, while swimming motility was reduced by up to 65.3% and 53.3%, respectively. Further studies are recommended to identify and characterize the biologically active constituents responsible for this antibacterial effectiveness. Furthermore, a future validation using an expanded clinical sample size is recommended.

The authors would like to thank Mr. Laith A. Ismael for his valuable guidance and support in the statistical review of this study.

This study utilized the ultrasound-assisted extraction (UAE) method, which serves as a pioneering investigation confirming the multi-targeted efficacy of these green botanical fractions, demonstrating up to 75.0% relative growth inhibition, 90.9% biofilm inhibition, and profound restrictions in motilities (up to 89.4% for swarming and 65.3% for swimming). Notably, within the available literature, there is a distinct lack of studies evaluating the targeted inhibitory effect of Melia azedarach extracts against bacterial violacein pigment production, where our findings demonstrated a remarkable 85.19% inhibition at sub-inhibitory concentrations.

IJK: Conceptualization, methodology, validation, investigation, resources, writing original draft, writing review and editing, supervision, funding acquisition, visualization, and project administration. AAJ: Methodology, formal analysis,investigation, data curation, writing original draft,writing review, editing, funding acquisition, and resources.

HAH: Conceptualization, methodology, validation, investigation, Software, resources, writing original draft, writing review, editing, and project administration.

All authors have read and agreed to the published version of this manuscript.

This research did not receive any specific grant from any funding agencies.

This study was conducted in accordance with the ethical approval granted by Al-Furat Al-Awsat Technical University, under Protocol No. 95/ ALMS dated August 16, 2025. Written consent of the participants was obtained.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors have declared no conflicts of interest.