Introduction

The emergence and rapid dissemination of multidrug-resistant (MDR) pathogens represent a significant global health concern, undermining the efficacy of current antimicrobial therapies1. Acinetobacter baumannii, a non-fermenting Gram-negative bacillus, has gained particular attention due to its remarkable ability to survive in hospital environments and acquire resistance determinants2. This opportunistic pathogen is frequently associated with severe nosocomial infections, including ventilator-associated pneumonia, catheter-related bloodstream infections, urinary tract infections, and complicated wound infections, particularly in critically ill or immunocompromised patients3. Its ability to resist multiple classes of antibiotics, especially carbapenems, and form biofilms contributes significantly to its persistence in hospital settings and its association with poor clinical outcomes4,5.

Biofilm formation is a key virulence factor of A. baumannii, providing protection against antibiotics and host immune defenses6. These biofilm-associated infections are notoriously challenging to treat, often resulting in prolonged hospital stays, elevated mortality rates, and significant healthcare costs7. Overexpression of biofilm-related genes such as bap (biofilm-associated protein) enhances biofilm formation, while resistance genes like blaOXA-51 (carbapenemase) and aacC1 (gentamicin resistance) further complicate treatment by conferring high-level antibiotic resistance8,9.

Advances in antimicrobial research have spotlighted small molecules with novel scaffolds as promising candidates for tackling resistant pathogens. Among these, 1,3,4-oxadiazoles have garnered attention for their potent antimicrobial properties and ability to disrupt bacterial biofilms10. The antibacterial activity of 1,3,4-oxadiazoles arises from their five-membered heterocyclic ring containing oxygen and nitrogen atoms, which facilitates strong interactions with bacterial targets and interferes with essential cellular functions11.

This study aims to investigate the antibacterial and antibiofilm efficacy of 1,3,4-oxadiazoles against the standard strain A. baumannii ATCC 19,606 and MDR clinical isolates. Key objectives include determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 1,3,4-oxadiazoles in comparison with imipenem and gentamicin, evaluating biofilm formation and structural changes before and after treatment through quantitative assays and scanning electron microscopy (SEM), and assessing the frequency and expression levels of critical genes (blaOXA-51, aacC1, and bap) associated with resistance and biofilm formation.

Material and method

All reagents and solvents were purchased from commercial suppliers (Merck, Germany and Fluka, Switzerland) and utilized as received without additional purification steps. Infrared spectroscopic analysis was conducted using a Shimadzu IR-460 spectrophotometer. Nuclear magnetic resonance data were recorded on a Bruker DRX-300 AVANCE instrument with 1H NMR measured at 300 MHz and 13C NMR at 75 MHz. All NMR samples were dissolved in deuterated chloroform with tetramethylsilane as the reference standard. The chemical shift values are expressed in parts per million (ppm), and coupling constants (J) are reported in Hertz. Column chromatographic purification was performed using silica gel powder obtained from Merck.

Oxadiazoles synthesis

In this study, (5-(3-methoxyphenyl-1, 3, 4-oxadiazol-2-yl) (pyridin-2-yl) methanol was synthesized via the reaction of 3-methoxybenzoic acid, 2-pyridinecarbaldehyde, and (N-isocyanimino) triphenylphosphorane in a 1:1:1 molar ratio, using acetonitrile as the reaction medium at room temperature. Triphenylphosphine oxide (Ph₃P=O) was obtained as a by-product.

Synthesis procedure for (5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-yl) (pyridin-2-yl) methanol

A solution of 3-methoxybenzoic acid (1 mmol) in acetonitrile (5 ml) was added slowly over a 15-min period to a reaction vessel containing N-isocyaniminotriphenylphosphorane (1 mmol) and pyridine-2-carbaldehyde (1 mmol) dissolved in acetonitrile (7 ml). The addition was performed at ambient temperature under continuous magnetic stirring. After complete addition, the reaction mixture was maintained with stirring for an additional 4 h. Following reaction completion, the acetonitrile was evaporated under vacuum. The resulting viscous material was subjected to purification via flash column chromatography using silica gel as the stationary phase and a 2:1 mixture of petroleum ether and ethyl acetate as the mobile phase. Subsequent solvent evaporation under reduced pressure yielded the desired products. The characterization data for the synthesized compounds are presented below:

White powder, m.p. 134 ºC, yield 80% (0.22g).

IR (KBr) (νmax, cm-1): 3424, 3054, 2914, 2837, 1658, 1573, 1525, 1522, 1483, 1424, 1386, 1280, 1216, 1037, 913.

1HNMR (300.13 MHz, CDCl3): δH = 3.92 (3H, OCH3, s), 6.15 (s, 1H, CH aliphatic), 6.52 (s, 1H, OH), 7.14–7.18 (m, 1H, CH Arom), 7.49 (t, 1H, 3JHH = 8.2 Hz, Arom), 7.62–7.67 (m, 1H, CH Arom), 7.74–7.76 (m, 1H, CH Arom), 7.84 ( d, 1H, 3JHH = 8.2 Hz, CH Arom), 7.87 (t, 2H, 3JHH = 7.7 Hz, CH Arom), 8.468 (d, 1H, 3JHH = 8.2 Hz, CH Arom), 8.927–8.941 (m, 1H, CH Arom).

13CNMR (75.467 MHz, CDCl3): δC = 55.66 (CH3), 68.42 (CH-OH), 112.21, 119.56, 120.40, 125.77, 128.14, 130.51, 137.31, 150.42 (8CH, Arom), 123.73, 151.68, 160.16, 160.74, 166.21 (5C).

Bacterial isolation

During a three-month period from February to April 2023, 13 A. baumannii isolates were collected from various inpatient wards at Besat Hospital in Hamadan, Iran. These isolates were sourced from clinical broncho alveolar lavage samples. Preliminary identification as A. baumannii was carried out through routine microbiological procedures, including cultivation on selective media and standard phenotypic assays such as colony morphology, oxidase and catalase tests, motility, citrate utilization, indole production, methyl red, and Voges-Proskauer tests. Molecular confirmation was performed using polymerase chain reaction (PCR) with established primers12. The isolates were subsequently preserved for further analysis by storing them in tryptic soy broth (TSB) containing 15% glycerol at -20 °C.

Antimicrobial susceptibility testing

The Kirby-Bauer disk diffusion method, following the guidelines established by the Clinical and Laboratory Standards Institute (CLSI), was used to assess antimicrobial susceptibility13. The tests were carried out on Mueller–Hinton agar plates with a selection of antibiotic disks: gentamicin (GM), ciprofloxacin (CIP), ceftazidime (CAZ), ceftazidime-clavulanate (CAC), imipenem (IMP), cefotaxime-clavulanate (CTX-C), tetracycline (TE), piperacillin (PIP), ampicillin-sulbactam (SAM), cefepime (FEP), cefotaxime (CTX), and cotrimoxazole (SXT). To ensure reliability, Escherichia coli ATCC 25,922 was used as the quality control strain. MDR was defined as resistance to at least one antibiotic in three or more distinct antibiotic classes14.

Screening of biofilm formation

The biofilm-forming capacity of A. baumannii isolates was evaluated using a microtiter plate (MTP) assay15. Cultures of the isolates were grown overnight in TSB enriched with 1% glucose and adjusted to a turbidity corresponding to a 0.5 McFarland standard. The prepared bacterial suspensions were then distributed into a 96-well microtiter plate and incubated at 37°C overnight to promote biofilm development.

After incubation, non-adherent cells were removed by washing the plate. The biofilms were fixed with absolute methanol and stained with 200 µl of a 1.5% (w/v) crystal violet solution for 15 min. Excess dye was aspirated, and the wells were rinsed three times to ensure all unbound stain was removed. The remaining stain, which was bound to the biofilm, was dissolved using 200 µl of 33% (v/v) acetic acid. The optical density (OD) of the solution was measured at 570 nm using a microtiter plate ELISA reader (BioTek, Germany).

Biofilm formation was quantitatively assessed by measuring the OD of stained biofilms in microtiter plates. To interpret the results, a cut-off OD (ODc) was calculated as the mean OD of the negative control plus three standard deviations. Based on the relationship between the OD of each isolate and the ODc, the level of biofilm production was classified into four categories: non-producer (OD ≤ ODc), weak producer (ODc < OD ≤ 2 × ODc), moderate producer (2 × ODc < OD ≤ 4 × ODc), and strong producer (OD > 4 × ODc)16.

Each isolate was tested three times to ensure reliability. A. baumannii ATCC 19,606, a known strong biofilm producer, was used as the positive control.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The CLSI broth microdilution protocol was utilized to assess the MIC and MBC of 1, 3, 4-oxadiazole, gentamicin, and imipenem against A. baumannii strains17. Briefly, serial twofold dilutions of each compound were prepared in Mueller–Hinton Broth (MHB) within the following concentration ranges: 1,3,4-oxadiazole (0.24–500 µg/mL), gentamicin (0.25–1024 µg/mL), and imipenem (0.25–1024 µg/mL). The prepared dilutions were dispensed into 96-well microtiter plates. Bacterial suspensions adjusted to a 0.5 McFarland standard were added to each well, and the plates were incubated at 37°C for 18–24 h.

The MIC was determined as the lowest concentration of the test compound that prevented observable bacterial growth following incubation. To establish the MBC, samples from wells showing no visible turbidity were aseptically subcultured onto Mueller–Hinton agar plates. After overnight incubation at 37 °C, the MBC was defined as the lowest concentration at which ≥ 99% of the initial bacterial population was eradicated, as indicated by the absence of colony formation.

All experiments were conducted in triplicate to ensure reliability. Positive controls included wells with bacterial inoculum, while wells containing uninoculated media served as negative controls.

PCR detection of biofilm-associated bap gene

DNA extraction was performed using a modified boiling method, as previously described4. Colonies grown on Mueller–Hinton agar were collected and suspended in 200 µl of sterile distilled water, followed by thorough vortexing. The suspension was centrifuged at 10,000 rpm for 12 min at 4°C, and the supernatant was discarded. The resulting pellet was resuspended in 200 µl of sterile distilled water, boiled in a water bath for 12 min, and centrifuged again at 10,000 rpm for 10 min. The supernatant containing the extracted DNA was transferred to a sterile tube and stored at -20°C for use as a template in PCR amplification.

The extracted DNA was screened for the presence of the biofilm-associated bap gene using PCR. Details of the primers used in this study are provided in Table 1.

Table 1 Primers used in this study for gene amplification.
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Biofilm inhibition assay

The inhibitory effects of 1,3,4-oxadiazole on biofilm formation were assessed using a 96-well microtiter plate assay18. Briefly, 200 µl of bacterial suspensions (10⁶ CFU/ml) diluted in TSB were dispensed into sterile wells. Serial dilutions of the test compound were then added to each well, and the plate was incubated overnight at 37 °C to allow biofilm formation.

Following incubation, the wells were washed to remove planktonic cells, and the remaining biofilms were fixed with absolute methanol. The fixed biofilms were stained using crystal violet, and the OD at 570 nm was measured using a microplate ELISA reader to quantify biofilm inhibition.

Biofilm gene expression

The effect of 1,3,4-oxadiazole on the expression of biofilm-associated genes was assessed using a real-time PCR assay. Isolates were first exposed to sub-MIC concentrations of the compound. Total RNA was then extracted using the RNX-Plus kit (SinaColon Co., Iran) and converted into cDNA through reverse transcription with random hexamer primers. The expression of the biofilm-associated gene (bap) was analyzed using SYBR Green qPCR Master Mix (SinaColon Co., Iran).

To normalize gene expression, the cycle threshold (CT) values of the bap gene were compared to those of the housekeeping gene 16S rRNA. Relative expression of the bap gene was calculated using the ΔΔCT method19.

Scanning electron microscopy (SEM) analysis

To investigate the effects of 1,3,4-oxadiazole on A. baumannii biofilms at MIC levels, scanning electron microscopy (SEM) was performed. The analysis focused on a single strain, A. baumannii 1, following a modified protocol from Nazari et al.20.

Fresh bacterial cultures were grown in TSB supplemented with 1% glucose (1% Glu TSB) at 37 °C for 24 h to promote biofilm formation. After incubation, the biofilms were exposed to 1,3,4-oxadiazole at concentrations corresponding to the MIC. The bacterial suspension, adjusted to 1.5 × 10⁷ CFU/mL, was incubated with the biofilms for another 24 h at 37 °C. Sterile glass slides, pre-cut to fit the wells, were introduced into the wells to facilitate biofilm development on their surfaces.

Following incubation, the slides were washed three times with sterile distilled water to remove non-adherent bacteria. Biofilms were then fixed in 2.5% glutaraldehyde in 1 × phosphate-buffered saline (PBS) for 3 h at room temperature. After fixation, the slides were rinsed three times with distilled water and post-fixed with 1.5% osmium tetroxide for 1 h.

The samples were dehydrated in a graded ethanol series (20%, 30%, 50%, 70%, 80%, 90%, and 100%), with each ethanol concentration applied for 10 min. The dehydrated slides were mounted on conductive copper tape for SEM analysis, coated with a thin gold nanoparticle layer to ensure electrical conductivity, and subsequently imaged using a MIRA3 scanning electron microscope (TESCAN Co., Czechia).

Cytotoxicity analysis

The cytotoxic effects of the synthesized 1,3,4-oxadiazole compound were assessed using the MTT assay on the A549 human lung carcinoma cell line, procured from the Pasteur Institute of Iran, Tehran21. A549 cells were seeded into 96-well plates at a density of 1 × 104 cells per well and allowed to adhere and grow under standard culture conditions (37 °C with 5% CO₂) for 24 h prior to treatment.

Following the initial incubation period, cells were treated with various concentrations of the test compound and incubated for an additional 24 h at 37 °C in a humidified incubator with 5% CO₂. After treatment, 15 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 4 h to allow for the formation of formazan crystals by metabolically active cells. Subsequently, 200 µL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the crystals. The absorbance was then measured at 570 nm using a microplate reader. Cell viability was determined using the following equation:

$${\text{Cell viability }}\left( \% \right)\, = \,\left( {{\text{OD sample }}/{\text{ OD negative control}}} \right)\, \times \,100.$$

All experiments were conducted in triplicate. Wells containing untreated A549 cells served as negative controls, while cells treated with DMSO were used as positive controls.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, USA). A t-test was employed to assess the significance of the anti-biofilm effects of 1,3,4-oxadiazole at different concentrations. Additionally, ANOVA was used to compare both the survival rates of A549 lung cancer cells treated with various concentrations of 1,3,4-oxadiazole and the gene expression levels between treated isolates and controls. Results are presented as mean ± standard deviation, with experiments conducted in triplicate. A confidence level of 95% was applied, and statistical significance was defined as p < 0.05. In addition, non-linear regression analysis was used to describe the correlation between compound concentration and the percentage of observed activity.

Results

Isolates and biofilm production assay

In this study, 13 A. baumannii strains were obtained from clinical samples for analysis. The strains demonstrated diverse abilities to form biofilms, with OD values varying between 0.3 and 2.2. According to these OD measurements, the isolates were classified into three categories: strong, moderate, and weak biofilm producers, as summarized in Table 1. Based on the established inclusion and exclusion criteria for identifying pathogenic isolates and assessing their biofilm-forming capabilities, a total of eight clinical isolates of A. baumannii were selected for further investigation. Additionally, the standard strain A. baumannii ATCC 19,606 was included in the analysis.

Susceptibility testing results of clinical A. baumannii isolates

Antibiotic susceptibility testing of eight A. baumannii clinical isolates showed varying resistance rates. Resistance was highest (100%) for GM, CIP, CAZ, CTX, FEP, TE, and trimethoprim-SXT. PIP showed a resistance rate of 50%, while IMP resistance was observed at 62.5%. All isolates were identified as MDR. Further details on the antimicrobial susceptibility testing are provided in Table 2.

Table 2 Antimicrobial susceptibility and biofilm formation analysis of Acinetobacter baumannii.
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MIC and MBC results

The findings revealed that 1,3,4-oxadiazole effectively inhibited the growth of all clinical A. baumannii isolates, with MIC ranging from 7.81 to 31.25 μg/ml. Furthermore, the compound demonstrated bactericidal activity, with MBC ranging from 7.81 to 62.5 μg/ml.

The geometric mean MIC values for 1,3,4-oxadiazole, gentamicin, and imipenem were calculated as 17.03, 20.74, and 5.65 μg/ml, respectively. Similarly, the geometric mean MBC values for these agents were 34.07, 49.35, and 12.33 μg/ml, respectively.

For the standard strain A. baumannii ATCC 19,606, the MIC and MBC of 1,3,4-oxadiazole were determined to be 7.81 μg/ml and 15.62 μg/ml, respectively. Comprehensive details of the MIC and MBC results are presented in Table 3.

Table 3 MIC and MBC ranges for gentamicin, imipenem, and 1,3,4-oxadiazole.
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PCR detection

Among the eight clinical isolates analyzed, the bap gene, associated with biofilm formation, was identified in six isolates, and its presence was significantly correlated with biofilm formation (p < 0.01). Notably, the two isolates exhibiting weak biofilm formation did not carry the bap gene, further underscoring the strong relationship between the bap gene and biofilm formation.

Biofilm inhibition

In this study, the inhibitory effects of 1,3,4-oxadiazole on A. baumannii biofilms were investigated. The MTP assay demonstrated that treatment with 1,3,4-oxadiazole resulted in a significant reduction in biofilm formation across all tested A. baumannii isolates (p < 0.05; Fig. 1).

Fig. 1
figure 1

Anti-biofilm activity of 1,3,4-oxadiazole against Acinetobacter baumannii isolates. Data are expressed as mean ± SD from three independent experiments (n = 3). A significant reduction in biofilm formation was observed in all treated isolates compared with the untreated control (p < 0.05).

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Activity of 1,3,4-oxadiazole on biofilm encoding genes

The effect of sub-MIC concentrations of 1,3,4-oxadiazole (ranging from 0.48 to 31.25 μg/ml) on the expression of the bap gene was evaluated in selected A. baumannii isolates after 24 h of exposure. The results, expressed as log2-fold changes, revealed a significant downregulation of bap expression in all tested isolates (p < 0.05; Fig. 2). Moreover, regression analysis indicated a dose-dependent relationship between 1,3,4-oxadiazole concentration and bap downregulation, with coefficients of determination (R2) of 0.91, 0.85, 0.88, and 0.74 for A. baumannii ATCC 19,606, isolate 1, isolate 3, and isolate 6, respectively.

Fig. 2
figure 2

Downregulation of the biofilm-associated bap gene in Acinetobacter baumannii isolates exposed to sub-inhibitory concentrations of 1,3,4-oxadiazole. Expression levels are presented as log2-fold change relative to untreated controls. Data represent mean ± SD from three independent experiments (n = 3).

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Scanning electron microscopy (SEM)

The anti-biofilm effects of 1,3,4-oxadiazole on A. baumannii biofilms were evaluated through SEM. The findings revealed that treatment with 1,3,4-oxadiazole at MIC concentrations significantly disrupted the structural integrity of the biofilm. This led to extensive bacterial lysis and a noticeable reduction in overall biofilm biomass. Intact and cohesive biofilm structures observed in untreated samples were replaced by fragmented, disorganized clusters, as illustrated in Fig. 3.

Fig. 3
figure 3

Effect of 1,3,4-Oxadiazole on Acinetobacter baumannii Biofilms (A) Untreated A. baumannii biofilm exhibiting substantial large biofilm biomass (LBB). (B and C) A. baumannii biofilm treated with 15.62 μg/mL of 1,3,4-oxadiazole, showing a significant reduction in biofilm biomass (small biofilm biomass, SBB).

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Cytotoxicity

The cytotoxicity analysis of 1,3,4-oxadiazole revealed dose-dependent effects on the A549 lung cancer cell line, with cytotoxicity rates of 27.62%, 56.33%, 89.81%, 92.11%, and 95.52% observed at concentrations of 500, 250, 125, 62.5, and 31.25 µg, respectively. Interestingly, at lower concentrations of 1,3,4-oxadiazole (15.62 and 7.81 µg), no cytotoxic effects were observed on the A549 cell line. Moreover, a t-test indicated no statistically significant difference in the survival rates between the samples treated with 15.62 and 7.81 µg of 1,3,4-oxadiazole and the control group (p = 0.085).

Discussion

The treatment of MDR pathogens poses a significant challenge, especially when these bacteria form biofilms, which further complicates antibiotic efficacy22. MDR pathogens are major contributors to severe infections, such as bronchoalveolar conditions, often associated with high mortality rates23. Current antimicrobial therapies primarily target planktonic bacteria, leaving biofilm-associated infections inadequately addressed24. This gap emphasizes the urgent need for novel agents capable of effectively targeting both biofilms and the embedded bacteria within them. Among emerging compounds, certain chemical entities, such as 1,3,4-oxadiazole derivatives, have demonstrated significant promise in tackling MDR bacterial infections and serve as potential candidates for antibiofilm therapeutics25.

In this study, 1,3,4-oxadiazole exhibited notable antibacterial activity against A. baumannii isolates, with MIC values ranging from 7.81 to 31.25 μg/ml and MBC values from 15.62 to 62.5 μg/ml. These findings are consistent with previous reports on the antibacterial properties of 1,3,4-oxadiazole against other MDR pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and E. coli26,27,28. The geometric mean MBC values for gentamicin and imipenem in this study were 49.35 μg/ml and 12.33 μg/ml, respectively, indicating that 1,3,4-oxadiazole exhibits comparable or superior efficacy against MDR A. baumannii. Mechanistically, 1,3,4-oxadiazole is thought to interact with bacterial membranes, forming pores that disrupt osmotic balance, ultimately leading to bacterial lysis29.

The genetic analysis revealed critical insights into the role of specific determinants in biofilm formation. Additionally, the strong correlation between the bap gene and robust biofilm formation underscores the importance of this gene in biofilm development. Notably, isolates lacking the bap gene exhibited weak biofilm formation, highlighting its pivotal role in biofilm integrity.

The inhibitory effects of 1,3,4-oxadiazole on biofilm formation were demonstrated using MTP assays, which showed a significant reduction in biofilm biomass across all tested isolates. These results align with prior studies reporting the antibiofilm efficacy of 1,3,4-oxadiazole against other MDR pathogens30,31. Furthermore, real-time PCR analysis revealed that the compound downregulated the expression of the bap gene, a critical determinant of biofilm formation in A. baumannii. This reduction in bap gene expression is consistent with similar studies that observed a decrease in biofilm-associated gene expression in other MDR pathogens treated with sub-MIC concentrations of 1,3,4-oxadiazole32,33.

SEM analysis provided further evidence of the antibiofilm activity of 1,3,4-oxadiazole. In the untreated state, dense and cohesive biofilm layers with well-structured bacterial aggregates were observed. However, exposure to the compound at MIC concentrations led to the disintegration of these cohesive layers, leaving bacteria mostly as dispersed individual cells with irregular morphology. Many cells showed clear signs of lysis, and the organized architecture typical of mature biofilms was no longer evident. These findings indicate that 1,3,4-oxadiazole not only prevents biofilm cohesion but also induces structural damage to bacterial cells, thereby supporting its potential as a therapeutic agent against biofilm-associated infections caused by A. baumannii.

Importantly, our findings provide insights into potential mechanisms for antibiofilm therapy development. By targeting both biofilm integrity and bacterial viability, 1,3,4-oxadiazole may offer dual-action efficacy, which is highly desirable in managing chronic infections where biofilms confer high resistance. The observed downregulation of biofilm-associated genes also suggests that this compound could potentially be combined with existing antibiotics to enhance their activity against MDR biofilm-forming pathogens.

Conclusion

This study demonstrates the significant antibacterial and antibiofilm potential of 1,3,4-oxadiazole against MDR A. baumannii. The compound exhibited robust activity, effectively reducing biofilm biomass, downregulating biofilm-associated genes, and disrupting biofilm structures. Its comparable efficacy to standard antibiotics, such as gentamicin and imipenem, highlights its promise as an alternative therapeutic agent. These findings highlight the potential of 1,3,4-oxadiazole as a dual-action therapeutic capable of addressing both planktonic and biofilm-associated MDR bacterial infections. By interfering with biofilm cohesion and bacterial viability, this compound may serve as a foundation for combination therapies that enhance the efficacy of conventional antibiotics. Further investigations, including in vivo studies and clinical trials, are warranted to fully explore its therapeutic potential and safety profile. Additionally, exploring structural analogs and optimization of pharmacological properties will be essential to advance 1,3,4-oxadiazole toward clinical application.