Mueller Hinton Broth (MHB), Nutrient Agar (NA), Gram Staining Kit, and Crystal Violet (cell culture tested) used in the study were supplied by HiMedia. Dimethyl sulfoxide (DMSO), Methanol, Glycerol [Aqueous] acetic acid, Sodium Chloride (NaCl), Isopropanol, Absolute Ethanol, Resazurin, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Merck (India). 4′,6-diamidino-2-phenylindole (DAPI) Stain and Propidium Iodide counter stain (PI) were purchased from Thermo Fischer Scientific. Foetal Bovine Serum (FBS), Bovine Serum Albumin (BSA), Dulbecco’s Modified Eagle Medium (DMEM), Trypsin, phosphate-buffered saline (PBS) were obtained from Gibco (Thermo Fischer Scientific). All chemicals used were of analytical research grade.

Gram (+) bacteria; Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 51299), Bacillus cereus (MTCC 121) and Gram (−) bacteria; Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 10145), Klebsiella pneumonia (NCTC 13440) were frozen as glycerol stocks and stored at −80°C. For each experiment, the frozen bacterial stock had been thawed and grown in MHB to the mid-logarithmic phase of growth under proper incubated conditions and then used.

To determine the MIC, the broth micro-dilution method was used, as described previously (Ali et al., 2022). The 96-well plates were labeled with the compound name, reference antibiotics (two wells/compound), and name of bacterial strains. 200 µL of MHB were dispensed into the wells except for the first column. 200 µL of the respective compounds (2a-2h) and drugs (CIP, AMK, and STR) were pipetted into the first column with a concentration equal to 128 μg/mL 2-fold serial dilution was performed so that the concentration from the first to 10th well ranges from 128–0.25 μg/mL. This was followed by inoculation of 50 µL of 10⁵ CFU/mL cells of S. aureus, E. faecalis, B. cereus, E. coli, P. aeruginosa, and K. pneumonia to wells in individual 96-well plates. The last two wells were taken as Growth Control (GC) and Media Control (MC), one lacking the drug and the other lacking both drug and compound as an indicator of contamination. The plates were sealed with parafilm. After 24 h of incubation at 37°C, plates were checked for the presence or absence of visible growth by comparing them with the Culture Control (CC) and MC Wells which also act as indictors of contamination. MIC was defined as the lowest concentration of compound at which no bacterial growth was visualized.

To ascertain if the compounds are bactericidal, the MBC assay was performed as per CSLI, 2016 guidelines (Mir et al., 2023). MBC is the lowest concentration of compound that leads to a 3 log10-fold decrease in CFU units or the lowest drug concentration that results in 99.9% killing of bacterial cells in initial inoculums. It is important to determine MBC as it provides insights into the optimal dosing regimen and identifies potential resistance mechanisms. The MBC was performed by broth micro dilution. The experiment was performed in duplicates. Briefly, 2 sterile 96-well plates were filled with MHB containing serial two-fold dilutions ranging from 1x- 4x MIC of compounds (2a-2h) and Ampicillin (4–16 μg/mL) as standard in a set of 2. Drug-free controls were also included. The plates were inoculated with 50 µL of S. aureus and E. faecalis respectively with a final density equal to 10⁵ CFU. The initial CFU of inoculums was verified by plating onto NA media. After 24 h, the drug-containing wells showing no sign of growth compared to growth culture tubes were appropriately diluted to sub-MIC levels by 10-fold serial dilution in MHB. 100 μL of each dilution was plated onto NA plates in duplicates. CFU was enumerated after 24 h of incubation at 37°C. CFU/mL was calculated using the formula: N o .   o f   C o l o n i e s × D i l u t i o n   F a c t o r ÷ V o l u m e   o f   C u l t u r e   P l a t e d (2)

Then log₁₀ values of an average number of colonies and log reduction were calculated using log calculator Omni Calculator to determine if there is a 3-log reduction in cell number. Ampicillin (AMP) was used as a control drug. Colonies ranging from 30–300 were considered countable. The lowest concentration of the drug which caused 99.9% killing with a 3-log reduction in CFU was taken as MBC of the drug.

To ascertain the bactericidal mechanism, i.e., the Time v/s Concentration dependent-killing of the lead compound 2d, the kill curve kinetics assay, was performed using the CFU/mL method as per CSLI guidelines with slight modifications. Another method adapted from the protocol described by Wang was also used to study the kill curve to provide insights into the degree of killing (concentration dependence) and rate of killing (time dependence) (Tsuji et al., 2008).

The PAE was determined as per the described protocol (Proma et al., 2020). Bacterial samples can have persistent subpopulations that remain dormant under antibiotic treatment and lead to disease recurrence following cessation of antibiotic therapy. The PAE was determined by the viable count method and the procedure adopted was the slight modification of protocol as reported previously. Briefly, an exponentially grown culture of E. faecalis and S. aureus with a final density of 10⁶ CFU/mL were exposed to 2d and control drug Ampicillin (AMP) at x, 2x, and 4xMIC for 2 h. After drug exposure, the drugs were removed by three cycles of centrifugation at 10,000 rpm for 10 min. The pellet obtained was resuspended in MHB. Antibiotics were removed immediately after drug exposure (2 h) and taken as 0-h readings and then after 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 h by centrifugation. Aliquots removed at predetermined time points were serially diluted 10-fold (N, 10^–1, 10^–2, 10^–3, 10^–4, 10^–5, and 10^–6) and plated on NA media in triplicates. Growth Controls with inoculums but no antibiotics were similarly processed and included to monitor the killing effect due to antibiotics to avoid any error in CFUs during processing.

The PAE was defined following Craig and Gudmundsson as P A E = T − C (3) where T is the time required for the viability count of an antibiotic-exposed culture to increase by 1 log10 above the count observed immediately after dilution and C is the corresponding time for the growth control.

DAPI and PI staining is commonly used to assess cell viability and the effect of antibiotics on cells. DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent dye that binds to DNA and emits blue fluorescence upon excitation by ultraviolet light. PI (propidium iodide) is also a fluorescent dye that binds to DNA, but it emits red fluorescence upon excitation by ultraviolet light. In live cells, the DAPI stain enters the nucleus and binds to DNA, whereas PI is excluded from the cell. However, in dead or damaged cells, the cell membrane becomes permeable to PI, which enters the cell and binds to DNA, causing it to emit red fluorescence. When using DAPI and PI staining to assess cell viability and the effect of antibiotics on cells, the cells are typically treated with the antibiotic and then stained with DAPI and PI. Live cells will appear blue, whereas dead cells will appear red. The staining was done as per the described protocol (Zotta et al., 2012). This staining method was used to study the effect of lead compound 2d on the viability of E. faecalis and S. aureus using DAPI and PI stains. Bacterial cells were grown to an OD600 of 0.5. 1 mL of the actively growing cultures was pipetted into Eppendorf tubes with MIC and 2xMIC of compound 2d. After incubation of 2 h at 37°C, the cultures in tubes containing 2d were centrifuged at 10,000 rpm for 10 min supernatant was decanted and the pellet was resuspended in 200 µL Normal Saline Solution (NSS). 20 µL of PI (5 μg/mL) were added to the tubes and incubated in the dark at 0°C for 15 min. The unbound PI was removed by centrifuging the tubes at 10,000 rpm for 10 min and washing the pellet thrice with 200 µL of NSS. This was followed by 15-min incubation with 2 µL of DAPI (10 μg/mL) at 0°C in the dark. Finally, 100 µL of cultures were mounted onto the clean well-labeled slides, left to dry, and methanol fixed. For negative controls, the corresponding bacterial cells that did not receive 2d treatment were processed under similar conditions. The stained bacterial cells were visualized under a fluorescent microscope and photographed using an inbuilt digital camera.

To determine whether compound 2d can inhibit the formation of biofilms, the Microtiter Plate Biofilm Assay was utilized as per described procedure (O’Toole, 2011; Mir et al., 2023). Firstly, the compound was prepared as a working stock (200 µL of compound in first well) and was serially diluted two-fold to obtain a concentration ranging from 0.25 to 128 μg/mL in 8 individual 96-well plates (1 plate for inoculation of each bacterium) already containing 100 µL MHB (final volume of 200 µL). 50 µL of bacterial suspension containing 10⁵ CFU/mL bacteria cells were added to the wells of their respective plates (S. aureus, E. faecalis, B. cereus, E. coli, P. aeruginosa, K. pneumonia). Culture control and Media control were included, in which only bacterial culture and growth media, respectively, were dispensed. The plates were then incubated at 37°C for 24 h to allow bacterial growth and understand if biofilm formation was inhibited in the presence of the compounds, as specified. No visible growth was observed in wells that correspond to the minimum inhibitory concentration (MIC) of the compound for the bacterial strains tested. After the incubation period, the planktonic bacterial cells in the wells were removed by washing three times with phosphate-buffered saline (PBS), and aliquots from the wells were serially diluted and plated onto NA to correlate the further findings. Next, 125 µL of 30% of 0.5% crystal violet (CV) that included methanol was added to all the wells of the 96-well plates. The plate was then incubated at 37°C for 20 min to stain the biofilm formed by microorganisms that are adherent to the abiotic surface of a microtiter plate. The unbound CV was removed by washing the wells with distilled water, and the wells were air-dried at room temperature. The wells were then visualized and photographed under a microscope to determine the extent of biofilm formation. To quantify the amount of biofilm formed, 200 µL of 30% [aqueous] acetic acid was added to each well to dissolve the crystal violet. The optical density (OD) of the dissolved CV was measured at 470 nm using a microplate reader. This protocol was repeated thrice to achieve optimal results and correct any background interference that may be present. The percentage of inhibition of biofilm formation was calculated using the following formula: 100 − TreatedOD 470 nm ÷  Growth  ControlOD 470 nm   × 100 (4)

This formula compares the optical density of the treated wells to that of the culture control wells and expresses the result as a percentage of inhibition.

A modified methodology incorporating diverse methods used earlier was implemented to determine the potential of compound 2d for biofilm eradication and assess the minimum concentration required for biofilm eradication (Kreth et al., 2005). 12 well plates were employed for this assay. To begin with, the bacterial strains (S.aureus, E. faecalis, B. cereus, P. aeruginosa, E. coli, and K. pneumonia) were grown to mid-log phase and diluted to obtain 10⁵ CFU/mL of cells. 3 mL of MHB was pipetted into the 12 well plates and subsequently inoculated with 100 µL of the bacterial suspension. Media Control well was taken as a positive control containing only MHB. For each bacterial strain, separate plates were utilized and subjected to incubation for 24, 48, and 72 h to assess the effect of different concentrations on various stages of bacterial biofilm formation. After the incubation at given time points the plates were washed thrice with PBS to remove the planktonic bacterial cells. After washing x, 2x, 4x, 8x, and 16x MIC concentrations of 2d compound were added to the wells and incubated for 24 h at 37°C. Culture Control well taken as a negative control was not incubated with the compound. After incubation plates the drug-containing plates were washed with PBS. This was followed by the addition of 30% of 0.5% CV stain. The plates were further incubated for 1 h at room temperature. In the next step, the CV was removed by thorough washing. Each well was observed under a microscope and photographed. To quantify the percentage of eradication 30% [aqueous] acetic acid was added to the wells to dissolve CV-staining adherent cells. This was done by recording the OD at 470 nm using a spectrophotometer. The percentage of biofilm eradication was calculated using formula (4). The percentages were calculated by taking the mean of three OD readings obtained for each concentration.

The critical factor that allows any compound to proceed successfully during drug discovery is selective toxicity to pathogens and non-toxicity to human cells. The assay was performed under the standards set by International Organization for Standardization (ISO) and Economic Cooperation and Development (OECD), to ensure accurate and reproducible results. The cytotoxicity of the lead compound 2d was evaluated against HEK-293. Before evaluating the cytotoxicity of the compound, the cells were grown to reach a confluency of 70%–80%. The culture techniques used were in accordance with the cell culture technique guidelines regulated by the International Cell Line Authentication Committee (ICLAC), American Type Culture Collection (ATCC), and the European Collection of Authenticated Cell Cultures (ECACC).

2-aminobenzothiazole (0.5 mmol) was dissolved in water (10 mL) in a 50 mL round bottom flask under a magnetic stirrer at room temperature; to this solution, hydrochloric acid was added dropwise until 2-aminobenzothiazole gets completely dissolved in water. In the next step solutions of sodium nitrite, (2 mmol), sodium acetate (2 mmol), and sodium azide (2 mmol) were added dropwise after every 5 min respectively and the reaction was stirred for further 30 min. The reaction progress was checked by thin layer chromatography which confirmed the completion of the reaction within 1 h. The solid precipitate was filtered, washed with water, and recrystallized from ethanol which did not require any further purification. (Note: NaNO ₂ , NaOAc, and NaN ₃ were first dissolved in water, and then added dropwise to the reaction mixture respectively. Also, the role of sodium acetate is to neutralize the acidic medium).

The Antibacterial Activity and MIC of all the synthesized compounds were determined using the Broth Micro dilution method as described in the methodology. All the synthesized compounds were tested for their antibacterial activity against the American Type Culture Collection (ATTC) strains of S. aureus, E. faecalis, B. cereus, K. pneumonia, P. aeruginosa and E. coli and clinical isolates of Methicillin Resistant S. aureus and Multidrug Resistant E. coli. It was found that compound 2d showed good antibacterial activity against the Gram (+) bacterial strains. It was elucidated that the compounds are more active against the Gram (+) bacterial strains, E. faecalis, and S. aureus with MIC of 8 μg/mL (Table 1). However, it was moderately active against Gram (−) bacteria P. aeruginosa and Gram (+) bacteria B. cereus with a MIC value of 64 μg/mL. Substantially the compound showed activity even against the Resistant strains, MRSA, and MDR E. coli of clinical origin with a MIC value of 128 μg/mL (Tables 1, 2). These results were found to be consistent with several other benzothiazole derivatives, which inhibited the growth of Gram (+) and Gram (−) bacteria (Catalano et al., 2013; Singh et al., 2013). The MIC values of control drugs of known MIC values against the bacterial strains were also similar to the range given by CSLI (Clinical And Laboratory Standards Institute).

As MIC only measures the ability of antimicrobial agents to inhibit bacterial growth, we performed an MBC assay which determines the lowest concentration of antimicrobial compound that can kill 99.9% of bacterial cells. MBC is required by regulatory agencies for approval of new antimicrobials. The MBC of the series of compounds 2a-h was assessed against the Gram (+) bacterial strains E. faecalis and S. aureus as the compounds exhibited comparatively better MIC values against these strains compared to other strains. The MBC values must not be more than four times the MIC, which is consistent with our results (Gonzalez et al., 2013). For both S. aureus and E. faecalis MBC of the lead compound 2d, was equal to 2xMIC, i.e., 16 μg/mL. The other compounds tested had bactericidal effects (led to 99.9% killing) rather than bacteriostatic effects, with MBC values of 128 μg/mL. Compound 2a showed bactericidal activity at 2xMIC (128 μg/mL) against E. faecalis. Compound 2b exhibited bactericidal activity at its MIC values, indicating their potential as promising antimicrobial agents that can effectively inhibit and kill bacterial pathogens. 2c showed bactericidal potential against both Gram (+) bacteria at 2xMIC.2e showed bactericidal potential only against E. faecalis at 4x MIC. Ampicillin was used as a control drug and its MIC and MBC values against S. aureus were found to be 4 and 8 μg/mL and for E. faecalis the MIC and MBC values were 2 and 4 μg/mL. Negative controls for both strains, devoid of compounds were used to validate the CFU counts. CFU enumeration indicated a 3-log reduction, 99.9% killing in both Gram (+) bacteria compared to the CFU count of Growth control which is indicative of the MBC. Overall, these findings suggest that the tested compounds have potent bactericidal activity against E. faecalis and S. aureus, which could be crucial in eradicating bacterial infections. The MBC values obtained after the enumeration of the CFU/mL are given in Table 3.

To study the dynamics of in vitro activity, we assessed the kill curve kinetics of our lead compound 2d against S. aureus and E. faecalis at concentrations of MIC (8 μg/mL), 2xMIC (16 μg/mL) and 4xMIC (32 μg/mL) for 24 h at seven time points (0, 4, 8, 12, 16, 20 and 24 h of exposure) using the two methods as described in methodology.

To investigate the effect of compound 2d, post its withdrawal, and assess whether the suppression in the growth of bacterial cells, S. aureus and E. faecalis persist after this brief exposure, the post-antibiotic effect (PAE) was calculated. PAE was deduced by the viable count method as described. Staphylococcus aureus and E. faecalis were individually treated with MIC (8 μg/mL), 2xMIC (16 μg/mL), and 4xMIC (32 μg/mL) concentrations of the compound 2d and MIC of Ampicillin for 2 h. The growth was monitored by CFU enumeration after a time interval of 1 h. The PAE results represented here are determined at a higher inoculum size of 1 × 10⁶ CFU/mL as against the routine inoculum size of 1 × 10⁵ CFU/mL more commonly used for susceptibility testing (MIC). To avoid the ambiguity in PAE results owing to the higher inoculum effect, MIC assay was first performed at a higher inoculum size (1 × 10⁶ CFU/mL) to select the correct concentrations of antibacterial agents (2d & AMP) for PAE determination. No change in MIC of 2d as well as Ampicillin was determined at higher inoculum size. PAE was calculated using formula (3) as described in the methodology.

To further explore the mechanism by which the compound targeted the bacterial cells, fluorescent microscopy was performed on the bacterial cells treated with the compound. Post 2d treatment, E. faecalis, and S. aureus were treated with two fluorescent dyes 4′,6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI). DAPI is a fluorescent dye that binds to DNA and emits blue fluorescence upon excitation by ultraviolet light and PI is also a fluorescent dye that binds to DNA, but it emits red fluorescence upon excitation by ultraviolet light. PI is a membrane-impermeable DNA binding fluorescent dye whereas DAPI is a membrane-permeable DNA binding dye. PI only enters cells with compromised membranes and stains the nucleic acid of dead cells. DAPI is permeable to the cell wall and fluoresces upon binding to chromosomal DNA, whether the cells are dead or alive. For control, the cells were treated with DAPI and PI without prior treatment with compound 2d. The DAPI/PI staining results revealed the presence of red fluorescence in both S. aureus and E. faecalis bacterial strains. The red fluorescence observed in the bacterial cells indicates the uptake and binding of PI, suggesting compromised cell membrane integrity. This red fluorescence was visually distinguishable from the blue fluorescence emitted by DAPI, which stains the DNA of both live and dead bacteria. The identification of compromised membrane integrity is of significance as it can indicate the susceptibility of S. aureus and E. faecalis strains to antimicrobial treatments. The presence of red fluorescence provides evidence of cell damage and potential loss of viability, suggesting that these strains may be more susceptible to the effects of 2d targeting the cell membrane. From Figures 5A,B, it is evident that the antibacterial activity of compound 2d was due to damage to the cell wall of the bacterial cells. These findings suggest that compound 2d targets the cell wall of bacterial cells, leading to compromised membrane integrity and ultimately cell death.

Conducting cell membrane-related studies on synthesized derivatives is a reasonable approach to understand their mechanisms of action. The cell membrane plays a critical role in regulating the passage of molecules and ions in and out of the cell, as well as in cell signalling and communication. Therefore, studying how the synthesized derivatives interact with the cell membrane can provide valuable insights into their potential mechanisms of action. However, we understand it is important to note that cell membrane-related studies alone may not provide a comprehensive understanding of the mechanisms of action of the synthesized derivatives. There could be additional mechanisms involved in their effects on cell viability that are independent of the cell membrane but DAPI and PI are commonly used fluorescent dyes that can be used to assess cell viability and membrane integrity. DAPI stains the nuclei of both live and dead cells, while PI is excluded from live cells but stains the DNA of dead or damaged cells with compromised membrane integrity. DAPI/PI staining allows for the differentiation between live cells (DAPI-positive, PI-negative). By using DAPI/PI staining, we wanted to assess the overall cell viability and determine whether the synthesized derivative have any detrimental effects on cell membrane integrity, which may lead to cell death.

The compound 2d, with the strong antibacterial activity, was tested against American Type Culture Collection (ATCC) strains of biofilm-forming Gram (+) bacteria, S. aureus, E. faecalis, B. cereus and Gram (−) bacteria, E. coli, P. aeruginosa, K. pneumonia. The Quantitative assessment of biofilm inhibition was done by calculating the percentage inhibition of the compound through spectrophotometric analysis for each bacterial strain by Microtiter Plate Biofilm Assay. The Qualitative assessment was done by CV staining and visualization under the microscope.

The percentage of biofilm inhibition was determined at various concentrations of compound 2d (0.25–128 μg/mL), and the values reported are the means of three optical density (OD) measurements to ensure more reliable and accurate results. Compound 2d exhibited significant biofilm inhibitory activity against all tested bacterial strains in a concentration-dependent manner. The percentage of biofilm inhibition varied across different concentrations and bacterial species. For S. aureus, compound 2d displayed increasing biofilm inhibition percentages with higher concentrations. At the highest tested concentration of 128 μg/mL, the biofilm inhibition was 70.30%. Similarly, for E. faecalis, the biofilm inhibition reached 73.04% at the same concentration. Bacillus cereus showed a distinct trend, with biofilm inhibition percentages of 61.23% and 53.09% at the highest concentration (128 μg/mL) and half that concentration (64 μg/mL), respectively. However, the inhibitory effect decreased significantly at lower concentrations. Among the Gram (−) bacteria, Pseudomonas aeruginosa demonstrated substantial biofilm inhibition, with percentages ranging from 62.07% at 128 μg/mL to 35.47% at 0.25 μg/mL. Escherichia coli and K. pneumonia exhibited lower biofilm inhibition percentages, ranging from 19.73% to 49.41% and from 14.07% to 45.59%, respectively, across the tested concentrations. Overall, these results indicate that compound 2d possesses significant biofilm inhibitory activity against a range of biofilm-forming bacterial strains with the most significant inhibitory potential against S. aureus and E. faecalis.

The percentage of biofilm inhibition at different concentrations of compound 2d was consistent with the qualitative analysis conducted using light microscopy. Microscopic examination revealed disrupted biofilm architecture and reduced biofilm biomass in the presence of compound 2d, supporting the quantitative findings. The microscopic analysis provided visual confirmation of the biofilm inhibitory potential of compound 2d. The growth control revealed a thick and compact biofilm structure, indicating the successful formation of mature biofilms by the tested bacterial strains. In contrast, the microscopic images of the biofilms treated with compound 2d at different concentrations displayed notable differences compared to the growth control. The biofilms exposed to compound 2d exhibited reduced biomass and disrupted biofilm architecture. The qualitative analysis corroborated the quantitative results, confirming that compound 2d has a significant impact on biofilm formation across different bacterial strains. The visual evidence of reduced biomass and disrupted biofilm architecture shown in the figures at various concentrations supports the notion that compound 2d interferes with the crucial steps of biofilm development (Figures 6–9).

The compound 2d exhibited strong biofilm inhibition potential against both E. faecalis and S. aureus that prompted the evaluation of Biofilm Eradication potential against these two strains. The antibiofilm effect of the compound was evaluated both qualitatively and quantitatively by Light Microscopy and Crystal Violet method at concentrations 8, 16, 32, 64, and 128 μg/mL to evaluate the effect of the compound on various stages of biofilm formed at 24, 48 and 72 h. Light microscopic images revealed that the growth control wells exhibited significant biofilm formation at all three-time points. At 24 h, cell attachment was observed, followed by cell aggregation at 36 h, and extensive biofilm formation or mat formation at 48 h. However, in wells incubated with compound 2d at different concentrations, non-aggregation of cells was observed during all stages of biofilm formation for both bacterial strains. This indicates the compound’s ability to inhibit cell aggregation, a critical step in biofilm development.

This qualitative finding was further investigated with the crystal violet staining method by comparing the absorbance between the growth control wells and the wells incubated with the compound. The percentage of biofilm Eradication at various concentrations is shown (Figures 10A, B) which indicates a strong antibiofilm potential of the compound eradicating up to more than 40% of the biofilm at all concentrations. The crystal violet staining method confirmed the qualitative findings, demonstrating the compound’s antibiofilm potential. The absorbance measurements showed that compound 2d eradicated over 40% of the biofilm at all tested concentrations. Notably, at higher concentrations, the compound exhibited substantial eradication potential against both E. faecalis and S. aureus, eliminating 70%–77% of the preformed biofilm at all three-time points. These results suggest that compound 2d has a strong biofilm eradication capability. The qualitative and quantitative evaluation of biofilm eradication correlated with each other. The eradication potential of the compound on the densely pre-biofilm at 72 h against both bacterial strains indicates that the compound can be a promising agent for biofilm eradication. The quantitative evaluation of the percentage of biofilm eradication was done as per the formula mentioned in the methodology and the results represented are the mean values of the three OD readings taken for each concentration in different wells. The compound effectively inhibited cell aggregation and eradicated preformed biofilms in a dose-dependent manner.

The biofilm eradication percentages of compound 2d against 24-h formed S. aureus biofilms were found to be 59% at 8 μg/mL, 67% at 16 μg/mL, 69% at 32 μg/mL, 74% at 64 μg/mL, and 76% at 128 μg/mL. For 48-h biofilms, the eradication percentages were 59%, 65%, 70%, 71%, and 74% at the corresponding concentrations. In the case of mature biofilms formed for 72 h, compound 2d exhibited eradication percentages of 50%, 54%, 62%, 68%, and 73% at the respective concentrations.

The biofilm eradication percentages of compound 2d against 24-h formed E. faecalis biofilms were found to be 42% at 8 μg/mL, 52% at 16 μg/mL, 61% at 32 μg/mL, 69% at 64 μg/mL, and 72% at 128 μg/mL. For 48-h biofilms, the eradication percentages were 58%, 59%, 67%, 70%, and 72% at the corresponding concentrations. In the case of mature biofilms formed for 72 h, compound 2d exhibited eradication percentages of 40%, 42%, 48%, 55%, and 66% at the respective concentrations.

These values represent the average mean of three independent experiments. The results of this study demonstrate the potent antibiofilm activity of compound 2d against S. aureus and E. faecalis biofilms. The compound exhibited remarkable efficacy in eradicating biofilms at various time points and concentrations. Compound 2d effectively eradicated a significant proportion of the 24-h formed biofilms. Even at the lowest concentration tested (8 μg/mL), the compound achieved a substantial eradication percentage. As the concentration increased the eradication percentages also increased, reaching 72%–76% at the highest concentration (128 μg/mL). These findings highlight the concentration-dependent efficacy of compound 2d in disrupting and eliminating early-stage S. aureus and E. faecalis biofilms. Furthermore, compound 2d demonstrated consistent efficacy in eradicating biofilms formed over 48 h. The eradication percentages indicated its ability to penetrate and disrupt biofilm structures at various concentrations. Notably, even at lower concentrations, the compound achieved eradication percentages above 50%–65%, demonstrating its potency against established biofilms in both bacteria. Compound 2d also exhibited substantial efficacy in eradicating mature biofilms formed over 72 h. The eradication percentages ranged from 50% to 73% for S. aureus and 40%–66% for E. faecalis, indicating its ability to target and disrupt the mature biofilm matrix. These results suggest that compound 2d possesses strong biofilm-eradicating properties, even in the presence of a mature biofilm structure. The findings from this study highlight the potential of compound 2d as an effective antibiofilm agent against S. aureus and E. faecalis. The ability of the compound to significantly reduce biofilm density and disrupt biofilm architecture is consistent with the qualitative observations made during the study. The strong correlation between the qualitative and quantitative evaluations supports the robustness of the antibiofilm activity of compound 2d. The compound effectively inhibited cell adhesion, auto-aggregation, and mat formation, as observed under light microscopy. These findings suggest that compound 2d may target key stages of biofilm development and inhibit biofilm formation (Figures 11–13).

The MTT-based cell viability assay performed on HEK-293 verified that cells exposed to 2d compound at varying concentrations for different time durations have a non-toxic effect on the human embryonic kidney cell lines. The results of the MTT-based cell viability assay were conducted on HEK-293 cells. Surprisingly, the findings indicate that the cells exposed to 2d exhibited a non-toxic response, demonstrating higher percentages of viable cells even at higher concentrations and longer exposure durations (Figure 14). This suggests that the 2d compound is cytocompatible and does not have a concentration- or time-dependent effect on cell viability. The MTT assay revealed that cell viability was significantly higher even at higher concentration ranges of 16–128 μg/mL. The percentage viability was calculated using formula (5).

The data presented in Table 6 demonstrate the favorable impact of the 2d compound on HEK-293 cell viability. At the lowest concentration tested (4 μg/mL), the average viability percentages remained consistently high across all concentration and time point combinations. At the highest concentration tested (128 μg/mL), the average viability percentage after 72 h of exposure was 88.15% ± 0.005774%, indicating that even at this relatively elevated concentration, the 2d compound did not significantly compromise cell viability. This trend of high viability percentages persisted throughout the experiment, irrespective of the exposure duration. It is important to note that these results are specific to HEK-293 cells and should not be generalized to other cell types without further experimentation.