2.2 In Vitro antibacterial activity

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.

The Optical Density (OD) of each well was measured at a wavelength of 600 nm using a microplate reader. The OD values for each well were recorded and the percentage of growth inhibition was calculated as described. In the calculation of percentage inhibition, the control well containing the bacterial culture but without any compound (GC) was used as the reference for comparison. This control well was used to determine the baseline OD value of the bacterial culture and considered as 100% growth or metabolic activity. The OD value of each well was then compared to the OD value of the control well to calculate the percentage inhibition of bacterial growth or metabolic activity. The percentage of growth inhibition in each well was calculated by comparing the OD of each well to that of the growth control wells using the following formula:

The data was plotted to determine the percentage of inhibition (plotting the percentage of inhibition versus the concentration of the antibiotic). The IC₅₀ value was determined from the curve as the concentration of the antibiotic that inhibits 50% of bacterial growth.

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:

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).

To determine the kill kinetics of lead 2d compound, E. faecalis and S. aureus cultures with cell density 1 × 10⁵ CFU/mL (confirmed by plate count) were exposed to 8 μg/mL MIC, 16 μg/mL (2xMIC) and 32 μg/mL (4xMIC) of 2d. A growth control CC was included as negative control (devoid of the compound). 2xMIC of AMP was taken as a standard drug. The log CFU/mL for all groups was determined at time 0 and at a subsequent time interval of 4 h (taken up to 24 h). Aliquots (100 µL) of drug-exposed cultures were pipetted out at predetermined time intervals of 4 h in 1 day, i.e., at 0, 4, 8, 9, 12, 16, 20, and 24 h and serially diluted, then plated onto NA for CFU enumeration. First, the colonies at different dilutions for each concentration were enumerated then the average colony count at dilutions 10^–1, 10^–2, 10^–3, and 10^–4 was calculated and then converting the value into log₁₀ (Note: the colonies on plates showing 30–300 colonies were only considered). The curve was generated by plotting Log₁₀ CFU values at each concentration against time. Time-kill curves were plotted and analyzed for the rate and extent of bacterial killing. The rate of killing was determined from the start of 0 h to maximal reduction in log₁₀ CFU/mL.

For this assay, working solutions of 2d were prepared in a series of conical tubes containing MIC, 2xMIC, and 4xMIC in 5 mL MHB. For each concentration, a set of tubes labeled with the time point were taken. 100 µL of 10 CFU/mL of E. faecalis and S. aureus were dispensed in their respective set of tubes and incubated at 37°C. At the various time points 0, 60, 120, 180, 240, 300, and 360 min, the incubated tubes containing culture-molecule mixtures were treated with 150 µL of MTT and incubated for 20 min. After incubation, the tubes were centrifuged at 10,000 rpm at 10°C for 10 min. The supernatant was discarded and the pellet containing formazan crystals at the bottom was dissolved in 1.5 mL of Isopropanol and absorption was measured at 550 nm. For positive control, the cells were treated with 2xMIC of STR. The untreated cultures were taken as a negative control. MTT can be reduced to purple formazan crystals by a dehydrogenase system of active cells which can be quantified through spectrophotometric analysis by dissolving in organic solvent Isopropanol. OD550 nm can be directly correlated to the number of metabolically active cells and this was used to determine the bacterial killing at various concentrations over time.

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

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:

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).

Human Embryonic Kidney Cell lines, HEK-293 were purchased from National Centre for Cell Sciences (NCCS), Pune, India. The cell lines were cultured in DMEM (supplemented with 10% foetal bovine serum) in a Carbon Dioxide incubator with 98% humidity and 5% CO₂ at 37°C.

Thawing of frozen cells: HEK-293 cells stored at −80°C were thawed by removing the frozen cryovial and thawing it in 37°C water for 1–2 min until no visible was seen in the vial. The vials were swirled gently to ensure that the cells are fully thawed. Once the cells were thawed, the vial was removed from the water bath and wiped with 70% ethanol to sterilize the outside of the vial.

Cell Culture and Expansion: The thawed HEK-293 cells were then transferred to a sterile hood and the contents of the vial were transferred into a sterile tube containing 9 mL of pre-warmed growth medium Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, and 1% penicillin-streptomycin. This was followed by centrifuging the tube at 1,000 rpm for 5 min to pellet the cells. The supernatant was aspirated and the cell pellet and resuspended in 2 mL of fresh pre-warmed growth medium. The cell suspension was transferred to a T75 culture flask and incubated at 37°C with 5% CO₂ until they reached the desired confluency of 70%–80%.

Harvesting of cells: Once the cells had reached the desired confluency, they were harvested using standard trypsinization techniques. A small volume of Trypsin-EDTA solution was added to the culture flask and incubated at 37°C for 2 min. During incubation, the Trypsin cleaves the proteins that attach the cells to the culture surface, causing the cells to detach from the surface. The culture medium was removed from the culture dish, and the cells were washed with phosphate-buffered saline (PBS) to remove any residual serum. After incubation, an equal volume of culture medium containing FBS and DMEM was added to neutralize the Trypsin and stop the enzymatic activity. The cells were then collected by gently tapping or swirling the culture dish and transferred to a centrifuge tube. The cells were again pelleted by centrifugation, and the supernatant is discarded. The cells were resuspended in a fresh culture medium, counted, and seeded into a new culture flask. The harvested cells were then counted using a hemocytometer. A predetermined number of cells were seeded into 96-well optimized for the cytotoxicity assay being performed.

The method was carried out as described earlier (Adan et al., 2016). The 100 µL of HEK-293 cells were seeded into the 96-well microtiter plate and allowed to grow for 24 h at 37°C with 5% CO₂. The cells were treated with 6 concentrations of the 2d compound 4, 8, 16, 32, 64, and 128 μg/mL dissolved in DMSO for time points 24, 48, and 72 h to determine the time and dose dependent effect of the compound on cell viability. Untreated control wells received only DMSO. After the given time points, the number of viable cells was determined. Briefly, the tissue culture medium was removed from the 96-well plate and replaced with 100 µL of fresh medium then 20 µL of the MTT stock solution (5 mg of MTT in 1 mL of PBS) was added to each well including the untreated controls. The 96-well plates were then incubated at 37°C and 5% CO₂ for 4 h. An aliquot (85 µL) of the medium was removed from the wells, and 150 µL of dimethyl sulfoxide was added to each well to dissolve the insoluble formazan crystals formed by viable cells and mixed thoroughly with the pipette and incubated at 37°C for 10 min. The optical density was measured at 590 nm with a microplate reader to determine the number of viable cells based on the selective ability of viable cells to reduce the tetrazolium component of MTT into purple-colored formazan crystals. The colored formazan intensity was measured at 570 nm absorbance in the micro-plate reader and growth percentage inhibition was calculated using the formula:

The results expressed were the average values of three experiments (±SD). Reduction of MTT to formazan crystals is mediated by the mitochondrial enzyme succinate dehydrogenase of cells the viable cells. The color of the formazan crystals that form after the reduction of MTT by viable cells indicates the level of cell viability and, hence, the cytotoxicity of the test compound. The formazan crystals appear as purple or dark blue precipitates in viable cells. The intensity of the color is directly proportional to the number of viable cells in the culture. Conversely, a decrease in the intensity of the color indicates a decrease in the number of viable cells and, hence, an increase in cytotoxicity. A complete absence of color indicates that all the cells in the culture have died, and the test compound is highly cytotoxic to the cells.

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).

Brown solid, yield: 93%; mp 170°C–173°C; IR (neat) ʋ: 2,124 cm⁻¹. ¹H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 7.98 (m, 1H), 7.63 (m, 1H). MS (m/z) 234.88 (M.F.:C₇H₂F₂N₄S).

Brown solid, yield: 93%; mp 155°C–158°C; IR (neat) ʋ: 2,113 cm⁻¹. ¹H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 8.25 (d, 1H), 7.94 (d, 1H), 7.35 (dd, 1H), 3.93 (s, 3H). MS (m/z) 207.03 (M.F.: C₈H₆N₄OS).

White solid, yield: 89%; mp 145°C–148°C; IR (neat) ʋ: 2,126 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.08 (d, 1H), 7.47 (dd, 1H), 7.39 (d, 1H), 3.74 (q, 2H), 1.28 (t, 3H).

Creamy white solid, yield: 95%; mp 190°C–193°C; IR (neat) ʋ: 2,120 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.77 (d, 1H), 8.40 (dd, 1H), 8.10 (d, 1H). MS (m/z) 222.03 (M.F.:C₇H₃N₅O₂S).

Brown solid, yield: 80%; mp 153°C–156°C; IR (neat) ʋ: 2,119 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.22 (d, 1H), 7.74 (d, 1H), 7.70–7.58 (m, 2H). MS (m/z) 176 (M.F.: C₇H₄N₄S).

Orange solid; yield: 80%; mp 117°C–120°C; IR (neat) ʋ: 2,120 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.73 (d, 1H), 7.30 (d, 1H), 7.18 (t, 1H), 2.70 (s, 3H). MS (m/z) 191 (M.F.: C₈H₆N₄S).

Brown solid, yield: 92%; mp 161°C–164°C; IR (neat) ʋ: 2,107 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.07 (d, 1H), 7.70 (d, 1H), 7.02 (dd, 1H), 4.06 (q, 2H), 1.44 (t, 3H). MS (m/z) 221 (M.F.: C₉H₈N₄OS).

Brown solid; yield: 82%; mp 180°C–183°C; IR (neat) ʋ: 2,110 cm⁻¹. ¹H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 8.32 (d, 1H), 8.16 (d, 1H), 7.66 (dd, 1H).