Evaluation of the Antibacterial Effect of Aurone-Derived Triazoles on Staphylococcus aureus

Infections caused by antibiotic-resistant bacteria continue to pose a significant public health threat despite their overall decreasing numbers in the last two decades. One group of compounds fundamental to the search for new agents is low-cost natural products. In this study, we explored a group of newly synthesized novel aurone-derived triazole compounds to identify those with pharmaceutical potential as inhibitors of antibiotic-resistant Staphylococcus aureus. Using the broth microdilution method, antibacterial activities against methicillin-resistant S. aureus ATCC 43300 (MRSA) and methicillin-sensitive S. aureus ATCC 29213 (MSSA) were identified for four aurone-derived triazole compounds, AT106, AT116, AT125, and AT137, using the half-maximal inhibitory concentrations for the bacteria (IC50) and mammalian cell lines (CC50). Compounds AT125 and AT137 were identified to have pharmaceutical potential as the IC50 values against MRSA were 5.412 µM and 3.870 µM, whereas the CC50 values measured on HepG2 cells were 50.57 µM and 39.81 µM, respectively, resulting in selectivity indexes (SI) > 10. Compounds AT106 and AT116 were also selected for further study. IC50 values for these compounds were 5.439 µM and 3.178 µM, and the CC50 values were 60.33 µM and 50.87 µM, respectively; however, SI values > 10 were for MSSA only. Furthermore, none of the selected compounds showed significant hemolytic activity for human erythrocytes. We also tested the four compounds against S. aureus biofilms. Although AT116 and AT125 successfully disrupted MSSA biofilms, there was no measurable potency against MRSA biofilms. Checkerboard antibiotic assays to identify inhibitory mechanisms for these compounds indicated activity against bacterial cell membranes and cell walls, supporting the pharmaceutical potential for aurone-derived triazoles against antibiotic-resistant bacteria. Examining structure–activity relationships between the four compounds in this study and other aurone-derived triazoles in our library suggest that substitution with a halogen on either the salicyl ring or triazole aryl group along with triazoles having nitrile groups improves anti-Staphylococcal activity with the location of the functionality being very important.


Introduction
Staphylococcus aureus and its antibiotic-resistant strains are well-known in medical and research communities. They are not only capable of causing illnesses with significant mortality [1,2], but with an annual hospitalization of 300,000 in the US alone [3], these pathogens, including methicillin-resistant S. aureus (MRSA), continue to pose a notable public health threat [4][5][6][7]. In addition to its healthcare system presence (HA-MRSA), MRSA strains can also be community-associated (CA-MRSA) and livestock-associated (LA-MRSA) [4,8,9]. Although these MRSA strains have been traditionally characterized as genetically distinct, interaction of individuals from the community with livestock and with those in healthcare environments has resulted in the intermingling of these strains [4,8,10], well as the maximal selectivity index (SI) ratios. All four AT compounds showed inhibition against MSSA with relatively low IC 50 concentrations, but only AT125 and AT137 were able to additionally inhibit MRSA with concentrations below 6 µM. Although AT106 exhibited less toxicity for the HepG2 and HeLa cells, such that higher concentrations (above 60 µM) of AT106 were less toxic compared to the other three AT compounds, higher concentrations of AT106 were required for anti-Staphylococcal activity. Conversely to their antibacterial activity where AT137 was the strongest and AT106 was the weakest performer, AT106 is the least toxic with CC 50 concentrations above 60 µM whereas AT137 is the most toxic with a 2.838 µM CC 50 . Still, all four compounds show pharmaceutical potential as they all have selectivity towards S. aureus. Unfortunately, these initial tests also show that only AT137 and AT125 were able to meet our selectivity criteria against MRSA, but we retained AT106 and AT116 for further testing as they could still be workable in mixtures with other bioactive compounds.
Antibiotics 2023, 12, x FOR PEER REVIEW

Aurone-Derived Triazole Anti-Staphylococcal Activity and Toxicity for Mammalian Ce
After screening the library of AT triazoles against S. aureus, four were chosen fo ther study (Figure 1). Table 1 and Table S1 depict the computed half-maximal conc tions as well as the maximal selectivity index (SI) ratios. All four AT compounds sh inhibition against MSSA with relatively low IC50 concentrations, but only AT12 AT137 were able to additionally inhibit MRSA with concentrations below 6 µM. Alth AT106 exhibited less toxicity for the HepG2 and HeLa cells, such that higher conc tions (above 60 µM) of AT106 were less toxic compared to the other three AT compo higher concentrations of AT106 were required for anti-Staphylococcal activity. Conv to their antibacterial activity where AT137 was the strongest and AT106 was the w performer, AT106 is the least toxic with CC50 concentrations above 60 µM whereas A is the most toxic with a 2.838 µM CC50. Still, all four compounds show pharmace potential as they all have selectivity towards S. aureus. Unfortunately, these initia also show that only AT137 and AT125 were able to meet our selectivity criteria a MRSA, but we retained AT106 and AT116 for further testing as they could still be wor in mixtures with other bioactive compounds.  In addition to the toxicity for mammalian cells, we assessed the hemolytic activ these AT compounds against human red blood cells. The results of the initial hem assay in Table 2 show that all but AT125 have no hemolytic activity at their half inhi concentrations, and all of them show less than 5% at 50 µM, confirming that thes appear to be safe for delivery through the bloodstream of patients.   In addition to the toxicity for mammalian cells, we assessed the hemolytic activity of these AT compounds against human red blood cells. The results of the initial hemolysis assay in Table 2 show that all but AT125 have no hemolytic activity at their half inhibitory concentrations, and all of them show less than 5% at 50 µM, confirming that these ATs appear to be safe for delivery through the bloodstream of patients.

Bacterial Growth in Aurone-Derived Triazoles
Growth curve experiments were conducted to gain insight into the dynamics of the inhibitory effect of the AT compounds. The graphs in Figure 2A-H show that all ATs successfully inhibited bacterial growth during the two-day period in the two highest AT In concentrations closer to the measured IC 50 values (5-25 µM), the graphs reflect our earlier distinction between strong inhibitors (AT125, AT137) and weaker inhibitors (AT106, AT116) of S. aureus growth. These results signify that AT125 ( Figure 2E,F) and AT137 ( Figure 2G,H) are successful in blocking bacterial growth at concentrations below 10 µM.

Bacterial Growth in Aurone-Derived Triazoles
Growth curve experiments were conducted to gain insight into the dynamics of the inhibitory effect of the AT compounds. The graphs in Figure 2A-H show that all ATs successfully inhibited bacterial growth during the two-day period in the two highest AT concentrations (25 µM and 100 µM). In concentrations closer to the measured IC50 values (5-25 µM), the graphs reflect our earlier distinction between strong inhibitors (AT125, AT137) and weaker inhibitors (AT106, AT116) of S. aureus growth. These results signify that AT125 ( Figure 2E,F) and AT137 ( Figure 2G,H) are successful in blocking bacterial growth at concentrations below 10 µM.

Biofilm Inhibition Microscopy
In addition to their inhibition potential against planktonic bacteria, the ATs were further investigated for disruption of S. aureus biofilms as these masses of bacteria pose a greater risk to patients [12,13,35]. Biofilm disruption was assessed by quantifying changes in bacterial cell numbers and the percentages of live and dead cells compared to the mocktreated control. As the imaging results in Figure 3 and Table S2 show, AT116 and AT125 treatments resulted in increased numbers of dead bacteria (2.42-3.95 fold increases) in pre-formed MSSA biofilms. All AT treatments resulted in 0.24-0.93 fold reductions of total bacterial cell numbers compared to the mock-treated control. AT116 and AT125 were the only two compounds with both reductions in total bacterial cell numbers and increases the dead cells. AT125 produced the largest fold increase in dead cells whereas AT137 had the largest decrease in total bacterial cells ( Figure 3, Table S2). Similar treatment and imaging were conducted for MRSA, but none of the ATs showed significant potency against MRSA biofilms with this assay.

Biofilm Inhibition Microscopy
In addition to their inhibition potential against planktonic bacteria, the ATs were further investigated for disruption of S. aureus biofilms as these masses of bacteria pose a greater risk to patients [12,13,35]. Biofilm disruption was assessed by quantifying changes in bacterial cell numbers and the percentages of live and dead cells compared to the mocktreated control. As the imaging results in Figure 3 and Table S2 show, AT116 and AT125 treatments resulted in increased numbers of dead bacteria (2.42-3.95 fold increases) in pre-formed MSSA biofilms. All AT treatments resulted in 0.24-0.93 fold reductions of total bacterial cell numbers compared to the mock-treated control. AT116 and AT125 were the only two compounds with both reductions in total bacterial cell numbers and increases the dead cells. AT125 produced the largest fold increase in dead cells whereas AT137 had the largest decrease in total bacterial cells ( Figure 3, Table S2). Similar treatment and imaging were conducted for MRSA, but none of the ATs showed significant potency against MRSA biofilms with this assay.

Checkerboard Assay
As an initial step to determine potential modes of action for the ATs, checkerboard assay experiments were conducted wherein mixtures of ATs and antibiotics used against S. aureus were tested for synergistic or antagonistic relationships depending on the fractional inhibitory concentration index (FICi) shown in Table 3. All ATs, except AT125, showed synergy with daptomycin against MSSA and MRSA as their FICi were below 0.5. AT125 was only synergistic with daptomycin against MSSA. AT116 and AT125 also showed synergy with vancomycin against MSSA. However, none of the compounds showed interaction with vancomycin against MRSA. Further testing with additional antibiotics and phenotypic assays will be necessary to further assess potential modes of action.  Following treatment, biofilms were stained with BacLight TM (Invitrogen, Carlsbad, CA, USA), a SYTO9 and propidium iodide mixture, such that bacteria with intact cell membranes stain fluorescent green, whereas those with damaged membranes (dead or dying bacterial cells) stain fluorescent red. In the images above, the bottom row shows dead cells (red), the center row depicts living bacteria (green), whereas the top row is the merged image combining both live and dead cells.

Checkerboard Assay
As an initial step to determine potential modes of action for the ATs, checkerboard assay experiments were conducted wherein mixtures of ATs and antibiotics used against S. aureus were tested for synergistic or antagonistic relationships depending on the fractional inhibitory concentration index (FICi) shown in Table 3. All ATs, except AT125, showed synergy with daptomycin against MSSA and MRSA as their FICi were below 0.5. AT125 was only synergistic with daptomycin against MSSA. AT116 and AT125 also showed synergy with vancomycin against MSSA. However, none of the compounds showed interaction with vancomycin against MRSA. Further testing with additional antibiotics and phenotypic assays will be necessary to further assess potential modes of action.
In this report, we have described the anti-Staphylococcal activity of aurone-derived 1,2,3-triazoles (ATs) formed by the reaction of aurones with sodium azide. The resulting ATs possess two sites for alkylation chemistry and the potential for modification of the ring system. To assess the pharmaceutical potential of the AT compounds, we used the standard method of calculating their selectivity index (SI), which is the ratio of their half-maximal toxicity concentration and their half-maximal inhibitory concentration. An SI higher than 10 shows that the compound has a large enough selectivity towards the bacterium, but not mammalian cells. The AT compounds chosen for this study (AT106, AT116, AT125, and AT137) all had SIs of >10 with mammalian cell lines and were not hemolytic for human erythrocytes indicating efficacy for delivery in the bloodstream. All four of the ATs were effective against MSSA, exhibiting IC 50 values between 3.09-5.44 µM (1.08-1.63 µg/mL). These concentrations are comparable to MIC ranges and reported IC 50 values for the reference antibiotics vancomycin and linezolid [52][53][54][55]. IC 50  Biofilms associated with S. aureus infections can be difficult to treat and eliminate due to multiple factors, including impermeability associated with chemical features of a drug or the physiology of the bacterial cells in the biofilm, such as reduced proliferation or metabolism, especially if dormant bacteria known as persisters are present [12]. For this reason, we treated pre-formed biofilms of MSSA or MRSA with the four AT compounds. Disruption of biofilm was assessed using the LIVE/DEAD BacLight TM assay which contains the dyes SYTO9 and propidium iodide. SYTO9 is transported across intact cell membranes and stains the nucleic acids, resulting in the green fluorescence of living cells. Propidium iodide cannot cross membranes of live cells, thus only staining nucleic acids of dead cells, producing a red fluorescence. Comparisons of live and dead cell numbers, as well as total bacterial cell numbers, to mock-treated control biofilms, were used to assess the efficacy of AT treatment. All ATs were effective against MSSA using the 100 µM concentration. This concurs with other observations that biofilms are more resistant to antibiotics, requiring concentrations up to 1000 times greater than required for planktonic bacteria [56].
Although the mode of action for these AT compounds is not yet clear, the synergism of ATs observed with vancomycin in checkerboard assays suggests the S. aureus cell wall as a potential target. S. aureus cell walls are composed of peptidoglycan, which is a layered polymer of alternating N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) molecules connected at the MurNAc residues by a short chain of amino acids [57]. The main EPS constituent of S. aureus polysaccharide biofilms is poly-N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA) [12]. If the ATs target cell wall polysaccharide synthesis, it is possible that they might also target the synthesis of biofilm polysaccharides. The antifungal triazole compound, itraconazole, has been shown to disrupt S. aureus biofilm, and although it blocks fungal ergosterol biosynthesis, it was proposed that the itraconazole might also target bacterial polysaccharide synthesis as does another antifungal drug, caspofungin [58]. Another study identified antifungal azoles as having S. aureus antibiofilm activity when combined with the antibiotic tetracycline. In this study, the authors demonstrate that the azoles act as competitive inhibitors of active efflux pumps in S. aureus biofilms, thus maintaining higher concentrations of the antibiotic in the bacterial cells by blocking the exit of the antibiotic [59]. This could also support the AT synergy observed with vancomycin or daptomycin in our study. An aurone-derived compound with anti-S. aureus biofilm activity suggests yet another potential mechanism of action for the ATs by the disruption of the quorum sensing [60]. The ATs also have the advantage of being small molecules (all <370 Da) which facilitates biofilm penetration [56]. Although all the AT compounds reduced total bacterial cell counts and decreased the numbers of viable bacterial cells for MSSA, none of the ATs successfully reduced MRSA biofilms, even at the highest concentration of AT compound. However, treatment success of biofilms may also be dependent on the strain of Staphylococcus, and we will continue to test AT anti-biofilm efficacy against other strains of S. aureus as well as biofilm-forming strains of Staphylococcus epidermidis [61].
Checkerboard assays with daptomycin and vancomycin were used to assess potential modes of action. For these assays, a computed FICi below 0.5 indicates synergy, whereas a score greater than 4 indicates antagonism. None of the ATs exhibited an antagonistic effect with daptomycin or vancomycin. All four ATs showed synergy with daptomycin against MRSA suggesting that these ATs target cell membranes [62]. Although many triazoles target fungal cell membrane synthesis, they can also exhibit other modes of action; thus, the daptomycin checkerboard assay is insufficient to draw conclusions about the anti-Staphylococcal mode of action for these molecules. Daptomycin acts by reorganizing Gram-positive bacterial membranes, leading to membrane permeabilization [62,63]. Membrane permeabilization could facilitate AT targeting of intracellular molecules. In fact, the bioavailability model of drug synergy proposes that two drugs can be synergistic if the action of one drug assists with another drug's availability to target cells, such as by increasing the second drug's entry into the cell [64]. We will investigate AT disruption of membrane integrity using phenotypic assays for bacterial cytoplasmic membrane damage, such as the LIVE/DEAD ® BacLight TM Bacterial Viability Kit or similar assays [65][66][67]. Synergy with vancomycin was only evident with AT116 and AT125 against MSSA, indicating the cell wall as a possible target of these ATs. Other 1,2,3-triazole-containing antibiotics, including cefatrizine and tazobactam, also target bacterial cell walls [47][48][49][50]. It is interesting that AT106 had FICi values between 0.5-1 for MSSA and MRSA as it has been suggested that values of 0.5 < FICi < 1 indicate an additive to synergistic effect for antimicrobials [68]. Again, it is possible that the synergy could result from vancomycin allowing entry of AT compounds into the bacterial cells. Future studies will use aurone combinations and expand antibiotics used for checkerboard assays to assess whether intracellular processes such as nucleic acid synthesis, protein synthesis, or metabolic pathways are affected. We have already shown that aurone antifungal activity is related to both oxidative stress and interference with amino acid and carbohydrate biosynthesis, and these ATs may also have multiple modes of action [30].
The structures of these aurone-derived triazoles may also be important to their anti-Staphylococcal activity. In a study evaluating fluoro-and trifluoromethyl-substituted salicylanilide derivatives against S. aureus [69], the authors report the significance of the 2-hydroxyl group on the benzoic acid portion for anti-Staphylococcal activity. All of the ATs in our study have a similar salicyl functional array. The authors of the salicylanilide derivatives study also reported the significance of halogen substitutions, especially bromine and trifluoromethyl groups to target Staphylococci [69]. All four of the ATs in this study are halogenated, with AT106 (chlorine) and AT116 (trifluoromethyl) having the halogen on the triazole phenyl group. Indeed, in the case of AT116, the trifluoromethyl group is meta, exactly as it is found in the optimal salicylanilides examined by Lal et al. [69]. AT125 and AT137, which were both effective against MRSA, contain bromine, although they differ in the location of bromine as well as the type of aromatic ring that contains them. The AT137 bromine is on a thiophene ring attached to the triazole, whereas the bromine of AT125 is on the salicyl portion, similar to that observed in triazole-substituted chalcones reported by Kant et al. [49]. One of the halogenated compounds described by Lal et al. [69] also had significant antibiofilm activity, although, similar to the results seen for our ATs, that activity required a 10-fold increase in the concentration of the compound. Other halogen- containing polyphenols, especially brominated furanones, have also been identified as having anti-biofilm properties for Gram-positive and Gram-negative bacteria, including Staphylococci. The proposed mechanism of action for these furanones is as quorum-sensing inhibitors [70][71][72][73][74][75]. Although the AT compounds no longer contain the aurone furan ring, the AT ring structures are halogenated with both AT125 and AT137 having a bromine either on the salicyl or the thiophene ring. This suggests a potential mechanism of action for the ATs in disrupting quorum sensing that will be further investigated.
Our AT library has additional compounds with structures comparable to AT125 and AT137 that possess bromines in other positions or are substituted with another halogen (Table S1). Although several of these were anti-Staphylococcal, they had lower SI indexes with mammalian cells and were not pursued; however, the structures of some of these compounds support the activity of the ATs in this study. ATs having halogens at the para position of the phenyl group had increased activity against S. aureus (AT106, AT113; Table S1), with a para halogen being less active against MSSA, but similar against MRSA strains (AT107 ; Table S1). Unlike AT137, AT135 (Table S1) has no substitution on the triazole thiophene ring, and without bromine, the anti-MRSA activity decreased by over 15-fold. ATs with meta or para halogens on the salicyl ring had increased anti-Staphylococcal activity (AT119, AT120, AT122-AT126; Table S1). These compounds all have a benzonitrile ring attached to the triazole, except for AT123 which has a methylbenzene ring (Table S1). In all cases, the carbonitrile or methyl group is para. AT100 (Table S1) also has a para methylbenzene ring on the triazole, but does not possess a halogen, yet exhibited significant inhibition against MRSA (>96%). Although the SI indexes were low, indicating toxicity for human cells, we plan to use these other ATs in phenotypic assays to determine modes of action for AT125 and AT137, as these compounds will help us to assess the structure-activity relationships between these molecules and their bioactivity. This expanded testing will provide a more comprehensive view of anti-Staphylococcal activity for the aurone-derived triazoles, which not only have the potential for multiple modes of antimicrobial activity but are also a cost-effective, explorable platform for antibiotic development.

Aurone-Derived Triazole Compounds
The syntheses and characterizations of the AT compounds used in this study have been previously described [34,76]. Purified powders of the AT compounds were solubilized in 100% dimethyl sulfoxide (DMSO) to a concentration of 10 mM. For bioactivity assays, these stock solutions were further diluted in Cation-Adjusted Mueller-Hinton Broth (CAMHB), RPMI-1640 (RPMI), or Brain Heart Infusion Broth (BHI) to achieve the final concentration of interest which ranged from 3.125 to 100 µM. AT stock solutions were stored at −20 • C.

Bacteria Culture Preparation
MRSA strain ATCC 43300 and MSSA strain S. aureus ATCC 29213 were grown on Tryptic Soy Agar (TSA) plates. Before use, an isolated colony was transferred from the TSA plate into Tryptic Soy Broth (TSB) and incubated 18-24 h in a humidified incubator at 37 • C. To adjust for a standard concentration of bacteria, the turbidity of the culture was measured at 600 nm, and its optical density (OD 600 ) adjusted to be in the range 0.08-0.12 which equates to a concentration of approximately 1 × 10 8 CFU/mL. This suspension was diluted 1:20 in TSB. The final mixture for experiments used the bacterial suspension and AT compound mixture as a 1:10 dilution which resulted in a final 5 × 10 5 CFU/mL bacterial concentration [77]. MRSA strain S. aureus ATCC 43300 and MSSA strain S. aureus ATCC 29213 were used for all testing in this study.

Antibacterial Assay
The assay for anti-Staphylococcal activity of AT compounds was prepared in 96-well transparent, U-bottom plates. A series of 2-fold dilutions of AT concentrations was tested in triplicate, with each well containing 10 µL of prepared bacterium and 90 µL of AT mixture in CAMHB. This resulted in 100 µL of volume in each well with a 5 × 10 5 CFU/mL bacterial concentration and 100, 50, 25, 12.5, 6.25, or 3.125 µM concentrations of the AT compound. Blank wells with only CAMHB, a negative control well with 10 µL of bacterium solution mixed into 90 µL CAMHB, a vehicle control well with bacteria mixed into 1% DMSO in CAMBH, and a positive cell death control with vancomycin (1 µL of stock 2 mg/mL solution in 89 µL CAMHB with 10 µL of bacteria) were included. The plate was incubated for 18-24 h at 37 • C in a humidified incubator. Before measurement, PrestoBlue TM dye (Invitrogen, Carlsbad, CA, USA) was added (10 µL per well) and incubated for 1 h with aluminum foil covering the plate. A CLARIOstar ® (BMG LABTECH, Cary, NC, USA) multi-mode plate reader was used to measure the fluorescence at wavelengths of 560 nm for excitation and 590 nm for emission. Inhibitory concentration (IC 50 ) values were defined as half inhibitory concentration and were computed using GraphPad Prism version 9.1 software [78].

Mammalian Cell Lines
Human HeLa epithelial (ATCC CCL-2) and HepG2 liver epithelial (ATCC HB-8065) cell lines were used to determine toxicity of AT compounds for mammalian cells. The cells were grown in a complete culture medium of RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS) at 37 • C in 5% CO 2 (95% room air) in a humidified incubator. Before use, attached cell monolayers, which had reached at least 90% confluency, were detached by trypsinization with trypsin-EDTA (0.25%) and resuspension in fresh complete culture media. For experiments, the stock concentration of 1 × 10 5 cells/mL (to achieve a final 1 × 10 4 cells/well concentration) was achieved by counting the trypsinized cells and adjusting to their final concentration for assays.

Cytotoxicity Assay
HeLa or HepG2 cells were pre-seeded into a 96-well transparent, flat bottom plate by adding a volume of 100 µL stock cell concentration (1 × 10 5 cells/mL) in complete culture media to each well. The plate was incubated overnight at 37 • C in a 5% CO 2 (95% room air) humidified incubator. After the incubation and before mixing in the AT compounds, a 100 µL volume of fresh complete culture media was added to the pre-seeded wells. The 2-fold dilutions of AT compounds in triplicates were prepared on a separate 96-well plate. Once the dilution plate was prepared, dilutions were transferred into the pre-seeded plate to achieve the same concentrations of ATs as for the inhibition assay described above. The final inoculated plate was then returned to the 5% CO 2 (95% room air) humidified incubator for an additional 24 h of incubation with the diluted ATs. Controls were blank wells containing only media, wells with cells and DMSO in complete culture media, and wells with cells and complete culture media only. Measurement of toxicity was analogous to the inhibition assay except that 20 µL of PrestoBlue TM was used and the incubation was 3-6 h before reading with the plate-reader. Cytotoxicity concentration (CC 50 ) was defined as the concentration where half of the cells remained viable and was computed using GraphPad Prism.

Hemolysis Assay
A single donor-specific blood sample was acquired from Innovative Research Inc. (Novi, MI, USA) for the assay (donor#:1WB3ALS40ML-36517). A volume of 1-2 mL of the human red blood cells (hRBCs in Alsevier's solution) was mixed with 8-10 mL of Phosphate-Buffered Saline (PBS) in a 15 mL centrifuge tube to pellet by centrifugation at 1400× g in a swinging bucket centrifuge for 10 min. The supernatant was then removed, and the cells were washed with 10 mL of PBS two additional times. The cells were resuspended in PBS to achieve a final 1% solution of hRBCs.
The wells of a 96-well U-bottom plate that contained AT compounds were prefilled with 50 µL of PBS. A 2-fold dilution of AT compounds was prepared. For each triplicate, an initial AT dilution was prepared using 6 µL of AT stock solution in 144 µL of PBS. This initial mixture was diluted 2-fold on a 96-well plate following the method described above for a final volume of 50 µL of diluted AT in each well. As a final step, 50 µL of the 1% hRBC solution was added to each well, except the blank wells. PBS and hRBCs were used for negative control, whereas hRBCs with 1% and 0.1% Triton-X100 were used as positive controls. After 1 h of incubation at 37 • C in a humidified incubator, the plate was centrifuged at 1000 rpm for 10 min in a Thermo Sorvall Legend XTR centrifuge with a TX-750 swinging bucket rotor (Thermo Fisher Scientific, Waltham, MA, USA) to pellet intact cells. After centrifugation, 50 µL of supernatant was transferred from each well into a new 96-well U-bottom plate and hemolysis of the hRBCs detected in a plate-reader for absorbance at 405 nm. Hemolytic activity for all concentrations of the ATs was computed following this formula:

Growth Curve Assay
For the growth curve assay, honeycomb plates (Growth Curves USA, Piscataway, NJ, USA) were used with TSB as the growth medium. Using bacterial cultures as described above, growth curve measurements were carried out in quintuplicates. For positive growth controls, wells with 10 µL of bacteria mixed into 90 µL of TSB were used. For quintuplicate treatment wells, 4-fold dilutions of AT compounds were used such that AT concentrations of 100, 25, 6.25, and 1.56 µM were tested. A 10 µL inoculum of bacteria prepared as described above was added to each dilution of the ATs. The growth in each well was measured in 15 min intervals for 48 h at 37 • C in the Bioscreen C instrument [79]. The results were processed and analyzed with Microsoft Excel (version 2201) and GraphPad Prism 9.3.1 software (GraphPad by Dotmatics, Boston, MA, USA).

Checkerboard Assay
This preparation for the checkerboard assay was analogous to the antibacterial assay described above in that the standard (5 × 10 5 CFU/mL) concentration of bacteria in media was mixed with 2-fold dilutions of antibacterial agents to verify their inhibition potential [80,81]. For this assay, the highest concentration of an agent was either 100 µM for the ATs or the minimal inhibitory concentration (MIC 90 -the concentration of the agent with at least 90% inhibition of the bacteria) for the antibiotics. Control wells contained 10 µL of bacteria with 90 µL of CAMHB, and the negative control was bacteria in 1% DMSO and CAMHB. Incubation, staining with PrestoBlue TM , and assessing cell viability are as described for the antibacterial assay above.

Biofilm Assay
To prepare AT-treated cultures, overnight bacterial cultures were used (adjusted to 1 × 10 8 CFU/mL) in BHI medium to grow biofilm in 35 mm MatTEK dishes with a 20 mm glass bottom well by adding 1500 µL of overnight bacteria per dish. The dishes were incubated at 37 • C in a humidified incubator for 48 h. The old media was refreshed by adding 1500 µL of BHI for the control, or the AT mixture (15 µL of AT stock with 1485 µL BHI to achieve 100 µM AT concentration), or DMSO mixture (15 µL DMSO with 1485 µL BHI) for negative control. The dishes were then incubated in a humidified incubator for an additional 24 h at 37 • C. The media was removed, and cells were washed twice with 500 µL PBS, followed by the addition of 50 µL BacLight TM with 250 µL PBS for 15 min. Biofilms were imaged using a Zeiss AxioObserver microscope with an LSM700 confocal module (Carl Zeiss Microscopy, White Plains, NY, USA). To assess biofilm disruption, Fiji [82] software version 2.14.0 was used. Initially, raw microscope images were imported, separating the color channels, as dead cells were stained fluorescent red and living cells were stained green. To allow particle counts, the color saturation threshold was set using the RenyiEntropy option [83] to clear the images from potential noise. This step was followed by watershed separation [84], which separates potentially overlapping cells, and then particle analysis was completed using the 0.5-1.5 micron size as a guide. Biofilm disruption was quantified both by reductions in total bacterial cell numbers and increases in dead cell numbers.