Novel antimony-based antimicrobial drug targets membranes of Gram-positive and Gram-negative bacterial pathogens

ABSTRACT Antimicrobial resistance (AMR) poses a significant worldwide public health crisis that continues to threaten our ability to successfully treat bacterial infections. With the decline in effectiveness of conventional antimicrobial therapies and the lack of new antibiotic pipelines, there is a renewed interest in exploring the potential of metal-based antimicrobial compounds. Antimony-based compounds with a long history of use in medicine have re-emerged as potential antimicrobial agents. We previously synthesized a series of novel organoantimony(V) compounds complexed with cyanoximates with a strong potential of antimicrobial activity against several AMR bacterial and fungal pathogens. Here, five selected compounds were studied for their antibacterial efficacy against three important bacterial pathogens: Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. Among five tested compounds, SbPh4ACO showed antimicrobial activity against all three bacterial strains with the MIC of 50–100 µg/mL. The minimum bactericidal concentration/MIC values were less than or equal to 4 indicating that the effects of SbPh4ACO are bactericidal. Moreover, ultra-thin electron microscopy revealed that SbPh4ACO treatment caused membrane disruption in all three strains, which was further validated by increased membrane permeability. We also showed that SbPh4ACO acted synergistically with the antibiotics, polymyxin B and cefoxitin used to treat AMR strains of P. aeruginosa and S. aureus, respectively, and that at synergistic MIC concentration 12.5 µg/mL, its cytotoxicity against the cell lines, Hela, McCoy, and A549 dropped below the threshold. Overall, the results highlight the antimicrobial potential of novel antimony-based compound, SbPh4ACO, and its use as a potentiator of other antibiotics against both Gram-positive and Gram-negative bacterial pathogens. IMPORTANCE Antibiotic resistance presents a critical global public health crisis that threatens our ability to combat bacterial infections. In light of the declining efficacy of traditional antibiotics, the use of alternative solutions, such as metal-based antimicrobial compounds, has gained renewed interest. Based on the previously synthesized innovative organoantimony(V) compounds, we selected and further characterized the antibacterial efficacy of five of them against three important Gram-positive and Gram-negative bacterial pathogens. Among these compounds, SbPh4ACO showed broad-spectrum bactericidal activity, with membrane-disrupting effects against all three pathogens. Furthermore, we revealed the synergistic potential of SbPh4ACO when combined with antibiotics, such as cefoxitin, at concentrations that exert no cytotoxic effects tested on three mammalian cell lines. This study offers the first report on the mechanisms of action of novel antimony-based antimicrobial and presents the therapeutic potential of SbPh4ACO in combating both Gram-positive and Gram-negative bacterial pathogens while enhancing the efficacy of existing antibiotics.

S ince their discovery, antibiotics have become an essential part of the modern healthcare, allowing treatment of otherwise fatal infections (1)(2)(3).Years of improper use of these compounds lead to antimicrobial resistance (AMR) in bacteria posing a significant and urgent threat to public health on a global scale (4)(5)(6).According to a global systematic analysis conducted in 2019, there were an estimated 4.95 million annual fatalities associated with bacterial AMR (6).This death toll was estimated to reach 10 million by 2050 emphasizing the pressing need for novel antimicrobials that are effective and safe (7)(8)(9).Despite the urgency, the current pipeline of developing new antibiotics remains insufficient to counter the rising resistance in bacterial pathogens (10,11).Only 12 new antimicrobials have been approved since 2017, and 10 of them belong to already existing classes, which undermines their effectiveness due to the risk of rapid resistance development (11,12).With the increasing demand for novel antimicrobial strategies, metals and metal-containing compounds have gained renewed interest as potential antimicrobials (13)(14)(15)(16).One of the advantages of using metal-based antimicrobials is the observed capacity to target multiple cellular processes in bacteria leading to pleiotropic effects, a property that decelerates the evolution of resistance (14,17).Furthermore, the ease with which the physicochemical properties of metal-based complexes can be fine-tuned by incorporating a variety of organic ligands makes them more attractive (13,18).
The two metals, antimony (Sb) and bismuth (Bi), have a long history of use in medicinal chemistry, which dates back to the 18th century (19).Sb and its salts have been used by ancient Egyptians and Assyrians to treat a variety of ailments including fevers and skin irritation (18,20,21).Since early 20th century, Sb-based drugs have been used to treat leishmaniasis, an infective parasitic disease caused by the proto zoan Leishmania (22).In addition to their antiparasitic activity, several organoantimony compounds have also been studied for their potential as antimicrobial, antifungal, antiviral, and anticancer agents (18,(23)(24)(25)(26)(27).Previously, we synthesized and characterized a series of novel organoantimony(V) cyanoximates.These compounds, denoted by the general formula Sb(C 6 H 5 ) 4 L, with L representing the ligand, were obtained by subject ing AgL (or TlL) and Sb(C 6 H 5 ) 4 Br in CH 3 CN to high-yield heterogeneous metathesis at room temperature (28).The eight cyanoximate ligands used in our previous study belong to a new subclass of small organic molecules that are chemically and thermally stable (29) and non-cytotoxic (30)(31)(32) and exhibit a range of biological activities (33)(34)(35)(36)(37)(38).When tested against a panel of drug-resistant bacterial and fungal species, including Gram-negative Escherichia coli and Pseudomonas aeruginosa, Gram-positive Staphylococ cus aureus, and fungal pathogens Cryptococcus neoformans and Candida albicans, these compounds varied in their antibacterial activity and showed promise as both broad-and narrow-spectrum antimicrobials (28,39).
Here, we studied in more detail the antibacterial efficacy of selected five organoanti mony cyanoximates and investigated the underlying mechanism of action for SbPh 4 ACO against three pathogens P. aeruginosa, E. coli, and S. aureus.

RESULTS AND DISCUSSION
The tetraphenyl organoantimony cyanoximate, SbPh 4 ACO, inhibits the growth of pathogenic strains of P. aeruginosa, E. coli, and S. aureus Our previous study describes the synthesis and characterization of five novel tetraphenyl organoantimony cyanoximates, namely, SbPh 4 ACO, SbPh 4 ECO, SbPh 4 MCO, SbPh 4 TCO, and SbPh 4 TDCO (Fig. 1A).These compounds share a tetraphenyl backbone with Sb attached to it but differ in their associated cyanoximate ligand groups (Fig. 1A).To evaluate their antibacterial activity, we determined the minimum inhibitory concentra tion of these compounds and their respective ligand controls H(ACO), H(ECO), and H(MCO) by standard methods.The ligand controls of these compounds did not show any antibacterial activity when tested at the concentration ranging from 0 to 200 µg/mL (Table 1).In contrast, three out of five Sb-based compounds showed antibacterial activity against at least two of the three pathogens tested.Among them, SbPh 4 (ACO) inhibited the growth of all three bacterial pathogens, P. aeruginosa, E. coli, and S. aureus with MIC values ranging from 50 to 100 µg/mL, implying a strong potential as a broad-spectrum antimicrobial.SbPh 4 ECO and SbPh 4 MCO only inhibited the growth of P. aeruginosa and S. aureus.
The two compounds SbPh 4 TCO and SbPh 4 TDCO did not show any antibacterial activity toward any of the tested pathogens.This lack of antibacterial activity of SbPh 4 TDCO differs from our previous results that were based on disc diffusion assays and where the compound showed a growth inhibitory effect against S. aureus (28).It is possible that the poor solubility of the drug in liquid medium prevents its activity.This can be circumvented by structural modifications in future studies.
It is also noteworthy that the growth of E. coli was inhibited by only one [SbPh 4 (ACO)] out of five compounds, suggesting that this pathogen may harbor mechanisms different to those in the other two pathogens to deter the activity of these compounds.It has been reported that although P. aeruginosa exhibits reduced outer membrane permeabil ity compared with E. coli (approximately 8% of the E. coli threshold), the size exclusion cut-off for its outer membrane porins is approximately 3,000 Da , markedly higher than that of E. coli, which is around 500 Da (40)(41)(42).This difference may contribute to the observed differences in the susceptibility to Sb-based antimicrobials between these two organisms.
To develop a more mechanistic understanding of the antibacterial activity of these compounds, we selected SbPh 4 ACO that showed growth inhibition against all three tested bacterial strains.ellipsoids for atoms are drawn at their 50% probability level.The compound crystallized as a clear colorless block-like specimen (Fig. S2).The cyanoxime moiety adopts the trans-anti conformation with respect to the position of the N-O of the oxime and pyridyl ring along the C2-C3 bond.The ACO-oxime anion is planar, signifying no dihedral angle, and agrees with previously mentioned literature data for this ligand.This crystal structure is stabilized by H-bonding between one of amide's hydrogens and the cyanoximes N from N-O-Sb (Fig. S3).The geometry of the coordination polyhedron of the Sb(V) atom was a distorted trigonal bipyramid.The cyano group of the oxime is linear with atoms C1-C2-N2 = 175.9°(Table S2).Bond distances for the cyano group C2-N2 = 1.144Å, oxime group N1-O1 = 1.337Å, and C1-N1 = 1.278Å.This crystal structure has been deposited at the Cambridge Crystallographic Data Centre under number 2011862 and is available upon request.

SbPh 4 ACO exerts bactericidal effect on P. aeruginosa, E. coli, and S. aureus
To determine whether SbPh 4 ACO has a bactericidal effect, time-kill assays were performed for three bacterial strains P. aeruginosa PAO1, E. coli S17, and S. aureus NRS70.The strains were grown in cation-adjusted Mueller-Hinton broth (CA-MHB), normalized to reach a standard inoculum size of 5 × 10 5 CFU/mL as established (43), and treated with different concentrations of SbPh 4 ACO ranging from 0.5×-4× of their respective MIC values.Following the treatment, bacterial survival was monitored for 6 h by counting CFU and compared with the untreated growth control (Fig. 2).
Exposure to SbPh 4 ACO at its MIC concentration of 100 µg/mL led to 99.9% killing of P. aeruginosa after 3 h of incubation (Fig. 2A).For E. coli, the applied amount of the compounds had to be 2× MIC (200 µg/mL) in order to achieve 100% killing that also occurred after 3 h of incubation (Fig. 2B).In the case of S. aureus, exposure to 4× MIC (200 µg/mL) of the compound for 1 h (Fig. 2C) also led to 100% killing.So, overall, 100-200 µg/mL SbPh 4 ACO resulted in at least 99.9% reduction in 5 × 10 5 CFU/mL, thus demonstrating its bactericidal activity against the three bacterial strains (Fig. 2).
For each bacterial strain, the MBC was determined as the concentration of the antimicrobial that eliminates 90% of the bacterial population within 6 h upon exposure which is equivalent to 99% killing at 24 h as established (44,45).According to our observations, the MBC of SbPh 4 ACO for P. aeruginosa, E. coli, and S. aureus are 100, 200 and 200 µg/mL, respectively (Table 2).At these concentrations, we observed 100% killing for the three bacterial strains upon 1-3 h of exposure to this compound.Further, according to published studies (46)(47)(48), a drug is considered to have bactericidal activity when its MBC/MIC ratio is ≤4 and bacteriostatic, when the MBC/MIC ratio ≥ 8.The respective MBC/MIC ratios we obtained for SbPh 4 ACO for P. aeruginosa, E. coli, and S. aureus are 0.5, 1, and 4 (Table 2), further confirming the bactericidal effect of SbPh 4 ACO on these pathogens.
It is generally regarded that bactericidal antimicrobials are more desirable than bacteriostatic drugs, as they prevent the regrowth of the bacteria (49).Hence, from a clinical viewpoint, the bactericidal properties of SbPh 4 ACO make it more suitable for applications to treat bacterial infections.

SbPh 4 ACO disrupts P. aeruginosa, E. coli, and S. aureus membranes
To determine the nature of SbPh 4 ACO impact on bacterial cells, we employed thin-layer TEM imaging.The three bacterial strains P. aeruginosa PAO1, E. coli S17, and S. aureus NRS70 were grown in CA-MHB for 5 h and exposed to the MIC level of the compound for 1 h.Following the treatment, their cell morphology was visualized by TEM.As a control, cells were treated with the appropriate volume of dimethyl sulfoxide (DMSO) used to dissolve SbPh 4 ACO (Fig. 3).The controls for the three strains did not show any visible signs of cellular damage.In contrast, SbPh 4 ACO-treated cells showed prominent a The reported numbers represent the average of three experiments.Each antimicrobial was subjected to standard CLSI broth dilution assays at concentrations 0-200 µg/mL.>200 indicates that the MIC for the given compound was not detectable at the range of concentrations tested.MICs are highlighted in bold.
membrane disruptions in all three species.Both P. aeruginosa and E. coli cells showed electron-light regions near the membrane indicating the separation of the cytoplasm from the membrane (shown with arrows in Fig. 3A and B).We also observed electronlight regions appearing within the cytoplasm.In S. aureus, in addition to apparent membrane damage, a majority of cells appeared to have stalled during the formation or separation of the septum indicating interrupted cell division (Fig. 3C).These observa tions suggest that SbPh 4 ACO targets the bacterial cell membrane, causing membrane disruption, with accompanying changes in cell morphology likely leading to lethal effects on bacteria.

SbPh 4 ACO causes bacterial membrane permeabilization
Since the TEM results indicated that SbPh 4 ACO targets bacterial membranes, we tested its impact on membrane permeability by using the fluorescent stains NPN and PI (50-53) as probes for outer and inner membranes, respectively.For this, all three strains were exposed to SbPh 4 ACO for 1 h at the sub-inhibitory levels (0.5× MIC) and assayed for membrane permeability.In agreement with the TEM, the inner membrane permeability showed a consistent but modest increase of 1.4-1.8-fold(P ≤ 0.01) vs the corresponding solvent control (Fig. 4B, D and E).However, the permeability of the outer membranes of Gram-negative P. aeruginosa and E. coli increased much greater, by 12.2 and 5.6-fold (P ≤ 0.01), respectively (Fig. 4A, B, C and D).It is important to acknowledge that P. aerugi nosa possess multiple efflux systems that may impact the membrane permeability, and therefore, all the permeability measurements were compared with the corresponding solvent controls.SbPh 4 ACO itself may be translocated by efflux or impact efflux of other compounds, which warrants future studies for insights into its mode of action and its potential synergy with other antimicrobials.
Our results indicate that the effect of SbPh 4 ACO on the permeability of outer membranes of Gram-negative P. aeruginosa and E. coli is more pronounced than that on the inner membranes.These differences may be attributed to lower accessibility of the inner membrane or the differences in the composition between the inner and outer membranes.For example, lipopolysaccharides in the outer leaflet of the outer membrane may be more susceptible to SbPh 4 ACO than the phospholipid bilayer that makes up the inner membrane.The outer membrane in Gram-negative bacteria is well known as a permeability barrier, particularly against hydrophobic antibiotics, detergents, and dyes (54,55) as well as large hydrophilic drugs (42,(56)(57)(58)(59).As a result, most Gram-negative bacteria display intrinsic resistance to several antibiotics such as oxacillin, rifampicin, erythromycin, and vancomycin (55,60).The composition of the outer membrane is also known to undergo a variety of modifications to provide resistance to a wide range of antibiotics including β-lactams, quinolones, and colistin (61).Therefore, the ability of SbPh 4 ACO to disrupt the outer membrane and overcome this barrier can be used to potentiate the efficacy of antibiotics that are otherwise blocked by this barrier.This may expand the available therapeutic options for infections caused by Gram-negative bacterial pathogens and assist in lowering the required doses of antibiotics thereby reducing their toxicity and lowering the risk of resistance development.

SbPh 4 ACO shows synergism with clinically used antibiotics
To explore the potential of SbPh 4 ACO to potentiate the efficacy of antibiotics and to reduce the effective dose of the compound itself, we performed a series of checkerboard assays conducted with SbPh 4 ACO in combination with polymyxin-B, meropenem and cefoxitin [an alternative to methicillin (62), used in clinic against P. aeruginosa, E. coli, and methicillin-susceptible S. aureus, respectively (63-66)].Synergy is commonly determined by calculating the fractional inhibitory concentra tion index (FIC I ) (67)(68)(69).FIC I ≤ 0.5 indicates synergistic relationships between drugs, whereas FIC I in the range 0.5-4.0indicates an additive or indifferent relationship, and FIC I > 4.0 is indicative of antagonism between the drugs (70).The FIC I value calculated for the combination SbPh 4 ACO and polymyxin-B against P. aeruginosa reached 0.396 ±   3).Similarly, a synergistic behavior was observed for the combination of SbPh 4 ACO and cefoxitin against S. aureus with the FIC index of 0.5.However, the interaction between SbPh 4 ACO and meropenem resulted in the FIC I of 1, which was indicative of additive/indifferent relationships between these two drugs.
The strong synergy observed between SbPh 4 ACO and polymyxin-B suggests that the compounds, although may affect related processes, do not share specific targets.Given that polymyxin-B disrupts the outer membrane by targeting the LPS (71, 72) and since SbPh 4 ACO also disrupts the outer membrane and increases its permeability, it is possible that SbPh 4 ACO may not target LPS specifically.Polymyxin B was shown to synergize with different types of antibiotics, including meropenem (63,73,74) targeting penicillin-binding proteins (PBP) (65); however, SbPh 4 ACO showed indifferent FIC for the latter.This suggests that SbPh 4 ACO does not target PBPs.The synergistic impact of SbPh 4 ACO with cefoxitin (an alternative to methicillin) for S. aureus, also targeting PBPs involved in cell wall synthesis (75)(76)(77), further supports that SbPh 4 ACO may damage the outer layers of the cell wall and provide a better access to but not targeting PBPs located in the cytoplasmic membrane (78).The results indicate that SbPh 4 ACO may help salvage cefoxitin as a treatment for MRSA and reduce the dosage and therefore the toxicity of polymyxin B. Further studies are needed to discover the specific mechanisms of action and to identify antibiotics that can be used in combination with SbPh 4 ACO to treat E. coli.

Compound SbPh 4 (ACO) is non-cytotoxic to mammalian cells at the synergis tic MIC concentration
To determine the cytotoxic effects of SbPh 4 ACO on mammalian cells, we performed cytotoxicity assays with SbPh 4 ACO concentrations ranging from 12.5 to 200 μg/mL on three mammalian cell lines, HeLa, McCoy, and A549.These concentrations were selected based on the previously determined MIC concentrations of SbPh 4 ACO when used alone or in combination with other antibiotics (Tables 2 and 3).
When used at 12.5 µg/mL, SbPh 4 ACO exhibited minimal cytotoxicity with mean values of −0.30%, 6.33%, and 3.24% in HeLa, McCoy, and A549 cells, respectively (Fig. 5A,  B and C).Notably, these percentages fall well below the previously established cytotoxic ity threshold of 30% (79), indicating the non-cytotoxic nature of SbPh 4 ACO toward these mammalian cell lines at this concentration.This lack of cytotoxicity of SbPh 4 ACO coupled with the synergistic effect with cefoxitin signifies the potential combinatory application of this antimicrobial against MRSA.
At 25 µg/mL, SbPh 4 ACO showed a mean cytotoxicity of 22.37% in HeLa cells and higher cytotoxicity in McCoy and A549 cells which exceeded the threshold of 30%.When used at concentrations above 50 µg/mL, the cytotoxicity of SbPh 4 ACO exceeded 30% in all three cell lines (Fig. 5; Fig. S1).It is noteworthy that the levels of SbPh 4 ACO cytotoxicity varied in different cell lines which highlights the importance of including several lines in testing.
The strategy of using non-antibiotic drugs as adjuvants to boost the effectiveness of existing antibiotics presents a unique method to address the crisis of antibiotic resistance and has gained interest over the past few years (13,(80)(81)(82)(83). Several metalbased compounds have been studied for their synergistic effects with other antibiotics against resistant bacterial strains including another group V metalloid, bismuth, which a FIC index and the MIC concentration of SbPh 4 ACO when used in combination with antibiotics are represented as an average of at least three replicates with the standard error.potentiates tigecycline and meropenem against resistant strains of E. coli and Helico bacter pylori (13,82,83).Therefore, the observed antibacterial potential of organoanti mony antimicrobial compound SbPh 4 ACO, especially in combination with other antibiotics at concentrations showing no (below the 30% threshold) cytotoxic effects, paves new ways in the development and future applications of metal-based antimicro bial compounds to combat the emerging bacterial resistance.The cytotoxicity of SbPh 4 ACO, although limiting its application as an antimicrobial, may generate novel opportunities in anticancer treatments.In agreement, previous reports have shown that antimony-based compounds such as C 12 H 20 N 2 O 4 SbCl and C 14 H 24 N 2 O 4 SbCl have anticancer properties, exhibiting cytotoxicity in the cancer cell line A549 (84).Future work will address both the antimicrobial and cytotoxic effects of these compounds in an in vivo animal model.

Conclusions
The present study confirmed the antibacterial activity of three tetraphenyl cyanoxi mates against Gram-negative or Gram-positive bacteria, P. aeruginosa, E. coli, and S. aureus.Among them, SbPH 4 ACO demonstrated bactericidal activity against all three pathogens tested.Exposure to SbPH 4 ACO causes membrane damage and increases membrane permeability.The combinatory treatments with SbPH 4 ACO and two clinically used antibiotics, polymyxin B and cefoxitin against P. aeruginosa and S. aureus, respec tively, showed synergistic impact.Furthermore, at the synergistic MIC concentration 12.5 µg/mL, SbPh 4 ACO showed no (below the 30% threshold) cytotoxicity against the mammalian cell lines HeLa, McCoy, and A549.The results indicate that SbPh 4 ACO holds promise as an effective treatment alone or in combination with antibiotics against resistant bacterial infections caused by important human pathogens such as P. aerugi nosa, E. coli, and S. aureus.

Media and antimicrobial compounds
Low-salt Luria-Bertani (LB) medium was used for antimicrobial susceptibility screening.For susceptibility testing and determination of minimum inhibitory concentrations for selected antimicrobial compounds, CA-MHB (Sigma-Aldrich, catalog no.90922) was used as per CLSI standards (85).

Bacterial strains
Three bacterial pathogens used include Shiga-toxin producing E. coli strain S17(O113:H4) isolated from a chick liver with septicemia (86), respiratory methicillin-resistant S. aureus strain NRS70 (87), and lab-adapted burn wound P. aeruginosa isolate PAO1 (88).All strains were maintained in 10% skim milk at −80°C.Before each experiment, the bacterial strains were inoculated onto LB agar from frozen stocks and grown overnight at 37°C from which isolated colonies were picked for precultures.

Synthesis and characterization of Sb-based antimicrobial compound
Preparation of organometallic compounds used in our investigation is shown in Fig. 6 where we used the heterogeneous metathesis reaction between the solid Silver(I) salt of the 2-oximino-2-cyano-acetamide (cyanoxime ACO) and solution of the tetraphenylanti mony(V) bromide.The cyanoxime was obtained according to our previous work (35,89), while the latter antimony source was prepared according to published procedures (90,91).Fine powder of silver(I) bromide can't be easily filtered, and we applied the centrifugation procedure to obtain clear and colorless solution of target SbPh 4 (ACO) over pellet of AgBr as shown in Fig. S2.The solution was carefully transferred in a small beaker and was placed in a vacuum desiccator containing a beaker with paraffin turnings Further characterization of SbPh 4 (ACO) included the 13 C{ 1 H} NMR spectroscopy and the X-ray single crystal analysis.Details of crystal and refinement data as well as the most important bands (Å) and valence angles in the structure can be viewed in Tables S1 and  S2 while brief description of the structure is given in Fig. 1B; Fig. S3.

Time-kill assays
Cultures of P. aeruginosa PAO1, E. coli S17, and S. aureus NRS70 grown in CA-MHB for 12 h shaking at 200 rpm, 37°C, were normalized to OD 600nm of 0.3 to be inoculated into 2 mL of fresh CA-MHB at 1% inoculum size.Thus, inoculated main cultures were grown under the same conditions for 5 h until the OD 600nm of 0.4-0.6 and then normalized to OD 0.1 (PAO1), 0.05 (S17), and 0.05 (NRS70) to achieve 1.05 × 10 7 CFU/mL.Then, 25 µL of the normalized cells was inoculated into a 24-well clear plate carrying 500 µL CA-MHB, and this dilution provides a cell density of 5 × 10 5 CFU/mL, recommended by CLSI for time-kill assays (92).CA-MHB is supplemented with SbPh 4 ACO in the concentrations ranging from 0.25× MIC to 4× MIC, corresponding to each strain.The control wells contained CA-MHB with no antimicrobial.The bacterial cultures were incubated at 37°C shaking at 200 rpm, and 10-µL aliquots were collected at time points 0, 1, 3, and 6 h post-SbPh 4 ACO treatment.The aliquots were serially diluted 10 −1 -10 −8 in sterile 0.9% NaCl, and 5 µL of each serial dilution was plated on LB agar for CFU count.For data representation, a CFU value of 0 was replaced with 1 to allow use of the logarithmic scale.Each experiment was based on at least three replicates and repeated at least once.The reported CFU values represent the mean value along with standard error.The MBC value was determined as the concentration of the antimicrobial that eliminates at least 90% of the bacterial population within 6 h upon exposure, which is equivalent to 99% killing of the inoculum (3 log 10 -fold decrease in colony-forming units) at 24 h as established in references (43,44,93).15 min each.The last step was repeated two more times.Cells were then washed three times in propylene oxide for 15 min each wash, and the final pellet was embedded into Embed 812 (Electron Microscopy Sciences).Ultra-thin sections were prepared using Leica EM UC6 ultramicrotome equipped with a DiATOME diamond knife and contrasted with 2.5% aqueous uranyl acetate and 3% lead citrate Reynold's stain.The sections were imaged using a JEOL JEM-2100 Transmission Electron Microscope (JEOL, Tokyo, Japan) at the OSU Microscopy Facility.

Inner and outer membrane permeability assays
The permeability of outer and inner membranes of bacterial cells was assessed using fluorescent probes N-phenyl-1-naphthylamine (NPN) and propidium iodide (PI), respectively, as previously described, with modifications (52).Briefly, bacterial cultures grown in CA-MHB for 12 h shaking at 200 rpm at 37°C were normalized to OD 600 nm 0.3 and used to inoculate three 5 mL of CA-MHB at a 1% inoculum size.These cultures were grown for 5 h under the same conditions and split into two tubes: one treated with 0.5× MIC concentration of SbPh 4 ACO and the other (control) with an equal volume of DMSO for 1 h.After 1 h of treatment, cells were collected by centrifugation at 2,012 g, washed with 5 mM HEPES (pH 7.2) buffer supplemented with 5 mM glucose, and normalized to OD 600 nm 0.5.Then, 100 µL of thus normalized cells was added to a black 96-well plate, to which NPN and PI were added to a final concentration of 10 µM and 5 µM, respectively.As a positive control, to a subset of DMSO-control cells, polymyxin B was added to a final concentration of 5 µg/mL.Buffer with NPN/ PI alone served as the blank controls.The final volume of each well was 200 µL.After mixing the contents, the OD 600 nm and fluorescence at 350 nm (excitation)/420 nm (emission) for NPN and 535 nm (excitation) /617 nm (emission) for PI were monitored for 30 min every 5 min by using a Biotek Synergy Mx plate reader.Each experiment was based on at least three biological replicates and was repeated at least three times.

Cytotoxicity to mammalian cells
To determine the cytotoxicity of the SbPh 4 ACO compound toward mammalian cells, each cell line (HeLa, McCoy, or A549) (ATCC, Manassas, VA) was grown in the appropriate cell culture media (Eagle's Minimum Essential Medium + 10% fetal bovine serum (FBS) for HeLa and McCoy, F-12K medium +10% FBS for A549) and incubated with cell culture media alone or with each compound diluted to a range of concentrations from 12.5 to 200 µg/mL for 24 h at 37 °C, 5% CO 2 .The Cyquant LDH Cytotoxicity Assay Kit (Thermo Fisher) protocol was followed according to the manufacturer's instructions to determine the percentage cytotoxicity of the compound for each cell line following the incubation period.

Statistical analysis and graphics
Two-tailed paired Student's t-test was performed on GraphPad Prism version 10.0.0 for Windows (GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com),to determine statistical significance.The graphics were created in BioRender (BioRen der.com).

FIG 1
FIG 1 The structure of organoantimony tetraphenyl cyanoximate compounds.(A) The compounds comprise of five ligands (indicated in the green box), attached to a tetraphenyl backbone (indicated in the blue box) and an antimony (created in BioRender).The focus of this study, SbPh 4 ACO, is highlighted in red.Created in BioRender.com.(B) Molecular structure of Sb(Ph) 4 (ACO) with numbering of principal atoms only and H-atoms being omitted for clarity.Thermal

FIG 2
FIG 2 Time-kill assay of SbPh 4 ACO on P. aeruginosa PAO1 (A), E. coli S17 (B), and S. aureus NRS70 (C).Each bacterial culture was normalized to a final concentration of 5 × 10 5 CFU/mL and exposed to different concentrations of SbPh 4 ACO; 0.5× (blue), 1× (orange), 2× (green), and 4× (yellow) of their respective MIC values.The solvent control for each strain is indicated in black.The cultures were incubated at 37°C shaking at 200 rpm for 6 h, and aliquots collected at different timepoints were used to determine the colony count.CFU/mL values represent an average of three replicates with standard error of mean (SEM).Each experiment was repeated at least once for reproducibility.

FIG 3
FIG 3 Ultra-structural changes in P. aeruginosa PAO1 (i), E. coli S17 (ii), and S. aureus NRS70 (iii) upon treatment with SbPh 4 ACO.Bacterial cultures were treated with SbPh 4 ACO at their MIC concentration for 1 h, pelleted, and subjected to thin-layer TEM imaging.In the DMSO-treated control (panel A) after 1 h, cells have uniform cytoplasmic density and intact cell membranes.After being treated with 1xMIC of SbPh 4 ACO (panel B) for 1 h, cells showed signs of membrane detaching from the cytoplasm.Aberrant membrane disruptions visible upon treatment are indicated by black arrows.In S. aureus, the apparent stalling of cell division is indicated by a blue arrow.The level of magnification and the scale bar are represented on the bottom of each image.At least four fields were examined per sample for each condition.

FIG 4
FIG4 The inner (A) and outer (B) membrane permeability of P. aeruginosa (i), E. coli (ii), and S. aureus (iii) treated with DMSO (gray, control) and SbPh 4 ACO (colored) determined by PI and NPN uptake assays, respectively.Bacterial cultures grown for 12 h were inoculated at 1:100 into main cultures in MHB.These cultures were treated with 0.5× MIC concentration of SbPh 4 ACO for 1 h and were harvested and subjected to permeability assays using PI and NPN to determine the inner (shown in blue) and outer (shown in green) membrane permeability.The fluorescence levels of PI and NPN were measured at 535/617 nm and 350/420 nm, respectively, and were normalized by optical density at 600 nm (OD 600nm ).The values represent an average of at least three replicates along with the SEM.*P value ≤ 0.05, **P value ≤ 0.01, and ***P value ≤ 0.001.

FIG 5
FIG 5 Cytotoxicity of SbPh 4 ACO compound toward three mammalian cell lines.Cytotoxicity was performed using HeLa (A), McCoy (B), and A549 (C) cells incubated (24 h, 37°C, 5% CO 2 ) in cell culture media along with SbPh 4 ACO at MIC concentrations identified in combination with polymyxin-B and cefoxitin as determined by the checkerboard assay (12.5 µg/mL, 25 µg/mL, and 50 µg/mL, shown in yellow, blue, and gray, respectively).Percent cytotoxicity was determined per the manufacturer's instructions.Each experiment was conducted in triplicate wells, and the data shown are the means ± SEM of the two independent experiments for each cell line.The compound exhibited low cytotoxicity (<30%) at the 12.5 µg/mL concentration in all cell lines.
to absorb the solvent (CH 3 CN) and after ~1-week colorless plates and blocks of the SbPh 4 (ACO) compound begin to form at the bottom.The compound was harvested, air-dried, and packed into a vial for storage and further characterization and studies.The composition of the SbPh 4 (ACO) was established by elemental analysis on C, H, and N contents by the combustion method with the help of Atlantic Microlab (Norcross, GA; USA).The % composition of each element in C 27 H 22 N 3 O 2 Sb is presented as theoreti cally calculated (experimentally detected): C -59.81 (58.39),H -4.09 (4.16), N -7.75 (7.29).SbPh 4 (ACO) melts at 199.5°C and decomposes rapidly after 235°C.The vibrational spectrum of the compound in KBr comprises of the following bands (cm −1 ): ν(NH 2 ) -3,439, 3,366; ν(C≡N) -2,222; ν(C = O, amide) -1,679; ν(C = N, oxime) -1,478; ν(N-O, oxime) -1,086; and ρ(NH 2 , amide) -1,580.The compound is well soluble in common organic solvents such as acetone, acetonitrile, alcohols, DMSO, and DMF but is not soluble in hydrocarbons and water.

FIG 6
FIG6 The most convenient route to target organoantimony(V) cyanoximate.

TABLE 1
Minimum inhibitory concentrations of Sb-based antimicrobials and ligand controls a

TABLE 2
MBC of SbPh 4 ACO against P. aeruginosa PAO1, E. coli S17, and S. aureus NRS70 a The MIC and MBC values represent the average of at least three replicates.MBC/MIC ratios ≤ 4 were considered as bactericidal. a

TABLE 3
FIC index of various antimicrobial agents in combination with SbPh 4 ACO against P. aeruginosa PAO1, E. coli S17, and S. aureus NRS70 as determined by broth dilution and checkerboard assays a