Elucidating the Antimicrobial Activity, Virulence, and Resistance Mechanisms of Pentabromophenol on Staphylococcus aureus

Elucidating the Antimicrobial Activity, Virulence, and Resistance Mechanisms of Pentabromophenol on Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Elucidating the Antimicrobial Activity, Virulence, and Resistance Mechanisms of Pentabromophenol on Staphylococcus aureus Olanrewaju Rauf Olalekan, Minhwi Sim, Boya Bharath Reddy, Yong-Guy Kim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7138037/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Staphylococcus aureus is a common pathogen that readily acquires antibiotic resistance and often forms biofilm, further reducing drug susceptibility. In this study, we found pentabromophenol (PBP) as an antibacterial agent with low resistance against S. aureus . PBP was identified and selected for further evaluation. Its MIC is lower than antibiotics ciprofloxacin (1 µg/mL) and tetracycline (2 µg/mL). Also, PBP dose-dependently inhibited S. aureus biofilm formation. At MIC, PBP significantly reduced bacterial growth and decreased toxin (hemolysin) production. Quantitative RT-PCR analysis revealed that PBP treatment at sub-inhibitory concentration downregulated the expression of toxin production and stress response ( hla , sigB, sarA , and psm-α ), and the two-component regulators responsible for autolysis and antibiotic resistance in S. aureus ( arlR and arlS ). PBP exposure decreased metabolic activity and increased NPN uptake, thereby decreasing cellular respiration and energy metabolism. This results in the disruption of membrane homeostasis, by proxy inhibition of the efflux system. PBP did not exhibit notable drug resistance (4-fold) for 30 passages in contrast to ciprofloxacin, with over a 1000-fold change in MIC. PBP and vancomycin combination also exhibited synergistic antimicrobial activity against S. aureus . PBP was non-toxic to HepG2 liver cells and Caenorhabditis elegans at concentrations up to 10 µg/mL (20 × MIC). These findings position PBP as a promising antimicrobial compound to combat antimicrobial resistance and biofilm-related infections owing to PBP’s high antimicrobial potency, low toxicity, and diminished propensity to develop resistance. Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Microbiology antimicrobial antibiofilm halogenation pentabromophenol Staphylococcus aureus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1.0 Introduction Antimicrobial resistance (AMR) poses a significant challenge for treating infectious diseases. In 2019, bacterial AMR was associated with an estimated 4.95 million deaths worldwide, highlighting the need for new treatments. Staphylococcus aureus is a common pathogen that causes a spectrum of illnesses from minor skin and soft-tissue infections to severe pneumonia and sepsis. 1 Many S. aureus strains, including methicillin-resistant S. aureus and vancomycin-resistant (MRSA and VRSA), are resistant to multiple antibiotics. MRSA infections tend to result in higher mortality, longer hospital stays, and increased healthcare costs compared to infections by drug-sensitive strains 2 , 3 . Moreover, S. aureus can evade host immune defenses; for example, it produces virulence factors that help it survive neutrophil attacks. These features promote high prevalence, resistance, and virulence, making S. aureus a particularly important target for new antimicrobial strategies 4 . Biofilm formation drastically heightens S. aureus ’ tolerance to antibiotics and host immune responses, often leading to chronic, relapsing infections on indwelling medical devices and host tissues 5 , 6 . Apart from reinfections, antimicrobial resistance due to S. aureus also leads to increased spending through prolonged hospital stays, complex treatments, and increased mortality 7 . Additionally, biofilm-grown bacteria are notoriously difficult to eradicate because the matrix limits antibiotic penetration, and cells within have altered physiology, enabling them to withstand higher drug concentrations. Studies reported cells in biofilm with 10-1000-fold increased tolerance compared to planktonic cells, emphasizing the need for the least explored antimicrobial compounds capable of targeting both planktonic cells and biofilms 8 – 10 . These trends underscore an urgent need for new antimicrobial strategies to combat biofilm-associated resistance, effectively disrupt and curtail S. aureus pathogenicity 5 , thereby reducing the overall impact of antimicrobial resistance globally. Recent studies have highlighted the potential of natural and synthetic small molecules in this regard, showing that it is possible to inhibit S. aureus biofilms, reduce virulence, and ameliorate persistence associated with re-infections 11 . While halogenation has long been used to enhance the performance of antimicrobial agents, its inherent ability in antiseptics, especially for promoting antimicrobial activity while reducing resistance, has not been fully elucidated because of toxicity concerns. Many modern antibiotics and antiseptics contain halogens, underscoring the general value of halogenation in drug design 12 . Halogen atoms like chlorine, bromine, or iodine enhance antimicrobial potency by increasing membrane permeability, reactivity, and metabolic stability 12 . Phenolic compounds are a well-known class of antiseptics and disinfectants, and chemical modifications to phenols have long been used to tune their antimicrobial properties. 13 Small antibacterial molecules have been reported to disrupt bacterial membranes and virulence factor production, and introducing halogen substituents can further boost these effects. 14 In particular, halogenated phenolic compounds have attracted interest for their antibacterial and antibiofilm activities owing to enhanced hydrophobicity and reactivity, which facilitate biofilm penetration and membrane disruption 12 . For example, in our previous study, 2,4,6-triiodophenol (2,4,6-TIP) 15 , inhibited S. aureus biofilm formation at 5 µg/mL, reduced hemolytic and proteolytic activity, and suppressed RNAIII expression. It showed lower in vivo toxicity than phenol, indicating that halogenation can enhance efficacy while minimizing toxicity. These findings illustrated how modifying phenolic compounds with halogens can yield candidates that effectively target biofilms and virulence. Such compounds could support next-generation therapies that suppress resistance evolution in S. aureus strains. Consequently, in this study, we report the antimicrobial and antivirulence potential of multiple halogenated phenols, particularly pentabromophenol (PBP), which exhibited an MIC of 0.5 µg/mL against S. aureus . Notably, we focused on identifying compounds with potent activity and minimal toxicity. PBP was screened alongside selected analogs at concentrations of 2–50 µg/mL and evaluated for biofilm inhibition, cell viability, and safety in HepG2 cells, C . elegans , and radish seed ( Raphanus sativus ) models. We elucidated the mechanism of action via membrane permeability, creating an imbalance in gradient ions, efflux systems inhibition, metabolic impairment, and gene expression changes via qRT-PCR. The resistance passage profile and combination strategies with reference antibiotics were also studied, while quantitative structure–activity relationship (QSAR) modelling identified structure-activity trends. These results may position PBP as a promising, low-toxicity candidate, worthy of further consideration for tackling antimicrobial resistance. 2.0 Materials and Methods 2.1 Microbial Strains, Chemical Compounds, and Culture Conditions Methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 6538), methicillin-resistant S. aureus (MRSA MW2), and Staphylococcus epidermidis (ATCC 14990) were used in this study. MSSA ATCC 6538 and S . epidermidis cultures were incubated in Luria–Bertani (LB) broth, while MRSA strains (MW2 and ATCC 33591) were cultivated in LB supplemented with 0.2% glucose at 37°C. All test compounds (Fig. 1 ), along with crystal violet and antibiotics, including vancomycin, ciprofloxacin, gentamicin, and tetracycline, were bought from Sigma-Aldrich (St. Louis, MO, USA) or Combi-Blocks Inc. (San Diego, CA, USA). Compounds were dissolved in dimethyl sulfoxide (DMSO), and 0.1% (v/v) DMSO was used as a control. This concentration of DMSO did not affect bacterial cell growth or biofilm formation. 2.2 Biofilm Quantification using Crystal-Violet Assay Biofilm formation was assessed using a crystal violet (CV) staining assay in 96-well plates as previously described 16 . Bacterial cultures were treated with or without halogenated phenols, ciprofloxacin, and tetracycline (0–50 µg/mL), dispensed into the 96-well plates, and incubated for 24 h at 37°C without shaking. Thereafter, planktonic cells were removed by washing the plates thrice with distilled water, dried briefly, and stained with 0.1% CV (300 µL) for 20 min. Excess dye was removed with three washes. Then, the CV-stained cells were irrigated with 300 µL of 95% ethanol. OD570 absorbance measurements were taken using a Multiskan plate reader, shaking vigorously (Thermo Fisher Scientific, Waltham, MA, USA). Results were obtained from three independent experiments; each was performed in six replicates. 2.3 Antimicrobial Susceptibility Testing by MIC The minimum inhibitory concentrations (MICs) of active halogenated phenols and reference antibiotics (ciprofloxacin and tetracycline) were determined using the broth microdilution method, following CLSI guidelines as previously reported 17 . Briefly, diluted S. aureus overnight culture (~ 10 7 CFU/mL) in the presence or absence of the compounds was aliquoted into 96-well plates and kept at 37°C under static conditions for 24 h. After incubation, the lowest concentration with no visible bacterial growth was established as the MIC and recorded. 2.4 Bacterial Growth Kinetics and Bactericidal Activity To assess impact on bacterial growth kinetics, 300 µL of diluted cultures in LB medium (1:100) with or without PBP (0, 0.1, 0.2, 0.5, 1, 10, and 100 µg/mL), two reference antibiotics, ciprofloxacin and tetracycline (1 and 10 µg/mL) was aliquoted into 96-well plates and incubated at 37 ℃ for 24 h without shaking. The growth (OD600) was measured every 4 h using a Multiskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA). For bactericidal activity, 2 mL of treated or untreated cultures with PBP, ciprofloxacin, and tetracycline at (0-100 µg/mL) in 14 mL tubes were incubated at 37 ℃ and 250 rpm shaking conditions. At varying time points (0, 6, 12, and 24 h), 100 µL of the samples were taken, diluted serially, plated on LB agar, and incubated for 24 h at 37 ℃. The colony-forming units (CFU) were counted and plotted as log10 values 8 . 2.5 Scanning Electron Microscopy (SEM) Cell morphology induced by PBP and reference antibiotic treatments was visualized by biofilms developed on sterile nylon membranes (0.3 × 0.3 cm, Merck Millipore, USA). The sterile membranes were placed into 96-well plates containing S. aureus cells treated with or without PBP, ciprofloxacin, and tetracycline (0–10 µg/mL), and incubated at 37°C for 7 h. Biofilm-adhered membrane surfaces were fixed with 2% formaldehyde and 2.5% glutaraldehyde for 12 h. Dehydrated through a graded ethanol series (50%, 70%, 90%, 95%, or 99%, 20 min each), coated with platinum, and imaged using a Hitachi S-4800 (Tokyo, Japan) scanning electron microscope. 2.6 Assessment of Hemolytic Activity Hemolytic activity was quantified using fresh sheep red blood cells (MBcell, Seoul, Korea) as previously described 18 . 2 mL of fresh LB broth diluted with S. aureus ATCC 6538 cells (~ 10 7 CFU/mL) were cultured with PBP, ciprofloxacin, and tetracycline (0.1, 0.2, 0.5, 0.7, and 1 µg/mL) for 24 h at 250 rpm shaking conditions. Red blood cells were harvested from fresh sheep blood by centrifugation at 3,000 × g for 2 min, washed three times with PBS, and resuspended to 3.3% (v/v) in PBS. Subsequently, 100 µL of treated bacterial culture was added to 1 mL of red blood cells and incubated for 4 h at 37°C for 4 h shaking at 250 rpm. After centrifugation (10,000 × g, 10 min), the supernatant absorbances were measured at 543 nm to quantify hemolysis. 2.7 Antimicrobial Impact on Cell Metabolic Activity (XTT Reduction Method) Cellular metabolic activity was evaluated using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) according to the manufacturer's instructions (Cell proliferation kit, Sigma-Aldrich). Biofilms were initially established in 96-well plates as described in Section 2.2 . After incubation, non-adherent and spent medium were removed by rinsing with sterile H 2 O. 100 µL of a freshly prepared mixture of XTT labeling reagent and electron-coupling reagent (phenazine methosulfate) at 50:1 (v/v) was added to each well to assay the metabolic activities. Plates were incubated in the dark at 37°C for 30 min, and the absorbance was measured by a Multiskan EX microplate reader at 490 nm. 2.8 Membrane Potential Assessment Using Reactive Oxidative Species (ROS) Assay To evaluate the mechanism of activity, the potential for reactive oxygen species (ROS) production was determined following the protocol by 19 as modified. Briefly, 25 mL of the previous overnight culture of S. aureus cells was reinoculated in a 250 mL flask and incubated for 4 h, cells were harvested in LB broth, resuspended in PBS, and adjusted to (~ 10 7 CFU/mL)(OD 600 ~ 0.5). Treatment was performed with or without PBP, ciprofloxacin, and tetracycline (0, 1, 10, and 100 µg/mL). A stock solution of 35% v/v H 2 O 2 was diluted to achieve 500 or 1000 µg/mL, and the MIC of H 2 O 2 against S. aureus was established as 1000 µg/mL 20 , as described in section 2.3 . The setup was incubated for 3 h at 250 rpm and 37°C, and then, 2′,7′-dichlorofluorescein diacetate (DCFCDA) (10 mM, 1:2) was added to the cell suspensions and incubated in the dark for 30 min at 25°C. Fluorescence was measured using a spectrophotometer F-7000 (Hitachi, Tokyo, Tokyo, Japan) equipped with a xenon arc lamp. The excitation wavelength was at 485 nm, and emission intensities were recorded at 535 nm. Untreated and H2O2-treated (500 or 1000 µg/mL) samples were analyzed under the same conditions and served as negative and positive controls, respectively. 2.9 Membrane Permeability Measurement via NPN Uptake Assay The outer membrane permeability of S. aureus cells was determined using an N-phenyl-1-naphthylamine (NPN) uptake assay as previously described 21 . Briefly, overnight cultures were reinoculated into fresh LB broth (1:100) and grown to mid-log phase (OD600 ~ 0.05). Bacterial cells (∼10 7 CFU/mL were harvested, washed, and resuspended in PBS. A total of 1 mL aliquots were treated with or without PBP, ciprofloxacin, and tetracycline (0, 0.5, and 1 µg/mL). This was incubated at 37°C, with shaking (250 rpm) for 3 h. After incubation, cell suspensions were centrifuged (7000 rpm, 10 min), washed, and resuspended in 2 mL PBS. NPN (150 µL 10 mM in EtOH 7.5%) was added to each suspension, samples were incubated at room temperature in the dark for 10 min by placing them in a box. Benzalkonium chloride (0.5, 1, and 10 µg/mL) serves as the positive control. Fluorescence was measured at 350 nm excitation and 450 nm emission using a Hitachi F-7000 (Tokyo, Japan) spectrofluorometer. 2.10 Bacterial Resistance Development Monitoring Changes in the MIC values were monitored to determine the potential of S. aureus to develop resistance against PBP and reference antibiotics. The procedure follows 22 as modified. The initial MICs of the compounds were confirmed using broth dilution in 14 mL tubes. Subsequently, the bacterium (1:100) was cocultured with a sub-inhibitory concentration (sub-MICs) and incubated at 37°C for 24 h. Post incubation, overnight cultures that received sub-MIC were diluted and used as the starting culture for the next passage. After each passage, changes in MIC of the compounds against the S. aureus were observed. The process was repeated for 30 passages, and the graph of MIC values against the days of passage was generated to determine resistance development over time. The fold change and cumulative fold change for each compound were calculated as: 2.11 Antibiotic Combinatorial Strategies Using Checkerboard Assay The in vitro interactions between PBP and the reference antibiotics (ciprofloxacin, tetracycline, vancomycin, and gentamicin) were evaluated following procedures as earlier reported 23 . A checkerboard assay containing two-fold dilutions of pentabromophenol (Drug A) in the vertical wells and reference antibiotics (Drug B) in the horizontal wells. 100 µL of overnight S. aureus culture diluted in 0.9% NaCl (∼10 7 CFU/mL) was added to each well and incubated for 24 h at 37°C. Planktonic growth was assessed visually and by OD600 readings, while the biofilm was quantified as in section 2.2 . The percentage of growth in each well is calculated as: $$\:\frac{OD\:drug\:combination\:well-OD\:background\:}{OD\:drug\:free\:well-OD\:background}\times\:\:100$$ The new MIC and minimum biofilm inhibitory concentration (MBIC) were recorded as the lowest concentration with no visible planktonic growth or biofilm, respectively. The fractional inhibitory concentration (FIC) for each agent was calculated by identifying the effective combinations, based on the first well in each row showing no visible growth, using the formula: Drug A FIC: \(\:\frac{drug\:A\:MIC\:in\:combination}{drug\:A\:MIC\:alone}\) and Drug FIC B: \(\:\frac{drug\:B\:MIC\:in\:combination}{drug\:B\:MIC\:alone}\) FIC interpretations were as reported by Bellio 24 . The FIC index (FICI) = Drug A FIC + Drug B FIC was interpreted as FICI ≤ 0.5 (Synergy), 0.5 < FICI ≤ 1 (Additive), 1 < FICI < 2 (Indifferent), and FICI ≥ 2 (Antagonistic). The checkboard representative images were generated using conditional formatting in Microsoft Excel 365. 2.12 Transcriptional Evidence of Antimicrobial Mechanism via qRT-PCR We investigated the transcriptional effect of PBP on S. aureus using quantitative real-time PCR (qRT-PCR). Briefly, 25 mL of diluted overnight culture (∼10 7 CFU/mL) was treated with or without the active compound (0.25 µg/mL) for 6 h. 700 µL RNAlater® (Ambion, TX, USA) was added to each culture before centrifugation to stabilize RNA, rinsed with PBS after, and Qiagen RNeasy Mini Kits (Valencia, CA, USA) were used to extract total RNA according to the manufacturer's instructions. cDNA and amplification were performed using the ABI StepOne Real-Time PCR system (Applied Biosystems, CA, USA) in the presence of SYBR℗ Green Master Mix (Thermo Fisher, Warrington, UK), appropriate primers, and ddH 2 O. Gene-specific primers (Supplementary Table S2) targeted pathways related to biofilm suppression, QS, cytotoxicity, stress response, and antibiotic resistance. 16 S rRNA served as the housekeeping gene, and relative gene expression was calculated using the 2 − ΔΔCt method 25 . 2.13 Chemical Toxicity Using Human, Nematode, Seed Models, and ADMET Profiling Human hepatocellular carcinoma HepG2 cells (Korean Cell Line bank) were cultured in DMEM (Thermo Scientific) containing 10% FBS (Gendepot), dispensed into a 96-well plate, and incubated in a 5% CO 2 incubator at 37 ℃ for 24 h. After incubation, the spent medium was removed, fresh medium treated with PBP and 2,4,6-TIP (None, 10, 20, 50, 100, and 200 µg/mL) was added, and incubated for 24 h more under the same conditions. For the MTT assay, the spent medium was removed using a vacuum. MTT (0.5 µg/mL) was mixed in a new medium at a 1:10 dilution ratio, each well received 100 µL of this mixture, and were incubated at 37 ℃ in a 5% CO 2 incubator for 4 h. Post incubation, removal of the medium followed, and 100 µL of DMSO was used to treat the cells in the dark to extract formazan. Absorbance was then measured at 540 nm. The chemical toxicity of PBP and phenol was assessed using the C . elegans fer-15(b26); fem-1(hc17) strains, following previously established protocols 26 . Synchronized nematodes were carefully washed twice with M9 buffer (3 g/L KH 2 PO 4 , 6 g/L Na 2 HPO 4 , 5 g/L NaCl, 1 mM MgSO 4 ). Approximately 30–35 worms were then transferred to each well of a 96-well plate containing M9 buffer (300 mL) and varying concentrations (None, 0.5, 1, 10, 100, and 200 µg/mL) of PBP. The plates were subsequently incubated for 7 days at 25°C. Four independent cultures were utilized. Nematodes' viability was assessed based on their response to LED light for 30 s in a Digital Cell Imaging System ( iRiS™ Logos BioSystems, South Korea). In the seed model, initially, radish plant ( Raphanus sativus) seeds were soaked in sterile dH 2 O for 16 h and rinsed with dH 2 O three times. Sterilized subsequently by soaking in 95% ethanol, 3% Sodium Hypochlorite at 15 min intervals, and rinsed with sterile dH 2 O three more times. 9–10 seeds per plate were placed on soft agar containing Murashige and Skoog (0.86 g/L MS and 0.7% agar), supplemented with PBP and phenol (None, 1, 10, 100, and 200 µg/mL). The plates were incubated at 25°C for 7 days. Germination rates and seedling lengths were recorded daily. Four independent cultures were utilized for this experiment. ADMET (absorption, distribution, metabolism, excretion, and toxicity) and drug-likeness parameters of phenol and selected multi-halogenated derivatives (Supplementary Table S6), including PBP and 2,4,6-TIP derivatives, were investigated in silico using PreADMET ( https://www.molinspiration.com/ ), Protox ( https://tox.charite.de/protox3/ ) GUSAR ( http://www.way2drug.com/gusar/ ), and SwissADME ( http://www.swissadme.ch/index ), assessed between 21–24 December 2024. 2.14 Quantitative Structural Activity Relationship (3D-QSAR) An atom-based 3D-QSAR model was developed for phenol and 17 halogenated phenol derivatives (Supplementary S5). These compounds 3D structures were downloaded from PubChem, and minimization of macromolecule energy was carried out using the LigPrep module with the OPLS3E force field. Common substructure alignment of all energy-minimized derivatives was performed using the base structure of phenol as a reference, utilizing the Ligand Alignment tool in Maestro 12.5 (Schrödinger Software Solutions, USA). MICs were converted to pMICs [-log (MIC)], and a 3D atom-based QSAR model with two partial least squares (PLS) factors was constructed using PHASE (Schrödinger Software Solutions, USA). PLS factor 2 was used for QSAR visualization and activity predictions. 2.15 Statistical Analysis All experiments were repeated thrice with two replicates unless mentioned otherwise, and data are presented as mean ± SD. SigmaPlot 14.0 was used to plot graphs. Student t-tests were used to calculate statistical significance, between untreated and treated samples denoted by asterisks (∗ p ≤ 0.05 ), and error bars represent the SD. 3.0 Results 3.1 Antibiofilm and Antibacterial Activities of Halogenated Phenols against S. aureus 3.1.1 Screening of Halogenated Phenols Against S. aureus Biofilms and Growth Twelve multi-halogenated phenols were screened at 2, 10, and 50 µg/mL concentrations to evaluate their antibiofilm activity against methicillin-susceptible S. aureus ATCC 6538. Among the tested compounds, pentabromophenol (PBP) and 2,4,6-triiodophenol (2,4,6-TIP) were the most effective, exhibiting a 91% and 97% inhibition of biofilm formation at 50 µg/mL, respectively. Other multi-halogenated derivatives, such as 2,4,6-tribromo-3-nitrophenol and 3,4,5-triiodobenzoic acid, showed 85% and 71% inhibition, respectively. In contrast, fluorinated compounds, including 2,3,4-trifluorophenol and 2,3,5,6-tetrafluorophenol, displayed minimal activity, with only 1% and 3% inhibitions, respectively. Expectedly, the addition of a bromine atom at C-4 in tetrafluorophenol (4-bromotetrafluorophenol) only slightly improved inhibition to 14%. These findings suggest that multiple bromine or iodine substitutions, particularly at the C-2 and C-6 positions of the phenol ring, may suggest more effective and enhanced antibiofilm activity than fluorinated substitutions (Fig. 1 ). In addition, the minimum inhibitory concentration (MIC) observation established that PBP exhibited the lowest inhibitory concentration among the screened halogenated phenols. PBP inhibited planktonic S. aureus growth at 0.5 µg/mL, significantly lower than other halogenated phenols, including 2,4,6-triiodophenol (2,4,6-TIP) 15 , previously reported. Several compounds, including 3,4,5-triiodobenzoic acid, 2,4,6-tribromo-3-nitrophenol, and 4-bromotetrafluorophenol, exhibited MICs of 100 µg/mL. In contrast, other compounds, particularly fluorinated analogs, had MICs ≥ 200 µg/mL while phenol showed poor activity with an MIC exceeding 1000 µg/mL (Fig. 1 ). 3.1.2 Comparison of PBP and Antibiotics Effects on S . aureus At 0.5 µg/mL, PBP inhibited biofilm formation by 70%, comparable to ciprofloxacin (MIC: 1 µg/mL, 75% inhibition), and superior to tetracycline (MIC: 2 µg/mL, 50% inhibition). In planktonic cell growth inhibition, PBP showed 85% suppression, moderately higher than ciprofloxacin and tetracycline with 70% and 29%, respectively. PBP at 0.5 µg/mL demonstrated comparable or superior performance at a lower effective dose (Fig. 2 A, B, and C). Further testing of PBP at various concentrations (0.1, 0.2, 0.5, 1, or 10 µg/mL) against other clinical strains of S. aureus , notably MRSA MW2, MRSA 33591, and S . epidermidis , showed consistent activity. In MRSA MW2, PBP inhibited cell growth formation by 78% at an MIC of 1 µg/mL and reduced biofilm formation at 10 µg/mL by 60%. Interestingly, in MRSA 33591 and S . epidermidis (MICs: 0.5 and 1 µg/mL, respectively), PBP exhibited significantly higher planktonic cell growth inhibition, with 89% and 83% inhibition in MRSA 33951 and S . epidermidis , respectively. At 10 µg/mL, it also suppressed biofilm formation by 80% and 93% in both strains (Supplementary Figure S1 A, B, and C). These results establish PBP as the most potent candidate among the screened halogenated phenols, with consistent efficacy across multiple Staphylococcus strains. It showed significant, comparable antibacterial and antibiofilm activity to ciprofloxacin and superior activity to tetracycline in both biofilm and planktonic growth inhibition and was therefore selected for further study. 3.2 Antimicrobial and Bactericidal Effects of PBP on Growth and Kinetic Studies The antimicrobial activity of pentabromophenol (PBP) on S. aureus ATCC 6538 was assessed through growth kinetics and time-kill assays. At sub-inhibitory concentrations (0.1 and 0.2 µg/mL), PBP had minimal effect on planktonic cell growth. However, at its MIC (0.5 µg/mL), PBP significantly inhibited bacterial growth, and this suppression was sustained throughout the 24 h incubation period (Fig. 2 A). In contrast, ciprofloxacin and tetracycline achieved comparable growth inhibition only at concentrations 5- to 10-fold higher than their respective MICs, highlighting the superior potency of PBP at lower doses. In time-kill assays (Figs. 2 E and 2 F), PBP demonstrated a bacteriostatic effect at 2 × MIC, yielding an approximately 2-log10 reduction in CFU after 24 h, comparable to the effect observed with tetracycline at its MIC. PBP exhibited bactericidal activity only at a higher concentration (200× MIC), achieving a > 4 log10 reduction. Ciprofloxacin demonstrated bactericidal effects at just 0.5× MIC, resulting in a 3-log10 reduction compared to the untreated control. These findings align with established criteria distinguishing bacteriostatic agents, such as tetracycline, which typically induce ≤ 2 log10 reductions, from bactericidal agents like ciprofloxacin, which achieve ≥ 3 log10 reductions under comparable conditions. Accordingly, PBP can be classified primarily as a bacteriostatic compound at minimum dosage, with dose-dependent bactericidal potential at elevated concentrations 27 , 28 . 3.3 Cell Density and Structural Disruption Assessed by Live Imaging and SEM Live-cell imaging and scanning electron microscopy (SEM) were employed to assess the effects of PBP and reference antibiotics on S. aureus biofilm formation, cell density, and colony morphology. In the untreated control, 3D color-coded reconstructions revealed the dense biofilm architecture characteristic of S. aureus (Fig. 3 A). PBP treatment at 0.2 µg/m resulted in a slight decrease in biofilm mass, and marked reductions were observed at 0.5 and 1 µg/mL, indicating dose-dependent antibiofilm activity. In comparison, ciprofloxacin and tetracycline at equivalent concentrations produced only minimal reductions (Fig. 3 B). SEM images corroborated these results, revealing a significant reduction of bacterial cell density following PBP treatment at 0.5 and 1 µg/mL, without evident structural disruption of cell morphology, consistent with a bacteriostatic mode of action (Fig. 3 C). Tetracycline-treated cells exhibited modest reductions in cell density, while ciprofloxacin-treated samples displayed swollen and partially lysed cells at 1 µg/mL, indicating bactericidal activity (Fig. 3 D). These microscopy-based observations support the distinct modes of action observed in growth kinetics and time kill assays for PBP and the reference antibiotics. 3.4 Effect of PBP on Virulence and Possible Mechanisms S. aureus secretes α-hemolysin ( hla ), a pore-forming cytotoxin that lyses red blood cells and contributes to immune evasion. To evaluate the antivirulence effect of PBP, we assessed its ability to suppress hemolytic activity. At sub-MICs (0.1 and 0.2 µg/mL), PBP had minimal effect, whereas at 0.5 µg/mL, it significantly reduced hemolysis by 89%, and to near-baseline levels at 1 µg/mL with 98% inhibition. In contrast, ciprofloxacin and tetracycline showed limited inhibition at 0.5 µg/mL (8% and 2%, respectively), with comparable effects at 1 µg/mL (84% and 94%). These findings demonstrate PBP’s dose-dependent antivirulence effect, with greater efficacy at lower concentrations than reference antibiotics (Fig. 4 A). Bacterial metabolic viability was assessed by XTT reduction, indicating NAD(P)H-dependent dehydrogenase activity. At 0.5 µg/mL, PBP reduced metabolic activity by 50%, compared to 29% and 39% inhibition by ciprofloxacin and tetracycline, respectively, while at 1 µg/mL, all PBP and reference antibiotics showed comparable effects (PBP: 70%, ciprofloxacin: 69%, tetracycline: 65%) (Fig. 4 B). Reactive oxygen species (ROS) generation, a hallmark of bactericidal activity, was assessed. Ciprofloxacin induced significant fluorescence at 10 µg/mL, consistent with its known bactericidal mechanism 29 , whereas tetracycline produced negligible ROS, supporting its classification as bacteriostatic. Similarly, PBP generated minimal ROS at concentrations up to 10 µg/mL, suggesting a comparable, non-lytic mode of action (Fig. 4 C). Outer membrane permeability was assessed using an NPN uptake assay. PBP at 0.5 and 1 µg/mL induced significantly greater fluorescence than tetracycline and ciprofloxacin, comparable to benzalkonium chloride, a known membrane disruptor (Fig. 4 D). The elevated NPN fluorescence indicates increased outer membrane permeability, suggesting that PBP may impair efflux or destabilize membrane integrity without causing lysis, consistent with its bacteriostatic activity 30 , 31 . 3.5 Combination Efficacy of PBP with Antibiotics and Drug-Resistance Development Antimicrobial combinations are known to enhance efficacy and delay resistance development in Staphylococcus infections 32 . The synergistic potential of PBP was evaluated in combination with reference antibiotics, vancomycin (VAN), ciprofloxacin (CIP), tetracycline (TET), and gentamicin (GEN) using checkerboard assays 23 . PBP-VAN showed strong synergy in planktonic growth inhibition (FICI: 0.31), enhancing potency 16-fold and lowering the MIC from 0.5 to 0.031 µg/mL (Fig. 5 A, Supplementary Table S1 ). PBP combined with ciprofloxacin, tetracycline, and gentamicin, displayed additive interactions, and reduced MICs to 0.125 µg/mL (Fig. 5 B-D, Supplementary Table S1 ). Similar interactions were observed in biofilm inhibition, with PBP-vancomycin maintaining synergistic interactions while the rest of the PBP-combinations showed an additive pattern (Supplementary Figure S2). A serial passage assay was conducted over 30 days to assess the resistance-suppressing potential of PBP. PBP showed only a modest 4-fold MIC increase, similar to tetracycline, indicating a stable resistance profile (Fig. 6 A). In contrast, ciprofloxacin exhibited over a 1000-fold MIC increase within 13 passages. As the MIC exceeded 1000 µg/mL, a fold change of 1000 33 , and ciprofloxacin became increasingly insoluble at the required concentrations, the assay was discontinued on day 13. This sharp rise reflects accelerated resistance development, likely driven by target-site mutations and efflux upregulation under drug pressure 34 , 35 . These findings highlight PBP’s ability to bypass conventional resistance pathways and enhance efficacy when combined with conventional antibiotics, particularly vancomycin, through additive or synergistic interactions. This allows a reduced effective dose while maintaining strong antimicrobial activity. 3.6 Impact of PBP on Gene Expression in S. aureus To elucidate the molecular mechanisms of PBP’s antimicrobial action, we analyzed its effects on gene expression related to biofilm formation, virulence, antibiotic resistance, and stress adaptation in S. aureus . Quantitative real-time PCR (qRT-PCR) showed that PBP at sub-inhibitory concentration (0.25 µg/mL) significantly downregulated key regulatory and effector genes. Biofilm-related genes agrA , nuc1 , and sigB , virulence factors hla , psm-α , and sarA , and resistance-associated regulators arlR and arlS were all suppressed. Notably, hla and nuc1 were reduced by 7.0- and 4.5-fold, respectively, while psm-α and sarA were reduced by 8.6- and 2.5-fold. In parallel, arlR and arlS showed transcriptional repression of 2.4- and 2.5-fold, respectively (Fig. 6 B). These findings suggest that PBP’s bacteriostatic and antivirulence effects may stem from its disruption of global regulatory circuits controlling cytotoxicity, stress tolerance, and resistance modulation. 3.7 Assessment of PBP Reveals Low Toxicity Potential Across Models and ADMET Profiling Toxicity profiling is essential for evaluating the therapeutic potential of antimicrobial candidates. In HepG2 cells, MTT assays revealed dose-dependent cytotoxicity for both pentabromophenol (PBP) and 2,4,6-triiodophenol (2,4,6-TIP), a previously reported halogenated phenol with potent antibiofilm activity against S. aureus 15 . PBP consistently showed lower cytotoxicity, maintaining 90% and 78% cell viability at 10 and 20 µg/mL, respectively, compared to 57% and 28% for TIP. At higher concentrations, PBP-treated cells exhibited nearly double the survival rates compared to 2,4,6-TIP. However, reported oral LD50 values indicate that TIP (LD50 ~ 2000 mg/kg) is substantially less toxic in rats than PBP (LD50 ~ 200 mg/kg) or phenol (LD50 ~ 317 mg/kg). These findings suggest that while PBP shows improved cellular tolerance in vitro , TIP presents a lower risk of acute systemic toxicity 36 – 38 (Fig. 7 A). In the C. elegans toxicity model, PBP demonstrated superior safety. At 5 and 10 µg/mL (10× and 20 × MIC), worm survival rates were 70% and 60%, respectively, comparable to previously reported values for 2,4,6-TIP at 10 µg/mL (Fig. 7 B). In contrast, phenol showed pronounced toxicity, with survival dropping to 25% and 16% at the same concentrations (Fig. 7 C). Similarly, in the Raphanus sativus seed germination assay, PBP treatment at 1 and 10 µg/mL resulted in 94% and 88% germination by day 7 (Supplementary Figure S3A), with normal root and shoot development (Fig. 7 D). Phenol-treated seeds showed slightly reduced germination (89% and 78%) (Supplementary Figure S3B) but with significant growth deficiencies, including yellowing of roots, shoots, and leaves (Fig. 7 E). In ADMET profiling, the parent phenol scaffold was classified as toxicity class III, indicating relatively high acute toxicity risk. Less active or inactive analogs, pentachlorophenol, pentafluorophenol, 4-chloro-2-iodophenol, and trifluorophenol, were assigned to toxicity class IV, suggesting moderate toxicity. Similarly, PBP was categorized as toxicity class IV, indicating moderate acute toxicity. Furthermore, PBP exhibited minimal aquatic toxicity in both Oryzias latipes (medaka) and Pimephales promelas (fathead minnow), outperforming phenol and inactive analogs. It also showed higher predicted blood-brain barrier (BBB) and Caco-2 permeability, improved oral bioavailability scores, and a predicted low risk of hERG inhibition, much improved than 2,4,6-TIP and phenol (Supplementary Table S3). Collectively, these profiles may support PBP’s development as a low-toxicity antimicrobial candidate. 3.8 Quantitative Structure-Activity Relationship (3D-QSAR) Understanding structural features that influence antimicrobial activity is crucial for optimizing drug candidates. QSAR models link chemical structure to biological activity, highlighting key scaffold positions that drive efficacy 39 . In this study, an atom-based 3D-QSAR model was developed for phenol and multi-halogenated derivatives. These were compounds screened for antibiofilm activity against S. aureus as in section 2.3 , and 17 of them were selected with their MICs ranging from 5 to 1000 µg/mL (Supplementary Table S4) to ascertain the optimal positions for halogen substitution on the phenol scaffold that enhance antimicrobial activity 40 – 42 . Partial Least Squares (PLS) Factor 2, highlighted in bold letters, was selected as the final model based on its superior statistics (higher R 2 and Q 2 , greater stability, and lower RMSE) (Fig. 8 A). PLS factor 2 has R^2 = 0.83 and Q^2 = 0.86, significantly higher than the thresholds of 0.6 and 0.5, respectively. These metrics and a high Pearson correlation (r ≈ 0.984) between observed and predicted activities demonstrate model reliability. The predicted pMIC and experimentally determined MIC values aligned closely with the linear regression curve, indicating relatively good model accuracy (Fig. 8 A). 3D visualization of the QSAR model was performed, wherein blue fields represent the most favorable regions, while red interaction field represent the unfavorable region. Favorability was determined based on hydrogen bond donation, electron-withdrawing effects, hydrophobicity, and non-polar characteristics. The visualization revealed that positions C-2 and C-6 were favorable for antimicrobial activity against S. aureus , whereas substitution at C-3 was unfavorable in the phenol scaffold (Fig. 8 B). 4.0 Discussion 4.1 Mechanism of Action Halogenation is a well-established medicinal chemistry strategy, widely employed for improving antimicrobial potency, membrane permeability, and metabolic resilience of small molecules 12 , 43 . Among halogenated scaffolds, brominated phenols have emerged as promising candidates, particularly against resilient pathogens like MRSA 44 . In this study, PBP demonstrates superior antimicrobial and antibiofilm efficacy, with MIC values nearly 10-fold lower and activity surpassing that of previously reported 2,4,6-triiodophenol (TIP) 15 . This enhancement reinforces the therapeutic potential of multi-brominated phenol derivatives as a robust antimicrobial agent against biofilm-associated infections. Beyond its growth inhibition, PBP exhibited significant antivirulence properties. It effectively reduced biofilm biomass and hemolytic activity, showing comparable efficacy to ciprofloxacin and tetracycline. Transcriptomic analysis revealed downregulation of key genes of virulence, including α-hemolysin ( hla ) and phenol-soluble modulins (psm-α), along with global regulators such as agrA , sarA , sigB , and the two-component system arlR and arlS , which governs autolysis and antibiotic resistance development (Figs. 4 and 6 ) 45 . These findings indicate that PBP achieves its bacteriostatic activity by impairing regulatory networks essential for growth, persistence, and pathogenesis. Such antivirulence targeting strategies are increasingly valued for their capacity to reduce pathogenicity while minimizing selective pressure for resistance. Microscopy of PBP-treated cells within biofilm was structurally intact, supporting a non-bacteriolytic mode of action. The static growth profile of PBP-treated cultures is consistent with this observation (Fig. 3 ). Increased PBP-induced uptake of the NPN probe indicates compromised membrane permeability (Fig. 4 ). A similar activity has been observed in bromophenoxyphenols that disrupt membrane-linked resistance mechanisms. These non-lytic and non-lethal perturbations likely destabilize membrane potential without full rupture, impair nutrient and ion transport, and suppress efflux systems, leading to metabolic stress and growth inhibition 31 , 44 , 46 . Additionally, PBP may impair metabolic activity more than or comparable to standard antibiotics at equivalent MICs (Fig. 4 ), reinforcing its effect on energy-dependent cellular processes 47 . Importantly, PBP generated negligible ROS levels, unlike fluoroquinolones, which induce significant ROS by targeting DNA gyrase, leading to damage and cell death 48 . This energy-based, non-lytic mechanism may enhance its safety profile and reduce resistance emergence. The multi-targeted nature of this mode of action makes single-step resistance mutations less likely 49 . 4.2 Resistance Development The resistance-limiting effect of PBP is supported by its multi-targeted mechanism of action against S. aureus . PBP compromises the cytoplasmic membrane, as indicated by increased NPN uptake, in a manner that is not easily overcome by target-specific mutations 27 , 50 . This disruption may suggest impairment of the membrane potential and ATP synthesis, reflected in reduced metabolic activity, which may hinder energy-dependent processes such as efflux and stress response activation. Additionally, qRT-PCR analysis showed downregulation of genes involved in resistance regulation, including the arlR / arlS two-component system, suggesting a link between membrane stress and transcriptional repression. These combined effects reduce the bacterial capacity for adaptive changes during serial exposure, which may account for the absence of resistance development observed in the passage assay. In contrast, ciprofloxacin rapidly induced high-level resistance 51 , consistent with the accumulation of gyrase mutations and efflux pump upregulation under fluoroquinolone pressure 52 , 53 . This resistance stability, along with PBP’s ability to repress master regulators of persistence and virulence, highlights its promise as a potential therapeutic option. 4.3 Synergistic Combinations PBP also demonstrated synergistic interaction with glycopeptides like vancomycin and additive interactions with other standard antibiotics tested (Fig. 5 ). Such combinations lower the required dosage and prevent resistance development 54 – 57 . These observations align with clinical trends favoring combination therapies to eradicate biofilm-associated infections, where monotherapies often fail due to incomplete eradication or adaptive resistance. 4.3 Toxicity In terms of safety, phenol has been reported even at low concentrations to cause significant cytotoxicity in human fibroblasts and keratinocytes, nematodes, aquatic and plant models 58 – 60 . However, this study revealed that PBP exhibited low toxicity across multiple eukaryotic models (Fig. 7 ). It was well tolerated by human liver cells (HepG2), nematodes ( C . elegans ), and radish plant seedling germination, significantly more than tri-substituted halogenated analogs like 2,4,6-TIP. PBP’s ability to achieve potent antimicrobial activity at concentrations far below toxicity thresholds supports its suitability for further development. These findings align with recent reports on marine-derived brominated phenols and synthetic derivatives like pentabromopseudilin and thiophenones, which also exhibit strong anti-MRSA activity with limited host toxicity 61 , 62 . Collectively, this study reinforces the value of targeting bacterial virulence and membrane integrity as a strategy to overcome resistance. One of the more common resistance strategies employed by S. aureus is heightened efflux pump activity and membrane impermeability. By targeting and shutting down key regulatory circuits, compromising biofilm and membrane barriers, PBP offers a promising alternative to conventional antibiotics by increasing membrane permeability and inhibiting efflux pumps. Its efficacy, low resistance development, favorable safety profile, and synergy with existing antibiotics position it as a strong candidate for future therapeutic considerations against resistant staphylococcal infections. 5.0 Conclusion Pentabromophenol exhibits exceptional antimicrobial and antibiofilm activity against S. aureus , matching or exceeding the performance of standard drugs at much lower doses. It achieves this through a multi-faceted mechanism: destabilizing membrane function, suppressing energy metabolism, and silencing key virulence regulators ( hla , psm-α , nuc1 , agrA , sarA , arlR , and arlS , etc.). Notably, PBP induces minimal resistance even after prolonged exposure, and it synergizes effectively with antibiotics like vancomycin. Its strong antibacterial effect is accompanied by low toxicity to mammalian cells and model organisms, indicating favorable and possible therapeutic consideration. Finally, PBP presents itself as a potential lead agent in the fight against antibiotic-resistant S. aureus , offering a non-traditional halogenated scaffold that effectively targets biofilms and virulence while minimizing resistance development. Declarations Contributors Olanrewaju Rauf Olalekan: Formal analysis; investigation; methodology; data curation; visualization; writing-original draft, review and editing, Jintae Lee : Conceptualization; project administration; resources; supervision; validation; visualization; writing-review and editing, Jin-Hyung Lee : Methodology; data curation; formal analysis; funding acquisition; administration; resources; supervision; validation; writing-review and editing, MinHwi Sim: Data curation , visualization, Boya Bharath Reddy: software; formal analysis; visualization, Yong-Guy Kim: formal analysis; resources, visualization. Declaration of Interests The authors have no conflicts of interest to declare. All authors have seen and agree with the contents of the manuscript. We certify that the submission is original work and is not under review at any other publication. Acknowledgements This research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (RS-2025-00513239 to J. Lee and RS-2025-00553409 to J.-H. Lee) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded .by the Ministry of Health & Welfare, Republic of Korea (RS-2024-00450423). Data Sharing Statement Data will be made available on request. Supplementary information Supplementary materials related to this article, including additional tables and primer sequences, are available online and can be accessed to support the findings of this study. References Murray, C. J. L. et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. 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Comprehensive Exploration of Bromophenol Derivatives: Promising Antibacterial Agents against SA and MRSA. ACS Omega 9, 40897–40906, doi: https://doi.org/10.1021/acsomega.4c06115 (2024). Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.pdf Onlinefloatimage2.png Graphical abstract showing a summary of the study. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7138037","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":488142365,"identity":"ca51af96-152f-4acc-8aab-b74961ba9419","order_by":0,"name":"Olanrewaju Rauf Olalekan","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Olanrewaju","middleName":"Rauf","lastName":"Olalekan","suffix":""},{"id":488142367,"identity":"f1625bdf-f469-4f9a-a03b-a3b816c5b65a","order_by":1,"name":"Minhwi Sim","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Minhwi","middleName":"","lastName":"Sim","suffix":""},{"id":488142369,"identity":"4d85fa69-7c85-43ff-a049-093e09246890","order_by":2,"name":"Boya Bharath Reddy","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Boya","middleName":"Bharath","lastName":"Reddy","suffix":""},{"id":488142371,"identity":"7f075757-d42a-41db-8ac1-cb647123a0d9","order_by":3,"name":"Yong-Guy Kim","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Yong-Guy","middleName":"","lastName":"Kim","suffix":""},{"id":488142374,"identity":"89d33b32-b77b-45ce-9334-0e679cd66975","order_by":4,"name":"Jin-Hyung Lee","email":"","orcid":"","institution":"Yeungnam University","correspondingAuthor":false,"prefix":"","firstName":"Jin-Hyung","middleName":"","lastName":"Lee","suffix":""},{"id":488142377,"identity":"552d6a36-48bb-41ca-be1c-2907e970d354","order_by":5,"name":"Jintae Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACxgbmBgaGChj3ADFa2hiBWs6QooWBDaiFsY0ULczzGxsffJx3OM/gAPPDDwxn7hHlsGbDmdsOFxscYDOWYLhRTJSWNmnebYcTNxxgMGNg+JBAlJb233/ngLSwfyNaSxszYwNICw/QlhtEaUlsluw5lp448zBPsUTCGSK0GDYfPvjhR411Yt/x9o0fPhwjRksDmGoGBjeQIkIDA4M8hKojRu0oGAWjYBSMVAAA5Tc+5CV68SoAAAAASUVORK5CYII=","orcid":"","institution":"Yeungnam University","correspondingAuthor":true,"prefix":"","firstName":"Jintae","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-07-16 09:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7138037/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7138037/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87320729,"identity":"7b7211cb-3182-4df1-8231-b36bc0d4a916","added_by":"auto","created_at":"2025-07-22 16:31:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":725411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibiofilm screening of halogenated phenols. Screening of phenol and its halogenated derivatives for antibiofilm activity against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC 6538\u003c/strong\u003e. The Biofilm formation (indicated as bars) was quantified after 24 h of culture in 96-well plates without shaking in the presence of each compound at 0, 2, 10, or 50 µg/mL. Compound 12 (PBP), identified as the most active, is highlighted in red in the bar chart and table, * p \u0026lt; 0.05 versus untreated controls (None).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/6f57e81312c0e711dd4f8522.jpg"},{"id":87323149,"identity":"72deae1c-04ff-4b21-a9cd-81a60254cd02","added_by":"auto","created_at":"2025-07-22 16:55:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":787228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial activity of PBP and antibiotics against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm and growth. \u003c/strong\u003eBiofilm formation by \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 in the presence of pentabromophenol (PBP) (A), ciprofloxacin (CIP) (B), and tetracycline (TET) (C). Effects of PBP on the growth dynamics of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 (D). Time-kill kinetics of \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 treated with PBP (E), antibiotics (CIP and TET) (F), * p \u0026lt; 0.05 versus. non-treated controls (None).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/f14a4a16baeafe736e59f892.jpg"},{"id":87322366,"identity":"2595d6fa-afc8-4ba3-8a53-7eaa0cec3536","added_by":"auto","created_at":"2025-07-22 16:47:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1769801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibiofilm effect of PBP and antibiotics on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eaureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm architecture and cell morphology\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003e3D biofilm representation (A) and Scanning Electron Microscopy (SEM) (B) of \u003cem\u003eS. aureus\u003c/em\u003ebiofilm following 24 h treatment with PBP. 3D biofilm representation (C) and SEM (D) of \u003cem\u003eS. aureus\u003c/em\u003e biofilm post-treatment with ciprofloxacin and tetracycline for 24 h. Untreated cells (None) served as negative controls. Yellow and red scale bars represent 50 and 3 µm, respectively.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/2715b0d576d677af1fb0183a.jpg"},{"id":87321745,"identity":"2b1b6a1c-bfd8-4ea8-80a1-2794f72c976e","added_by":"auto","created_at":"2025-07-22 16:39:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":643438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntivirulence effects and mechanisms of action of PBP and standard antibiotics. \u003c/strong\u003eHemolytic activity (A), metabolic activity (B), reactive oxygen species (ROS) production (C), and NPN uptake assay (D) were assessed in \u003cem\u003eS. aureus\u003c/em\u003e post-treatment with pentabromophenol (PBP), ciprofloxacin (CIP), and tetracycline (TET), * p \u0026lt; 0.05 versus. untreated controls (None).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/e56fa4c7b58ed617bf3a8851.jpg"},{"id":87320735,"identity":"11458320-066d-4b93-8a4d-a88b80daba34","added_by":"auto","created_at":"2025-07-22 16:31:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":580832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombinatory therapeutic strategies of PBP with standard antibiotics\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eCheckerboard assay heatmaps visualizing interactions involving combinations of pentabromophenol with vancomycin (A), ciprofloxacin (B), tetracycline (C), and gentamicin (D) against \u003cem\u003eS. aureus\u003c/em\u003e. The color scale indicates OD600-based growth inhibition, transitioning from green (lowest inhibition) to white (highest inhibition). FICI represents fractional inhibitory concentration index wherein FICI ≤ 0.5 (Synergy), 0.5\u0026lt; FICI ≤ 1 (Additive), 1 \u0026lt; FICI\u0026lt; 2 (Indifferent), and FICI ≥ 2 (Antagonistic).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/cc049da23e7fd9954955455c.jpg"},{"id":87320737,"identity":"4d0e330f-9a21-4fd7-8c5e-136f54042212","added_by":"auto","created_at":"2025-07-22 16:31:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":968768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistance development and gene expression analysis. \u003c/strong\u003eDrug resistance assay in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus,\u003c/em\u003eshowing changes in MICs of pentabromophenol, ciprofloxacin, and tetracycline over 30 passages (A), gene expression analysis of biofilm-related genes in \u003cem\u003eS. aureus\u003c/em\u003e with pentabromophenol (PBP) at 0.25 μg/mL for 6 h at 250 rpm under aerobic shaking conditions (B). Fold change indicates gene expression differences relative to the untreated control (None) as determined by qRT-PCR. * p \u0026lt; 0.05 vs. non-treated controls. \u003cem\u003e16S rRNA\u003c/em\u003e was used as the housekeeping gene.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/c6800b90eca139e8f2e52d76.jpg"},{"id":87322368,"identity":"9b593ed7-198f-47ee-aa11-66a913af1993","added_by":"auto","created_at":"2025-07-22 16:47:45","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1135739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eToxicity assay of PBP and phenol in the HepG2 cell, nematode, and seed (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRaphanus sativus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) models.\u003c/strong\u003e HepG2 cell viability after treatment with PBP and 2,4,6-TIP, assessed using MTT assay (A), survival percentages of \u003cem\u003eC.\u003c/em\u003e \u003cem\u003eelegans\u003c/em\u003eexposed to PBP (B) and phenol (C) for 7 days. Seedling growth of \u003cem\u003eR. sativus\u003c/em\u003ecultured with or without various concentrations of PBP (d), phenol (E), at 25°C for 7 days. The red scale bar in (D) and (E) represents 5 cm.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/82cc05d626ddfcf93526b9c8.jpg"},{"id":87321755,"identity":"3695f591-e827-4493-8959-df95c89edc6f","added_by":"auto","created_at":"2025-07-22 16:39:45","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":791198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThree-dimensional quantitative structure-activity (3D-QSAR) relationship of halogenated phenols\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eScatter plot showing correlation between predicted pMIC values and the experimentally determined MIC of 17 halogenated phenols, including PBP and phenol scaffold (A). 3D-QSAR visualization of favorability contours showing favorable substitutions (blue contours) at C-2 and C-6 and the unfavorable substitutions (red contours) at C-3 (B).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/7ec3cabbb97ab95e567b6a43.jpg"},{"id":89313515,"identity":"e17d3738-5dc8-40c0-ba03-7599c1c77fcb","added_by":"auto","created_at":"2025-08-18 16:38:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9212737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/c3953068-f95f-40f3-b310-5a13d1f48a3d.pdf"},{"id":87321746,"identity":"023bea90-c7ea-41ab-a798-725ccc48c0f7","added_by":"auto","created_at":"2025-07-22 16:39:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":883699,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/3163c9067cd2aeeb41f92f0f.pdf"},{"id":87320743,"identity":"d90e6eb6-3f66-401a-a608-c4a55721efd5","added_by":"auto","created_at":"2025-07-22 16:31:45","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":144543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract showing a summary of the study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7138037/v1/d68b91ac621afa80c8db5e1f.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elucidating the Antimicrobial Activity, Virulence, and Resistance Mechanisms of Pentabromophenol on Staphylococcus aureus","fulltext":[{"header":"1.0 Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) poses a significant challenge for treating infectious diseases. In 2019, bacterial AMR was associated with an estimated 4.95\u0026nbsp;million deaths worldwide, highlighting the need for new treatments. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a common pathogen that causes a spectrum of illnesses from minor skin and soft-tissue infections to severe pneumonia and sepsis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Many \u003cem\u003eS. aureus\u003c/em\u003e strains, including methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e and vancomycin-resistant (MRSA and VRSA), are resistant to multiple antibiotics. MRSA infections tend to result in higher mortality, longer hospital stays, and increased healthcare costs compared to infections by drug-sensitive strains\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMoreover, \u003cem\u003eS. aureus\u003c/em\u003e can evade host immune defenses; for example, it produces virulence factors that help it survive neutrophil attacks. These features promote high prevalence, resistance, and virulence, making \u003cem\u003eS. aureus\u003c/em\u003e a particularly important target for new antimicrobial strategies\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBiofilm formation drastically heightens \u003cem\u003eS. aureus\u003c/em\u003e\u0026rsquo; tolerance to antibiotics and host immune responses, often leading to chronic, relapsing infections on indwelling medical devices and host tissues\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Apart from reinfections, antimicrobial resistance due to \u003cem\u003eS. aureus\u003c/em\u003e also leads to increased spending through prolonged hospital stays, complex treatments, and increased mortality\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, biofilm-grown bacteria are notoriously difficult to eradicate because the matrix limits antibiotic penetration, and cells within have altered physiology, enabling them to withstand higher drug concentrations. Studies reported cells in biofilm with 10-1000-fold increased tolerance compared to planktonic cells, emphasizing the need for the least explored antimicrobial compounds capable of targeting both planktonic cells and biofilms\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These trends underscore an urgent need for new antimicrobial strategies to combat biofilm-associated resistance, effectively disrupt and curtail \u003cem\u003eS. aureus\u003c/em\u003e pathogenicity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, thereby reducing the overall impact of antimicrobial resistance globally.\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the potential of natural and synthetic small molecules in this regard, showing that it is possible to inhibit \u003cem\u003eS. aureus\u003c/em\u003e biofilms, reduce virulence, and ameliorate persistence associated with re-infections\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While halogenation has long been used to enhance the performance of antimicrobial agents, its inherent ability in antiseptics, especially for promoting antimicrobial activity while reducing resistance, has not been fully elucidated because of toxicity concerns. Many modern antibiotics and antiseptics contain halogens, underscoring the general value of halogenation in drug design\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Halogen atoms like chlorine, bromine, or iodine enhance antimicrobial potency by increasing membrane permeability, reactivity, and metabolic stability\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhenolic compounds are a well-known class of antiseptics and disinfectants, and chemical modifications to phenols have long been used to tune their antimicrobial properties.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Small antibacterial molecules have been reported to disrupt bacterial membranes and virulence factor production, and introducing halogen substituents can further boost these effects.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In particular, halogenated phenolic compounds have attracted interest for their antibacterial and antibiofilm activities owing to enhanced hydrophobicity and reactivity, which facilitate biofilm penetration and membrane disruption\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. For example, in our previous study, 2,4,6-triiodophenol (2,4,6-TIP)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, inhibited \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation at 5 \u0026micro;g/mL, reduced hemolytic and proteolytic activity, and suppressed \u003cem\u003eRNAIII\u003c/em\u003e expression. It showed lower in vivo toxicity than phenol, indicating that halogenation can enhance efficacy while minimizing toxicity. These findings illustrated how modifying phenolic compounds with halogens can yield candidates that effectively target biofilms and virulence. Such compounds could support next-generation therapies that suppress resistance evolution in \u003cem\u003eS. aureus\u003c/em\u003e strains.\u003c/p\u003e\u003cp\u003eConsequently, in this study, we report the antimicrobial and antivirulence potential of multiple halogenated phenols, particularly pentabromophenol (PBP), which exhibited an MIC of 0.5 \u0026micro;g/mL against \u003cem\u003eS. aureus\u003c/em\u003e. Notably, we focused on identifying compounds with potent activity and minimal toxicity. PBP was screened alongside selected analogs at concentrations of 2\u0026ndash;50 \u0026micro;g/mL and evaluated for biofilm inhibition, cell viability, and safety in HepG2 cells, \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eelegans\u003c/em\u003e, and radish seed (\u003cem\u003eRaphanus sativus\u003c/em\u003e) models. We elucidated the mechanism of action via membrane permeability, creating an imbalance in gradient ions, efflux systems inhibition, metabolic impairment, and gene expression changes via qRT-PCR. The resistance passage profile and combination strategies with reference antibiotics were also studied, while quantitative structure\u0026ndash;activity relationship (QSAR) modelling identified structure-activity trends. These results may position PBP as a promising, low-toxicity candidate, worthy of further consideration for tackling antimicrobial resistance.\u003c/p\u003e"},{"header":"2.0 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Microbial Strains, Chemical Compounds, and Culture Conditions\u003c/h2\u003e\u003cp\u003eMethicillin-susceptible \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MSSA; ATCC 6538), methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA MW2), and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (ATCC 14990) were used in this study. MSSA ATCC 6538 and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eepidermidis\u003c/em\u003e cultures were incubated in Luria\u0026ndash;Bertani (LB) broth, while MRSA strains (MW2 and ATCC 33591) were cultivated in LB supplemented with 0.2% glucose at 37\u0026deg;C. All test compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), along with crystal violet and antibiotics, including vancomycin, ciprofloxacin, gentamicin, and tetracycline, were bought from Sigma-Aldrich (St. Louis, MO, USA) or Combi-Blocks Inc. (San Diego, CA, USA). Compounds were dissolved in dimethyl sulfoxide (DMSO), and 0.1% (v/v) DMSO was used as a control. This concentration of DMSO did not affect bacterial cell growth or biofilm formation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Biofilm Quantification using Crystal-Violet Assay\u003c/h2\u003e\u003cp\u003eBiofilm formation was assessed using a crystal violet (CV) staining assay in 96-well plates as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Bacterial cultures were treated with or without halogenated phenols, ciprofloxacin, and tetracycline (0\u0026ndash;50 \u0026micro;g/mL), dispensed into the 96-well plates, and incubated for 24 h at 37\u0026deg;C without shaking. Thereafter, planktonic cells were removed by washing the plates thrice with distilled water, dried briefly, and stained with 0.1% CV (300 \u0026micro;L) for 20 min. Excess dye was removed with three washes. Then, the CV-stained cells were irrigated with 300 \u0026micro;L of 95% ethanol. OD570 absorbance measurements were taken using a Multiskan plate reader, shaking vigorously (Thermo Fisher Scientific, Waltham, MA, USA). Results were obtained from three independent experiments; each was performed in six replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.3 Antimicrobial Susceptibility Testing by MIC\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe minimum inhibitory concentrations (MICs) of active halogenated phenols and reference antibiotics (ciprofloxacin and tetracycline) were determined using the broth microdilution method, following CLSI guidelines as previously reported\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Briefly, diluted \u003cem\u003eS. aureus\u003c/em\u003e overnight culture (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) in the presence or absence of the compounds was aliquoted into 96-well plates and kept at 37\u0026deg;C under static conditions for 24 h. After incubation, the lowest concentration with no visible bacterial growth was established as the MIC and recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Bacterial Growth Kinetics and Bactericidal Activity\u003c/h2\u003e\u003cp\u003eTo assess impact on bacterial growth kinetics, 300 \u0026micro;L of diluted cultures in LB medium (1:100) with or without PBP (0, 0.1, 0.2, 0.5, 1, 10, and 100 \u0026micro;g/mL), two reference antibiotics, ciprofloxacin and tetracycline (1 and 10 \u0026micro;g/mL) was aliquoted into 96-well plates and incubated at 37 ℃ for 24 h without shaking. The growth (OD600) was measured every 4 h using a Multiskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA). For bactericidal activity, 2 mL of treated or untreated cultures with PBP, ciprofloxacin, and tetracycline at (0-100 \u0026micro;g/mL) in 14 mL tubes were incubated at 37 ℃ and 250 rpm shaking conditions. At varying time points (0, 6, 12, and 24 h), 100 \u0026micro;L of the samples were taken, diluted serially, plated on LB agar, and incubated for 24 h at 37 ℃. The colony-forming units (CFU) were counted and plotted as log10 values\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eCell morphology induced by PBP and reference antibiotic treatments was visualized by biofilms developed on sterile nylon membranes (0.3 \u0026times; 0.3 cm, Merck Millipore, USA). The sterile membranes were placed into 96-well plates containing \u003cem\u003eS. aureus\u003c/em\u003e cells treated with or without PBP, ciprofloxacin, and tetracycline (0\u0026ndash;10 \u0026micro;g/mL), and incubated at 37\u0026deg;C for 7 h. Biofilm-adhered membrane surfaces were fixed with 2% formaldehyde and 2.5% glutaraldehyde for 12 h. Dehydrated through a graded ethanol series (50%, 70%, 90%, 95%, or 99%, 20 min each), coated with platinum, and imaged using a Hitachi S-4800 (Tokyo, Japan) scanning electron microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Assessment of Hemolytic Activity\u003c/h2\u003e\u003cp\u003eHemolytic activity was quantified using fresh sheep red blood cells (MBcell, Seoul, Korea) as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. 2 mL of fresh LB broth diluted with \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 cells (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) were cultured with PBP, ciprofloxacin, and tetracycline (0.1, 0.2, 0.5, 0.7, and 1 \u0026micro;g/mL) for 24 h at 250 rpm shaking conditions. Red blood cells were harvested from fresh sheep blood by centrifugation at 3,000 \u0026times; g for 2 min, washed three times with PBS, and resuspended to 3.3% (v/v) in PBS. Subsequently, 100 \u0026micro;L of treated bacterial culture was added to 1 mL of red blood cells and incubated for 4 h at 37\u0026deg;C for 4 h shaking at 250 rpm. After centrifugation (10,000 \u0026times; g, 10 min), the supernatant absorbances were measured at 543 nm to quantify hemolysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.7 Antimicrobial Impact on Cell Metabolic Activity (XTT Reduction Method)\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eCellular metabolic activity was evaluated using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) according to the manufacturer's instructions (Cell proliferation kit, Sigma-Aldrich). Biofilms were initially established in 96-well plates as described in Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. After incubation, non-adherent and spent medium were removed by rinsing with sterile H\u003csub\u003e2\u003c/sub\u003eO. 100 \u0026micro;L of a freshly prepared mixture of XTT labeling reagent and electron-coupling reagent (phenazine methosulfate) at 50:1 (v/v) was added to each well to assay the metabolic activities. Plates were incubated in the dark at 37\u0026deg;C for 30 min, and the absorbance was measured by a Multiskan EX microplate reader at 490 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Membrane Potential Assessment Using Reactive Oxidative Species (ROS) Assay\u003c/h2\u003e\u003cp\u003eTo evaluate the mechanism of activity, the potential for reactive oxygen species (ROS) production was determined following the protocol by\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e as modified. Briefly, 25 mL of the previous overnight culture of \u003cem\u003eS. aureus\u003c/em\u003e cells was reinoculated in a 250 mL flask and incubated for 4 h, cells were harvested in LB broth, resuspended in PBS, and adjusted to (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e CFU/mL)(OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;0.5). Treatment was performed with or without PBP, ciprofloxacin, and tetracycline (0, 1, 10, and 100 \u0026micro;g/mL). A stock solution of 35% v/v H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was diluted to achieve 500 or 1000 \u0026micro;g/mL, and the MIC of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e against \u003cem\u003eS. aureus\u003c/em\u003e was established as 1000 \u0026micro;g/mL\u003csup\u003e20\u003c/sup\u003e, as described in section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e. The setup was incubated for 3 h at 250 rpm and 37\u0026deg;C, and then, 2\u0026prime;,7\u0026prime;-dichlorofluorescein diacetate (DCFCDA) (10 mM, 1:2) was added to the cell suspensions and incubated in the dark for 30 min at 25\u0026deg;C. Fluorescence was measured using a spectrophotometer F-7000 (Hitachi, Tokyo, Tokyo, Japan) equipped with a xenon arc lamp. The excitation wavelength was at 485 nm, and emission intensities were recorded at 535 nm. Untreated and H2O2-treated (500 or 1000 \u0026micro;g/mL) samples were analyzed under the same conditions and served as negative and positive controls, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Membrane Permeability Measurement via NPN Uptake Assay\u003c/h2\u003e\u003cp\u003eThe outer membrane permeability of \u003cem\u003eS. aureus\u003c/em\u003e cells was determined using an N-phenyl-1-naphthylamine (NPN) uptake assay as previously described\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Briefly, overnight cultures were reinoculated into fresh LB broth (1:100) and grown to mid-log phase (OD600\u0026thinsp;~\u0026thinsp;0.05). Bacterial cells (\u0026sim;10\u003csup\u003e7\u003c/sup\u003e CFU/mL were harvested, washed, and resuspended in PBS. A total of 1 mL aliquots were treated with or without PBP, ciprofloxacin, and tetracycline (0, 0.5, and 1 \u0026micro;g/mL). This was incubated at 37\u0026deg;C, with shaking (250 rpm) for 3 h. After incubation, cell suspensions were centrifuged (7000 rpm, 10 min), washed, and resuspended in 2 mL PBS. NPN (150 \u0026micro;L 10 mM in EtOH 7.5%) was added to each suspension, samples were incubated at room temperature in the dark for 10 min by placing them in a box. Benzalkonium chloride (0.5, 1, and 10 \u0026micro;g/mL) serves as the positive control. Fluorescence was measured at 350 nm excitation and 450 nm emission using a Hitachi F-7000 (Tokyo, Japan) spectrofluorometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Bacterial Resistance Development Monitoring\u003c/h2\u003e\u003cp\u003eChanges in the MIC values were monitored to determine the potential of \u003cem\u003eS. aureus\u003c/em\u003e to develop resistance against PBP and reference antibiotics. The procedure follows\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e as modified. The initial MICs of the compounds were confirmed using broth dilution in 14 mL tubes. Subsequently, the bacterium (1:100) was cocultured with a sub-inhibitory concentration (sub-MICs) and incubated at 37\u0026deg;C for 24 h. Post incubation, overnight cultures that received sub-MIC were diluted and used as the starting culture for the next passage. After each passage, changes in MIC of the compounds against the \u003cem\u003eS. aureus\u003c/em\u003e were observed. The process was repeated for 30 passages, and the graph of MIC values against the days of passage was generated to determine resistance development over time. The fold change and cumulative fold change for each compound were calculated as:\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1753201752.png\"\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Antibiotic Combinatorial Strategies Using Checkerboard Assay\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e interactions between PBP and the reference antibiotics (ciprofloxacin, tetracycline, vancomycin, and gentamicin) were evaluated following procedures as earlier reported\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A checkerboard assay containing two-fold dilutions of pentabromophenol (Drug A) in the vertical wells and reference antibiotics (Drug B) in the horizontal wells. 100 \u0026micro;L of overnight \u003cem\u003eS. aureus\u003c/em\u003e culture diluted in 0.9% NaCl (\u0026sim;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) was added to each well and incubated for 24 h at 37\u0026deg;C. Planktonic growth was assessed visually and by OD600 readings, while the biofilm was quantified as in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. The percentage of growth in each well is calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{OD\\:drug\\:combination\\:well-OD\\:background\\:}{OD\\:drug\\:free\\:well-OD\\:background}\\times\\:\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe new MIC and minimum biofilm inhibitory concentration (MBIC) were recorded as the lowest concentration with no visible planktonic growth or biofilm, respectively. The fractional inhibitory concentration (FIC) for each agent was calculated by identifying the effective combinations, based on the first well in each row showing no visible growth, using the formula: Drug A FIC: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{drug\\:A\\:MIC\\:in\\:combination}{drug\\:A\\:MIC\\:alone}\\)\u003c/span\u003e\u003c/span\u003e and Drug FIC B: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{drug\\:B\\:MIC\\:in\\:combination}{drug\\:B\\:MIC\\:alone}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eFIC interpretations were as reported by Bellio\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The FIC index (FICI)\u0026thinsp;=\u0026thinsp;Drug A FIC\u0026thinsp;+\u0026thinsp;Drug B FIC was interpreted as FICI\u0026thinsp;\u0026le;\u0026thinsp;0.5 (Synergy), 0.5\u0026thinsp;\u0026lt;\u0026thinsp;FICI\u0026thinsp;\u0026le;\u0026thinsp;1 (Additive), 1\u0026thinsp;\u0026lt;\u0026thinsp;FICI\u0026thinsp;\u0026lt;\u0026thinsp;2 (Indifferent), and FICI\u0026thinsp;\u0026ge;\u0026thinsp;2 (Antagonistic). The checkboard representative images were generated using conditional formatting in Microsoft Excel 365.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Transcriptional Evidence of Antimicrobial Mechanism via qRT-PCR\u003c/h2\u003e\u003cp\u003eWe investigated the transcriptional effect of PBP on \u003cem\u003eS. aureus\u003c/em\u003e using quantitative real-time PCR (qRT-PCR). Briefly, 25 mL of diluted overnight culture (\u0026sim;10\u003csup\u003e7\u003c/sup\u003e CFU/mL) was treated with or without the active compound (0.25 \u0026micro;g/mL) for 6 h. 700 \u0026micro;L RNAlater\u0026reg; (Ambion, TX, USA) was added to each culture before centrifugation to stabilize RNA, rinsed with PBS after, and Qiagen RNeasy Mini Kits (Valencia, CA, USA) were used to extract total RNA according to the manufacturer's instructions. cDNA and amplification were performed using the ABI StepOne Real-Time PCR system (Applied Biosystems, CA, USA) in the presence of SYBR℗ Green Master Mix (Thermo Fisher, Warrington, UK), appropriate primers, and ddH\u003csub\u003e2\u003c/sub\u003eO. Gene-specific primers (Supplementary Table S2) targeted pathways related to biofilm suppression, QS, cytotoxicity, stress response, and antibiotic resistance. 16\u003cem\u003eS rRNA\u003c/em\u003e served as the housekeeping gene, and relative gene expression was calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Chemical Toxicity Using Human, Nematode, Seed Models, and ADMET Profiling\u003c/h2\u003e\u003cp\u003eHuman hepatocellular carcinoma HepG2 cells (Korean Cell Line bank) were cultured in DMEM (Thermo Scientific) containing 10% FBS (Gendepot), dispensed into a 96-well plate, and incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37 ℃ for 24 h. After incubation, the spent medium was removed, fresh medium treated with PBP and 2,4,6-TIP (None, 10, 20, 50, 100, and 200 \u0026micro;g/mL) was added, and incubated for 24 h more under the same conditions. For the MTT assay, the spent medium was removed using a vacuum. MTT (0.5 \u0026micro;g/mL) was mixed in a new medium at a 1:10 dilution ratio, each well received 100 \u0026micro;L of this mixture, and were incubated at 37 ℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 4 h. Post incubation, removal of the medium followed, and 100 \u0026micro;L of DMSO was used to treat the cells in the dark to extract formazan. Absorbance was then measured at 540 nm. The chemical toxicity of PBP and phenol was assessed using the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eelegans\u003c/em\u003e fer-15(b26); fem-1(hc17) strains, following previously established protocols\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Synchronized nematodes were carefully washed twice with M9 buffer (3 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 6 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 5 g/L NaCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e). Approximately 30\u0026ndash;35 worms were then transferred to each well of a 96-well plate containing M9 buffer (300 mL) and varying concentrations (None, 0.5, 1, 10, 100, and 200 \u0026micro;g/mL) of PBP. The plates were subsequently incubated for 7 days at 25\u0026deg;C. Four independent cultures were utilized. Nematodes' viability was assessed based on their response to LED light for 30 s in a Digital Cell Imaging System ( iRiS\u0026trade; Logos BioSystems, South Korea). In the seed model, initially, radish plant (\u003cem\u003eRaphanus sativus)\u003c/em\u003e seeds were soaked in sterile dH\u003csub\u003e2\u003c/sub\u003eO for 16 h and rinsed with dH\u003csub\u003e2\u003c/sub\u003eO three times. Sterilized subsequently by soaking in 95% ethanol, 3% Sodium Hypochlorite at 15 min intervals, and rinsed with sterile dH\u003csub\u003e2\u003c/sub\u003eO three more times. 9\u0026ndash;10 seeds per plate were placed on soft agar containing Murashige and Skoog (0.86 g/L MS and 0.7% agar), supplemented with PBP and phenol (None, 1, 10, 100, and 200 \u0026micro;g/mL). The plates were incubated at 25\u0026deg;C for 7 days. Germination rates and seedling lengths were recorded daily. Four independent cultures were utilized for this experiment. ADMET (absorption, distribution, metabolism, excretion, and toxicity) and drug-likeness parameters of phenol and selected multi-halogenated derivatives (Supplementary Table S6), including PBP and 2,4,6-TIP derivatives, were investigated \u003cem\u003ein silico\u003c/em\u003e using PreADMET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.molinspiration.com/\u003c/span\u003e\u003cspan address=\"https://www.molinspiration.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Protox (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tox.charite.de/protox3/\u003c/span\u003e\u003cspan address=\"https://tox.charite.de/protox3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) GUSAR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.way2drug.com/gusar/\u003c/span\u003e\u003cspan address=\"http://www.way2drug.com/gusar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and SwissADME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/index\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), assessed between 21\u0026ndash;24 December 2024.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Quantitative Structural Activity Relationship (3D-QSAR)\u003c/h2\u003e\u003cp\u003eAn atom-based 3D-QSAR model was developed for phenol and 17 halogenated phenol derivatives (Supplementary S5). These compounds 3D structures were downloaded from PubChem, and minimization of macromolecule energy was carried out using the LigPrep module with the OPLS3E force field. Common substructure alignment of all energy-minimized derivatives was performed using the base structure of phenol as a reference, utilizing the Ligand Alignment tool in Maestro 12.5 (Schr\u0026ouml;dinger Software Solutions, USA). MICs were converted to pMICs [-log (MIC)], and a 3D atom-based QSAR model with two partial least squares (PLS) factors was constructed using PHASE (Schr\u0026ouml;dinger Software Solutions, USA). PLS factor 2 was used for QSAR visualization and activity predictions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were repeated thrice with two replicates unless mentioned otherwise, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. SigmaPlot 14.0 was used to plot graphs. Student t-tests were used to calculate statistical significance, between untreated and treated samples denoted by asterisks (\u0026lowast;\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/em\u003e), and error bars represent the SD.\u003c/p\u003e\u003c/div\u003e"},{"header":"3.0 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Antibiofilm and Antibacterial Activities of Halogenated Phenols against \u003cem\u003eS. aureus\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Screening of Halogenated Phenols Against \u003cem\u003eS. aureus\u003c/em\u003e Biofilms and Growth\u003c/h2\u003e\u003cp\u003eTwelve multi-halogenated phenols were screened at 2, 10, and 50 \u0026micro;g/mL concentrations to evaluate their antibiofilm activity against methicillin-susceptible \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538. Among the tested compounds, pentabromophenol (PBP) and 2,4,6-triiodophenol (2,4,6-TIP) were the most effective, exhibiting a 91% and 97% inhibition of biofilm formation at 50 \u0026micro;g/mL, respectively. Other multi-halogenated derivatives, such as 2,4,6-tribromo-3-nitrophenol and 3,4,5-triiodobenzoic acid, showed 85% and 71% inhibition, respectively. In contrast, fluorinated compounds, including 2,3,4-trifluorophenol and 2,3,5,6-tetrafluorophenol, displayed minimal activity, with only 1% and 3% inhibitions, respectively. Expectedly, the addition of a bromine atom at C-4 in tetrafluorophenol (4-bromotetrafluorophenol) only slightly improved inhibition to 14%. These findings suggest that multiple bromine or iodine substitutions, particularly at the C-2 and C-6 positions of the phenol ring, may suggest more effective and enhanced antibiofilm activity than fluorinated substitutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, the minimum inhibitory concentration (MIC) observation established that PBP exhibited the lowest inhibitory concentration among the screened halogenated phenols. PBP inhibited planktonic \u003cem\u003eS. aureus\u003c/em\u003e growth at 0.5 \u0026micro;g/mL, significantly lower than other halogenated phenols, including 2,4,6-triiodophenol (2,4,6-TIP)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, previously reported. Several compounds, including 3,4,5-triiodobenzoic acid, 2,4,6-tribromo-3-nitrophenol, and 4-bromotetrafluorophenol, exhibited MICs of 100 \u0026micro;g/mL. In contrast, other compounds, particularly fluorinated analogs, had MICs\u0026thinsp;\u0026ge;\u0026thinsp;200 \u0026micro;g/mL while phenol showed poor activity with an MIC exceeding 1000 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 Comparison of PBP and Antibiotics Effects on \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eAt 0.5 \u0026micro;g/mL, PBP inhibited biofilm formation by 70%, comparable to ciprofloxacin (MIC: 1 \u0026micro;g/mL, 75% inhibition), and superior to tetracycline (MIC: 2 \u0026micro;g/mL, 50% inhibition). In planktonic cell growth inhibition, PBP showed 85% suppression, moderately higher than ciprofloxacin and tetracycline with 70% and 29%, respectively. PBP at 0.5 \u0026micro;g/mL demonstrated comparable or superior performance at a lower effective dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, and C). Further testing of PBP at various concentrations (0.1, 0.2, 0.5, 1, or 10 \u0026micro;g/mL) against other clinical strains of \u003cem\u003eS. aureus\u003c/em\u003e, notably MRSA MW2, MRSA 33591, and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eepidermidis\u003c/em\u003e, showed consistent activity. In MRSA MW2, PBP inhibited cell growth formation by 78% at an MIC of 1 \u0026micro;g/mL and reduced biofilm formation at 10 \u0026micro;g/mL by 60%. Interestingly, in MRSA 33591 and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eepidermidis\u003c/em\u003e (MICs: 0.5 and 1 \u0026micro;g/mL, respectively), PBP exhibited significantly higher planktonic cell growth inhibition, with 89% and 83% inhibition in MRSA 33951 and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eepidermidis\u003c/em\u003e, respectively. At 10 \u0026micro;g/mL, it also suppressed biofilm formation by 80% and 93% in both strains (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A, B, and C). These results establish PBP as the most potent candidate among the screened halogenated phenols, with consistent efficacy across multiple \u003cem\u003eStaphylococcus\u003c/em\u003e strains. It showed significant, comparable antibacterial and antibiofilm activity to ciprofloxacin and superior activity to tetracycline in both biofilm and planktonic growth inhibition and was therefore selected for further study.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Antimicrobial and Bactericidal Effects of PBP on Growth and Kinetic Studies\u003c/h2\u003e\u003cp\u003eThe antimicrobial activity of pentabromophenol (PBP) on \u003cem\u003eS. aureus\u003c/em\u003e ATCC 6538 was assessed through growth kinetics and time-kill assays. At sub-inhibitory concentrations (0.1 and 0.2 \u0026micro;g/mL), PBP had minimal effect on planktonic cell growth. However, at its MIC (0.5 \u0026micro;g/mL), PBP significantly inhibited bacterial growth, and this suppression was sustained throughout the 24 h incubation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, ciprofloxacin and tetracycline achieved comparable growth inhibition only at concentrations 5- to 10-fold higher than their respective MICs, highlighting the superior potency of PBP at lower doses. In time-kill assays (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), PBP demonstrated a bacteriostatic effect at 2 \u0026times; MIC, yielding an approximately 2-log10 reduction in CFU after 24 h, comparable to the effect observed with tetracycline at its MIC. PBP exhibited bactericidal activity only at a higher concentration (200\u0026times; MIC), achieving a\u0026thinsp;\u0026gt;\u0026thinsp;4 log10 reduction. Ciprofloxacin demonstrated bactericidal effects at just 0.5\u0026times; MIC, resulting in a 3-log10 reduction compared to the untreated control. These findings align with established criteria distinguishing bacteriostatic agents, such as tetracycline, which typically induce\u0026thinsp;\u0026le;\u0026thinsp;2 log10 reductions, from bactericidal agents like ciprofloxacin, which achieve\u0026thinsp;\u0026ge;\u0026thinsp;3 log10 reductions under comparable conditions. Accordingly, PBP can be classified primarily as a bacteriostatic compound at minimum dosage, with dose-dependent bactericidal potential at elevated concentrations\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cell Density and Structural Disruption Assessed by Live Imaging and SEM\u003c/h2\u003e\u003cp\u003eLive-cell imaging and scanning electron microscopy (SEM) were employed to assess the effects of PBP and reference antibiotics on \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation, cell density, and colony morphology. In the untreated control, 3D color-coded reconstructions revealed the dense biofilm architecture characteristic of \u003cem\u003eS. aureus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). PBP treatment at 0.2 \u0026micro;g/m resulted in a slight decrease in biofilm mass, and marked reductions were observed at 0.5 and 1 \u0026micro;g/mL, indicating dose-dependent antibiofilm activity. In comparison, ciprofloxacin and tetracycline at equivalent concentrations produced only minimal reductions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). SEM images corroborated these results, revealing a significant reduction of bacterial cell density following PBP treatment at 0.5 and 1 \u0026micro;g/mL, without evident structural disruption of cell morphology, consistent with a bacteriostatic mode of action (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Tetracycline-treated cells exhibited modest reductions in cell density, while ciprofloxacin-treated samples displayed swollen and partially lysed cells at 1 \u0026micro;g/mL, indicating bactericidal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These microscopy-based observations support the distinct modes of action observed in growth kinetics and time kill assays for PBP and the reference antibiotics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Effect of PBP on Virulence and Possible Mechanisms\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e secretes α-hemolysin (\u003cem\u003ehla\u003c/em\u003e), a pore-forming cytotoxin that lyses red blood cells and contributes to immune evasion. To evaluate the antivirulence effect of PBP, we assessed its ability to suppress hemolytic activity. At sub-MICs (0.1 and 0.2 \u0026micro;g/mL), PBP had minimal effect, whereas at 0.5 \u0026micro;g/mL, it significantly reduced hemolysis by 89%, and to near-baseline levels at 1 \u0026micro;g/mL with 98% inhibition. In contrast, ciprofloxacin and tetracycline showed limited inhibition at 0.5 \u0026micro;g/mL (8% and 2%, respectively), with comparable effects at 1 \u0026micro;g/mL (84% and 94%). These findings demonstrate PBP\u0026rsquo;s dose-dependent antivirulence effect, with greater efficacy at lower concentrations than reference antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Bacterial metabolic viability was assessed by XTT reduction, indicating NAD(P)H-dependent dehydrogenase activity. At 0.5 \u0026micro;g/mL, PBP reduced metabolic activity by 50%, compared to 29% and 39% inhibition by ciprofloxacin and tetracycline, respectively, while at 1 \u0026micro;g/mL, all PBP and reference antibiotics showed comparable effects (PBP: 70%, ciprofloxacin: 69%, tetracycline: 65%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Reactive oxygen species (ROS) generation, a hallmark of bactericidal activity, was assessed. Ciprofloxacin induced significant fluorescence at 10 \u0026micro;g/mL, consistent with its known bactericidal mechanism\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, whereas tetracycline produced negligible ROS, supporting its classification as bacteriostatic. Similarly, PBP generated minimal ROS at concentrations up to 10 \u0026micro;g/mL, suggesting a comparable, non-lytic mode of action (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Outer membrane permeability was assessed using an NPN uptake assay. PBP at 0.5 and 1 \u0026micro;g/mL induced significantly greater fluorescence than tetracycline and ciprofloxacin, comparable to benzalkonium chloride, a known membrane disruptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The elevated NPN fluorescence indicates increased outer membrane permeability, suggesting that PBP may impair efflux or destabilize membrane integrity without causing lysis, consistent with its bacteriostatic activity\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Combination Efficacy of PBP with Antibiotics and Drug-Resistance Development\u003c/h2\u003e\u003cp\u003eAntimicrobial combinations are known to enhance efficacy and delay resistance development in \u003cem\u003eStaphylococcus\u003c/em\u003e infections\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The synergistic potential of PBP was evaluated in combination with reference antibiotics, vancomycin (VAN), ciprofloxacin (CIP), tetracycline (TET), and gentamicin (GEN) using checkerboard assays \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. PBP-VAN showed strong synergy in planktonic growth inhibition (FICI: 0.31), enhancing potency 16-fold and lowering the MIC from 0.5 to 0.031 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). PBP combined with ciprofloxacin, tetracycline, and gentamicin, displayed additive interactions, and reduced MICs to 0.125 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D, Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Similar interactions were observed in biofilm inhibition, with PBP-vancomycin maintaining synergistic interactions while the rest of the PBP-combinations showed an additive pattern (Supplementary Figure S2).\u003c/p\u003e\u003cp\u003eA serial passage assay was conducted over 30 days to assess the resistance-suppressing potential of PBP. PBP showed only a modest 4-fold MIC increase, similar to tetracycline, indicating a stable resistance profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, ciprofloxacin exhibited over a 1000-fold MIC increase within 13 passages. As the MIC exceeded 1000 \u0026micro;g/mL, a fold change of 1000\u003csup\u003e33\u003c/sup\u003e, and ciprofloxacin became increasingly insoluble at the required concentrations, the assay was discontinued on day 13. This sharp rise reflects accelerated resistance development, likely driven by target-site mutations and efflux upregulation under drug pressure\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These findings highlight PBP\u0026rsquo;s ability to bypass conventional resistance pathways and enhance efficacy when combined with conventional antibiotics, particularly vancomycin, through additive or synergistic interactions. This allows a reduced effective dose while maintaining strong antimicrobial activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Impact of PBP on Gene Expression in \u003cem\u003eS. aureus\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanisms of PBP\u0026rsquo;s antimicrobial action, we analyzed its effects on gene expression related to biofilm formation, virulence, antibiotic resistance, and stress adaptation in \u003cem\u003eS. aureus\u003c/em\u003e. Quantitative real-time PCR (qRT-PCR) showed that PBP at sub-inhibitory concentration (0.25 \u0026micro;g/mL) significantly downregulated key regulatory and effector genes. Biofilm-related genes \u003cem\u003eagrA\u003c/em\u003e, \u003cem\u003enuc1\u003c/em\u003e, and \u003cem\u003esigB\u003c/em\u003e, virulence factors \u003cem\u003ehla\u003c/em\u003e, \u003cem\u003epsm-α\u003c/em\u003e, and \u003cem\u003esarA\u003c/em\u003e, and resistance-associated regulators \u003cem\u003earlR\u003c/em\u003e and \u003cem\u003earlS\u003c/em\u003e were all suppressed. Notably, \u003cem\u003ehla\u003c/em\u003e and \u003cem\u003enuc1\u003c/em\u003e were reduced by 7.0- and 4.5-fold, respectively, while \u003cem\u003epsm-α\u003c/em\u003e and \u003cem\u003esarA\u003c/em\u003e were reduced by 8.6- and 2.5-fold. In parallel, \u003cem\u003earlR\u003c/em\u003e and \u003cem\u003earlS\u003c/em\u003e showed transcriptional repression of 2.4- and 2.5-fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These findings suggest that PBP\u0026rsquo;s bacteriostatic and antivirulence effects may stem from its disruption of global regulatory circuits controlling cytotoxicity, stress tolerance, and resistance modulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Assessment of PBP Reveals Low Toxicity Potential Across Models and ADMET Profiling\u003c/h2\u003e\u003cp\u003eToxicity profiling is essential for evaluating the therapeutic potential of antimicrobial candidates. In HepG2 cells, MTT assays revealed dose-dependent cytotoxicity for both pentabromophenol (PBP) and 2,4,6-triiodophenol (2,4,6-TIP), a previously reported halogenated phenol with potent antibiofilm activity against \u003cem\u003eS. aureus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. PBP consistently showed lower cytotoxicity, maintaining 90% and 78% cell viability at 10 and 20 \u0026micro;g/mL, respectively, compared to 57% and 28% for TIP. At higher concentrations, PBP-treated cells exhibited nearly double the survival rates compared to 2,4,6-TIP. However, reported oral LD50 values indicate that TIP (LD50\u0026thinsp;~\u0026thinsp;2000 mg/kg) is substantially less toxic in rats than PBP (LD50\u0026thinsp;~\u0026thinsp;200 mg/kg) or phenol (LD50\u0026thinsp;~\u0026thinsp;317 mg/kg). These findings suggest that while PBP shows improved cellular tolerance \u003cem\u003ein vitro\u003c/em\u003e, TIP presents a lower risk of acute systemic toxicity\u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In the \u003cem\u003eC. elegans\u003c/em\u003e toxicity model, PBP demonstrated superior safety. At 5 and 10 \u0026micro;g/mL (10\u0026times; and 20 \u0026times; MIC), worm survival rates were 70% and 60%, respectively, comparable to previously reported values for 2,4,6-TIP at 10 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In contrast, phenol showed pronounced toxicity, with survival dropping to 25% and 16% at the same concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Similarly, in the \u003cem\u003eRaphanus sativus\u003c/em\u003e seed germination assay, PBP treatment at 1 and 10 \u0026micro;g/mL resulted in 94% and 88% germination by day 7 (Supplementary Figure S3A), with normal root and shoot development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Phenol-treated seeds showed slightly reduced germination (89% and 78%) (Supplementary Figure S3B) but with significant growth deficiencies, including yellowing of roots, shoots, and leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eIn ADMET profiling, the parent phenol scaffold was classified as toxicity class III, indicating relatively high acute toxicity risk. Less active or inactive analogs, pentachlorophenol, pentafluorophenol, 4-chloro-2-iodophenol, and trifluorophenol, were assigned to toxicity class IV, suggesting moderate toxicity. Similarly, PBP was categorized as toxicity class IV, indicating moderate acute toxicity. Furthermore, PBP exhibited minimal aquatic toxicity in both \u003cem\u003eOryzias latipes\u003c/em\u003e (medaka) and \u003cem\u003ePimephales promelas\u003c/em\u003e (fathead minnow), outperforming phenol and inactive analogs. It also showed higher predicted blood-brain barrier (BBB) and Caco-2 permeability, improved oral bioavailability scores, and a predicted low risk of hERG inhibition, much improved than 2,4,6-TIP and phenol (Supplementary Table S3). Collectively, these profiles may support PBP\u0026rsquo;s development as a low-toxicity antimicrobial candidate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Quantitative Structure-Activity Relationship (3D-QSAR)\u003c/h2\u003e\u003cp\u003eUnderstanding structural features that influence antimicrobial activity is crucial for optimizing drug candidates. QSAR models link chemical structure to biological activity, highlighting key scaffold positions that drive efficacy\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In this study, an atom-based 3D-QSAR model was developed for phenol and multi-halogenated derivatives. These were compounds screened for antibiofilm activity against \u003cem\u003eS. aureus\u003c/em\u003e as in section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e, and 17 of them were selected with their MICs ranging from 5 to 1000 \u0026micro;g/mL (Supplementary Table S4) to ascertain the optimal positions for halogen substitution on the phenol scaffold that enhance antimicrobial activity\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Partial Least Squares (PLS) Factor 2, highlighted in bold letters, was selected as the final model based on its superior statistics (higher R\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and Q\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, greater stability, and lower RMSE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). PLS factor 2 has R^2\u0026thinsp;=\u0026thinsp;0.83 and Q^2\u0026thinsp;=\u0026thinsp;0.86, significantly higher than the thresholds of 0.6 and 0.5, respectively. These metrics and a high Pearson correlation (r\u0026thinsp;\u0026asymp;\u0026thinsp;0.984) between observed and predicted activities demonstrate model reliability. The predicted pMIC and experimentally determined MIC values aligned closely with the linear regression curve, indicating relatively good model accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). 3D visualization of the QSAR model was performed, wherein blue fields represent the most favorable regions, while red interaction field represent the unfavorable region. Favorability was determined based on hydrogen bond donation, electron-withdrawing effects, hydrophobicity, and non-polar characteristics. The visualization revealed that positions C-2 and C-6 were favorable for antimicrobial activity against \u003cem\u003eS. aureus\u003c/em\u003e, whereas substitution at C-3 was unfavorable in the phenol scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e"},{"header":"4.0 Discussion","content":"\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Mechanism of Action\u003c/h2\u003e\u003cp\u003eHalogenation is a well-established medicinal chemistry strategy, widely employed for improving antimicrobial potency, membrane permeability, and metabolic resilience of small molecules\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong halogenated scaffolds, brominated phenols have emerged as promising candidates, particularly against resilient pathogens like MRSA\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this study, PBP demonstrates superior antimicrobial and antibiofilm efficacy, with MIC values nearly 10-fold lower and activity surpassing that of previously reported 2,4,6-triiodophenol (TIP)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This enhancement reinforces the therapeutic potential of multi-brominated phenol derivatives as a robust antimicrobial agent against biofilm-associated infections.\u003c/p\u003e\u003cp\u003eBeyond its growth inhibition, PBP exhibited significant antivirulence properties. It effectively reduced biofilm biomass and hemolytic activity, showing comparable efficacy to ciprofloxacin and tetracycline. Transcriptomic analysis revealed downregulation of key genes of virulence, including α-hemolysin (\u003cem\u003ehla\u003c/em\u003e) and phenol-soluble modulins (psm-α), along with global regulators such as \u003cem\u003eagrA\u003c/em\u003e, \u003cem\u003esarA\u003c/em\u003e, \u003cem\u003esigB\u003c/em\u003e, and the two-component system \u003cem\u003earlR\u003c/em\u003e and \u003cem\u003earlS\u003c/em\u003e, which governs autolysis and antibiotic resistance development (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. These findings indicate that PBP achieves its bacteriostatic activity by impairing regulatory networks essential for growth, persistence, and pathogenesis. Such antivirulence targeting strategies are increasingly valued for their capacity to reduce pathogenicity while minimizing selective pressure for resistance.\u003c/p\u003e\u003cp\u003eMicroscopy of PBP-treated cells within biofilm was structurally intact, supporting a non-bacteriolytic mode of action. The static growth profile of PBP-treated cultures is consistent with this observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Increased PBP-induced uptake of the NPN probe indicates compromised membrane permeability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A similar activity has been observed in bromophenoxyphenols that disrupt membrane-linked resistance mechanisms. These non-lytic and non-lethal perturbations likely destabilize membrane potential without full rupture, impair nutrient and ion transport, and suppress efflux systems, leading to metabolic stress and growth inhibition\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, PBP may impair metabolic activity more than or comparable to standard antibiotics at equivalent MICs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), reinforcing its effect on energy-dependent cellular processes\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly, PBP generated negligible ROS levels, unlike fluoroquinolones, which induce significant ROS by targeting DNA gyrase, leading to damage and cell death\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This energy-based, non-lytic mechanism may enhance its safety profile and reduce resistance emergence. The multi-targeted nature of this mode of action makes single-step resistance mutations less likely\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Resistance Development\u003c/h2\u003e\u003cp\u003eThe resistance-limiting effect of PBP is supported by its multi-targeted mechanism of action against \u003cem\u003eS. aureus\u003c/em\u003e. PBP compromises the cytoplasmic membrane, as indicated by increased NPN uptake, in a manner that is not easily overcome by target-specific mutations\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This disruption may suggest impairment of the membrane potential and ATP synthesis, reflected in reduced metabolic activity, which may hinder energy-dependent processes such as efflux and stress response activation. Additionally, qRT-PCR analysis showed downregulation of genes involved in resistance regulation, including the \u003cem\u003earlR\u003c/em\u003e/\u003cem\u003earlS\u003c/em\u003e two-component system, suggesting a link between membrane stress and transcriptional repression. These combined effects reduce the bacterial capacity for adaptive changes during serial exposure, which may account for the absence of resistance development observed in the passage assay. In contrast, ciprofloxacin rapidly induced high-level resistance\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, consistent with the accumulation of gyrase mutations and efflux pump upregulation under fluoroquinolone pressure\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This resistance stability, along with PBP\u0026rsquo;s ability to repress master regulators of persistence and virulence, highlights its promise as a potential therapeutic option.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Synergistic Combinations\u003c/h2\u003e\u003cp\u003ePBP also demonstrated synergistic interaction with glycopeptides like vancomycin and additive interactions with other standard antibiotics tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Such combinations lower the required dosage and prevent resistance development\u003csup\u003e\u003cspan additionalcitationids=\"CR55 CR56\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. These observations align with clinical trends favoring combination therapies to eradicate biofilm-associated infections, where monotherapies often fail due to incomplete eradication or adaptive resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Toxicity\u003c/h2\u003e\u003cp\u003eIn terms of safety, phenol has been reported even at low concentrations to cause significant cytotoxicity in human fibroblasts and keratinocytes, nematodes, aquatic and plant models\u003csup\u003e\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHowever, this study revealed that PBP exhibited low toxicity across multiple eukaryotic models (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). It was well tolerated by human liver cells (HepG2), nematodes (\u003cem\u003eC\u003c/em\u003e. \u003cem\u003eelegans\u003c/em\u003e), and radish plant seedling germination, significantly more than tri-substituted halogenated analogs like 2,4,6-TIP. PBP\u0026rsquo;s ability to achieve potent antimicrobial activity at concentrations far below toxicity thresholds supports its suitability for further development. These findings align with recent reports on marine-derived brominated phenols and synthetic derivatives like pentabromopseudilin and thiophenones, which also exhibit strong anti-MRSA activity with limited host toxicity\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Collectively, this study reinforces the value of targeting bacterial virulence and membrane integrity as a strategy to overcome resistance. One of the more common resistance strategies employed by \u003cem\u003eS. aureus\u003c/em\u003e is heightened efflux pump activity and membrane impermeability. By targeting and shutting down key regulatory circuits, compromising biofilm and membrane barriers, PBP offers a promising alternative to conventional antibiotics by increasing membrane permeability and inhibiting efflux pumps. Its efficacy, low resistance development, favorable safety profile, and synergy with existing antibiotics position it as a strong candidate for future therapeutic considerations against resistant staphylococcal infections.\u003c/p\u003e\u003c/div\u003e"},{"header":"5.0 Conclusion","content":"\u003cp\u003ePentabromophenol exhibits exceptional antimicrobial and antibiofilm activity against \u003cem\u003eS. aureus\u003c/em\u003e, matching or exceeding the performance of standard drugs at much lower doses. It achieves this through a multi-faceted mechanism: destabilizing membrane function, suppressing energy metabolism, and silencing key virulence regulators (\u003cem\u003ehla\u003c/em\u003e, \u003cem\u003epsm-α\u003c/em\u003e, \u003cem\u003enuc1\u003c/em\u003e, \u003cem\u003eagrA\u003c/em\u003e, \u003cem\u003esarA\u003c/em\u003e, \u003cem\u003earlR\u003c/em\u003e, and \u003cem\u003earlS\u003c/em\u003e, etc.). Notably, PBP induces minimal resistance even after prolonged exposure, and it synergizes effectively with antibiotics like vancomycin. Its strong antibacterial effect is accompanied by low toxicity to mammalian cells and model organisms, indicating favorable and possible therapeutic consideration. Finally, PBP presents itself as a potential lead agent in the fight against antibiotic-resistant \u003cem\u003eS. aureus\u003c/em\u003e, offering a non-traditional halogenated scaffold that effectively targets biofilms and virulence while minimizing resistance development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOlanrewaju Rauf Olalekan:\u003c/strong\u003e Formal analysis; investigation; methodology; data curation; visualization; writing-original draft, review and editing, \u003cstrong\u003eJintae Lee\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eConceptualization; project administration; resources; supervision; validation; visualization; writing-review and editing, \u003cstrong\u003eJin-Hyung Lee\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMethodology; data curation; formal analysis; funding acquisition; administration; resources; supervision; validation; writing-review and editing, \u003cstrong\u003eMinHwi Sim:\u0026nbsp;\u003c/strong\u003eData curation\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003evisualization,\u003cstrong\u003e\u0026nbsp;Boya Bharath Reddy:\u0026nbsp;\u003c/strong\u003esoftware; formal analysis; visualization,\u003cstrong\u003e\u0026nbsp;Yong-Guy Kim:\u0026nbsp;\u003c/strong\u003eformal analysis; resources, visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare. All authors have seen and agree with the contents of the manuscript. We certify that the submission is original work and is not under review at any other publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (RS-2025-00513239 to J. Lee and\u0026nbsp;RS-2025-00553409 to J.-H. Lee) and by a grant of the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded .by the Ministry of Health \u0026amp; Welfare, Republic of Korea (RS-2024-00450423).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary materials related to this article, including additional tables and primer sequences, are available online and can be accessed to support the findings of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMurray, C. J. 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Comprehensive Exploration of Bromophenol Derivatives: Promising Antibacterial Agents against SA and MRSA. \u003cem\u003eACS Omega\u003c/em\u003e 9, 40897\u0026ndash;40906, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsomega.4c06115\u003c/span\u003e\u003cspan address=\"10.1021/acsomega.4c06115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial, antibiofilm, halogenation, pentabromophenol, Staphylococcus aureus","lastPublishedDoi":"10.21203/rs.3.rs-7138037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7138037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a common pathogen that readily acquires antibiotic resistance and often forms biofilm, further reducing drug susceptibility. In this study, we found pentabromophenol (PBP) as an antibacterial agent with low resistance against \u003cem\u003eS. aureus\u003c/em\u003e. PBP was identified and selected for further evaluation. Its MIC is lower than antibiotics ciprofloxacin (1 \u0026micro;g/mL) and tetracycline (2 \u0026micro;g/mL). Also, PBP dose-dependently inhibited \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation. At MIC, PBP significantly reduced bacterial growth and decreased toxin (hemolysin) production. Quantitative RT-PCR analysis revealed that PBP treatment at sub-inhibitory concentration downregulated the expression of toxin production and stress response (\u003cem\u003ehla\u003c/em\u003e, \u003cem\u003esigB, sarA\u003c/em\u003e, and \u003cem\u003epsm-α\u003c/em\u003e), and the two-component regulators responsible for autolysis and antibiotic resistance in \u003cem\u003eS. aureus\u003c/em\u003e (\u003cem\u003earlR\u003c/em\u003e and \u003cem\u003earlS\u003c/em\u003e). PBP exposure decreased metabolic activity and increased NPN uptake, thereby decreasing cellular respiration and energy metabolism. This results in the disruption of membrane homeostasis, by proxy inhibition of the efflux system. PBP did not exhibit notable drug resistance (4-fold) for 30 passages in contrast to ciprofloxacin, with over a 1000-fold change in MIC. PBP and vancomycin combination also exhibited synergistic antimicrobial activity against \u003cem\u003eS. aureus\u003c/em\u003e. PBP was non-toxic to HepG2 liver cells and \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e at concentrations up to 10 \u0026micro;g/mL (20 \u0026times; MIC). These findings position PBP as a promising antimicrobial compound to combat antimicrobial resistance and biofilm-related infections owing to PBP\u0026rsquo;s high antimicrobial potency, low toxicity, and diminished propensity to develop resistance.\u003c/p\u003e","manuscriptTitle":"Elucidating the Antimicrobial Activity, Virulence, and Resistance Mechanisms of Pentabromophenol on Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 16:31:40","doi":"10.21203/rs.3.rs-7138037/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bd879f4-cad4-4854-a38f-2bd03416db98","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51815459,"name":"Health sciences/Diseases"},{"id":51815460,"name":"Biological sciences/Drug discovery"},{"id":51815461,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-08-18T16:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 16:31:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7138037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7138037","identity":"rs-7138037","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}