Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria

Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria | 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 Research Article Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria Zhichao Xu, Yuanyuan Tang, Junhua Ma, Peiyu Li, Zewen Wen, Zhijian Yu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8617708/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2026 Read the published version in BMC Microbiology → Version 1 posted 11 You are reading this latest preprint version Abstract The escalating prevalence of multidrug-resistant (MDR) and biofilm-forming Staphylococcus aureus has created an urgent demand for alternative therapeutic strategies beyond conventional antibiotics. In this study, we report for the first time the potent antibacterial and antibiofilm activities of MLS0315771, a competitive phosphomannose isomerase (MPI) inhibitor, against a broad range of Gram-positive bacteria. MLS0315771 exhibited strong inhibitory and bactericidal effects against both MSSA and MRSA, as well as Enterococcus faecium strains resistant to linezolid, with MIC₅₀/₉₀ values of 6.25/6.25 μM. This compound also significantly delayed bacterial proliferation at subinhibitory concentrations and eradicated mature biofilms in vitro. Time-kill assays revealed that MLS0315771 displayed time-dependent bactericidal activity comparable or superior to vancomycin. Proteomic profiling of S. aureus exposed to MLS0315771 and functional assays confirmed that MLS0315771 disrupts bacterial membrane integrity and induces depolarization, thereby impairing essential membrane functions. Checkerboard analyses further indicated that unsaturated long-chain fatty acids antagonized its antibacterial activity, supporting a membrane-targeting antibacterial mechanism. Moreover, MLS0315771 displayed low hemolytic activity and minimal cytotoxicity toward mammalian cells, suggesting a favorable therapeutic index. Collectively, these findings demonstrate that MLS0315771 acts as a novel membrane-targeting antibacterial agent with potent activity against MDR and biofilm-forming Gram-positive pathogens, highlighting its potential as a promising lead compound for future antimicrobial development. MLS0315771 Staphylococcus aureus multidrug resistance biofilm inhibition membrane-targeting antibacterial agent Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The continuous rise and global dissemination of antibiotic-resistant bacteria have become one of the most critical threats to modern healthcare [1]. Among these pathogens, Staphylococcus aureus —a small, spherical, Gram-positive bacterium belonging to the phylum Firmicutes—stands out as a major clinical concern [2]. As a commensal organism, S. aureus is commonly found colonizing the human nasal cavity, skin, and gastrointestinal tract without causing harm under normal circumstances [3]. However, when the integrity of host barriers is compromised, this seemingly benign bacterium can rapidly transition into an opportunistic pathogen capable of triggering a spectrum of diseases, ranging from mild skin and soft tissue infections to severe and life-threatening conditions such as bacteremia and endocarditis [4]. The pathogenicity of S. aureus is largely attributed to its arsenal of virulence factors, which facilitate adhesion, immune evasion, and tissue invasion [5]. Notably, the bacterium can effectively escape innate and adaptive immune responses either by secreting a variety of toxins and enzymes or by forming robust biofilms on host tissues and medical device surfaces [6, 7]. These biofilms provide a protective niche that enhances bacterial survival and contributes to chronic and recurrent infections [8]. Driven by environmental selection pressure and genetic adaptability, S. aureus has developed resistance to multiple classes of antibiotics [9]. The emergence of methicillin-resistant S. aureus (MRSA) in particular has posed a formidable clinical challenge, rendering many first-line β-lactam antibiotics ineffective [4, 10]. According to the 2024 CHINET study (https://www.chinets.com/Data/AntibioticDrugFast), the detection rate of MRSA in clinical S. aureus isolates has steadily decreased since 2005, reaching 29.2% in 2024. Despite a gradual decline in MRSA detection rates in recent years, S. aureus remains among the leading causes of both community- and hospital-acquired infections worldwide [11]. The continuing evolution of multidrug-resistant (MDR) strains, especially those resistant to last-resort antibiotics such as vancomycin, linezolid, and daptomycin, highlights the urgent need for novel therapeutic agents that can effectively target both planktonic and biofilm-associated forms of this pathogen [12, 13]. The discovery and development of new antibiotics remain time-consuming, resource-intensive, and often unsuccessful, as many candidates fail in clinical trials due to safety or efficacy concerns [14]. In contrast, drug repurposing by identifying new antimicrobial uses for existing compounds offers a practical and efficient alternative [15]. This strategy capitalizes on the established pharmacological and toxicological profiles of approved drugs, thereby reducing both development cost and risk [16, 17]. As a result, repurposing has become an increasingly attractive approach in the search for novel antimicrobial agents. MLS0315771 is a potent competitive phosphomannose isomerase (MPI) inhibitor that can divert Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation [18, 19]. MLS0315771 also increases mannose metabolic flux toward glycosylation in zebrafish embryos [18]. However, the potential of MLS0315771 in antimicrobial therapy has not been adequately investigated. In this study, we present the first evidence that MLS0315771 possesses potent and broad-spectrum antibacterial activity against both planktonic and biofilm-associated Gram-positive bacteria. Using Staphylococcus aureus and Enterococcus faecalis as representative models, we found that MLS0315771 not only inhibits bacterial growth but also exerts bactericidal effects and markedly suppresses biofilm development, while exhibiting minimal hemolytic activity and limited cytotoxicity toward mammalian cells. To further uncover its underlying antibacterial mechanism, a series of complementary analyses were conducted, including quantitative proteomic profiling, membrane permeability and depolarization assays and checkerboard assays with phospholipids and fatty acids. It was found that MLS0315771 effectively kills S. aureus in vitro by targeting bacterial membranes. Taken together, these results highlight MLS0315771 as a promising candidate for the development of new therapeutic strategies targeting multidrug-resistant and biofilm-forming Gram-positive pathogens. Results MLS0315771 Exhibits Potent Antibacterial Activity Against Multiple Gram-Positive Bacteria, Including MRSA and Linezolid-Resistant Strains A total of 158 clinical isolates of Gram-positive bacteria were included in this study. Among S. aureus isolates, methicillin-susceptible S. aureus (MSSA) strains were most frequently recovered from wound specimens (30.77%), whereas methicillin-resistant S. aureus (MRSA) isolates were predominantly derived from sputum samples (52.94%) ( Figure S1 ). For the 37 E. faecium clinical isolates, including 5 isolates intermediate to and 14 isolates resistant to linezolid, urine was the most common source (42.86%).. MLS0315771 exhibited strong antibacterial activity against both MSSA and MRSA isolates, with MIC₅₀/₉₀ values of 25/25 μM for each ( Table 1 ). Notably, MLS0315771 remained active against E. faecium isolates displaying intermediate or complete resistance to linezolid ( Table S1 ). In addition, MLS0315771 demonstrated comparable or even superior antibacterial activity against other clinically isolated Gram-positive bacteria, including S. haemolyticus , S. hominis , S. capitis , S. lugdunensis , S. epidermidis , S. agalactiae , and E. faecalis ( Table 1 ), collectively indicating that MLS0315771 might possess broad-spectrum activity against a wide range of clinically relevant Gram-positive bacteria. To further evaluate the antibacterial effect of MLS0315771 at subinhibitory concentrations, growth curve analyses were performed on four MRSA isolates, four S. aureus isolates, and two E. faecium isolates under MLS0315771 pressure ( Figure 1 ). Within the 24-hour observation period, bacterial growth was completely inhibited at 1 × MIC of MLS0315771. At subinhibitory concentrations, MLS0315771 prolonged the lag phase of bacterial growth by 5-16 hours, indicating a concentration-dependent inhibitory effect of this chemical on bacterial proliferation. Table 1. The MIC distribution of MLS0315771 against Gram-positive bacteria. Bacteria species Total MLS0315771 MIC values (μM) MIC 50 /MIC 90 1.56 3.125 6.25 12.5 25 MRSA 54 0 2 14 23 15 12.5/12.5 MSSA 15 0 0 0 4 11 25/25 S. haemolyticus 4 0 2 1 1 0 6.25/12.5 S. hominis 5 0 0 2 3 0 12.5/12.5 S. capitis 2 0 0 2 0 0 6.25/6.25 S. lugdunensis 1 0 0 1 0 0 6.25/6.25 S. agalactiae 11 2 1 2 6 0 12.5/12.5 S. epidermids 26 0 5 15 6 0 6.25/12.5 E. faecalis 37 0 7 17 13 0 6.25/12.5 E. faecium 3 0 1 2 0 0 6.25/6.25 Total 158 2 18 56 56 26 12.5/25 MRSA, methicillin-resistant Staphylococcus aureus ; MSSA, methicillin-susceptible Staphylococcus aureus ; MIC50/MIC90, the MIC values for 50% or 90% of bacterial growth inhibition. MLS0315771 Exhibits In Vitro Bactericidal and Bacteriostatic Activity Comparable to Vancomycin In vitro time-kill assays were conducted using vancomycin as a positive control to evaluate the bactericidal potency of MLS0315771 ( Figure 2 ). Against the standard strain Staphylococcus aureus ATCC29213, MLS0315771 exhibited bacteriostatic activity within the first 8 hours at concentrations of 4 × and 8 × MIC. By 24 hours, both concentrations of MLS0315771 reduced the initial bacterial count by more than 3 log₁₀ (CFU/mL), corresponding to a ≥ 99.9% reduction in viable cells. Moreover, at 24 hours, the residual bacterial count in the MLS0315771-treated groups was lower than that observed with 8 μg/mL vancomycin ( Figure 2A ). Similarly, in the clinical MRSA isolate YUSA145, MLS0315771 demonstrated bacteriostatic activity during the first 8 hours, followed by a ≥2 log₁₀ (CFU/mL) reduction in bacterial load at 24 hours when treated with 4 × and 8 × MIC MLS0315771, indicating more than 90% bacterial killing. Under identical conditions, vancomycin exhibited sustained bacteriostatic rather than bactericidal activity throughout the 24-hour period ( Figure 2B ). For the Enterococcus faecium clinical isolate EF16C51, both MLS0315771 and vancomycin produced comparable inhibitory effects, maintaining persistent bacteriostasis over the 24-hour duration ( Figure 2C ). In conclusion, based on the observed time-kill kinetics, MLS0315771 appears to act as a time-dependent rather than a concentration-dependent antimicrobial agent. MLS0315771 Exhibits Excellent Anti-Biofilm Activity Based on the MIC values obtained from broth microdilution assays and the bacterial growth curve results, five MRSA and four MSSA biofilm-positive Staphylococcus aureus isolates were selected for in vitro biofilm inhibition and eradication assays. When subinhibitory concentrations of MLS0315771 were added at the initial stage of biofilm formation, MLS031577 exhibited an inhibitory effect on S. aureus biofilm formation ( Figure 3A and Figure 3B ). In addition to its effect on S. aureus , MLS0315771 also inhibited biofilm formation of E.faecium ( Figure S2 ). In our assays, MLS0315771 could significantly inhibit the biofilm formation of S. aureus and its inhibitory effect on S. aureus biofilm formation was stronger than that observed against E. faecium . Furthermore, the effect of MLS0315771 on mature S. aureus biofilms was investigated. After biofilms were allowed to mature for 24 hours, the culture medium was replaced with fresh medium containing 8 × MIC of MLS0315771 and incubated for an additional 24 hours. Crystal violet staining revealed that MLS0315771 effectively disrupted and removed established S. aureus biofilms ( Figure 3C ). Moreover, confocal laser scanning microscopy (CLSM) was employed to visualize biofilm thickness and the spatial distribution of live and dead cells. The result showed that treatment with 8 × MIC of MLS0315771 resulted in a thinner biofilm of S. aureus YUSA145, YUSA218 and CHS655 ( Figure 3D ). These findings show that MLS0315771 has potent antibiofilm activity against S. aureus . Proteomic analysis of S. aureus treated with MLS0315771 Quantitative label-free proteomic analysis investigated the global proteomic response of S. aureus YUSA145 treated with 1/2 × MIC MLS0315771 during the exponential growth phase. The results were obtained after performing principal component analysis (PCA) on the raw data and excluding two replicates that showed substantial variation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD073535 [20, 21]. When compared to the control group, 236 proteins exhibited significantly different expression levels (≥|1.5|-fold change, p≤0.05), including 63 up-regulated and 173 down-regulated proteins in the MLS0315771 treatment group ( Figure 4 A and 4 B ). Detailed information of the functional proteins with significantly different expression levels is listed in Table S2. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The KEGG results showed that the differential proteins were predominantly associated with pathways related to nitrogen metabolism, riboflavin metabolism, purine metabolism, S. aureus infection, microbial metabolism in diverse environments, arginine biosynthesis and pyruvate metabolism ( Figure 4C ). In addition, Gene Ontology (GO) analysis of the differentially expressed proteins was also conducted using the OmicsBean online platform, classifying them into three major categories: biological processes, molecular functions, and cellular components ( Figure 4D ). Based on KEGG data, a protein-protein interaction (PPI) network was constructed for the differentially expressed proteins ( Figure 4E ). The analysis revealed that interactions among downregulated proteins were mainly centered on Uncharacterized leukocidin-like protein 1, Delta-hemolysin, RNase H type-1 domain-containing protein, Global transcriptional regulator Spx, Staphylococcal secretory antigen ssaA2 and Type VII secretion system extracellular protein A. These proteins were primarily involved in biological process such as cytolysis, hemolysis, virulence, nucleic acid binding, regulation of DNA-templated transcription and hydrolase activity. Notably, one major virulence determinant associated with membrane dynamics, hld (3.875-fold downregulated) were markedly repressed. Hld, an amphipathic peptide under the control of the agr quorum-sensing system, facilitates membrane curvature remodeling through pore formation and promotes membrane vesicle (MV) biogenesis, both of which are vital for bacterial cytotoxicity and intercellular signaling [22]. Its downregulation is therefore consistent with impaired MV-mediated virulence transmission [22]. Loss of Hld weakens offensive membrane-disruptive capacity and the proteomic finding highlight membrane perturbation as a principal mechanism underlying the bactericidal action of MLS0315771 against S. aureus , thereby providing molecular evidence supporting its membrane-targeting antibacterial potential. Antibacterial mechanism of MLS0315771 against S. aureus Membrane permeability and membrane potential homeostasis are essential for bacterial growth and proliferation. To investigate the primary antibacterial mechanism of MLS0315771, we first examined its effects on membrane depolarization and permeability. Following different concentrations of MLS0315771 treatment, the MSSA strain SA113 exhibited pronounced disruption of both membrane permeability and membrane depolarization compared with the control group and MLS0315771 treatment led to dose-dependent increases in the uptake of propidium iodide (PI), and DiBAC₄(3), as reflected by elevated fluorescence intensity ( Figure 5 A-D ). These findings demonstrate that MLS0315771 compromises the integrity of S. aureus cell membranes. Given that MLS0315771 damages S. aureus membrane integrity, and considering the complex lipid composition of bacterial membranes, we further explored potential membrane lipid targets of MLS0315771 by exogenously supplementing abundant membrane lipids. Four major membrane phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL), were tested for their intrinsic antibacterial activity. None of the phospholipids inhibited bacterial growth for their MICs all >128 μg/mL ( Table S 3 ). Moreover, supplementation with these lipids did not alter the MIC values of MLS0315771 ( Table S 4 ), indicating that MLS0315771 does not exert its antibacterial effect through direct interaction with these four membrane lipids. In addition, checkerboard assay results ( Figure 5 E-G ) uncovered that the unsaturated long-chain fatty acids linoleic acid, γ-linoleic acid and arachidonic acid significantly increased the MIC values of MLS0315771 and reduced its antibacterial activity against S. aureus SA113 in a concentration-dependent manner. Based on the proteomic results, we examined whether MLS0315771 interferes with α-hemolysin activity. The findings revealed that MLS0315771 strongly inhibited α-hemolysin production in the S. aureus USA300 strain ( Figure 5 H ). This result is consistent with the hypothesis derived from our proteomic analysis. Together, these findings confirm that MLS0315771 interacts with bacterial cell membrane, affecting its membrane integrity through affecting the fatty acid-related pathway. These results provide further evidence supporting our earlier hypothesis that MLS0315771 targets cell membrane-associated processes, thereby offering an important mechanistic basis for its antibacterial activity. Moreover, ClpX is a gene that has been reported to play a pivotal role in S. aureus growth and virulence [23]. A clpX deletion mutant of S. aureus SA113 was generated using a gene knockout strategy. To characterize the antimicrobial susceptibility of the mutant strain, minimum inhibitory concentration (MIC) assays and growth curve analyses were performed ( Figure 5I-J and Table S5 ). Compared with the wild-type SA113 strain, deletion of clpX resulted in a twofold increase in the MIC of MLS0315771, from 6.25 μM to 12.5 μM. Significant differences in overall growth kinetics were observed between the wild-type and ΔClpx strains under standard culture conditions. These results indicate that loss of clpX reduces the susceptibility of S. aureus to MLS0315771 and we reasonably assume that ClpX may be related to the antibacterial activity of MLS0315771 against S. aureus . MLS0315771 safety evaluation The cytotoxic potential of MLS0315771 was assessed through CCK-8 and hemolysis assays. In mammalian cell lines, including LX-2, HEK-293T, Beas-2b, J774A.1, A549 and Huvec cells ( Figure 6 A-F ), cell viability dynamics were monitored over a 24-hour exposure period and the measurable cytotoxic effects were observed starting at 100 μM compared with untreated controls for most of the cell lines except LX-2 cells. And the CC 50 values for J774A.1 and A549 cells were over 100 μM. Notably, these concentrations exceeded the effective antibacterial levels, suggesting that MLS0315771 possesses a reasonable therapeutic window. Hemolysis analysis revealed that MLS0315771 caused negligible human and sheep erythrocyte lysis, even at concentrations as high as 50-200 μM, indicating good biocompatibility ( Figure 6 G and 6 H ). Considering that many high-efficacy antibiotics are often accompanied by severe side effects such as hepatotoxicity, nephrotoxicity, and immune suppression, these findings collectively support that MLS0315771 exhibits low cytotoxicity and an overall favorable safety profile. Discussion The continuous rise of multidrug-resistant (MDR) Gram-positive pathogens, particularly Staphylococcus aureus and Enterococcus faecium , poses a persistent global health challenge [24]. In order to address the growing number of drug-resistant strains and the scarcity of current treatment choices, it is imperative to develop alternative antimicrobial techniques to tackle drug-resistant bacteria, including the repurposing of existing medications [25]. In this regard, we effectively identified MLS0315771 with antibacterial activity by screening the compound library of clinical trials. MLS0315771, a competitive MPI inhibitor from the benzoisothiazolone class, could shift mannose flux to glycosylation in various cell lines, including certain CDG-Ia fibroblast lines [18]. It was found that MLS0315771 had the potential for the development and optimization of molecules that may eventually result in a novel medication against Lyme disease [26]. In this study, we identified MLS0315771 as a promising antibacterial compound exhibiting potent activity against a broad spectrum of clinically relevant Gram-positive bacteria, including MSSA, MRSA and linezolid-resistant E. faecium . The MIC data demonstrated that MLS0315771 displayed comparable or superior activity to vancomycin across multiple isolates, underscoring its potential as a novel lead compound for the treatment of refractory infections caused by resistant strains. Consistent with its low MIC values, time-kill assays revealed that MLS0315771 exerted rapid and sustained bactericidal effects against both S. aureus and E. faecalis isolates. Compared to vancomycin, MLS0315771 achieved a greater reduction in bacterial burden within 24 hours, suggesting that it acts through a time-dependent killing mechanism. This finding is significant given the increasing number of MRSA strains that have developed reduced susceptibility to vancomycin. The prolonged lag phase observed in bacterial growth curve analyses under subinhibitory concentrations of MLS0315771 further supports a concentration-dependent inhibition of bacterial proliferation, reflecting its robust antibacterial potency. Biofilm formation is one of the most critical virulence mechanisms of S. aureus and E. faecium , contributing to chronic and device-associated infections [27, 28]. Our findings demonstrated that MLS0315771 not only prevented biofilm formation at early developmental stages but also effectively disrupted mature biofilms. These results are particularly noteworthy since most conventional antibiotics exhibit poor efficacy against biofilm-embedded bacterial cells [29]. Confocal microscopy further confirmed the marked reduction in biofilm thickness and viability following MLS0315771 treatment, indicating that this compound may effectively combat persistent infections driven by biofilm-forming pathogens. However, more research is required to confirm MLS0315771's therapeutic effectiveness as an antibacterial drug against S. aureus infection, including in vivo trials and thorough clinical validation. To gain mechanistic insights into the antibacterial action of MLS0315771, proteomic profiling of S. aureus following exposure revealed extensive reprogramming of bacterial metabolism and virulence networks. Among the 236 differentially expressed proteins, downregulation of key virulence determinants such as delta-hemolysin (hld), staphylococcal secretory antigen ssaA2, and type VII secretion system components suggested a suppression of cytolytic activity and intercellular communication. The inhibition of hld, a central factor in membrane vesicle biogenesis, aligns with the observed disruption of membrane integrity, indicating that MLS0315771 may impair S. aureus pathogenicity by targeting membrane-associated processes [30]. In addition, the type VII secretion system (T7SS) has been recognized as a critical determinant of pathogenicity in S. aureus , functioning as a specialized apparatus for the translocation of virulence factors across the thick cell envelope [31]. In S. aureus , this system mediates the secretion of the nuclease toxin EsaD through a coordinated interaction with the accessory proteins EsaE and EsaG. Suppression of T7SS-related proteins, including EsaD, EsaE, and EssC, by MLS0315771 therefore implies impaired secretion of virulence factors and a disruption of membrane-coupled protein export machinery [32]. This observation reinforces the notion that MLS0315771 perturbs bacterial viability not only by compromising membrane integrity but also by attenuating T7SS-dependent virulence delivery, a dual action that could synergistically weaken S. aureus pathogenic potential. And Enrichment analysis highlighted pathways associated with nitrogen metabolism, purine biosynthesis, and pyruvate metabolism, implying that MLS0315771 not only compromises membrane function but also interferes with essential metabolic circuits necessary for bacterial survival. Proteomics can only identify the possible mechanism in a preliminary manner. More validation research is required by creating mutant strains and overexpressing elevated proteins for enzymatic assays. In general, this finding demonstrate the complex effects of MLS0315771 on bacterial physiology. Mechanistic assays provided further evidence that MLS0315771 targets the bacterial membrane. Fluorescence-based analyses revealed marked depolarization and increased permeability in S. aureus treated with MLS0315771, confirming membrane disruption as a primary antibacterial mechanism. Furthermore, phospholipids are crucial components of the bacterial membrane, and studies have shown that some antimicrobial peptides can permeate the membrane, influence enzyme activity inside the bacteria, disrupt substance metabolism, and eventually cause bacterial death [33]. However, the absence of changes in MICs upon supplementation with common membrane phospholipids (PC, PE, PG, and CL) suggested that MLS0315771 does not act through direct lipid binding. However, the attenuation of antibacterial activity in the presence of unsaturated long-chain fatty acids, including linoleic and arachidonic acids, indicates interference with fatty acid-related metabolic pathways. This result supports a model in which MLS0315771 perturbs bacterial membrane stability and function indirectly, possibly through modulation of lipid homeostasis or disruption of membrane-associated protein functions. Hemolysin, a major virulence factor secreted by Staphylococcus aureus , is classified into five types (α, β, γ, δ, ε) according to antigenicity. Among these, α-hemolysin exhibits the strongest pathogenic activity and has been most extensively studied [34]. The inhibition of α-hemolysin production further corroborates this membrane-targeting effect and suggests a secondary suppression of virulence expression. It is conceivable that MLS0315771 causes damage to bacterial cell membranes, which eventually results in bacterial death. Antibacterial compounds that target the bacterial cell membrane are anticipated to be a potential target for antibacterial agent screening because they have the potential to rapidly kill bacteria and provide advantages in lowering the tendency of bacteria to develop resistance [35, 36]. This offers important information about MLS0315771's possible function in rupturing bacterial cell membranes. It is plausible that MLS0315771 causes damage to bacterial cell membranes, which eventually results in bacterial death. In this study, although our primary analyses suggest that MLS0315771 exerts its antibacterial activity predominantly through disruption of the bacterial membrane, the altered susceptibility observed in the clpX deletion mutant indicates that additional intracellular factors may also contribute to the antibacterial response. ClpX is a member of the Clp/Hsp100 subfamily of AAA+ ATPases and plays a central role in protein quality control, stress adaptation, and growth regulation in S. aureus [37]. Functionally, ClpX recognizes specific peptide motifs on substrate proteins and cooperates with the ClpP protease to mediate ATP-dependent unfolding and degradation of target polypeptides [38]. Given the essential role of ClpX in maintaining cellular homeostasis, loss of clpX may indirectly affect bacterial tolerance to MLS0315771-induced stress, including membrane perturbation. However, our current data are limited to phenotypic observations, and no direct evidence supports a specific molecular interaction between MLS0315771 and ClpX. Therefore, it is more plausible that ClpX contributes to the adaptive or stress-response pathways that modulate bacterial susceptibility to MLS0315771 rather than serving as a direct antibacterial target. Further studies focusing on proteostasis regulation and stress signaling pathways will be required to clarify the precise role of ClpX in the antibacterial activity of MLS0315771 against S. aureus . Equally important, the cytotoxicity and hemolysis assays demonstrated that MLS0315771 possesses a favorable safety profile. Minimal hemolytic activity was observed even at high concentrations, and the cytotoxic effects toward mammalian cell lines were only detectable at doses substantially exceeding antibacterial levels. These findings indicate a promising therapeutic window and suggest that MLS0315771 could achieve effective antibacterial concentrations without exerting substantial toxicity on host tissues. Given that many potent antibiotics are associated with nephrotoxicity or hepatotoxicity, the low cytotoxicity of MLS0315771 enhances its potential for further development [39, 40]. Collectively, our findings suggest that MLS0315771 exerts multifaceted antibacterial effects, characterized by strong activity against MDR Gram-positive bacteria, potent antibiofilm capacity, and disruption of bacterial membrane integrity and virulence pathways. The integrated proteomic and mechanistic evidence indicates that membrane perturbation and inhibition of fatty acid-associated processes are central to its mode of action. Future studies focusing on identifying direct molecular targets and assessing in vivo pharmacokinetics will be critical for advancing MLS0315771 toward clinical translation. Overall, this study highlights MLS0315771 as a compelling candidate for further optimization and development as a novel antimicrobial agent capable of addressing the growing challenge of multidrug-resistant and biofilm-associated Gram-positive infections. Materials and Methods Bacterial Identification and Growth Conditions A total of 158 Gram-positive coccal isolates were included in this study, comprising Staphylococcus aureus (n = 69; MRSA, n = 44; MSSA, n = 25), Staphylococcus epidermidis (n = 26), and Enterococcus faecium (n = 37), along with other species as listed in Table 1. All clinical isolates were collected and identified from various specimens of 534 patients admitted to Shenzhen Nanshan People’s Hospital between 2011 and 2015 [41, 42]. Prior to experimental use, both clinical and reference strains were reconfirmed using a MALDI-TOF mass spectrometry (IVD MALDI Biotyper, Bruker, Karlsruhe, Germany). Verified isolates were preserved in tryptic soy broth (TSB) supplemented with 20% glycerol and stored at -80 °C for subsequent analyses. Determination of Minimum Inhibitory Concentrations (MICs) The MICs of antimicrobial compounds were determined using the standard broth microdilution method in cation-adjusted Mueller-Hinton broth (CAMHB), following the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100, 29 th ed.). Twofold serial dilutions of MLS0315771 were prepared and exposed to a series of half dilutions of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μM, and bacterial suspensions were adjusted to the appropriate inoculum density. The MIC was defined as the lowest concentration of compound completely inhibiting visible growth after incubation. S. aureus ATCC 29213, E. faecalis ATCC 29212, and E. faecalis OG1RF were used as quality control strains. MLS0315771 and vancomycin (VAN) were purchased from MCE (Princeton, United States). Bacterial Growth Curves Bacterial growth kinetics were monitored following a previously described method [43]. Single colonies of S. aureus and E. faecalis were inoculated into TSB and cultured overnight at 37 °C with shaking at 220 rpm. Overnight cultures (16-20 h) were diluted in fresh TSB containing appropriate concentrations of MLS0315771 and transferred to a honeycomb microplate. The optical density at 600 nm (OD₆₀₀) was continuously measured for 24 h using a Bioscreen C automated growth curve analyzer (Turku, Finland). Growth curves were plotted based on OD₆₀₀ readings over time. Time-Kill Assay Time-kill kinetics were determined according to a previously described method [44]. Single colonies were inoculated into 4-5 mL CAMHB and grown overnight at 37 °C with shaking at 220 rpm. The following day, cultures were diluted 1:100 into 6 mL fresh CAMHB and grown to the logarithmic phase (approximately 4-6 h). Log-phase cells were adjusted to ~1 × 10⁶ CFU/mL. MLS0315771 solutions at twice the desired final concentrations were prepared in CAMHB and mixed 1:1 with bacterial suspensions to yield a final bacterial density of ~5 × 10⁵ CFU/mL. Cultures were incubated statically at 37 °C, and aliquots (100 μL) were collected at 0, 3, 6, 8, and 24 h. Samples were serially diluted 10-fold in saline, plated (50 μL) onto CAMHB agar, and incubated for 24 h at 37 °C for colony CFU counting. Antibiofilm Activity Assay The antibiofilm efficacy of MLS0315771 was evaluated using a crystal violet staining method as described previously [45], with slight modifications. Briefly, overnight cultures of S. aureus and E. faecalis were diluted in fresh TSB supplemented with 2% glucose (TSBG) to a final concentration of approximately 1 × 10⁶ CFU/mL. Aliquots of 200 μL were dispensed into sterile 96-well polystyrene microplates, followed by treatment with MLS0315771 at concentrations corresponding to 1/2×, 1/4×, and 1/8× of the MIC. Plates were incubated statically at 37 °C for 24 h to allow biofilm formation. After incubation, planktonic cells were removed, and wells were gently rinsed with PBS to eliminate non-adherent bacteria. The remaining biofilms were stained with 0.1% crystal violet, washed, and solubilized in ethanol. The absorbance was then measured at 570 nm using a microplate reader (BioTek, USA) to quantify biofilm biomass. Confocal Laser Scanning Microscopy (CLSM) Analysis The structural changes of S. aureus biofilms following MLS0315771 exposure were visualized using confocal laser scanning microscopy (CLSM) as described in previous studies [46]. Overnight cultures of the S. aureus strains YUSA145, YUSA218 and CHS655 were diluted 1:200 in fresh TSBG and seeded into confocal dishes, followed by incubation for 24 h at 37 °C to establish mature biofilms. Subsequently, the biofilms were gently washed three times with sterile saline (0.9% NaCl) and treated with fresh TSBG medium containing MLS0315771 at 8 × MIC or an equivalent volume of DMSO as a control. After a further 24 h of incubation, biofilms were washed with sterile PBS and stained using the Live/Dead BacLight viability kit containing SYTO9 and propidium iodide (PI) for 30 min in the dark. Stained samples were observed using a confocal laser scanning microscope, and biofilm architecture and bacterial viability were analyzed from the acquired images. LC–MS/MS Proteomics Analysis Quantitative proteomic profiling of S. aureus was performed using LC–MS/MS, following procedures modified from a previously described protocol [47]. Briefly, S. aureus strain YUSA145 was grown in TSB to the logarithmic growth phase and subsequently treated with MLS0315771 at 1/2 × MIC or an equivalent volume of DMSO for 2 h at 37 °C with agitation at 220 rpm. Following treatment, bacterial cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C, and washed twice with pre-chilled PBS. Total proteins were extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail (Beyotime, Shanghai, China). Cell disruption was achieved by mechanical agitation with 0.1 mm glass beads at 70 Hz, and the lysates were clarified by centrifugation (12,000 rpm, 15 min, 4 °C). Protein concentrations were determined using the BCA assay kit (Thermo Fisher Scientific, USA). For digestion, equal amounts of protein were reduced with 10 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA) at 70 °C for 1 h, followed by alkylation with 50 mM iodoacetamide (IAA; Sigma-Aldrich) for 15 min in the dark. Samples were then desalted and concentrated using Amicon Ultra centrifugal filters, and digested overnight with trypsin (Promega, Madison, WI, USA) at 37 °C. The resulting peptides were separated on a C18 reverse-phase column (75 μm × 250 mm, Acclaim PepMap RSLC, 2 μm) prior to analysis. Mass spectrometric detection was carried out on a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). The obtained raw spectra were processed and analyzed for protein identification and quantification using standard bioinformatic pipelines. Membrane Permeability and Depolarization Assay The effects of MLS0315771 on bacterial membrane integrity and potential were evaluated based on a previously reported protocol [44], with minor modifications. Staphylococcus aureus SA113 was first cultured overnight in TSB medium at 37 °C. The bacterial suspension was then adjusted to 1 × 10⁷ CFU/mL in HEPES buffer (5 mM HEPES, 20 mM glucose, pH 7.4) and incubated with either 1 µM propidium iodide (PI) or 1 µM Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol [DiBAC₄(3)] for 2 h to allow dye uptake. Subsequently, KCL was introduced to a final concentration of 0.1 M to equilibrate intracellular and extracellular potassium levels. Fluorescence measurements were performed in a 96-well microplate after allowing the samples to reach a stable baseline. MLS0315771 was then added to achieve final concentrations equivalent to 1×, 2×, 4× and 8× MIC, and fluorescence signals were continuously monitored using a Cytation 1 Cell Imaging Multimode Reader (BioTek, USA). The excitation/emission wavelengths used were 217/340 nm for PI and 540/560 nm for DiBAC₄(3). Untreated cells served as the negative control, while 0.1% Triton X-100 was included as a positive control to indicate complete membrane disruption. Antibacterial Activity of MLS0315771 in the Presence of Phospholipids and Fatty Acids To evaluate the influence of phospholipids and fatty acids on the antibacterial efficacy of MLS0315771, the compounds were first dissolved in methanol. Arachidonic acid (A5837, Sigma-Aldrich, USA) and other related lipids were tested using a checkerboard microdilution approach as previously described [48]. Briefly, S. aureus SA113 was cultured in TSB medium, and assays were performed with phospholipids at concentrations ranging from 8-128 μg/mL and fatty acids at 3.9-500 μM. Serial dilutions of fatty acids and phospholipids were arrayed along the abscissa and ordinate, respectively, in 96-well plates, followed by inoculation with bacterial suspensions at 1 × 10⁶ CFU/mL. After incubation at 37 °C for 24 h, bacterial growth was quantified by measuring the optical density at 600 nm (OD₆₀₀) using a microplate reader. The resulting data were used to determine the effect of lipid components on the MIC of MLS0315771. Construction of the ClpX Deletion Mutant The ClpX-deficient strain of Staphylococcus aureus SA113 was constructed using the temperature-sensitive shuttle vector pKOR1 as previously described [49]. Briefly, DNA fragments flanking the ClpX locus were amplified from the SA113 genomic DNA by PCR and subsequently cloned into the pKOR1 plasmid to generate the allelic exchange construct. The recombinant plasmid was introduced into S. aureus SA113 by electroporation. Transformants were initially selected on tryptic soy agar (TSA) plates supplemented with chloramphenicol (10 μg/mL). Putative ClpX knockout clones were screened by PCR using genomic DNA as the template, and correct allelic replacement was further confirmed by DNA sequencing. Finally, the pKOR1 plasmid was eliminated from the mutant strain following established plasmid-curing procedures. α-Hemolysin Secretion Assay The inhibitory effect of MLS0315771 on S. aureus α-hemolysin secretion was assessed using rabbit red blood cells (RBCs), following a modified protocol from a previous study [50]. Overnight cultures of S. aureus USA300 were diluted 1:200 into TSB containing 1/8×, 1/4×, 1/2× and 1× MIC concentrations of MLS0315771 and incubated at 37 °C for 12 h. The supernatants were harvested by centrifugation and sterilized through 0.22 μm filters. Subsequently, 100 μL of the sterile supernatant was combined with an equal volume of 2% rabbit RBC suspension and incubated at 37 °C for 30 min. After centrifugation at 1500 rpm, 100 μL of the supernatant was transferred to a 96-well plate for absorbance measurement at 540 nm. PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. The hemolytic activity was calculated using the formula: Hemolysis rate (%) = [(OD sample -OD blank )/(OD positive -OD blank )] × 100. All assays were performed in triplicate to ensure reproducibility. Hemolytic Activity The hemolytic potential of MLS0315771 was evaluated using fresh human and sheep erythrocytes as described previously [51]. Briefly, erythrocytes were washed three times with sterile phosphate-buffered saline (PBS) and resuspended to a final concentration of 4% (v/v). Aliquots of 100 μL erythrocyte suspension were distributed into 96-well plates and treated with serial dilutions of MLS0315771 (0.195–100 μM for human and 0.75–200 μM for sheep). The mixtures were incubated at 37 °C for 30 min, followed by centrifugation at 1000 × g for 5 min. Subsequently, 100 μL of the supernatant was carefully transferred to a new 96-well flat-bottom plate, and the absorbance at 540 nm (OD₅₄₀) was measured using a microplate reader. PBS and 1% Triton X-100 served as the negative and positive controls, respectively. The percentage of hemolysis was calculated using the following formula: Hemolysis rate (%) = [(OD sample -OD negative )/(OD positive -OD negative )] × 100. Cytotoxicity Assay The cytotoxic effects of MLS0315771 on mammalian cells were determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s protocol. LX-2, HEK-293T, Beas-2b, J774A.1, A549 and Huvec cells were seeded into 96-well plates at a density of 6 × 10³ cells/well in complete Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight incubation at 37 °C under 5% CO₂, the cells were exposed to fresh medium containing varying concentrations of MLS0315771 (0–100 μM, including 0.78, 1.56, 3.125, 12.5, 25, 50, and 100 μM) for 24 h. Following treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were further incubated for 1 h under the same conditions. Absorbance was recorded at 450 nm (OD₄₅₀) using a microplate reader. Cell viability was expressed as a ratio of experimental to control groups. The CC50 values were determined using the GraphPad Prism software (version 10.1.2). The data reported here are from three different experiments. Declarations Ethics approval and consent to participate All methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital and the 1964 Helsinki Declaration and its later amendments, or comparable ethical standards. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines in China (Clinical trial number: not applicable). Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People’s Hospital. Authors’ Contributions + Z.X., Y.T., J.M., and P.L. contributed equally to this work. S.H., Z.C., and T.H. conceived and designed the study. Z.X., Y.T., J.M., and P.L. performed the main experiments, including antibacterial activity assays, antibiofilm assays, and mechanistic studies. Z.W. and Z.Y. assisted with bacterial strain collection, data acquisition, and experimental validation. B.B. contributed to data analysis and interpretation. Y.T. and Z.X. drafted the manuscript. B.B., Z.C., S.H., and T.H. critically revised the manuscript for important intellectual content. Z.C., S.H., and T.H. acquired funding and supervised the project. All authors read and approved the final manuscript. Consent for publication Not applicable. Competing interests The authors declare that they have no conflicts of interest. Data Availability Statement The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD073535. Funding This work was supported by the following grants: National Natural Science Foundation of China (82572621); Sanming Project of Medicine in Shenzhen (SMZ202303037); Shenzhen Key Medical Discipline Construction Fund (SZXK06162); Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20240813114518024, KJZD20240903103500002) and the Shenzhen Nanshan District Scientific Research Program of the People’s Republic of China (NS2024044Y, NS2024006Y, NS2024007Y, NSZD2024036, NSZD2024023, NSZD2024032, NSZD2025001, NSZD2025005). References Salam MA, Al-Amin MY, Salam MT, Pawar JS, Akhter N, Rabaan AA, Alqumber MA: Antimicrobial resistance: a growing serious threat for global public health . In: Healthcare: 2023 . MDPI: 1946. Bashabsheh RH, AL-Fawares Ol, Natsheh I, Bdeir R, Al-Khreshieh RO, Bashabsheh HH: Staphylococcus aureus epidemiology, pathophysiology, clinical manifestations and application of nano-therapeutics as a promising approach to combat methicillin resistant Staphylococcus aureus . Pathogens and Global Health 2024, 118 (3):209-231. Pal M, Shuramo MY, Tewari A, Srivastava JP, Steinmetz CH: Staphylococcus aureus from a commensal to zoonotic pathogen: a critical appraisal . International Journal of Clinical and Experimental Medicine Research 2023, 7 (2). Touaitia R, Mairi A, Ibrahim NA, Basher NS, Idres T, Touati A: Staphylococcus aureus: A Review of the Pathogenesis and Virulence Mechanisms . Antibiotics 2025, 14 (5):470. Parsons JB, Mourad A, Conlon BP, Kielian T, Fowler Jr VG: Methicillin-resistant and susceptible Staphylococcus aureus: tolerance, immune evasion and treatment . Nature Reviews Microbiology 2025:1-19. Ramírez-Larrota JS, Eckhard U: An introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion . Biomolecules 2022, 12 (2):306. Aboelnaga N, Elsayed SW, Abdelsalam NA, Salem S, Saif NA, Elsayed M, Ayman S, Nasr M, Elhadidy M: Deciphering the dynamics of methicillin-resistant Staphylococcus aureus biofilm formation: from molecular signaling to nanotherapeutic advances . Cell Communication and Signaling 2024, 22 (1):188. Howden BP, Giulieri SG, Wong Fok Lung T, Baines SL, Sharkey LK, Lee JY, Hachani A, Monk IR, Stinear TP: Staphylococcus aureus host interactions and adaptation . Nature Reviews Microbiology 2023, 21 (6):380-395. Panneerselvam VP, Vajravelu LK, Thulukanam J, Lathakumari RH, Vimala PB, Nair DM: Genomic adaptations of methicillin-resistant Staphylococcus aureus in healthcare environments . Ecological Genetics and Genomics 2025, 34 :100331. Kaushik A, Kest H, Sood M, Steussy BW, Thieman C, Gupta S: Biofilm producing methicillin-resistant Staphylococcus aureus (MRSA) infections in humans: Clinical implications and management . Pathogens 2024, 13 (1):76. Alsolami A, ALGhasab NS, Alharbi MS, Bashir AI, Saleem M, Syed Khaja AS, Aldakheel DF, Rakha E, Alshammari JA, Taha TE: Community-acquired methicillin-resistant Staphylococcus aureus in hospitals: Age-specificity and potential zoonotic–zooanthroponotic transmission dynamics . Diagnostics 2023, 13 (12):2089. Rajput P, Nahar KS, Rahman KM: Evaluation of antibiotic resistance mechanisms in gram-positive bacteria . Antibiotics 2024, 13 (12):1197. Yarahmadi A, Najafiyan H, Yousefi MH, Khosravi E, Shabani E, Afkhami H, Aghaei SS: Beyond antibiotics: exploring multifaceted approaches to combat bacterial resistance in the modern era: a comprehensive review . Frontiers in Cellular and Infection Microbiology 2025, 15 :1493915. Muteeb G, Rehman MT, Shahwan M, Aatif M: Origin of antibiotics and antibiotic resistance, and their impacts on drug development: A narrative review . Pharmaceuticals 2023, 16 (11):1615. Barbarossa A, Rosato A, Corbo F, Clodoveo ML, Fracchiolla G, Carrieri A, Carocci A: Non-antibiotic drug repositioning as an alternative antimicrobial approach . Antibiotics 2022, 11 (6):816. Mishra AS, Vasanthan M, Malliappan SP: Drug repurposing: A leading strategy for new threats and targets . ACS pharmacology & translational science 2024, 7 (4):915-932. Krishnamurthy N, Grimshaw AA, Axson SA, Choe SH, Miller JE: Drug repurposing: a systematic review on root causes, barriers and facilitators . BMC health services research 2022, 22 (1):970. Sharma V, Ichikawa M, He P, Bravo Y, Dahl R, Ng BG, Cosford ND, Freeze HH: Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts . Journal of Biological Chemistry 2011, 286 (45):39431-39438. Liang R, Ye Z-W, Qin Z, Xie Y, Yang X, Sun H, Du Q, Luo P, Tang K, Hu B: PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness . Nature Communications 2024, 15 (1):2144. Ma J, Chen T, Wu S, Yang C, Bai M, Shu K, Li K, Zhang G, Jin Z, He F: iProX: an integrated proteome resource . Nucleic acids research 2019, 47 (D1):D1211-D1217. Chen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, Fu Y, Yang C, Li M, Wu S: iProX in 2021: connecting proteomics data sharing with big data . Nucleic acids research 2022, 50 (D1):D1522-D1527. Verdon J, Girardin N, Lacombe C, Berjeaud J-M, Héchard Y: δ-hemolysin, an update on a membrane-interacting peptide . Peptides 2009, 30 (4):817-823. Conlon BP, Nakayasu ES, Fleck LE, LaFleur MD, Isabella VM, Coleman K, Leonard SN, Smith RD, Adkins JN, Lewis K: Activated ClpP kills persisters and eradicates a chronic biofilm infection . Nature 2013, 503 (7476):365-370. Alara JA, Alara OR: An overview of the global alarming increase of multiple drug resistant: a major challenge in clinical diagnosis . Infectious Disorders-Drug TargetsDisorders) 2024, 24 (3):26-42. Tarin-Pello A, Suay-Garcia B, Perez-Gracia M-T: Antibiotic resistant bacteria: current situation and treatment options to accelerate the development of a new antimicrobial arsenal . Expert review of anti-infective therapy 2022, 20 (8):1095-1108. Dutta S, Rana VS, Backstedt BT, Shakya AK, Kitsou C, Yas OB, Smith AA, Ronzetti MH, Lipman RM, Araujo-Aris S: Borrelial phosphomannose isomerase as a cell surface localized protein that retains enzymatic activity and promotes host-pathogen interaction . Mbio 2025, 16 (3):e03609-03624. Lanka S, Katta A, Kovvali M, Pandrangi S: Enterococcus faecium Virulence Factors and Biofilm Components: Synthesis, Structure, Function, and Inhibitors . In: ESKAPE Pathogens: Detection, Mechanisms and Treatment Strategies. Springer; 2024: 209-226. Nikolic P, Mudgil P: The cell wall, cell membrane and virulence factors of Staphylococcus aureus and their role in antibiotic resistance . Microorganisms 2023, 11 (2):259. Ciarolla AA, Lapin N, Williams D, Chopra R, Greenberg DE: Physical approaches to prevent and treat bacterial biofilm . Antibiotics 2022, 12 (1):54. Chen J, Lv Y, Shang W, Yang Y, Wang Y, Hu Z, Huang X, Zhang R, Yuan J, Huang J: Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles . Frontiers in Microbiology 2023, 14 :1254367. Bowman L, Palmer T: The type VII secretion system of Staphylococcus . Annual review of microbiology 2021, 75 (1):471-494. Cao Z, Casabona MG, Kneuper H, Chalmers JD, Palmer T: The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria . Nature microbiology 2016, 2 (1):1-11. Huang X, Li G: Antimicrobial peptides and cell-penetrating peptides: non-antibiotic membrane-targeting strategies against bacterial infections . Infection and Drug Resistance 2023:1203-1219. Eltwisy HO, Twisy HO, Hafez MH, Sayed IM, El-Mokhtar MA: Clinical infections, antibiotic resistance, and pathogenesis of Staphylococcus haemolyticus . Microorganisms 2022, 10 (6):1130. Ganesan N, Mishra B, Felix L, Mylonakis E: Antimicrobial peptides and small molecules targeting the cell membrane of Staphylococcus aureus . Microbiology and Molecular Biology Reviews 2023, 87 (2):e00037-00022. Veiko AG, Olchowik-Grabarek E, Sekowski S, Roszkowska A, Lapshina EA, Dobrzynska I, Zamaraeva M, Zavodnik IB: Antimicrobial activity of quercetin, naringenin and catechin: Flavonoids inhibit Staphylococcus aureus-induced hemolysis and modify membranes of bacteria and erythrocytes . Molecules 2023, 28 (3):1252. Camberg JL, Hoskins JR, Wickner S: ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics . Proceedings of the National Academy of Sciences 2009, 106 (26):10614-10619. Gersch M, Famulla K, Dahmen M, Göbl C, Malik I, Richter K, Korotkov VS, Sass P, Rübsamen-Schaeff H, Madl T: AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control . Nature communications 2015, 6 (1):6320. Sharma V, Singh TG: Drug induced nephrotoxicity-A mechanistic approach . Molecular Biology Reports 2023, 50 (8):6975-6986. Doß S, Blessing C, Haller K, Richter G, Sauer M: Influence of antibiotics on functionality and viability of liver cells in vitro . Current issues in molecular biology 2022, 44 (10):4639-4657. Wen Z, Chen C, Shang Y, Fan K, Li P, Li C, Zheng J, Deng Q, Yu Z: Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ . Phytomedicine 2024, 130 :155590. Chen C, Li D, Shang Y, Lin Z, Wen Z, Li P, Yu Z, Chen Z, Liu X: Antibacterial Activity and Mechanism of Candesartan Cilexetil against Enterococcus faecalis . ACS omega 2024, 9 (19):21510-21519. Liu X, Xiong Y, Shi Y, Deng X, Deng Q, Liu Y, Yu Z, Li D, Zheng J, Li P: In vitro activities of licochalcone A against planktonic cells and biofilm of Enterococcus faecalis . Frontiers in Microbiology 2022, 13 :970901. Xiong Y, Chen Z, Bai B, Peng Y, Liu S, Fang D, Wen Z, Shang Y, Lin Z, Han S: Thiazolopyrimidinone Derivative H5–23 Enhances Daptomycin Activity against Linezolid-Resistant Enterococcus faecalis by Disrupting the Cell Membrane . ACS Infectious Diseases 2023, 9 (12):2523-2537. Zheng J, Shang Y, Wu Y, Zhao Y, Chen Z, Lin Z, Li P, Sun X, Xu G, Wen Z: Loratadine inhibits Staphylococcus aureus virulence and biofilm formation . IScience 2022, 25 (2). Zheng J, Liu X, Xiong Y, Meng Q, Li P, Zhang F, Liu X, Lin Z, Deng Q, Wen Z: AMXT-1501 targets membrane phospholipids against Gram-positive and-negative multidrug-resistant bacteria . Emerging Microbes & Infections 2024, 13 (1):2321981. Meng Q, Wang X, Huang X, Li C, Yu Z, Li P, Liu X, Wen Z: Repurposing Benzbromarone as an Antibacterial Agent against Gram-Positive Bacteria . ACS Infectious Diseases 2024, 10 (12):4208-4221. Lee AS, De Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, Harbarth S: Methicillin-resistant Staphylococcus aureus . Nature reviews Disease primers 2018, 4 (1):1-23. Bae T, Schneewind O: Allelic replacement in Staphylococcus aureus with inducible counter-selection . Plasmid 2006, 55 (1):58-63. Goc A, Sumera W, Rath M, Niedzwiecki A: Inhibition of α-hemolysin activity of Staphylococcus aureus by theaflavin 3, 3’-digallate . Plos one 2023, 18 (8):e0290904. Chen Z, Song K, Shang Y, Xiong Y, Lyu Z, Chen J, Zheng J, Li P, Wu Y, Gu C: Selection and identification of novel antibacterial agents against planktonic growth and biofilm formation of Enterococcus faecalis . Journal of Medicinal Chemistry 2021, 64 (20):15037-15052. Additional Declarations No competing interests reported. Supplementary Files TableS2.xlsx MLS.docx Cite Share Download PDF Status: Published Journal Publication published 29 Apr, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 26 Mar, 2026 Reviews received at journal 17 Mar, 2026 Reviewers agreed at journal 17 Feb, 2026 Reviews received at journal 17 Feb, 2026 Reviewers agreed at journal 15 Feb, 2026 Reviewers agreed at journal 30 Jan, 2026 Reviewers invited by journal 28 Jan, 2026 Editor assigned by journal 28 Jan, 2026 Editor invited by journal 26 Jan, 2026 Submission checks completed at journal 26 Jan, 2026 First submitted to journal 26 Jan, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8617708","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581776303,"identity":"fc0db0d1-c5be-4b38-b7df-ec3df9e09e03","order_by":0,"name":"Zhichao Xu","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Xu","suffix":""},{"id":581776304,"identity":"ffcd275a-00f3-4338-9cde-ccdf28a8397f","order_by":1,"name":"Yuanyuan Tang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Tang","suffix":""},{"id":581776305,"identity":"14cff348-ae88-4da1-af3c-198be0919e46","order_by":2,"name":"Junhua Ma","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Ma","suffix":""},{"id":581776306,"identity":"c6b9b2f1-770c-41b3-8d32-3be11aac44bf","order_by":3,"name":"Peiyu Li","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Peiyu","middleName":"","lastName":"Li","suffix":""},{"id":581776307,"identity":"ed4b1f13-93d2-4586-87fb-84b74218502f","order_by":4,"name":"Zewen Wen","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Zewen","middleName":"","lastName":"Wen","suffix":""},{"id":581776308,"identity":"cfd11da1-29fd-46b9-9322-c1ea874ed1bd","order_by":5,"name":"Zhijian Yu","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Zhijian","middleName":"","lastName":"Yu","suffix":""},{"id":581776309,"identity":"1ae7e2c3-1c8b-4125-b4df-45e4ab1d9fb4","order_by":6,"name":"Bing Bai","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Bai","suffix":""},{"id":581776310,"identity":"25e1bb0e-75e3-47a8-aeef-43fa11964bcc","order_by":7,"name":"Zhong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIie3RIQvCQBTA8RsHZzldfSviR3iyojDwg1h2CEuzGxYmCyuCdd9jYB4+WBKsC4Yls3HB4GEzyJ3NcL/8/nD3HmOO84d87u17GCLpc069VRKUBeHykEyDUiRoleClTWAnziFe5QzsXtalCJ1sVE2SIcuitbHwqhTn1eKmTjRuetYm29yUcEhxA/Kuk0mMXk7mROiEnoJUXUgEq0TKVuUgKERum8CoIBboJQPpJcc2f1mRVw7vUx6J+kcWmZNP8W/jjuM4zjcvkh5BJeg7stQAAAAASUVORK5CYII=","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":true,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Chen","suffix":""},{"id":581776311,"identity":"b9a0a456-67b9-474e-95d2-f65f90d535f3","order_by":8,"name":"Shiqing Han","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"prefix":"","firstName":"Shiqing","middleName":"","lastName":"Han","suffix":""},{"id":581776314,"identity":"8395eacf-bb9f-4225-aee3-f1dc312e602c","order_by":9,"name":"Tieying Hou","email":"","orcid":"","institution":"Shenzhen Nanshan People’s Hospital and Affiliated Nanshan Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Tieying","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2026-01-16 11:09:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8617708/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8617708/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-026-05074-9","type":"published","date":"2026-04-29T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101504772,"identity":"b0bc767e-b06a-40eb-8034-08008e04331d","added_by":"auto","created_at":"2026-01-30 14:19:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":169264,"visible":true,"origin":"","legend":"\u003cp\u003eMLS0315771 significantly inhibited the growth of Gram-positive bacteria\u0026nbsp; \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e aureus \u003c/em\u003eand \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A to D) The planktonic cells of \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e aureus\u003c/em\u003e CHS101, YUSA6, YUSA103 and YUSA129, and (E and F) \u003cem\u003eE. faecalis\u003c/em\u003e OG1RF and EF16C166 were treated with 1/8 ×, 1/4 ×, 1/2 ×, and 1 × MIC of MLS0315771. The OD\u003csub\u003e600\u003c/sub\u003e value of bacterial cells was measured at 1-h intervals for 24 h by automatic bacterial growth instrument. TSB without MLS0315771 was used as a negative control. Data are shown as Mean ± SEM (n = 3). All experiments were performed in triplicate.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/1d7ef32a59cb18e531250169.png"},{"id":101504767,"identity":"c73be404-2a84-4a0f-9e96-5dfb8975ad7a","added_by":"auto","created_at":"2026-01-30 14:19:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31799,"visible":true,"origin":"","legend":"\u003cp\u003eTime-killing assay of MLS0315771 with various concentrations against the planktonic growth of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis \u003c/em\u003eisolates.\u003c/p\u003e\n\u003cp\u003e(A-B) \u003cem\u003eS. aureus\u003c/em\u003e ATCC29213 and YUSA145 and (C) \u003cem\u003eE. faecalis \u003c/em\u003eEF16C51 were challenged with 4 × and 8 × MIC of MLS0315771 as well as 8 × MIC of vancomycin. Bacteria growth in TSB without MLS0315771 was used as an untreated control. Data are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/88de7f0cb6aa7ae548913240.jpeg"},{"id":101752460,"identity":"4bcf7123-1030-430e-963d-1f50df914fe9","added_by":"auto","created_at":"2026-02-03 10:27:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109838,"visible":true,"origin":"","legend":"\u003cp\u003eAntibiofilm activity of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e. (A-B) MSSA and MRSA isolates were tested for the inhibitory effect of MLS0315771 on the biofilm formation under different concentrations (1/16 ×, 1/8 ×, 1/4 ×, 1/2 × MIC). (C) \u003cem\u003eS. aureus\u003c/em\u003eisolates were tested for the eradication effect of MLS0315771. (D) \u003cem\u003eS. aureus\u003c/em\u003e YUSA145, YUSA218 and CHS655 treated with 8 × MIC of MLS0315771 for 24 hours were stained with SYTO9/PI nucleic acid dye and observed under CLSM to visualize biofilm cells. Data are represented as means ± SD, n=3. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/6abc58b969da82883bc63ed2.jpeg"},{"id":101504774,"identity":"0ad2cf7c-c832-45dc-90dc-58fde0857cb3","added_by":"auto","created_at":"2026-01-30 14:19:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123727,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic response of \u003cem\u003eS. aureus\u003c/em\u003eYUSA145 to MLS0315771. (A-B) Differential protein volcano plot and statistical analysis after treatment with a sub-inhibitory concentration of MLS0315771. The x-axis represents the fold change of differentially expressed proteins between the MLS0315771 treated group and the untreated group of \u003cem\u003eS. aureus\u003c/em\u003e, and the y-axis represents the significance of the difference (p-value). Red dots represent upregulated proteins after treatment, while blue dots represent downregulated proteins. (C) KEGG analysis of \u003cem\u003eS. aureus\u003c/em\u003e after treatment with MLS0315771. (D) GO biological process analysis of differentially expressed proteins. (E) PPI network analysis of differentially expressed proteins between the MLS0315771 treatment group and the DMSO group in \u003cem\u003eS. aureus\u003c/em\u003e. The PPI analysis was based on the KEGG database. Rectangles represent GO/KEGG pathways, with colors indicating the significance of the p-values. Circles represent proteins or genes, with colors representing fold changes.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/82408c0095a4b0d8f8b75444.jpeg"},{"id":101752459,"identity":"e8041174-1bf7-4e52-bdfc-bf55c90b77ff","added_by":"auto","created_at":"2026-02-03 10:27:36","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101251,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of MLS0315771 on \u003cem\u003eS. aureus\u003c/em\u003e cell membrane. (A-D) Membrane permeability (A-B) and depolarization (C-D) by MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e SA113. (E to G) Changes in the MIC values of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e SA113 after the addition of different concentrations of fatty acids linoleic acid, γ-linoleic acid and arachidonic acid. (H) Impact of α-hemolysin production in \u003cem\u003eS. aureus\u003c/em\u003e USA300 after the addition of different concentrations of MLS0315771. (I-J) The planktonic cells of WT and ΔClpx \u003cem\u003eS. aureus\u003c/em\u003e were treated with 1.56, 3.125, 6.25, 12.5 and 25 μM of MLS0315771.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/db008d6c48ee38f2ecf9e1f2.jpeg"},{"id":101504769,"identity":"4e0b6b8c-7be2-42ab-8385-c63703755364","added_by":"auto","created_at":"2026-01-30 14:19:58","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":160958,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity and hemolysis activity of MLS0315771. (A to F) The cytotoxicity of MLS0315771 on LX-2, HEK-293T, Beas-2b, J774A.1, A549 and Huvec cells. These cells were incubated overnight in 96-well plates and treated with different concentrations (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μg/ml) of MLS0315771. After 24 h of treatment, the viability of the cells was assessed using the CCK-8 assay, and the optical density at 450 nm was measured using a Microplate Reader. CC\u003csub\u003e50\u003c/sub\u003e represents the half-maximal inhibitory concentration for cell death, and it was calculated by GraphPad Prism version 10.1.2 software. (G-H) Hemolysis of MLS0315771 of human and sheep erythrocytes. The concentration of MLS0315771 ranged 0.195 to 100 μM for human and ranged 0.78 to 200 μM for sheep. Data are represented as means ± SD, n = 3. ***P \u0026lt; 0.001 and ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/91aeec421269d12855a0e6e6.jpeg"},{"id":108437780,"identity":"c97a7c77-5f97-4363-90d1-3ae6749e8453","added_by":"auto","created_at":"2026-05-04 16:03:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1164089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/352f65ee-725e-4241-b639-9913dab855df.pdf"},{"id":101504773,"identity":"b7a6e111-2307-4238-9f83-03457156a00c","added_by":"auto","created_at":"2026-01-30 14:19:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":104034,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/e2be6bcf6f31e6edcedf5a96.xlsx"},{"id":101752181,"identity":"c441d73f-338f-470b-955e-f26ca8adcaec","added_by":"auto","created_at":"2026-02-03 10:25:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":122367,"visible":true,"origin":"","legend":"","description":"","filename":"MLS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8617708/v1/8bfc1d3c74d7374ca138299d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe continuous rise and global dissemination of antibiotic-resistant bacteria have become one of the most critical threats to modern healthcare [1]. Among these pathogens, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u0026mdash;a small, spherical, Gram-positive bacterium belonging to the phylum Firmicutes\u0026mdash;stands out as a major clinical concern [2]. As a commensal organism, \u003cem\u003eS. aureus\u003c/em\u003e is commonly found colonizing the human nasal cavity, skin, and gastrointestinal tract without causing harm under normal circumstances [3]. However, when the integrity of host barriers is compromised, this seemingly benign bacterium can rapidly transition into an opportunistic pathogen capable of triggering a spectrum of diseases, ranging from mild skin and soft tissue infections to severe and life-threatening conditions such as bacteremia and endocarditis [4]. The pathogenicity of \u003cem\u003eS. aureus\u003c/em\u003e is largely attributed to its arsenal of virulence factors, which facilitate adhesion, immune evasion, and tissue invasion [5]. Notably, the bacterium can effectively escape innate and adaptive immune responses either by secreting a variety of toxins and enzymes or by forming robust biofilms on host tissues and medical device surfaces [6, 7]. These biofilms provide a protective niche that enhances bacterial survival and contributes to chronic and recurrent infections [8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDriven by environmental selection pressure and genetic adaptability, \u003cem\u003eS. aureus\u003c/em\u003e has developed resistance to multiple classes of antibiotics [9]. The emergence of methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) in particular has posed a formidable clinical challenge, rendering many first-line \u0026beta;-lactam antibiotics ineffective [4, 10]. According to the 2024 CHINET study (https://www.chinets.com/Data/AntibioticDrugFast), the detection rate of MRSA in clinical \u003cem\u003eS. aureus\u003c/em\u003e isolates has steadily decreased since 2005, reaching 29.2% in 2024. Despite a gradual decline in MRSA detection rates in recent years, \u003cem\u003eS. aureus\u003c/em\u003e remains among the leading causes of both community- and hospital-acquired infections worldwide [11]. The continuing evolution of multidrug-resistant (MDR) strains, especially those resistant to last-resort antibiotics such as vancomycin, linezolid, and daptomycin, highlights the urgent need for novel therapeutic agents that can effectively target both planktonic and biofilm-associated forms of this pathogen [12, 13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe discovery and development of new antibiotics remain time-consuming, resource-intensive, and often unsuccessful, as many candidates fail in clinical trials due to safety or efficacy concerns [14]. In contrast, drug repurposing by identifying new antimicrobial uses for existing compounds offers a practical and efficient alternative [15]. This strategy capitalizes on the established pharmacological and toxicological profiles of approved drugs, thereby reducing both development cost and risk [16, 17]. As a result, repurposing has become an increasingly attractive approach in the search for novel antimicrobial agents. MLS0315771 is a potent competitive phosphomannose isomerase (MPI) inhibitor that can divert Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation [18, 19]. MLS0315771 also increases mannose metabolic flux toward glycosylation in zebrafish embryos [18]. However, the potential of MLS0315771 in antimicrobial therapy has not been adequately investigated. In this study, we present the first evidence that MLS0315771 possesses potent and broad-spectrum antibacterial activity against both planktonic and biofilm-associated Gram-positive bacteria. Using \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e as representative models, we found that MLS0315771 not only inhibits bacterial growth but also exerts bactericidal effects and markedly suppresses biofilm development, while exhibiting minimal hemolytic activity and limited cytotoxicity toward mammalian cells. To further uncover its underlying antibacterial mechanism, a series of complementary analyses were conducted, including quantitative proteomic profiling, membrane permeability and depolarization assays and checkerboard assays with phospholipids and fatty acids. It was found that MLS0315771 effectively kills \u003cem\u003eS. aureus\u003c/em\u003e in vitro by targeting bacterial membranes. Taken together, these results highlight MLS0315771 as a promising candidate for the development of new therapeutic strategies targeting multidrug-resistant and biofilm-forming Gram-positive pathogens.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMLS0315771 Exhibits Potent Antibacterial Activity Against Multiple Gram-Positive Bacteria, Including MRSA and Linezolid-Resistant Strains\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 158 clinical isolates of Gram-positive bacteria were included in this study. Among \u003cem\u003eS. aureus\u003c/em\u003e isolates, methicillin-susceptible \u003cem\u003eS. aureus\u003c/em\u003e (MSSA) strains were most frequently recovered from wound specimens (30.77%), whereas methicillin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (MRSA) isolates were predominantly derived from sputum samples (52.94%) (\u003cstrong\u003eFigure S1\u003c/strong\u003e). For the 37 \u003cem\u003eE. faecium\u003c/em\u003e clinical isolates, including 5 isolates intermediate to and 14 isolates resistant to linezolid, urine was the most common source (42.86%)..\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMLS0315771\u0026nbsp;exhibited strong antibacterial activity against both MSSA and MRSA isolates, with MIC₅₀/₉₀ values of 25/25 \u0026mu;M for each (\u003cstrong\u003eTable 1\u003c/strong\u003e). Notably, MLS0315771 remained active against \u003cem\u003eE. faecium\u003c/em\u003e isolates displaying intermediate or complete resistance to linezolid (\u003cstrong\u003eTable S1\u003c/strong\u003e). In addition, MLS0315771 demonstrated comparable or even superior antibacterial activity against other clinically isolated Gram-positive bacteria, including \u003cem\u003eS. haemolyticus\u003c/em\u003e, \u003cem\u003eS. hominis\u003c/em\u003e, \u003cem\u003eS. capitis\u003c/em\u003e, \u003cem\u003eS. lugdunensis\u003c/em\u003e, \u003cem\u003eS. epidermidis\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e, and \u003cem\u003eE. faecalis\u003c/em\u003e (\u003cstrong\u003eTable 1\u003c/strong\u003e), collectively indicating that MLS0315771 might possess broad-spectrum activity against a wide range of clinically relevant Gram-positive bacteria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further evaluate the antibacterial effect of MLS0315771 at subinhibitory concentrations, growth curve analyses were performed on four MRSA isolates, four \u003cem\u003eS. aureus\u003c/em\u003e isolates, and two \u003cem\u003eE. faecium\u003c/em\u003e isolates under MLS0315771 pressure (\u003cstrong\u003eFigure 1\u003c/strong\u003e). Within the 24-hour observation period, bacterial growth was completely inhibited at 1 \u0026times; MIC of MLS0315771. At subinhibitory concentrations, MLS0315771 prolonged the lag phase of bacterial growth by 5-16 hours, indicating a concentration-dependent inhibitory effect of this chemical on bacterial proliferation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. The MIC distribution of MLS0315771 against Gram-positive bacteria.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 134px;\"\u003e\n \u003cp\u003eBacteria species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 71px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 277px;\"\u003e\n \u003cp\u003eMLS0315771 MIC values (\u0026mu;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 86px;\"\u003e\n \u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e/MIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e3.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMRSA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e12.5/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eMSSA\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e25/25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. haemolyticus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. hominis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e12.5/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. capitis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. lugdunensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. agalactiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e12.5/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. epidermids\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecium\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6.25/6.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 71px;\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e12.5/25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eMRSA, methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e; MSSA, methicillin-susceptible \u003cem\u003eStaphylococcus aureus\u003c/em\u003e;\u003cem\u003e\u0026nbsp;\u003c/em\u003eMIC50/MIC90, the MIC values for 50% or 90% of bacterial growth inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLS0315771 Exhibits \u003cem\u003eIn Vitro\u003c/em\u003e Bactericidal and Bacteriostatic Activity Comparable to Vancomycin\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e time-kill assays were conducted using vancomycin as a positive control to evaluate the bactericidal potency of MLS0315771 (\u003cstrong\u003eFigure 2\u003c/strong\u003e). Against the standard strain \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC29213, MLS0315771 exhibited bacteriostatic activity within the first 8 hours at concentrations of 4 \u0026times; and 8 \u0026times; MIC. By 24 hours, both concentrations of MLS0315771 reduced the initial bacterial count by more than 3 log₁₀ (CFU/mL), corresponding to a \u0026ge; 99.9% reduction in viable cells. Moreover, at 24 hours, the residual bacterial count in the MLS0315771-treated groups was lower than that observed with 8 \u0026mu;g/mL vancomycin (\u003cstrong\u003eFigure 2A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilarly, in the clinical MRSA isolate YUSA145,\u0026nbsp;MLS0315771\u0026nbsp;demonstrated bacteriostatic activity during the first 8 hours, followed by a \u0026ge;2 log₁₀ (CFU/mL) reduction in bacterial load at 24 hours when treated with 4 \u0026times; and 8 \u0026times; MIC\u0026nbsp;MLS0315771, indicating more than 90% bacterial killing. Under identical conditions, vancomycin exhibited sustained bacteriostatic rather than bactericidal activity throughout the 24-hour period (\u003cstrong\u003eFigure 2B\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003eEnterococcus faecium\u003c/em\u003e clinical isolate EF16C51, both MLS0315771 and vancomycin produced comparable inhibitory effects, maintaining persistent bacteriostasis over the 24-hour duration (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). In conclusion, based on the observed time-kill kinetics,\u0026nbsp;MLS0315771\u0026nbsp;appears to act as a time-dependent rather than a concentration-dependent antimicrobial agent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLS0315771 Exhibits Excellent Anti-Biofilm Activity\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the MIC values obtained from broth microdilution assays and the bacterial growth curve results, five MRSA and four MSSA biofilm-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e isolates were selected for \u003cem\u003ein vitro\u003c/em\u003e biofilm inhibition and eradication assays. When subinhibitory concentrations of MLS0315771 were added at the initial stage of biofilm formation, MLS031577 exhibited an inhibitory effect on \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation (\u003cstrong\u003eFigure 3A and Figure 3B\u003c/strong\u003e). In addition to its effect on \u003cem\u003eS. aureus\u003c/em\u003e, MLS0315771 also inhibited biofilm formation of \u003cem\u003eE.faecium\u003c/em\u003e (\u003cstrong\u003eFigure S2\u003c/strong\u003e). In our assays, MLS0315771 could significantly inhibit the biofilm formation of \u003cem\u003eS. aureus\u003c/em\u003e and its inhibitory effect on \u003cem\u003eS. aureus\u003c/em\u003e biofilm formation was stronger than that observed against \u003cem\u003eE. faecium\u003c/em\u003e. Furthermore, the effect of MLS0315771 on mature \u003cem\u003eS. aureus\u003c/em\u003e biofilms was investigated. After biofilms were allowed to mature for 24 hours, the culture medium was replaced with fresh medium containing 8 \u0026times; MIC of MLS0315771 and incubated for an additional 24 hours. Crystal violet staining revealed that MLS0315771 effectively disrupted and removed established \u003cem\u003eS. aureus\u003c/em\u003e biofilms (\u003cstrong\u003eFigure 3C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, confocal laser scanning microscopy (CLSM) was employed to visualize biofilm thickness and the spatial distribution of live and dead cells. The result showed that treatment with 8 \u0026times; MIC of MLS0315771 resulted in a thinner biofilm of \u003cem\u003eS. aureus\u003c/em\u003e YUSA145, YUSA218 and CHS655 (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). These findings show that MLS0315771 has potent antibiofilm activity against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic analysis of \u003cem\u003eS. aureus\u003c/em\u003e treated with MLS0315771\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative label-free proteomic analysis investigated the global proteomic response of \u003cem\u003eS. aureus\u003c/em\u003e YUSA145 treated with 1/2 \u0026times; MIC MLS0315771 during the exponential growth phase. The results were obtained after performing principal component analysis (PCA) on the raw data and excluding two replicates that showed substantial variation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD073535 [20, 21]. When compared to the control group, 236 proteins exhibited significantly different expression levels (\u0026ge;|1.5|-fold change, p\u0026le;0.05), including 63 up-regulated and 173 down-regulated proteins in the MLS0315771 treatment group (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003eA and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e). Detailed information of the functional proteins with significantly different expression levels is listed in Table S2. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The KEGG results showed that the differential proteins were predominantly associated with pathways related to nitrogen metabolism, riboflavin metabolism, purine metabolism, \u003cem\u003eS. aureus\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003einfection, microbial metabolism in diverse environments, arginine biosynthesis and pyruvate metabolism (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). In addition, Gene Ontology (GO) analysis of the differentially expressed proteins was also conducted using the OmicsBean online platform, classifying them into three major categories: biological processes, molecular functions, and cellular components (\u003cstrong\u003eFigure 4D\u003c/strong\u003e). Based on KEGG data, a protein-protein interaction (PPI) network was constructed for the differentially expressed proteins (\u003cstrong\u003eFigure 4E\u003c/strong\u003e). The analysis revealed that interactions among downregulated proteins were mainly centered on Uncharacterized leukocidin-like protein 1, Delta-hemolysin, RNase H type-1 domain-containing protein, Global transcriptional regulator Spx, Staphylococcal secretory antigen ssaA2 and Type VII secretion system extracellular protein A. These proteins were primarily involved in biological process such as cytolysis, hemolysis, virulence, nucleic acid binding, regulation of DNA-templated transcription and hydrolase activity. Notably, one major virulence determinant associated with membrane dynamics, hld (3.875-fold downregulated) were markedly repressed. Hld, an amphipathic peptide under the control of the agr quorum-sensing system, facilitates membrane curvature remodeling through pore formation and promotes membrane vesicle (MV) biogenesis, both of which are vital for bacterial cytotoxicity and intercellular signaling [22]. Its downregulation is therefore consistent with impaired MV-mediated virulence transmission [22]. Loss of Hld weakens offensive membrane-disruptive capacity and the proteomic finding highlight membrane perturbation as a principal mechanism underlying the bactericidal action of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e, thereby providing molecular evidence supporting its membrane-targeting antibacterial potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial mechanism of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembrane permeability and membrane potential homeostasis are essential for bacterial growth and proliferation. To investigate the primary antibacterial mechanism of MLS0315771, we first examined its effects on membrane depolarization and permeability. Following different concentrations of MLS0315771 treatment, the MSSA strain SA113 exhibited pronounced disruption of both membrane permeability and membrane depolarization compared with the control group and MLS0315771 treatment led to dose-dependent increases in the uptake of propidium iodide (PI), and DiBAC₄(3), as reflected by elevated fluorescence intensity (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003eA-D\u003c/strong\u003e). These findings demonstrate that MLS0315771 compromises the integrity of \u003cem\u003eS. aureus\u003c/em\u003e cell membranes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that MLS0315771 damages \u003cem\u003eS. aureus\u003c/em\u003e membrane integrity, and considering the complex lipid composition of bacterial membranes, we further explored potential membrane lipid targets of MLS0315771 by exogenously supplementing abundant membrane lipids. Four major membrane phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL), were tested for their intrinsic antibacterial activity. None of the phospholipids inhibited bacterial growth for their MICs all \u0026gt;128 \u0026mu;g/mL (\u003cstrong\u003eTable S\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e). Moreover, supplementation with these lipids did not alter the MIC values of\u0026nbsp;MLS0315771\u0026nbsp;(\u003cstrong\u003eTable S\u003c/strong\u003e\u003cstrong\u003e4\u003c/strong\u003e), indicating that\u0026nbsp;MLS0315771\u0026nbsp;does not exert its antibacterial effect through direct interaction with these four membrane lipids. In addition, checkerboard assay results (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003eE-G\u003c/strong\u003e) uncovered that the unsaturated long-chain fatty acids linoleic acid, \u0026gamma;-linoleic acid and arachidonic acid significantly increased the MIC values of MLS0315771 and reduced its antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e SA113 in a concentration-dependent manner. Based on the proteomic results, we examined whether MLS0315771 interferes with \u0026alpha;-hemolysin activity. The findings revealed that MLS0315771 strongly inhibited \u0026alpha;-hemolysin production in the \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eUSA300 strain (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e). This result is consistent with the hypothesis derived from our proteomic analysis. Together, these findings confirm that MLS0315771 interacts with bacterial cell membrane, affecting its membrane integrity through affecting the fatty acid-related pathway. These results provide further evidence supporting our earlier hypothesis that MLS0315771 targets cell \u0026nbsp; membrane-associated processes, thereby offering an important mechanistic basis for its antibacterial activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, ClpX is a gene that has been reported to play a pivotal role in \u003cem\u003eS. aureus\u003c/em\u003e growth and virulence [23]. A clpX deletion mutant of \u003cem\u003eS. aureus\u003c/em\u003e SA113 was generated using a gene knockout strategy. To characterize the antimicrobial susceptibility of the mutant strain, minimum inhibitory concentration (MIC) assays and growth curve analyses were performed (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5I-J and Table S5\u003c/strong\u003e). Compared with the wild-type SA113 strain, deletion of clpX resulted in a twofold increase in the MIC of MLS0315771, from 6.25 \u0026mu;M to 12.5 \u0026mu;M. Significant differences in overall growth kinetics were observed between the wild-type and \u0026Delta;Clpx strains under standard culture conditions. These results indicate that loss of clpX reduces the susceptibility of \u003cem\u003eS. aureus\u003c/em\u003e to MLS0315771 and we reasonably assume that ClpX may be related to the antibacterial activity of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMLS0315771 safety evaluation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxic potential of MLS0315771 was assessed through CCK-8 and hemolysis assays. In mammalian cell lines, including LX-2, HEK-293T, Beas-2b, J774A.1, A549 and Huvec cells (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003eA-F\u003c/strong\u003e), cell viability dynamics were monitored over a 24-hour exposure period and the measurable cytotoxic effects were observed starting at 100 \u0026mu;M compared with untreated controls for most of the cell lines except LX-2 cells. And the CC\u003csub\u003e50\u003c/sub\u003e values for J774A.1 and A549 cells were over 100 \u0026mu;M. Notably, these concentrations exceeded the effective antibacterial levels, suggesting that MLS0315771 possesses a reasonable therapeutic window. Hemolysis analysis revealed that MLS0315771 caused negligible human and sheep erythrocyte lysis, even at concentrations as high as 50-200 \u0026mu;M, indicating good biocompatibility (\u003cstrong\u003eFigure\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003eG and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e). Considering that many high-efficacy antibiotics are often accompanied by severe side effects such as hepatotoxicity, nephrotoxicity, and immune suppression, these findings collectively support that MLS0315771 exhibits low cytotoxicity and an overall favorable safety profile.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe continuous rise of multidrug-resistant (MDR) Gram-positive pathogens, particularly \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEnterococcus faecium\u003c/em\u003e, poses a persistent global health challenge [24]. In order to address the growing number of drug-resistant strains and the scarcity of current treatment choices, it is imperative to develop alternative antimicrobial techniques to tackle drug-resistant bacteria, including the repurposing of existing medications [25]. In this regard, we effectively identified MLS0315771 with antibacterial activity by screening the compound library of clinical trials. MLS0315771, a competitive MPI inhibitor from the benzoisothiazolone class, could shift mannose flux to glycosylation in various cell lines, including certain CDG-Ia fibroblast lines [18]. It was found that MLS0315771 had the potential for the development and optimization of molecules that may eventually result in a novel medication against Lyme disease [26]. In this study, we identified MLS0315771 as a promising antibacterial compound exhibiting potent activity against a broad spectrum of clinically relevant Gram-positive bacteria, including MSSA, MRSA and linezolid-resistant \u003cem\u003eE. faecium\u003c/em\u003e. The MIC data demonstrated that MLS0315771 displayed comparable or superior activity to vancomycin across multiple isolates, underscoring its potential as a novel lead compound for the treatment of refractory infections caused by resistant strains. Consistent with its low MIC values, time-kill assays revealed that MLS0315771 exerted rapid and sustained bactericidal effects against both \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003efaecalis\u003c/em\u003e isolates. Compared to vancomycin, MLS0315771 achieved a greater reduction in bacterial burden within 24 hours, suggesting that it acts through a time-dependent killing mechanism. This finding is significant given the increasing number of MRSA strains that have developed reduced susceptibility to vancomycin. The prolonged lag phase observed in bacterial growth curve analyses under subinhibitory concentrations of MLS0315771 further supports a concentration-dependent inhibition of bacterial proliferation, reflecting its robust antibacterial potency. Biofilm formation is one of the most critical virulence mechanisms of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. faecium\u003c/em\u003e, contributing to chronic and device-associated infections [27, 28]. Our findings demonstrated that MLS0315771 not only prevented biofilm formation at early developmental stages but also effectively disrupted mature biofilms. These results are particularly noteworthy since most conventional antibiotics exhibit poor efficacy against biofilm-embedded bacterial cells [29]. Confocal microscopy further confirmed the marked reduction in biofilm thickness and viability following MLS0315771 treatment, indicating that this compound may effectively combat persistent infections driven by biofilm-forming pathogens. However, more research is required to confirm MLS0315771\u0026apos;s therapeutic effectiveness as an antibacterial drug against \u003cem\u003eS. aureus\u003c/em\u003e infection, including \u003cem\u003ein vivo\u003c/em\u003e trials and thorough clinical validation.\u003c/p\u003e\n\u003cp\u003eTo gain mechanistic insights into the antibacterial action of MLS0315771, proteomic profiling of \u003cem\u003eS. aureus\u003c/em\u003e following exposure revealed extensive reprogramming of bacterial metabolism and virulence networks. Among the 236 differentially expressed proteins, downregulation of key virulence determinants such as delta-hemolysin (hld), staphylococcal secretory antigen ssaA2, and type VII secretion system components suggested a suppression of cytolytic activity and intercellular communication. The inhibition of hld, a central factor in membrane vesicle biogenesis, aligns with the observed disruption of membrane integrity, indicating that MLS0315771 may impair \u003cem\u003eS. aureus\u003c/em\u003e pathogenicity by targeting membrane-associated processes [30]. In addition, the type VII secretion system (T7SS) has been recognized as a critical determinant of pathogenicity in \u003cem\u003eS. aureus\u003c/em\u003e, functioning as a specialized apparatus for the translocation of virulence factors across the thick cell envelope [31]. In \u003cem\u003eS. aureus\u003c/em\u003e, this system mediates the secretion of the nuclease toxin EsaD through a coordinated interaction with the accessory proteins EsaE and EsaG. Suppression of T7SS-related proteins, including EsaD, EsaE, and EssC, by MLS0315771 therefore implies impaired secretion of virulence factors and a disruption of membrane-coupled protein export machinery [32]. This observation reinforces the notion that MLS0315771 perturbs bacterial viability not only by compromising membrane integrity but also by attenuating T7SS-dependent virulence delivery, a dual action that could synergistically weaken \u003cem\u003eS. aureus\u003c/em\u003e pathogenic potential. And Enrichment analysis highlighted pathways associated with nitrogen metabolism, purine biosynthesis, and pyruvate metabolism, implying that MLS0315771 not only compromises membrane function but also interferes with essential metabolic circuits necessary for bacterial survival. Proteomics can only identify the possible mechanism in a preliminary manner. More validation research is required by creating mutant strains and overexpressing elevated proteins for enzymatic assays. In general, this finding \u0026nbsp;demonstrate the complex effects of MLS0315771 on bacterial physiology.\u003c/p\u003e\n\u003cp\u003eMechanistic assays provided further evidence that MLS0315771 targets the bacterial membrane. Fluorescence-based analyses revealed marked depolarization and increased permeability in \u003cem\u003eS. aureus\u003c/em\u003e treated with MLS0315771, confirming membrane disruption as a primary antibacterial mechanism. Furthermore, phospholipids are crucial components of the bacterial membrane, and studies have shown that some antimicrobial peptides can permeate the membrane, influence enzyme activity inside the bacteria, disrupt substance metabolism, and eventually cause bacterial death [33]. However, the absence of changes in MICs upon supplementation with common membrane phospholipids (PC, PE, PG, and CL) suggested that MLS0315771 does not act through direct lipid binding. However, the attenuation of antibacterial activity in the presence of unsaturated long-chain fatty acids, including linoleic and arachidonic acids, indicates interference with fatty acid-related metabolic pathways. This result supports a model in which MLS0315771 perturbs bacterial membrane stability and function indirectly, possibly through modulation of lipid homeostasis or disruption of membrane-associated protein functions. Hemolysin, a major virulence factor secreted by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, is classified into five types (\u0026alpha;, \u0026beta;, \u0026gamma;, \u0026delta;, \u0026epsilon;) according to antigenicity. Among these, \u0026alpha;-hemolysin exhibits the strongest pathogenic activity and has been most extensively studied [34]. The inhibition of \u0026alpha;-hemolysin production further corroborates this membrane-targeting effect and suggests a secondary suppression of virulence expression. It is conceivable that MLS0315771 causes damage to bacterial cell membranes, which eventually results in bacterial death. Antibacterial compounds that target the bacterial cell membrane are anticipated to be a potential target for antibacterial agent screening because they have the potential to rapidly kill bacteria and provide advantages in lowering the tendency of bacteria to develop resistance [35, 36]. This offers important information about MLS0315771\u0026apos;s possible function in rupturing bacterial cell membranes. It is plausible that MLS0315771 causes damage to bacterial cell membranes, which eventually results in bacterial death. In this study, although our primary analyses suggest that MLS0315771 exerts its antibacterial activity predominantly through disruption of the bacterial membrane, the altered susceptibility observed in the clpX deletion mutant indicates that additional intracellular factors may also contribute to the antibacterial response. ClpX is a member of the Clp/Hsp100 subfamily of AAA+ ATPases and plays a central role in protein quality control, stress adaptation, and growth regulation in \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003e[37]. Functionally, ClpX recognizes specific peptide motifs on substrate proteins and cooperates with the ClpP protease to mediate ATP-dependent unfolding and degradation of target polypeptides [38]. Given the essential role of ClpX in maintaining cellular homeostasis, loss of clpX may indirectly affect bacterial tolerance to MLS0315771-induced stress, including membrane perturbation. However, our current data are limited to phenotypic observations, and no direct evidence supports a specific molecular interaction between MLS0315771 and ClpX. Therefore, it is more plausible that ClpX contributes to the adaptive or stress-response pathways that modulate bacterial susceptibility to MLS0315771 rather than serving as a direct antibacterial target. Further studies focusing on proteostasis regulation and stress signaling pathways will be required to clarify the precise role of ClpX in the antibacterial activity of MLS0315771 against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eEqually important, the cytotoxicity and hemolysis assays demonstrated that MLS0315771 possesses a favorable safety profile. Minimal hemolytic activity was observed even at high concentrations, and the cytotoxic effects toward mammalian cell lines were only detectable at doses substantially exceeding antibacterial levels. These findings indicate a promising therapeutic window and suggest that MLS0315771 could achieve effective antibacterial concentrations without exerting substantial toxicity on host tissues. Given that many potent antibiotics are associated with nephrotoxicity or hepatotoxicity, the low cytotoxicity of MLS0315771 enhances its potential for further development [39, 40].\u003c/p\u003e\n\u003cp\u003eCollectively, our findings suggest that MLS0315771 exerts multifaceted antibacterial effects, characterized by strong activity against MDR Gram-positive bacteria, potent antibiofilm capacity, and disruption of bacterial membrane integrity and virulence pathways. The integrated proteomic and mechanistic evidence indicates that membrane perturbation and inhibition of fatty acid-associated processes are central to its mode of action. Future studies focusing on identifying direct molecular targets and assessing in vivo pharmacokinetics will be critical for advancing MLS0315771 toward clinical translation. Overall, this study highlights MLS0315771 as a compelling candidate for further optimization and development as a novel antimicrobial agent capable of addressing the growing challenge of multidrug-resistant and biofilm-associated Gram-positive infections.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eBacterial Identification and Growth Conditions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 158 Gram-positive coccal isolates were included in this study, comprising \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (n = 69; MRSA, n = 44; MSSA, n = 25), \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e (n = 26), and \u003cem\u003eEnterococcus faecium\u003c/em\u003e (n = 37), along with other species as listed in Table 1. All clinical isolates were collected and identified from various specimens of 534 patients admitted to Shenzhen Nanshan People\u0026rsquo;s Hospital between 2011 and 2015 [41, 42]. Prior to experimental use, both clinical and reference strains were reconfirmed using a MALDI-TOF mass spectrometry (IVD MALDI Biotyper, Bruker, Karlsruhe, Germany). Verified isolates were preserved in tryptic soy broth (TSB) supplemented with 20% glycerol and stored at -80 \u0026deg;C for subsequent analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetermination of Minimum Inhibitory Concentrations (MICs)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe MICs of antimicrobial compounds were determined using the standard broth microdilution method in cation-adjusted Mueller-Hinton broth (CAMHB), following the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100, 29\u003csup\u003eth\u003c/sup\u003e ed.). Twofold serial dilutions of MLS0315771 were prepared and exposed to a series of half dilutions of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 \u0026mu;M, and bacterial suspensions were adjusted to the appropriate inoculum density. The MIC was defined as the lowest concentration of compound completely inhibiting visible growth after incubation. \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213, \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212, and \u003cem\u003eE. faecalis\u003c/em\u003e OG1RF were used as quality control strains. MLS0315771 and vancomycin (VAN) were purchased from MCE (Princeton, United States).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBacterial Growth Curves\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBacterial growth kinetics were monitored following a previously described method [43]. Single colonies of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e were inoculated into TSB and cultured overnight at 37 \u0026deg;C with shaking at 220 rpm. Overnight cultures (16-20 h) were diluted in fresh TSB containing appropriate concentrations of MLS0315771 and transferred to a honeycomb microplate. The optical density at 600 nm (OD₆₀₀) was continuously measured for 24 h using a Bioscreen C automated growth curve analyzer (Turku, Finland). Growth curves were plotted based on OD₆₀₀ readings over time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTime-Kill Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTime-kill kinetics were determined according to a previously described method [44]. Single colonies were inoculated into 4-5 mL CAMHB and grown overnight at 37 \u0026deg;C with shaking at 220 rpm. The following day, cultures were diluted 1:100 into 6 mL fresh CAMHB and grown to the logarithmic phase (approximately 4-6 h). Log-phase cells were adjusted to ~1 \u0026times; 10⁶ CFU/mL. MLS0315771 solutions at twice the desired final concentrations were prepared in CAMHB and mixed 1:1 with bacterial suspensions to yield a final bacterial density of ~5 \u0026times; 10⁵ CFU/mL. Cultures were incubated statically at 37 \u0026deg;C, and aliquots (100 \u0026mu;L) were collected at 0, 3, 6, 8, and 24 h. Samples were serially diluted 10-fold in saline, plated (50 \u0026mu;L) onto CAMHB agar, and incubated for 24 h at 37 \u0026deg;C for colony CFU counting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntibiofilm Activity Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe antibiofilm efficacy of MLS0315771 was evaluated using a crystal violet staining method as described previously [45], with slight modifications. Briefly, overnight cultures of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e were diluted in fresh TSB supplemented with 2% glucose (TSBG) to a final concentration of approximately 1 \u0026times; 10⁶ CFU/mL. Aliquots of 200 \u0026mu;L were dispensed into sterile 96-well polystyrene microplates, followed by treatment with MLS0315771 at concentrations corresponding to 1/2\u0026times;, 1/4\u0026times;, and 1/8\u0026times; of the MIC. Plates were incubated statically at 37 \u0026deg;C for 24 h to allow biofilm formation. After incubation, planktonic cells were removed, and wells were gently rinsed with PBS to eliminate non-adherent bacteria. The remaining biofilms were stained with 0.1% crystal violet, washed, and solubilized in ethanol. The absorbance was then measured at 570 nm using a microplate reader (BioTek, USA) to quantify biofilm biomass.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConfocal Laser Scanning Microscopy (CLSM) Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe structural changes of \u003cem\u003eS. aureus\u003c/em\u003e biofilms following MLS0315771 exposure were visualized using confocal laser scanning microscopy (CLSM) as described in previous studies [46]. Overnight cultures of the \u003cem\u003eS. aureus\u003c/em\u003e strains YUSA145, YUSA218 and CHS655 were diluted 1:200 in fresh TSBG and seeded into confocal dishes, followed by incubation for 24 h at 37 \u0026deg;C to establish mature biofilms. Subsequently, the biofilms were gently washed three times with sterile saline (0.9% NaCl) and treated with fresh TSBG medium containing MLS0315771 at 8 \u0026times; MIC or an equivalent volume of DMSO as a control. After a further 24 h of incubation, biofilms were washed with sterile PBS and stained using the Live/Dead BacLight viability kit containing SYTO9 and propidium iodide (PI) for 30 min in the dark. Stained samples were observed using a confocal laser scanning microscope, and biofilm architecture and bacterial viability were analyzed from the acquired images.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLC\u0026ndash;MS/MS Proteomics Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantitative proteomic profiling of \u003cem\u003eS. aureus\u003c/em\u003e was performed using LC\u0026ndash;MS/MS, following procedures modified from a previously described protocol [47]. Briefly, \u003cem\u003eS. aureus\u003c/em\u003e strain YUSA145 was grown in TSB to the logarithmic growth phase and subsequently treated with MLS0315771 at 1/2 \u0026times; MIC or an equivalent volume of DMSO for 2 h at 37 \u0026deg;C with agitation at 220 rpm. Following treatment, bacterial cells were harvested by centrifugation at 5000 rpm for 10 min at 4 \u0026deg;C, and washed twice with pre-chilled PBS. Total proteins were extracted using RIPA lysis buffer supplemented with a protease inhibitor cocktail (Beyotime, Shanghai, China). Cell disruption was achieved by mechanical agitation with 0.1 mm glass beads at 70 Hz, and the lysates were clarified by centrifugation (12,000 rpm, 15 min, 4 \u0026deg;C). Protein concentrations were determined using the BCA assay kit (Thermo Fisher Scientific, USA). For digestion, equal amounts of protein were reduced with 10 mM dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA) at 70 \u0026deg;C for 1 h, followed by alkylation with 50 mM iodoacetamide (IAA; Sigma-Aldrich) for 15 min in the dark. Samples were then desalted and concentrated using Amicon Ultra centrifugal filters, and digested overnight with trypsin (Promega, Madison, WI, USA) at 37 \u0026deg;C. The resulting peptides were separated on a C18 reverse-phase column (75 \u0026mu;m \u0026times; 250 mm, Acclaim PepMap RSLC, 2 \u0026mu;m) prior to analysis. Mass spectrometric detection was carried out on a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). The obtained raw spectra were processed and analyzed for protein identification and quantification using standard bioinformatic pipelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMembrane Permeability and Depolarization Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effects of MLS0315771 on bacterial membrane integrity and potential were evaluated based on a previously reported protocol [44], with minor modifications. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SA113 was first cultured overnight in TSB medium at 37 \u0026deg;C. The bacterial suspension was then adjusted to 1 \u0026times; 10⁷ CFU/mL in HEPES buffer (5 mM HEPES, 20 mM glucose, pH 7.4) and incubated with either 1 \u0026micro;M propidium iodide (PI) or 1 \u0026micro;M Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol [DiBAC₄(3)] for 2 h to allow dye uptake. Subsequently, KCL was introduced to a final concentration of 0.1 M to equilibrate intracellular and extracellular potassium levels. Fluorescence measurements were performed in a 96-well microplate after allowing the samples to reach a stable baseline. MLS0315771 was then added to achieve final concentrations equivalent to 1\u0026times;, 2\u0026times;, 4\u0026times; and 8\u0026times; MIC, and fluorescence signals were continuously monitored using a Cytation 1 Cell Imaging Multimode Reader (BioTek, USA). The excitation/emission wavelengths used were 217/340 nm for PI and 540/560 nm for DiBAC₄(3). Untreated cells served as the negative control, while 0.1% Triton X-100 was included as a positive control to indicate complete membrane disruption.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAntibacterial Activity of MLS0315771 in the Presence of Phospholipids and Fatty Acids\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the influence of phospholipids and fatty acids on the antibacterial efficacy of MLS0315771, the compounds were first dissolved in methanol. Arachidonic acid (A5837, Sigma-Aldrich, USA) and other related lipids were tested using a checkerboard microdilution approach as previously described [48]. Briefly, \u003cem\u003eS. aureus\u003c/em\u003e SA113 was cultured in TSB medium, and assays were performed with phospholipids at concentrations ranging from 8-128 \u0026mu;g/mL and fatty acids at 3.9-500 \u0026mu;M. Serial dilutions of fatty acids and phospholipids were arrayed along the abscissa and ordinate, respectively, in 96-well plates, followed by inoculation with bacterial suspensions at 1 \u0026times; 10⁶ CFU/mL. After incubation at 37 \u0026deg;C for 24 h, bacterial growth was quantified by measuring the optical density at 600 nm (OD₆₀₀) using a microplate reader. The resulting data were used to determine the effect of lipid components on the MIC of MLS0315771.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConstruction of the\u0026nbsp;ClpX\u0026nbsp;Deletion Mutant\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ClpX-deficient strain of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e SA113 was constructed using the temperature-sensitive shuttle vector pKOR1 as previously described [49]. Briefly, DNA fragments flanking the ClpX locus were amplified from the SA113 genomic DNA by PCR and subsequently cloned into the pKOR1 plasmid to generate the allelic exchange construct. The recombinant plasmid was introduced into \u003cem\u003eS. aureus\u003c/em\u003e SA113 by electroporation. Transformants were initially selected on tryptic soy agar (TSA) plates supplemented with chloramphenicol (10 \u0026mu;g/mL). Putative ClpX knockout clones were screened by PCR using genomic DNA as the template, and correct allelic replacement was further confirmed by DNA sequencing. Finally, the pKOR1 plasmid was eliminated from the mutant strain following established plasmid-curing procedures.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-Hemolysin Secretion Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe inhibitory effect of MLS0315771 on \u003cem\u003eS. aureus\u003c/em\u003e \u0026alpha;-hemolysin secretion was assessed using rabbit red blood cells (RBCs), following a modified protocol from a previous study [50]. Overnight cultures of \u003cem\u003eS. aureus\u003c/em\u003e USA300 were diluted 1:200 into TSB containing 1/8\u0026times;, 1/4\u0026times;, 1/2\u0026times; and 1\u0026times; MIC concentrations of MLS0315771 and incubated at 37 \u0026deg;C for 12 h. The supernatants were harvested by centrifugation and sterilized through 0.22 \u0026mu;m filters. Subsequently, 100 \u0026mu;L of the sterile supernatant was combined with an equal volume of 2% rabbit RBC suspension and incubated at 37 \u0026deg;C for 30 min. After centrifugation at 1500 rpm, 100 \u0026mu;L of the supernatant was transferred to a 96-well plate for absorbance measurement at 540 nm. PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. The hemolytic activity was calculated using the formula: Hemolysis rate (%) = [(OD\u003csub\u003esample\u003c/sub\u003e-OD\u003csub\u003eblank\u003c/sub\u003e)/(OD\u003csub\u003epositive\u003c/sub\u003e-OD\u003csub\u003eblank\u003c/sub\u003e)] \u0026times;\u0026nbsp;100. All assays were performed in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003eHemolytic Activity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hemolytic potential of MLS0315771 was evaluated using fresh human and sheep erythrocytes as described previously [51]. Briefly, erythrocytes were washed three times with sterile phosphate-buffered saline (PBS) and resuspended to a final concentration of 4% (v/v). Aliquots of 100 \u0026mu;L erythrocyte suspension were distributed into 96-well plates and treated with serial dilutions of MLS0315771 (0.195\u0026ndash;100 \u0026mu;M for human and 0.75\u0026ndash;200 \u0026mu;M for sheep). The mixtures were incubated at 37 \u0026deg;C for 30 min, followed by centrifugation at 1000 \u0026times; g for 5 min. Subsequently, 100 \u0026mu;L of the supernatant was carefully transferred to a new 96-well flat-bottom plate, and the absorbance at 540 nm (OD₅₄₀) was measured using a microplate reader. PBS and 1% Triton X-100 served as the negative and positive controls, respectively. The percentage of hemolysis was calculated using the following formula:\u0026nbsp;Hemolysis\u0026nbsp;rate\u0026nbsp;(%) = [(OD\u003csub\u003esample\u003c/sub\u003e-OD\u003csub\u003enegative\u003c/sub\u003e)/(OD\u003csub\u003epositive\u003c/sub\u003e-OD\u003csub\u003enegative\u003c/sub\u003e)] \u0026times; 100.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCytotoxicity Assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe cytotoxic effects of MLS0315771 on mammalian cells were determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer\u0026rsquo;s protocol. LX-2, HEK-293T, Beas-2b, J774A.1, A549 and Huvec cells were seeded into 96-well plates at a density of 6 \u0026times; 10\u0026sup3; cells/well in complete Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After overnight incubation at 37 \u0026deg;C under 5% CO₂, the cells were exposed to fresh medium containing varying concentrations of MLS0315771 (0\u0026ndash;100 \u0026mu;M, including 0.78, 1.56, 3.125, 12.5, 25, 50, and 100 \u0026mu;M) for 24 h. Following treatment, 10 \u0026mu;L of CCK-8 reagent was added to each well, and the plates were further incubated for 1 h under the same conditions. Absorbance was recorded at 450 nm (OD₄₅₀) using a microplate reader. Cell viability was expressed as a ratio of experimental to control groups. The CC50 values were determined using the GraphPad Prism software (version 10.1.2). The data reported here are from three different experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations and were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital and the 1964 Helsinki Declaration and its later amendments, or comparable ethical standards. All experimental procedures involving human subjects were approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital. Bacterial strains were collected as part of the routine clinical management of patients, according to the national guidelines in China (Clinical trial number: not applicable). Therefore, informed consent was not sought, and informed consent waiver was approved by the institutional ethical committee of Shenzhen Nanshan People\u0026rsquo;s Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e+\u003c/sup\u003eZ.X., Y.T., J.M., and P.L. contributed equally to this work. S.H., Z.C., and T.H. conceived and designed the study. Z.X., Y.T., J.M., and P.L. performed the main experiments, including antibacterial activity assays, antibiofilm assays, and mechanistic studies. Z.W. and Z.Y. assisted with bacterial strain collection, data acquisition, and experimental validation. B.B. contributed to data analysis and interpretation. Y.T. and Z.X. drafted the manuscript. B.B., Z.C., S.H., and T.H. critically revised the manuscript for important intellectual content. Z.C., S.H., and T.H. acquired funding and supervised the project. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD073535.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following grants: National Natural Science Foundation of China (82572621); Sanming Project of Medicine in Shenzhen (SMZ202303037); Shenzhen Key Medical Discipline Construction Fund (SZXK06162); Science, Technology and Innovation Commission of Shenzhen Municipality of basic research funds (JCYJ20240813114518024, KJZD20240903103500002) and the Shenzhen Nanshan District Scientific Research Program of the People\u0026rsquo;s Republic of China (NS2024044Y, NS2024006Y, NS2024007Y, NSZD2024036, NSZD2024023, NSZD2024032, NSZD2025001, NSZD2025005).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSalam MA, Al-Amin MY, Salam MT, Pawar JS, Akhter N, Rabaan AA, Alqumber MA: \u003cstrong\u003eAntimicrobial resistance: a growing serious threat for global public health\u003c/strong\u003e. In: \u003cem\u003eHealthcare: 2023\u003c/em\u003e. MDPI: 1946.\u003c/li\u003e\n\u003cli\u003eBashabsheh RH, AL-Fawares Ol, Natsheh I, Bdeir R, Al-Khreshieh RO, Bashabsheh HH: \u003cstrong\u003eStaphylococcus aureus epidemiology, pathophysiology, clinical manifestations and application of nano-therapeutics as a promising approach to combat methicillin resistant Staphylococcus aureus\u003c/strong\u003e. \u003cem\u003ePathogens and Global Health \u003c/em\u003e2024, \u003cstrong\u003e118\u003c/strong\u003e(3):209-231.\u003c/li\u003e\n\u003cli\u003ePal M, Shuramo MY, Tewari A, Srivastava JP, Steinmetz CH: \u003cstrong\u003eStaphylococcus aureus from a commensal to zoonotic pathogen: a critical appraisal\u003c/strong\u003e. \u003cem\u003eInternational Journal of Clinical and Experimental Medicine Research \u003c/em\u003e2023, \u003cstrong\u003e7\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eTouaitia R, Mairi A, Ibrahim NA, Basher NS, Idres T, Touati A: \u003cstrong\u003eStaphylococcus aureus: A Review of the Pathogenesis and Virulence Mechanisms\u003c/strong\u003e. \u003cem\u003eAntibiotics \u003c/em\u003e2025, \u003cstrong\u003e14\u003c/strong\u003e(5):470.\u003c/li\u003e\n\u003cli\u003eParsons JB, Mourad A, Conlon BP, Kielian T, Fowler Jr VG: \u003cstrong\u003eMethicillin-resistant and susceptible Staphylococcus aureus: tolerance, immune evasion and treatment\u003c/strong\u003e. \u003cem\u003eNature Reviews Microbiology \u003c/em\u003e2025:1-19.\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-Larrota JS, Eckhard U: \u003cstrong\u003eAn introduction to bacterial biofilms and their proteases, and their roles in host infection and immune evasion\u003c/strong\u003e. \u003cem\u003eBiomolecules \u003c/em\u003e2022, \u003cstrong\u003e12\u003c/strong\u003e(2):306.\u003c/li\u003e\n\u003cli\u003eAboelnaga N, Elsayed SW, Abdelsalam NA, Salem S, Saif NA, Elsayed M, Ayman S, Nasr M, Elhadidy M: \u003cstrong\u003eDeciphering the dynamics of methicillin-resistant Staphylococcus aureus biofilm formation: from molecular signaling to nanotherapeutic advances\u003c/strong\u003e. \u003cem\u003eCell Communication and Signaling \u003c/em\u003e2024, \u003cstrong\u003e22\u003c/strong\u003e(1):188.\u003c/li\u003e\n\u003cli\u003eHowden BP, Giulieri SG, Wong Fok Lung T, Baines SL, Sharkey LK, Lee JY, Hachani A, Monk IR, Stinear TP: \u003cstrong\u003eStaphylococcus aureus host interactions and adaptation\u003c/strong\u003e. \u003cem\u003eNature Reviews Microbiology \u003c/em\u003e2023, \u003cstrong\u003e21\u003c/strong\u003e(6):380-395.\u003c/li\u003e\n\u003cli\u003ePanneerselvam VP, Vajravelu LK, Thulukanam J, Lathakumari RH, Vimala PB, Nair DM: \u003cstrong\u003eGenomic adaptations of methicillin-resistant Staphylococcus aureus in healthcare environments\u003c/strong\u003e. \u003cem\u003eEcological Genetics and Genomics \u003c/em\u003e2025, \u003cstrong\u003e34\u003c/strong\u003e:100331.\u003c/li\u003e\n\u003cli\u003eKaushik A, Kest H, Sood M, Steussy BW, Thieman C, Gupta S: \u003cstrong\u003eBiofilm producing methicillin-resistant Staphylococcus aureus (MRSA) infections in humans: Clinical implications and management\u003c/strong\u003e. \u003cem\u003ePathogens \u003c/em\u003e2024, \u003cstrong\u003e13\u003c/strong\u003e(1):76.\u003c/li\u003e\n\u003cli\u003eAlsolami A, ALGhasab NS, Alharbi MS, Bashir AI, Saleem M, Syed Khaja AS, Aldakheel DF, Rakha E, Alshammari JA, Taha TE: \u003cstrong\u003eCommunity-acquired methicillin-resistant Staphylococcus aureus in hospitals: Age-specificity and potential zoonotic\u0026ndash;zooanthroponotic transmission dynamics\u003c/strong\u003e. \u003cem\u003eDiagnostics \u003c/em\u003e2023, \u003cstrong\u003e13\u003c/strong\u003e(12):2089.\u003c/li\u003e\n\u003cli\u003eRajput P, Nahar KS, Rahman KM: \u003cstrong\u003eEvaluation of antibiotic resistance mechanisms in gram-positive bacteria\u003c/strong\u003e. \u003cem\u003eAntibiotics \u003c/em\u003e2024, \u003cstrong\u003e13\u003c/strong\u003e(12):1197.\u003c/li\u003e\n\u003cli\u003eYarahmadi A, Najafiyan H, Yousefi MH, Khosravi E, Shabani E, Afkhami H, Aghaei SS: \u003cstrong\u003eBeyond antibiotics: exploring multifaceted approaches to combat bacterial resistance in the modern era: a comprehensive review\u003c/strong\u003e. \u003cem\u003eFrontiers in Cellular and Infection Microbiology \u003c/em\u003e2025, \u003cstrong\u003e15\u003c/strong\u003e:1493915.\u003c/li\u003e\n\u003cli\u003eMuteeb G, Rehman MT, Shahwan M, Aatif M: \u003cstrong\u003eOrigin of antibiotics and antibiotic resistance, and their impacts on drug development: A narrative review\u003c/strong\u003e. \u003cem\u003ePharmaceuticals \u003c/em\u003e2023, \u003cstrong\u003e16\u003c/strong\u003e(11):1615.\u003c/li\u003e\n\u003cli\u003eBarbarossa A, Rosato A, Corbo F, Clodoveo ML, Fracchiolla G, Carrieri A, Carocci A: \u003cstrong\u003eNon-antibiotic drug repositioning as an alternative antimicrobial approach\u003c/strong\u003e. \u003cem\u003eAntibiotics \u003c/em\u003e2022, \u003cstrong\u003e11\u003c/strong\u003e(6):816.\u003c/li\u003e\n\u003cli\u003eMishra AS, Vasanthan M, Malliappan SP: \u003cstrong\u003eDrug repurposing: A leading strategy for new threats and targets\u003c/strong\u003e. \u003cem\u003eACS pharmacology \u0026amp; translational science \u003c/em\u003e2024, \u003cstrong\u003e7\u003c/strong\u003e(4):915-932.\u003c/li\u003e\n\u003cli\u003eKrishnamurthy N, Grimshaw AA, Axson SA, Choe SH, Miller JE: \u003cstrong\u003eDrug repurposing: a systematic review on root causes, barriers and facilitators\u003c/strong\u003e. \u003cem\u003eBMC health services research \u003c/em\u003e2022, \u003cstrong\u003e22\u003c/strong\u003e(1):970.\u003c/li\u003e\n\u003cli\u003eSharma V, Ichikawa M, He P, Bravo Y, Dahl R, Ng BG, Cosford ND, Freeze HH: \u003cstrong\u003ePhosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts\u003c/strong\u003e. \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e2011, \u003cstrong\u003e286\u003c/strong\u003e(45):39431-39438.\u003c/li\u003e\n\u003cli\u003eLiang R, Ye Z-W, Qin Z, Xie Y, Yang X, Sun H, Du Q, Luo P, Tang K, Hu B: \u003cstrong\u003ePMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness\u003c/strong\u003e. \u003cem\u003eNature Communications \u003c/em\u003e2024, \u003cstrong\u003e15\u003c/strong\u003e(1):2144.\u003c/li\u003e\n\u003cli\u003eMa J, Chen T, Wu S, Yang C, Bai M, Shu K, Li K, Zhang G, Jin Z, He F: \u003cstrong\u003eiProX: an integrated proteome resource\u003c/strong\u003e. \u003cem\u003eNucleic acids research \u003c/em\u003e2019, \u003cstrong\u003e47\u003c/strong\u003e(D1):D1211-D1217.\u003c/li\u003e\n\u003cli\u003eChen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, Fu Y, Yang C, Li M, Wu S: \u003cstrong\u003eiProX in 2021: connecting proteomics data sharing with big data\u003c/strong\u003e. \u003cem\u003eNucleic acids research \u003c/em\u003e2022, \u003cstrong\u003e50\u003c/strong\u003e(D1):D1522-D1527.\u003c/li\u003e\n\u003cli\u003eVerdon J, Girardin N, Lacombe C, Berjeaud J-M, H\u0026eacute;chard Y: \u003cstrong\u003e\u0026delta;-hemolysin, an update on a membrane-interacting peptide\u003c/strong\u003e. \u003cem\u003ePeptides \u003c/em\u003e2009, \u003cstrong\u003e30\u003c/strong\u003e(4):817-823.\u003c/li\u003e\n\u003cli\u003eConlon BP, Nakayasu ES, Fleck LE, LaFleur MD, Isabella VM, Coleman K, Leonard SN, Smith RD, Adkins JN, Lewis K: \u003cstrong\u003eActivated ClpP kills persisters and eradicates a chronic biofilm infection\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e2013, \u003cstrong\u003e503\u003c/strong\u003e(7476):365-370.\u003c/li\u003e\n\u003cli\u003eAlara JA, Alara OR: \u003cstrong\u003eAn overview of the global alarming increase of multiple drug resistant: a major challenge in clinical diagnosis\u003c/strong\u003e. \u003cem\u003eInfectious Disorders-Drug TargetsDisorders) \u003c/em\u003e2024, \u003cstrong\u003e24\u003c/strong\u003e(3):26-42.\u003c/li\u003e\n\u003cli\u003eTarin-Pello A, Suay-Garcia B, Perez-Gracia M-T: \u003cstrong\u003eAntibiotic resistant bacteria: current situation and treatment options to accelerate the development of a new antimicrobial arsenal\u003c/strong\u003e. \u003cem\u003eExpert review of anti-infective therapy \u003c/em\u003e2022, \u003cstrong\u003e20\u003c/strong\u003e(8):1095-1108.\u003c/li\u003e\n\u003cli\u003eDutta S, Rana VS, Backstedt BT, Shakya AK, Kitsou C, Yas OB, Smith AA, Ronzetti MH, Lipman RM, Araujo-Aris S: \u003cstrong\u003eBorrelial phosphomannose isomerase as a cell surface localized protein that retains enzymatic activity and promotes host-pathogen interaction\u003c/strong\u003e. \u003cem\u003eMbio \u003c/em\u003e2025, \u003cstrong\u003e16\u003c/strong\u003e(3):e03609-03624.\u003c/li\u003e\n\u003cli\u003eLanka S, Katta A, Kovvali M, Pandrangi S: \u003cstrong\u003eEnterococcus faecium Virulence Factors and Biofilm Components: Synthesis, Structure, Function, and Inhibitors\u003c/strong\u003e. In: \u003cem\u003eESKAPE Pathogens: Detection, Mechanisms and Treatment Strategies.\u003c/em\u003e Springer; 2024: 209-226.\u003c/li\u003e\n\u003cli\u003eNikolic P, Mudgil P: \u003cstrong\u003eThe cell wall, cell membrane and virulence factors of Staphylococcus aureus and their role in antibiotic resistance\u003c/strong\u003e. \u003cem\u003eMicroorganisms \u003c/em\u003e2023, \u003cstrong\u003e11\u003c/strong\u003e(2):259.\u003c/li\u003e\n\u003cli\u003eCiarolla AA, Lapin N, Williams D, Chopra R, Greenberg DE: \u003cstrong\u003ePhysical approaches to prevent and treat bacterial biofilm\u003c/strong\u003e. \u003cem\u003eAntibiotics \u003c/em\u003e2022, \u003cstrong\u003e12\u003c/strong\u003e(1):54.\u003c/li\u003e\n\u003cli\u003eChen J, Lv Y, Shang W, Yang Y, Wang Y, Hu Z, Huang X, Zhang R, Yuan J, Huang J: \u003cstrong\u003eLoaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles\u003c/strong\u003e. \u003cem\u003eFrontiers in Microbiology \u003c/em\u003e2023, \u003cstrong\u003e14\u003c/strong\u003e:1254367.\u003c/li\u003e\n\u003cli\u003eBowman L, Palmer T: \u003cstrong\u003eThe type VII secretion system of Staphylococcus\u003c/strong\u003e. \u003cem\u003eAnnual review of microbiology \u003c/em\u003e2021, \u003cstrong\u003e75\u003c/strong\u003e(1):471-494.\u003c/li\u003e\n\u003cli\u003eCao Z, Casabona MG, Kneuper H, Chalmers JD, Palmer T: \u003cstrong\u003eThe type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria\u003c/strong\u003e. \u003cem\u003eNature microbiology \u003c/em\u003e2016, \u003cstrong\u003e2\u003c/strong\u003e(1):1-11.\u003c/li\u003e\n\u003cli\u003eHuang X, Li G: \u003cstrong\u003eAntimicrobial peptides and cell-penetrating peptides: non-antibiotic membrane-targeting strategies against bacterial infections\u003c/strong\u003e. \u003cem\u003eInfection and Drug Resistance \u003c/em\u003e2023:1203-1219.\u003c/li\u003e\n\u003cli\u003eEltwisy HO, Twisy HO, Hafez MH, Sayed IM, El-Mokhtar MA: \u003cstrong\u003eClinical infections, antibiotic resistance, and pathogenesis of Staphylococcus haemolyticus\u003c/strong\u003e. \u003cem\u003eMicroorganisms \u003c/em\u003e2022, \u003cstrong\u003e10\u003c/strong\u003e(6):1130.\u003c/li\u003e\n\u003cli\u003eGanesan N, Mishra B, Felix L, Mylonakis E: \u003cstrong\u003eAntimicrobial peptides and small molecules targeting the cell membrane of Staphylococcus aureus\u003c/strong\u003e. \u003cem\u003eMicrobiology and Molecular Biology Reviews \u003c/em\u003e2023, \u003cstrong\u003e87\u003c/strong\u003e(2):e00037-00022.\u003c/li\u003e\n\u003cli\u003eVeiko AG, Olchowik-Grabarek E, Sekowski S, Roszkowska A, Lapshina EA, Dobrzynska I, Zamaraeva M, Zavodnik IB: \u003cstrong\u003eAntimicrobial activity of quercetin, naringenin and catechin: Flavonoids inhibit Staphylococcus aureus-induced hemolysis and modify membranes of bacteria and erythrocytes\u003c/strong\u003e. \u003cem\u003eMolecules \u003c/em\u003e2023, \u003cstrong\u003e28\u003c/strong\u003e(3):1252.\u003c/li\u003e\n\u003cli\u003eCamberg JL, Hoskins JR, Wickner S: \u003cstrong\u003eClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics\u003c/strong\u003e. \u003cem\u003eProceedings of the National Academy of Sciences \u003c/em\u003e2009, \u003cstrong\u003e106\u003c/strong\u003e(26):10614-10619.\u003c/li\u003e\n\u003cli\u003eGersch M, Famulla K, Dahmen M, G\u0026ouml;bl C, Malik I, Richter K, Korotkov VS, Sass P, R\u0026uuml;bsamen-Schaeff H, Madl T: \u003cstrong\u003eAAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control\u003c/strong\u003e. \u003cem\u003eNature communications \u003c/em\u003e2015, \u003cstrong\u003e6\u003c/strong\u003e(1):6320.\u003c/li\u003e\n\u003cli\u003eSharma V, Singh TG: \u003cstrong\u003eDrug induced nephrotoxicity-A mechanistic approach\u003c/strong\u003e. \u003cem\u003eMolecular Biology Reports \u003c/em\u003e2023, \u003cstrong\u003e50\u003c/strong\u003e(8):6975-6986.\u003c/li\u003e\n\u003cli\u003eDo\u0026szlig; S, Blessing C, Haller K, Richter G, Sauer M: \u003cstrong\u003eInfluence of antibiotics on functionality and viability of liver cells in vitro\u003c/strong\u003e. \u003cem\u003eCurrent issues in molecular biology \u003c/em\u003e2022, \u003cstrong\u003e44\u003c/strong\u003e(10):4639-4657.\u003c/li\u003e\n\u003cli\u003eWen Z, Chen C, Shang Y, Fan K, Li P, Li C, Zheng J, Deng Q, Yu Z: \u003cstrong\u003eBaohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ\u003c/strong\u003e. \u003cem\u003ePhytomedicine \u003c/em\u003e2024, \u003cstrong\u003e130\u003c/strong\u003e:155590.\u003c/li\u003e\n\u003cli\u003eChen C, Li D, Shang Y, Lin Z, Wen Z, Li P, Yu Z, Chen Z, Liu X: \u003cstrong\u003eAntibacterial Activity and Mechanism of Candesartan Cilexetil against Enterococcus faecalis\u003c/strong\u003e. \u003cem\u003eACS omega \u003c/em\u003e2024, \u003cstrong\u003e9\u003c/strong\u003e(19):21510-21519.\u003c/li\u003e\n\u003cli\u003eLiu X, Xiong Y, Shi Y, Deng X, Deng Q, Liu Y, Yu Z, Li D, Zheng J, Li P: \u003cstrong\u003eIn vitro activities of licochalcone A against planktonic cells and biofilm of Enterococcus faecalis\u003c/strong\u003e. \u003cem\u003eFrontiers in Microbiology \u003c/em\u003e2022, \u003cstrong\u003e13\u003c/strong\u003e:970901.\u003c/li\u003e\n\u003cli\u003eXiong Y, Chen Z, Bai B, Peng Y, Liu S, Fang D, Wen Z, Shang Y, Lin Z, Han S: \u003cstrong\u003eThiazolopyrimidinone Derivative H5\u0026ndash;23 Enhances Daptomycin Activity against Linezolid-Resistant Enterococcus faecalis by Disrupting the Cell Membrane\u003c/strong\u003e. \u003cem\u003eACS Infectious Diseases \u003c/em\u003e2023, \u003cstrong\u003e9\u003c/strong\u003e(12):2523-2537.\u003c/li\u003e\n\u003cli\u003eZheng J, Shang Y, Wu Y, Zhao Y, Chen Z, Lin Z, Li P, Sun X, Xu G, Wen Z: \u003cstrong\u003eLoratadine inhibits Staphylococcus aureus virulence and biofilm formation\u003c/strong\u003e. \u003cem\u003eIScience \u003c/em\u003e2022, \u003cstrong\u003e25\u003c/strong\u003e(2).\u003c/li\u003e\n\u003cli\u003eZheng J, Liu X, Xiong Y, Meng Q, Li P, Zhang F, Liu X, Lin Z, Deng Q, Wen Z: \u003cstrong\u003eAMXT-1501 targets membrane phospholipids against Gram-positive and-negative multidrug-resistant bacteria\u003c/strong\u003e. \u003cem\u003eEmerging Microbes \u0026amp; Infections \u003c/em\u003e2024, \u003cstrong\u003e13\u003c/strong\u003e(1):2321981.\u003c/li\u003e\n\u003cli\u003eMeng Q, Wang X, Huang X, Li C, Yu Z, Li P, Liu X, Wen Z: \u003cstrong\u003eRepurposing Benzbromarone as an Antibacterial Agent against Gram-Positive Bacteria\u003c/strong\u003e. \u003cem\u003eACS Infectious Diseases \u003c/em\u003e2024, \u003cstrong\u003e10\u003c/strong\u003e(12):4208-4221.\u003c/li\u003e\n\u003cli\u003eLee AS, De Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, Harbarth S: \u003cstrong\u003eMethicillin-resistant Staphylococcus aureus\u003c/strong\u003e. \u003cem\u003eNature reviews Disease primers \u003c/em\u003e2018, \u003cstrong\u003e4\u003c/strong\u003e(1):1-23.\u003c/li\u003e\n\u003cli\u003eBae T, Schneewind O: \u003cstrong\u003eAllelic replacement in Staphylococcus aureus with inducible counter-selection\u003c/strong\u003e. \u003cem\u003ePlasmid \u003c/em\u003e2006, \u003cstrong\u003e55\u003c/strong\u003e(1):58-63.\u003c/li\u003e\n\u003cli\u003eGoc A, Sumera W, Rath M, Niedzwiecki A: \u003cstrong\u003eInhibition of \u0026alpha;-hemolysin activity of Staphylococcus aureus by theaflavin 3, 3\u0026rsquo;-digallate\u003c/strong\u003e. \u003cem\u003ePlos one \u003c/em\u003e2023, \u003cstrong\u003e18\u003c/strong\u003e(8):e0290904.\u003c/li\u003e\n\u003cli\u003eChen Z, Song K, Shang Y, Xiong Y, Lyu Z, Chen J, Zheng J, Li P, Wu Y, Gu C: \u003cstrong\u003eSelection and identification of novel antibacterial agents against planktonic growth and biofilm formation of Enterococcus faecalis\u003c/strong\u003e. \u003cem\u003eJournal of Medicinal Chemistry \u003c/em\u003e2021, \u003cstrong\u003e64\u003c/strong\u003e(20):15037-15052.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MLS0315771, Staphylococcus aureus, multidrug resistance, biofilm inhibition, membrane-targeting antibacterial agent","lastPublishedDoi":"10.21203/rs.3.rs-8617708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8617708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The escalating prevalence of multidrug-resistant (MDR) and biofilm-forming Staphylococcus aureus has created an urgent demand for alternative therapeutic strategies beyond conventional antibiotics. In this study, we report for the first time the potent antibacterial and antibiofilm activities of MLS0315771, a competitive phosphomannose isomerase (MPI) inhibitor, against a broad range of Gram-positive bacteria. MLS0315771 exhibited strong inhibitory and bactericidal effects against both MSSA and MRSA, as well as Enterococcus faecium strains resistant to linezolid, with MIC₅₀/₉₀ values of 6.25/6.25 μM. This compound also significantly delayed bacterial proliferation at subinhibitory concentrations and eradicated mature biofilms in vitro. Time-kill assays revealed that MLS0315771 displayed time-dependent bactericidal activity comparable or superior to vancomycin. Proteomic profiling of S. aureus exposed to MLS0315771 and functional assays confirmed that MLS0315771 disrupts bacterial membrane integrity and induces depolarization, thereby impairing essential membrane functions. Checkerboard analyses further indicated that unsaturated long-chain fatty acids antagonized its antibacterial activity, supporting a membrane-targeting antibacterial mechanism. Moreover, MLS0315771 displayed low hemolytic activity and minimal cytotoxicity toward mammalian cells, suggesting a favorable therapeutic index. Collectively, these findings demonstrate that MLS0315771 acts as a novel membrane-targeting antibacterial agent with potent activity against MDR and biofilm-forming Gram-positive pathogens, highlighting its potential as a promising lead compound for future antimicrobial development.","manuscriptTitle":"Drug repurposing: Antimicrobial and antibiofilm effects of MLS0315771 against Gram-positive bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 14:19:53","doi":"10.21203/rs.3.rs-8617708/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-26T19:51:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T12:39:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93303392646828599171688274704200142237","date":"2026-02-17T20:52:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T13:16:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180109153163809173239664225341097885655","date":"2026-02-15T10:02:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335325030232413704070614119967596974477","date":"2026-01-30T09:21:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T09:04:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T09:01:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T02:32:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-26T11:31:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-01-26T11:22:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e399e4fa-678c-414b-846d-4219e64ebd15","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:02:14+00:00","versionOfRecord":{"articleIdentity":"rs-8617708","link":"https://doi.org/10.1186/s12866-026-05074-9","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-04-29 15:56:59","publishedOnDateReadable":"April 29th, 2026"},"versionCreatedAt":"2026-01-30 14:19:53","video":"","vorDoi":"10.1186/s12866-026-05074-9","vorDoiUrl":"https://doi.org/10.1186/s12866-026-05074-9","workflowStages":[]},"version":"v1","identity":"rs-8617708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8617708","identity":"rs-8617708","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}