Enhanced antibacterial activity of organic acids via gallium chelation: a promising antibiotic alternative

Enhanced antibacterial activity of organic acids via gallium chelation: a promising antibiotic alternative | 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 Enhanced antibacterial activity of organic acids via gallium chelation: a promising antibiotic alternative Yuhuan Qin, Xian Liu, Wei Luo, Xia Li, Yong Meng, Hui Qin, Xuepin Liao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5970012/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As an alternative to antibiotics, acidifiers have gained widespread application in the feed industry. However, current acidifier products often suffer from limited antibacterial efficacy. To tackle this issue, we synthesized a series of organic acid - gallium complexes (Ga-OA) using organic acids (OA) and Ga 3+ as precursors, via a liquid-phase synthesis method. The antimicrobial activity of Ga-OA against Escherichia coli , Staphylococcus aureus , and Salmonella spp. was assessed using the Oxford cup and agar dilution methods to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. It was found that the Ga-OA complexes showed markedly higher antibacterial activity than each individually, and the antibacterial activity of Ga-OA complexes followed the order: Ga-Lac (lactic acid) > Ga-Ac (acetic acid) > Ga-BA (butyric acid). Furthermore, The MIC values of Ga-Lac against Escherichia coli, Staphylococcus aureus, and Salmonella spp were 2.84, 0.18, and 2.84 mmol/L, respectively, meanwhile, the MBC values of Ga-Lac against these three bacteria were 5.68, 1.42, and 5.68 mmol/L, respectively. Transcriptome analysis revealed that the antibacterial mechanism of Ga-OA is initiated by organic acid (OA) binding to bacterial membranes, which promotes Ga 3+ entry into the cell. This intracellular Ga³⁺ then disrupts iron transport, ultimately resulting in bacterial death. These results suggest that Ga-OA complexes have the potential to be a promising, safe, and effective antibacterial agent in animal husbandry, providing a solution to antibiotic resistance concerns. organic acids (OA) gallium (Ga³⁺) complexes antibacterial activity transcriptome analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1.Introduction The widespread use of antibiotics as growth promoters in livestock feed [1] has resulted in antibiotic overuse, contributing to food safety concerns and the emergenceof antibiotic-resistant bacteria in animals [2][3][4] . This situation poses a significant threat to global human health [5][6] . Consequently, the livestock industry is undergoing a global shift towards "antibiotic-free farming", demanding the urgent development of novel alternatives to antibiotics. Currently recognized novel and safe antibacterial agents that can serve as alternatives to antibiotics include acidifiers, oligosaccharides, plant extracts, antimicrobial peptides, enzyme preparations, etc [7] . Among these, acidifiers are particularly notable for their diverse benefits. They create an acidic environment in the livestock gastrointestinal tract, reducing inflammation, inhibits pathogen growth and reproduction, preventing excessive gastrointestinal dysfunction, and enhancing livestock resilience [8][9] . Furthermore, acidifiers improve palatability and digestibility, increase digestive enzyme activity and antioxidant properties, and optimize the gastrointestinal microbiota and nutrient absorption, leading to improved digestion and absorption [10] . Acidifiers also participate in the biosynthesis and metabolism of substances, promoting the absorption of minerals and other nutrients. These compounds can be broadly classified into three primary classes : inorganic acids, organic acids and composite acidifiers. Numerous studies have consistently shown that organic acidifiers exhibit greater effectiveness than their inorganic counterparts [11][12][13] . Specifically, inorganic acids undergo rapid dissociation leading to a marked decrease in pH values within the animal's esophagus and stomach, potentially causing tissue damage and developmental problems; Conversely, organic acids display a more controlled release of protons within the stomach. Organic acids serve as effective feed growth promoters in livestock and poultry by inhibiting the proliferation of pathogenic microorganisms. These benefits include antimicrobial activity, immune modulation, and enhanced mineral and vitamin absorption [14] . Research has shown that broiler chickens treated with organic acidifiers gained weight and exhibited lower levels of low-density lipoprotein and higher levels of triglycerides, indicating that acidifiers can serve as effective alternatives to antibiotics [15] . Qiu et al. found that incorporating 0.8% acidifier into feed can enhance the immune system, antioxidant capacity, and liver lipid metabolism in broiler chickens, ultimately promoting their growth and production [16] . Consequently, organic acids contribute to improve the live weight and growth performance of livestock while maintaining high animal safety. However, acidifiers often suffered the following problems, including limited specificity, poor efficacy against certain pathogens and inability to replace all antibiotics. Accordingly, a significant focus of future research and development on acidifiers should be to broaden their antimicrobial spectrum and enhance their antibacterial efficacy. In recent years, Ga(III) has emerged as a promising antibacterial agent, exhibiting both bacteriostatic and bactericidal effects. Study by Harrington et al . demonstrated that gallium nitrate can inhibit and kill Streptococcus pneumoniae , while oral administration of gallium maltophenol in mice effectively cures infections caused by this bacterium [17] . Olakanmi et al . found that gallium nitrate can inhibit the activity of Mycobacterium tuberculosis ribonucleotide reductase, a key enzyme for DNA replication, in a mouse pulmonary tuberculosis model, thereby inhibiting the growth of Mycobacterium tuberculosis [18] . These findings highlight the potential of Ga(III) as a therapeutic agent against various bacterial pathogens. It was suggested the significant chemical resemblance between Ga(III) and Fe(III) makes it difficult for bacteria to effectively discriminate between the two metals [19][20] . Upon cellular uptake, Ga(III) disrupts bacterial iron homeostasis by interfering with the iron transport system, resulting in iron deficiency and ultimately bacterial death [21][22][23][24] . This process can also lead to decreased or inactivated iron-dependent enzyme activities [25] , disruption of the bacterial electron transport chain [26] , and the induction of oxidative stress [27] . However, the high aqueous solubility of gallium salts results in weak cellular affinity, which limits their efficacy as monotherapies due to the challenge of intracellular delivery required for their antibacterial effects [28] . Nevertheless, the coordination chemistry of Ga(III) presents a promising avenue to overcome this limitation. Herein, gallium-organic acid complexes (Ga-OA) with enhanced antibacterial effects were synthesized through autonomous assembly and coordination. Organic acids, capable of associating with phospholipids and proteins in bacterial cell walls, act as both antibacterial agents and efficient carriers for Ga(III) entry into cells. This dual role amplifies the inhibitory effects of both Ga(III) and the organic acid, leading to enhanced antibacterial activity in the Ga(III)- OA complexes. We investigated the antibacterial activity of Ga-OA against three common pathogenic bacteria in livestock and poultry: Escherichia coli, Staphylococcus aureus , and Salmonella spp . Furthermore, transcriptomic analysis was used to explore the antibacterial mechanisms of these complexes. 2. Experimental Section 2.1. Materials Gallium nitrate (Ga (NO 3 ) 3 ·6H 2 O), analytical pure, purchased from Aladdin. L-lactic acid (Lac), acetic acid (Ac), and butyric acid (BA) are all analytical pure and purchased from Aladdin. NaCl, Agar powder, glutaraldehyde, methanol and other chemicals used in the study were all of analytical grade. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 23656, and Salmonella SIIA 235 were provided by the Tanning Biotechnology Laboratory of Sichuan University. Medium: The liquid medium is Mueller Hinton (MH) broth medium, while the solid agar plate and test tube slanted medium are both supplemented with 1.5% agar in nutrient broth (NB) medium. 2.2 Synthesis of Gallium-Organic Acid Complex (Ga-OA) A series of gallium-organic acid complex were synthesized via a liquid-phase method. Optimally, precise quantities of organic acids (OA) and gallium nitrate Ga(NO 3 ) 3 were weighed based on the molar ratios of 0.5:1 and 0.25:1, respectively. Each compound was individually dissolved in purified water. Subsequently, the Ga(NO 3 ) 3 solution was added dropwise to the OA solution. The mixture was stirred at room temperature for 8 hours (Fig. 1 ) to facilitate complex formation. 2.3 Characterization The chemical structure of Ga-OA complexes were characterized using FT-IR spectroscopy in the wavenumber range of 500–4000 cm − 1 . The surface elemental composition and binding energy of the prepared samples were determined through X-ray photoelectron spectroscopy (XPS) with AI Kα X-rays source. The chemical structure of the Ga-OA was further analyzed by Raman spectroscopy (Roman), and the sample was scanned by 532 nm laser in the range of 200–2000 cm − 1 . 2.4 Preparation of bacterial suspension. To prepare bacterial suspensions, each test strain was initially activated and cultured on nutrient broth (NB) slant medium for 24 hours, undergoing two generations of activation. Subsequently, several loops of well-growing colonies were scraped and inoculated into MH liquid medium for expansion culture. The cultures were incubated in a shaker at 37°C and 180 r/min for 24 hours. Finally, an appropriate volume of the expanded culture broth was collected, washed with sterile physiological saline, and diluted to a concentration of 10 7 CFU/mL. 2.5 Antibacterial activity test of Ga-OA Inhibition circle diameter: The diameter of the zone of inhibition of the Ga-OA against three common poultry and livestock pathogens was determined using the horizontal diffusion method (Oxford cup method). ConFig.d plates were incubated at 37 ℃ for 24 h, and the diameter of the zone of inhibition on each plate was measured by vernier caliper (mm). MIC and MBC determination. The minimum inhibitory concentration (MIC) of Ga-OA against the pathogenic bacteria was determined using the twofold dilution method. Samples were incubated at 37℃ for 24 h, and the growth of bacteria was visually observed. The lowest concentration at which the solution became clear and transparent was defined as the minimum inhibitory concentration (MIC). To determine the minimum bactericidal concentration (MBC), the lowest inhibitory concentration (MIC) and a concentration higher than the MIC from the first three groups were inoculated onto sterile agar plates and incubated at 37 o C for 24 hours. The lowest concentration that resulted in no bacterial growth on the agar plate was defined as the minimum bactericidal concentration (MBC). Growth dynamics: S. aureus was selected to determine its growth kinetic curve. Following 8 hours of expansion culture, S. aureus cells were centrifuged and diluted to prepare a bacterial suspension at a concentration of 10 7 CFU/mL. The suspension was then combined with the corresponding MIC concentration of Ga-OA and MH medium mixture. Samples were collected every 3 h, and measure the OD 600 . A curve was plotted according to the results to observe the effect of the complex on the growth of bacteria during action times. The content of Ga 3+ in cells: To determine the content of Ga 3+ in the cells, inductively coupled plasma spectrometry (ICP-OES) was used. S. aureus after extended activation culture was prepared into a suspension of 10 7 CFU/mL. It was added into the MH medium with Ga-OA and Ga 3+ at the concentration of MIC and cultured for 24 h. Bacteria were sampled and collected every 3 h and digested with concentrated nitric acid and hydrogen peroxide. 2.6 Antibacterial mechanism of Ga-OA Field Emission Scanning Electron Microscopy (FESEM) Analysis: SEM was employed to examine the microscopic morphology of S. aureus cells treated with the Ga-OA complexes. Following treatment by Ga-OA, S. aureus cells were fixed with 2.5% glutaraldehyde, centrifugation with ethanol gradient, and finally dried at room temperature. TEM transmission electron microscopy (TEM) analysis: In order to investigate the effect of Ga-OA complexes on the internal structure of S. aureus cells, the bacteria were treated with Ga-OA and equimolar concentrations of Ga 3+ and OA, respectively. The organisms were the collected, fixed, dehydrated, embedded, and stained to make ultrathin sections. Ultrastructural changes of the cells were subsequently observed using a Hitachi-HT7700 transmission electron microscope. Transcriptome sequencing analysis: To further investigate the genetic effects of the Ga-OA complex on S. aureus , RNA sequencing was conducted. Accordingly, S. aureus cells in the logarithmic growth phase were inoculated in MH medium containing Ga 3+ or Ga-Acsolution and incubated at 37 ℃ for 4 h. Then, bacteria were collected and stored frozen, and the total RNA was extracted from the bacterial samples, and sequencing libraries were prepared using the NovaSeqXPlus platform at Shanghai Meiji Biotechnology Co. High-quality reads were aligned to the genome of S. aureus GCF_000756205.1 using Bowtie software. Transcripts per million (TPM) values were used to quantify gene expression levels in the samples. Differential expression analysis was performed using DEGSeq2 software to identify differentially expressed genes (DEGs) based on the criteria of |log 2 FC| ≥ 1 and p-adjusted value < 0.05. Functional enrichment analysis was then conducted on the DEGs to elucidate the inhibitory mechanisms of the Ga-OA complexes against S. aureus . 3. Result and discuss 3.1 Chemical Structural Characterization of Ga-OA. 3.1.1 FT-IR analysis The chemical structures of the organic acids and their gallium complexes was characterized by FT-IR. The FT-IR spectra of the Ga-OA complexes showed substantial differences from those of the individual organic acids (Fig. 2 a-c). All three OAs exhibit a pronounced broad absorption peak around 3450 cm − 1 , which corresponds to the stretching vibration of υ O-H in the carboxylic acid moiety of OA molecule [29] . As shown in Fig. 2 (a), lactic acid (Lac) exhibited a distinct single peak near 1652 cm⁻¹, characteristic of the C = O bond in the carboxyl group [30] . Additionally, a stretching vibration peak of the C-O bond was observed at 1243 cm − 1[31] . In the Ga-Lac complex, these two peaks were significantly weakened, suggesting the involvement of the carbonyl group participates in the coordination reaction. This observation was further supported by the decrease in peak intensity upon complex formation. Furthermore, the principles of organic acid-metal ion interactions indicate that complexation leads to alterations in the C-O bond energy within the carboxylic acid [32][33][34][35] . Similarly, in Fig. 2 (b), acetic acid (Ac) exhibits a characteristic C-O stretching vibration peak at 1278 cm − 1 , which was significantly weakened in the complexes, and the peaks at 1652 cm − 1 for the complexes are shifted to higher wavenumbers. Figure 2 (c) shows that butyric acid (BA) displayed a characteristic C-O stretching vibration peak at 1203 cm − 1 , which also diminished upon complexation. These findings indicate that the gallium ion coordinates with the oxygen atom of the carbonyl group in the organic acid to form the organic acid-gallium ion complexes. 3.1.2 XPS spectral analysis X-ray photoelectron spectroscopy (XPS) analysis of the O1s spectra of OA and Ga-OA (Fig. 2 d-f) provided further evidence of complex formation. Compared to OA, the characteristic peak positions of the Ga-OA complex exhibited notable shifts. The O1s spectrum of Lac displays characteristic peaks at binding energies of 531.93 eV and 532.67 eV, corresponding to C = O and C-O (Fig. S1 ), respectively [36][37] . In contrast, the O1s spectrum of the Ga-Lac complex Fig. 2 (d) revealed a new characteristic peak at a binding energy of 533.18 eV, attributed to the bond of Ga-O. This shift arises from the coordination reaction between the carbonyl group of Lac and Ga 3+ . Specifically, the electrons on the carbonyl oxygen shift to the empty orbitals under the influence of the central Ga 3+ ion [38] , resulting in a decrease in the electron cloud density of the ligand Lac. Therefore, the bond energy of the carbonyl oxygen shifted towards higher binding energies, forming a new Ga-O coordination bond. Similarly, upfield shifts in the O1s spectra of acetic acid (Ac) (Fig. S2) and butyric acid (BA) (Fig. S3) and their respective Ga 3+ complexes (Fig. 2 e and Fig. 2 f) confirmed the coordination reaction leading to the upfield shift of the carbonyl groups in OA. In general, these XPS findings provide further evidence of the successful coordination and bonding of Ga 3+ with the carbonyl groups of OA. 3.1.3 Raman spectral analysis Figure 2 (g-i) shows the Raman spectra of OA and Ga-OA. Compared to OA, the Raman spectra of Ga-OA complexes in the range of 200–2000 cm − 1 are significantly different. The Raman spectrum of OA lacks discernible characteristic absorption peaks, whereas the Ga-OA spectrum exhibits a prominent novel characteristic peak near 1050 cm − 1 , attributable to the bending vibration of the carboxyl group C-O [39] . This peak is absent in the spectrum of OA, definitively indicating a coordination reaction between OA and Ga 3+ . 3.2 Antimicrobial activity of the Ga-OA 3.2.1 Inhibitory circle diameter The diameter of the inhibition zone serves as a preliminary indicator of antibacterial activity, exhibiting a positive correlation with antibacterial potency within a defined range. Figure 3 (a-c) depicts the diameters of inhibition zones generated by organic acids, gallium, and organic acid-gallium complexes with varying molar ratios against three common livestock pathogens: E. coli , S. aureus , and Salmonella . As shown in Fig. 3 (a-c), the three organic acids (lactic acid, acetic acid, and butyric acid) exhibit moderate inhibitory effects against these pathogens, with inhibition zone diameters ranging from approximately 14 to 17 mm. Gallium ions also demonstrate some inhibitory activity, with inhibition zone diameters of approximately 8 to 13 mm. Significantly, the antibacterial activity of the Ga-OA surpasses that of both the individual organic acids and gallium ions. At a coordination molar ratio of 0.5:1 (Ga/OA), Ga-OA exhibits inhibition zone diameters of approximately 16–20 mm against E. coli , 20–24 mm against S. aureus , and 15–18 mm against Salmonella . Even at lower coordination molar ratios (as shown in Fig. S4-S6), the Ga-OA demonstrate higher antibacterial activity compared to their individual components. 3.2.2 MIC and MBC We further validated the enhanced antibacterial efficacy of Ga-OA by assessing the MIC and MBC values against pathogenic bacteria using both the monomer and its complex. As presented in Table 1 , the MIC values of Ga-OA ( molar ratio of Ga:OA was 0.5:1) were significantly lower than those of Ga³⁺ and OA alone, indicating a substantial enhancement in its antibacterial performance. Similarly, the MBC values of Ga-OA ( molar ratio of Ga:OA was 0.5:1), as shown in Table 2 , were also reduced compared to the individual use of Ga³⁺ and OA, further supporting the notion of a synergistic antibacterial effect. A comparison of the MIC and MBC values (Tables 1 and 2 ) reveals that Ga-OA exhibited the most potent inhibitory effect against S. aureus , which aligns with the findings from the inhibition zone assays. The lower MBC values for Ga-OA, compared to Ga³⁺ and OA alone, suggest an improved bactericidal efficacy of the complex, thereby highlighting its potential as an effective antimicrobial agent. The enhanced antibacterial activity of Ga-OA complexes arises from a muti-faceted mechanism. Upon coordination with OA, a portion of the positive charge on the Ga 3+ ion is transferred to the OA molecule, inducing electron delocalization within the resulting complex. This electron delocalization effect reduces the polarity of Ga 3+ while enhancing the its ability to permeate the lipid bilayer of cell membranes [40][41][42] . Consequently, the complex readily penetrates the cell interior, disrupting cellular processes and leading to enhanced antibacterial potency. Notably, Ga-OA exhibits varying degrees of inhibitory activities against different pathogenic bacteria, with S. aureus exhibiting the most pronounced inhibitory effect. This variation in susceptibility could be attributed to differences in the cell wall structure of different microorganisms. Ga-OA complexes are more likely to penetrate the cell wall and accumulate intracellularly, potentially affecting the synthesis of intracellular DNA replication-related enzymes, hindering base replication, and ultimately inhibiting DNA synthesis, thereby inhibiting bacterial growth [40] . 3.2.3 Growth kinetics of S. aureus To further investigate the inhibitory effects of Ga-OA against S. aureus , the Ga-Lac complex was selected to study its growth inhibition. Figure 3 (c) presents the growth kinetic curves of S. aureus after treatment with Ga-Lac and Lac, respectively. Compared to the blank control group, S. aureus in the experimental groups (Ga-Lac, Lac) exhibited a degree of growth, however, their growth rates were lower than that of the blank control. The order of inhibitory strength was Ga-Lac > Lac > blank control, indicating that the gallium organic acid complex effectively suppressed the growth of S. aureus . Table 1 MIC of OA, Ga 3+ and Ga-OA against three poultry pathogenic bacteria. Samples MIC (mmol/L) Escherichia coli Staphylococcus aureus Salmonella Lactic acid 15.00 3.13 22.74 Acetic acid 7.50 3.13 8.53 Butyric acid 7.50 3.13 11.37 Ga 3+ 4.00 4.00 4.00 Ga-Lactic acid 2.84 0.18 2.84 Ga-Acetic acid 2.13 1.07 2.13 Ga-Butyric acid 2.91 0.73 2.91 Table 2 MBC of OA, Ga 3+ and Ga-OA against three poultry pathogenic bacteria. Samples MBC (mmol/L) Escherichia coli Staphylococcus aureus Salmonella Lactic acid 15.00 3.13 22.74 Acetic acid 7.50 3.13 8.53 Butyric acid 7.50 3.13 11.37 Ga 3+ 8.00 8.00 8.00 Ga-Lactic acid 5.68 1.42 5.68 Ga-Acetic acid 4.26 4.26 4.26 Ga-Butyric acid 5.81 2.14 5.81 3.3 Antimicrobial mechanism Antibacterial activity investigations revealed that Lac, Ga 3+ , and Ga-OA all exhibited inhibitory effects against three bacterial species, leading to alterations in bacterial cell morphology. To assess these morphological alterations, SEM was employed to observe the changes in S. aureus cells following treatment with Lac, Ga 3+ , and Ga-Lac, respectively. The results are shown in Fig. 4 (a-d). Compared to the control group (sterile saline), significant alterations in cellular morphology were observed in the experimental groups treated with Lac, Ga 3+ and Ga-Lac with varying degrees of impact among Lac, Ga 3+ , and Ga-Lac. As shown in Fig. 4 d, typically S. aureus cells exhibit a spherical shape with a smooth surface. Following Lac treatment, cells displayed slight deformation with partial inward concavity on the surface. This could be attributed to Lac's small molecular size and lipophilic nature, allowing it to traverse the cell wall and membrane [41] , interacting with intracellular active substances and causing morphological changes [43] . Cells treated with Ga 3+ also underwent mild deformation and displayed a rougher surface (Fig. 4 b). However, comparing Lac and Ga 3+ , cells exposed to Ga-OA demonstrated more pronounced concavity. As shown in Fig. 4 c, the cell was severely deformed, the cell wall structure collapsed, and the cell membrane ruptured, indicating Ga-OA showed stronger synergistic bacterial inhibition. This heightened efficacy arises from the Ga-Lac complex inheriting the liposolubility of Lac, facilitating its penetration through the cell wall and membrane into the cell interior to exert its antibacterial function. Further, TEM was employed to investigate alterations in the intracellular ultrastructure of bacteria, with the results presented in Fig. 4 e-h. As shown in Fig. 4 h, S. aureus bacterium in the blank control group maintained a smooth spherical shape, featuring relatively intact cell walls, cell membranes, a plump and evenly distributed cytoplasm, and abundant organelles. In contrast, while the cell walls and membranes remained relatively intact in Lac-treated cells, the cytoplasm exhibited slight coagulation, and weaker plasmolysis was observed at the cell termini (Fig. 4 e). Integrating the SEM findings, it can be inferred that Lac displays good affinity towards cells but exerts limited effects. Similarly, in Ga 3+ -treated cells, the cell walls and membranes remained intact, while slight surface deformation was observed (Fig. 4 f), accompanied by cytoplasmic condensation and vacuolation, indicating that the primary target of Ga 3+ lies within the cytoplasm. However, due to its weak affinity with cells, only a small amount of Ga 3+ entered the cytoplasm to exert its effects. Notably, the most pronounced morphological changes were observed in Ga-OA treated cells (Fig. 4 g), characterized by a roughened cell surface, distinct ruptures in the cell wall and membrane, cytoplasmic leakage, uneven cytoplasmic distribution, severe coagulation and vacuolation, and incomplete cell morphology. These findings further suggest that the active site of the complex primarily resides in the cytoplasm. Combined with the SEM results, it can be postulated that upon forming a complex with OA, Ga 3+ utilizes OA as a carrier to enhance its liposolubility, facilitating easier penetration into cells and subsequently causing cellular damage or even apoptosis, thereby synergistically potentiating the antibacterial efficacy. To verify whether gallium ions in the complex enter cells to exert their effects, the accumulation of gallium ions in S. aureus cells at different time points was analyzed. As shown in Fig. S7, the experimental group was Ga-Lac, while the control group was Lac. Within 0 to 12 hours, the content of Ga in cells from the control group remained essentially zero, whereas in the experimental group, the Ga content initially increased and then decreased with prolonged treatment time. This indicates that Ga-Lac in its complex form more readily penetrates the cell membrane, entering the interior. As the treatment duration lengthened, the inhibitory effects of both Ga-Lac and Ga³⁺ prompted bacterial cells to secrete lysozyme, leading to autolysis, membrane rupture, and solute leakage, thereby decreasing the Ga content. These findings suggest that Ga-Lacexhibits a higher affinity for cells, manifesting in a stronger inhibitory capacity, consistent with observations from SEM images and TEM images. 3.4 Transcriptome Analysis RNA-seq technique was used to analyze the gene expressions of three groups (Con, Ga 3+ and Ga-Lac) to further illustrate the molecular mechanism of complex inhibition. The transcriptome sequencing results revealed 1565 and 1576 DEGs expressed by S. aureus that treated with Ga 3+ vs Con and Ga-Lac vs Con groups (Fig. 5 a-b), with 761 genes up-regulated and 815 genes down-regulated in Ga-Lac vs Con. The differences between the groups were significant, especially the Ga-Lac vs Con group (Fig. 5 c). To elucidate the functional implications of these DEGs, a Clusters of Orthologous Groups (COG) analysis was performed, categorizing the DEGs into eight predicted functional categories in S. aureus (Fig. 6 a). The results suggested that the repression of genes associated with metabolism had a greater impact on S. aureus growth. To further explore these metabolic pathways, KEGG annotations analysis was conducted on DEGs in the Ga-Lac vs Con group. Downregulated genes were primarily concentrated in metabolic and environmental information processing pathways (Fig. 6 b). KEGG enrichment analysis indicated that the phosphotransferase system and galactose metabolism pathways were significantly enriched (Fig. 6 c), suggesting that metabolism-related and transport system-related genes were significantly affected by Ga-Lac treatment. 3.4.1 Carbohydrate transport and metabolism The down-regulated DEGs in Ga-Lac vs Con involved in carbohydrate transport and metabolism were shown in Fig. 6 d. Gene CEP64_RS14610 encoding proteins involved in pentose and gluconate interconversion pathway as well as the synthesis of the PTS fructose transporter subunit was down-regulated. Genes ( furA, furB, pfkA ) involved in fructose and mannose metabolism were down-regulated. The expression of galactose metabolism-related genes ( bgaB, lacE, CEP64_RS14625, CEP64_RS14615, lacA, CEP64_RS14710, CEP64_RS1470 ) was distinctively repressed. Furthermore, the down-regulation of pentose phosphate pathway-related genes ( fbaA, CEP64_RS15195, CEP64_RS18360, hxlA, CEP64_RS2514 ) were significant. Additionally, genes involved in glycolysis/gluconeogenesis, including CEP64_RS24405, CEP64_RS8920, CEP64_RS15110 were also remarkably down-regulated. The above results indicated that Ga-Lac inhibits the growth of S. aureus by disrupting its carbohydrate transport and metabolism. 3.4.2 Coenzyme transport and metabolism The down-regulated DEGs in Ga-Lac vs Con involved in coenzyme transport and metabolism were shown in Fig. 6 e. Cofactors synthesis are essential for a range of intracellular chemical reactivity and specificity[43]. Down-regulation of NAD-dependent aldehyde dehydrogenase-related genes ( CEP64_RS15110 ) inhibited intracellular redox reactions. The gene ( CEP64_RS14395 ) encoding the NAD (P)-binding protein in siroheme synthase, which was involved in porphyrin metabolism, was down-regulated, as were ilvD, panC , genes related to the biosynthesis pathway of pantothenate and coenzyme A. Synthesis inhibition of these coenzymes in anabolic and catabolic pathways leads to bacterial metabolic disruption. 3.4.3 Amino acid transport and metabolism The down-regulated DEGs in Ga-Lac vs Con involved in amino acid transport and metabolism were shown in Fig. 6 f. These genes are mainly involved in the biosynthesisof alanine, aspartic acid, and glutamic acid ( gabT, CEP64_RS12660 ), lysine ( pepV ), cysteine ( metC, CEP64_RS26510 ), and tryptophan ( CEP64_RS20535 ). This down-regulation resulted in the inhibition of cellular components synthesis and intracellular processes of S. aureus , including signal transduction and molecular recognition. These results suggest that Ga-Lac may induce S. aureus apoptosis by deterring amino acid metabolism, potentially leading to apoptosis. 3.4.4 Environmental information processing 3.4.4.1 ABC transporter system Down-regulated DEGs involved in membrane transport in Ga-Lac vs Con were mainly concentrated in the ABC transporter system and PTS transporter system (Fig S8). The expression of genes involved in the ABC-type transporter system is shown in Table 3 . The genes ( CEP64_RS16540 ) encoding ABC transporter proteases were down-regulated, which inhibited periplasmic protein synthesis and galactose transport. Similarly, down-regulation of CEP64_RS24125 , encoding the amino acid ABC transporter protein permease, inhibited the ABC-type amino acid transport system. Additionally, the down-regulated DEGs ( CEP64_RS24130, CEP64_RS25245 ) inhibited the substrate-binding structural domain proteins of transporter. These findings suggest that inhibition of the ABC-type iron transport system disrupts the bacterial protein transport system as well as the para-energetic conversion system, thereby blocking intracellular protein transport as well as energy conversion in S. aureus. 3.4.4.2 PTS transport system The carbohydrate-phosphotransferase system (PTS) is a key carbohydrate transport system that recognizes both intracellular and extracellular signals, transferring the high-energy phosphate of phosphoenolpyruvate (PEP) to other enzymes and proteins [44] . Down-regulation DEGs ( CEP64_RS24390 ) involved in the synthesis of cellulose-specific component related genes were observed, Galactitol-specific component related genes ( CEP64_RS14615, CEP64_RS24780 ) and fructose-specific component related genes ( CEP64_RS25080, CEP64_RS2509 ) were significantly down-regulated, which deterred phosphotransferase system (PTS). These results suggest that Ga-Lac inhibits S. aureus by disrupting the ABC transporter system and PTS transporter systems, which are essential for disrupts the bacterial protein transport system and energy conversion system. Table 3 Expression of DEGs related to Environmental information processing to S. aureus in Ga-Lac vs Con ABC transporter system and PTS transporter system Gene ID Gene name Description Log 2 FC (Lac vs Con) CEP64_RS16540 CEP64_RS16540 extracellular solute-binding protein -3.3711 CEP64_RS16545 CEP64_RS16545 sugar ABC transporter permease -2.5588 CEP64_RS24125 CEP64_RS24125 amino acid ABC transporter permease -3.7373 CEP64_RS24130 CEP64_RS24130 transporter substrate-binding domain-containing protein -4.3453 CEP64_RS24135 CEP64_RS24135 amino acid ABC transporter ATP-binding protein -3.7349 CEP64_RS25245 CEP64_RS25245 transporter substrate-binding domain-containing protein -3.3968 CEP64_RS25230 CEP64_RS25230 amino acid ABC transporter permease -4.7213 CEP64_RS22820 Opp3b oligopeptide ABC transporter permease -3.2234 CEP64_RS14515 CEP64_RS14515 Fe(3+) dicitrate ABC transporter substrate-binding protein -3.5025 CEP64_RS24390 CEP64_RS24390 PTS cellobiose transporter subunit IIA -5.2356 CEP64_RS14615 CEP64_RS14615 PTS galactitol transporter subunit IIC -3.1063 CEP64_RS24780 CEP64_RS24780 PTS sugar transporter subunit IIB -5.7316 CEP64_RS25080 CEP64_RS25080 PTS fructose transporter subunit IIC -3.2642 CEP64_RS25090 CEP64_RS2509 PTS fructose transporter subunit IIB -2.0612 4. Conclusion This study reports the novel synthesis and characterization of Ga-OA, an antimicrobial agent formed by complexing organic acids with gallium ion (Ga 3+ ). While the organic acids exhibit broad-spectrum antibacterial activity, but they are often suffered the problems of limited specificity, poor efficacy against certain pathogens and inability to replace all antibiotics. In contrast, Ga 3+ exhibit strong antibacterial activity, but the high aqueous solubility has limited its application. These problems were effectively addressed in this study by the chelation of organic acids with Ga 3+ . Ga-OA significantly enhances the lipid solubility of Ga 3+ , facilitating greater cell penetration and disruption of cellular metabolism. Transcriptome sequencing revealed downregulation of genes involved in the glycolysis pathway in S. aureus treated with Ga-OA, highlighting its potential to target critical metabolic pathways. Ga-OA holds promising potential as a novel antimicrobial agent due to its superior antibacterial activity and low toxicity profile. It presents a viable alternative to traditional antibiotics for controlling bacterial infections in poultry and livestock farming. Declarations Founding mianynag habio bioengineering co., Ltd, and Horizontal Science and Technology Project（No．NJGS-2023000594）, Luzhou Laojiao Co., Ltd. Appendix A. Supplementary data Supplementary data to this article can be found online. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics declaration The authors declare that this study was conducted in accordance with ethical standards and guidelines. Consent to publish The participant has consented to the submission of the case report to the journal. Consent to participate Yuhuan Qin: Supervision, Conceptualization. Xian Liu: Investigation. Wei Luo: Supervision, Conceptualization. Xia Li: Investigation. Mengyang Huang: Investigation. Hui Qin: Investigation. Xuepin Liao: Supervision. Bi Shi: Investigation. Informed consent was obtained from all individual participants included in the study. Acknowledgement We sincerely thank the financial supports provided by the mianynag habio bioengineering co., Ltd, and Horizontal Science and Technology Project（No．NJGS-2023000594）, Luzhou Laojiao Co., Ltd. Author Contribution QYH completed the experimental content and wrote the article, All authors reviewed the manuscript. References T. Pulingam, T. Parumasivam, A. M. Gazzali, A. M. Sulaiman, J. Y. Chee, M. j Lakshmanan, C. F. Chin, K. Sudesh. (2022)Antimicrobial resistance: prevalence, economic burden, mechanisms of resistance and strategies to overcome, Eur J Pharm. 170106103. Dibner J J, Richards J D, Antibiotic growth promoters in agriculture: history and mode of action, Poultry Science. 84(2005) 634-643. Ronquillo M G, Hernandez J C A, Antibiotic and synthetic growth promoters in animal diets: review of impact and an alytical methods, Food Control. 72(2017)255-267. Brown K, Uwiera RRE Uwiera, ML Kalmokoff, SPJ Brooks, GD Inglis, Antimicrobial growth promoter use in livestock: a requirement to unde rstand their modes of action to develop effective alternatives, International Journal of Antimicrobial Agents. 49(2017)12-24. A. Pormohammad , R. J Turner, Silver Antibacterial Synergism Activities with Eight Other Metal(loid)-Based Antimicrobials against Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus , Antibiotics . 9(2020)853. V. M. Dcosta, C. E. King, L. Kalan, M. Morar, W. W. L. Sung, C. Schwarz, D. Froese, G. Zazula, F. Calmels, R. Debruyne, G. B. Golding, H. N. Poinar & G. D. Wright, Antibiotic resistance is ancient, Nature. 477(2011)457–461. H. Ayalew, H. Zhang, J. Wang, S. Wu, K. Qiu,G. Qi, Potential Feed Additives as Antibiotic Alternatives in Broiler Production, Frontiers in Veterinary Science. 17(2022)9164732022. E. Kowalczyk, E. Patyra, K. Kwiatek, Organic acids and their importance in animal husbandry, Medycyna Weterynaryjna-Veterinary Medicine-Science and Practice. 69(2013)269-273. Y. Sun, A. Ni, Y. Jiang, Y. l. Li, Z. Huang, L. Shi, H. Xu, C. Chen, D. l. Li, Y. Han, J. Chen, Effects of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative statuses of broiler chickens under clostridium perfringens type a challenge, Avian Dis. 64(2020)393-400. W. Y. Bing, W. Y, L. X. Jing, F. Qiu, Potential effects of acidifier and amylase as substitutes for antibiotic on the growth performance, nutrient digestion and gut microbiota in yellow-feathered broilers, Animals. 10(2020) 1858. D. Li, X. Huang, F. Hao,Y. Lv, H. Chen, W. Xiong, P. Liu, H Luo,S. Wu, Preparation of organic-inorganic composites with high antibacterial activity based on sepiolite, chitosan and zinc: The study of the active antibacterial sites of chitosan‑zinc oxide structure, Applied Clay Science. 216(2022)106325. S. Brul, P. Coote, Preservative agents in foods: Mode of action and microbial resistance mechanisms, International Journal of Food Microbiology. 50(1999)1-17. K. S. Agrawal, A. V. Sarda, R. Shrotriya, M. Bachhav, G. Nataraj, Acetic acid dressings: finding the Holy Grail for infected wound management Indian, Plast Surg. 50( 2017)273-280. C. Q. Gao, H. Q. Shi, W. Y. Xie, L. Zhao, J. Zhang, C. Ji, Q. G. Ma, Dietary supplementation with acidifiers improves the growth performance, meat quality and intestinal health of broiler chickens, Anim Nutr. 7(2021)762–769. S. K. Nath , M. T. Hossain, M. Ferdous, A. Siddika, A. Hossain, A. A. Maruf, A. T. Chowdhory, T. C. Nath, Effects of antibiotic, acidifier, and probiotic supplementation on mortality rates, lipoprotein profile, and carcass traits of broiler chickens, Veterinary and Animal Science. 22(2023) 22100325. W. Pelyuntha, A. Yafa, B. Charoenwong, K. Vongkamjan, Effectiveness of the Organic Acid-Based Antimicrobial Agent to Prevent Bacterial Contamination in Fish Meal, Animals . 12(2022)3367. J. R. Harrington, R. J. Martens, D. CohenN, L. R. Bernstein, Antimicrobial activity of gallium against virulent Rhodococcus equiin vitro an in vivo, J Vet Pharmacol Ther. 29(2006)121-7. O. Olakanmi, B. Kesavalu, R. Pasula, Y. Maher, L. Abdalla, B. Schlesinger, B. Britigan, The transition metal gallium nitrate is efficacious in murine models of tuberculousis and inhibits key bacterial Fe-dependent enzymes, Antimicrob Agents Chemother. 57(2013)6074-80. R. García-Contreras, B. Pérez-Eretza, E. Lira-Silva, R. Jasso-Chávez, R. Coria-Jiménez, A. Rangel-Vega, T. Maeda, T. K Wood, Gallium induces the production of virulence factors in Pseudomonas aeruginosa , Pathogens and Disease. 70(2014)95–98. G. M. Rodriguez, R. Gardner, N. Kaur, O. Phanstiel, Utilization of Fe3+ -acinetoferrin analogs as an iron source by Mycobacterium tuberculosis, Bio Metals. 21(2008)93-103. K. Nikoleta, D. Todor, Gallium as an antibacterial agent: a DFT/ SMD study of the Ga3+/Fe3+ competition for binding bacterial siderophores, Inorg Chem. 59(2020)6242-6254. T. Olczak, D. Maszczak-S, J. W. Smalley, M. Olczak, Gallium (III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells, Arch Microbiol. 194(2012)719-724. M. Piatek, D. M. Griffith, K. Kavanagh, Quantitative proteomic reveals gallium maltolate induces an iron-limited stress response and reduced quorum-sensing in Pseudomonas aeruginosa, J Biol Inorg Chem. CH Goss，Y Kaneko，L Khuu，GD Anderson，S Ravishankar，ML Aitken，N Lechtzin，G Zhou，DM Czyz，K Mclean, Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections, Sci Transl Med. 10(2018)7520. S. R. Choi, B. E. Britigan, P. Narayanasamy, Iron/Heme metabolism-targeted gallium(iii) nanoparticles are active against extracellular and intracellular pseudomonas aeruginosa and acinetobacter baumannii, Antimicrob Agents Chemother. 63(2019)e02643-18. L. Lei, C. Hao, Y. Nie, M. Li, Y. Hou, W. Rao, Superior antibacterial activity of gallium based liquid metals due to Ga3+ induced intracellular ROS generation, J Mater Chem B. 9(2020)85-93. I. Stojiljkovic, B D. Evavold, V. Kumar, Antimicrobial properties of porphyrins, Expert Opin Investig Drugs. 10(2001)309-320. É. A Enyedy, O. Dömötör, B. Krisztina, A. Hetényi, T. Tuccinardi, B. K Keppler, Interaction of the anticancer gallium(III) complexes of 8-hydroxyquinoline and maltol with human serum proteins, Journal of biological inorganic chemistry. 20(2015)77-88. NI. Gorshkov, А Y Murko, II Gavrilova, М А Bezrukova 1, А I Kipper 1, S V Shatik 2, А V Tokarev 2, V D Krasikov 1, Е F Panarin 1, Metal-Polymer Complexes of Gallium/Gallium-68 with Copolymers of N-Vinylpyrrolidonewith N-Vinylformamideand N-Vinyliminodiacetic Acid: A Hint for Radiolabeling of Water-Soluble Synthetic Flexible Chain Macromolecules, Polymers. 12(2020)2889. C. Zhu, H X Yang, Y Z Wang, Y Z Wanng,D Q Zhang.Y Chen, L X Gao, Synergistic effect between glutamic acid and rare earth cerium (III) as corrosion inhibitors on AA5052 aluminum alloy in neutral chloride medium, Ionics. 25(2019)1395-1406. B Kabak, E. Kendüzler, Europium metal-organic frameworks: Synthesis, characterization, and application as fluorescence sensors for the detection of Cu 2+ , Ni2+ cations and T3, T4 hormones, Talanta. 266(2024)124944. L. Todorov, L. Saso, K. Benarous, M. Traykova, Synthesis, Structure and Impact of 5-Aminoorotic Acid and Its Complexes with Lanthanum(III) and Gallium(III) on the Activity of Xanthine Oxidase, Molecules. 26(2021)4503. El Habeeb Abeer A, Novel Gallium(III), Germanium(IV), and Hafnium(IV) Folate Complexes and Their Spectroscopic, Thermal Decomposition, Morphological, and Biological Characteristics, Bioinorganic Chemistry and Applications. 2020, 2020: 6678688. M. A. Akhtar, Z. Hadzhieva, I, Dlouhý, A. Boccaccini, Electrophoretic Deposition and Characterization of Functional Coatings Based on an Antibacterial Gallium (III)-Chitosan Complex, Coatings. 10(2020)483. S. P. Valappil, E. A. Abou Neel, K. M. Zakir Hossain, W. Paul, Durgadas Cherukaraveedu, B. Wade, Tahera I. Ansari, Christopher K. Hope, S. M. Higham, Chandra P, Sharma f Novel lactoferrin-conjugated gallium complex to treat Pseudomonas aeruginosa wound infection, International Journal of Biological Macromolecules, 258(2024)128838. S. Saini, Haritha Sree Yaddanapudi, K. Tian, Yinong Yin, D. Magginetti, A. Tiwari, Terbium Ion Doping in Ca3Co4O9: A Step towards High-Performance Thermoelectric Materials, Scientific Reports. 7(2017)44621. M Jovaní, A Sanz, H Beltrán-Mir, E. Cordoncillo, New red-shade environmental-friendly multifunctional pigment based on Tb and Fe doped Y2Zr2O7 for ceramic applications and cool roof coatings, Dyes Pigments. 133(2016)33-40. R. Lapuente, C. Quijada, F. Huerta, F. Cases & José L Vázquez, X-ray photoelectron spectroscopy study of the composition of polyphenol films formed on Pt by electropolymerisation of phenol in the presence of sulphide in carbonate medium, Polymer Journal. 35(2003)911- 916. Ściana B, W. Dawidowski, D. Radziewicz, J. Jadczak, M. C López-Escalante 3, V. González de la Cruz, M. Gabás, Influence of As-N Interstitial Complexes on Strain Generated in GaAsN Epilayers Grown by AP-MOVPE, Energies. 15(2022)3036. S R Choi, B. Switzer, B E Britigan, P. Narayanasamy, Gallium Porphyrin and Gallium Nitrate Synergistically Inhibit Mycobacterial Species by Targeting Different Aspects of Iron/Heme Metabolism[J]. ACS Infect Dis , 26(2020)2582-2591. C. Auger, J. Lemire, V. Appanna, V. Appanna, Gallium in bacteria, metabolic and medical implications, Springer New York. (2013)800-807. A Stinzi, C Barnes, J Xu, K N Raymond, Raymond, Microbial iron transport via a siderophore shuttle: a membrane iron transport paradigm, Proc Natl Acad Sci. 97(2000)10691-10696. G. Rhys, G. Chris, Cofactor F420: an expanded view of its distribution, biosynthesis, and roles in bacteria and archaea, FEMS microbiology reviews. 45(2021)465-478. Pflüger-Grau K, Lorenzo V, From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida, FEMS Microbiology Letters. 356(2014)144-153. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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-5970012","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":414626657,"identity":"db6a4e08-b143-4604-943f-80fd2c296e84","order_by":0,"name":"Yuhuan Qin","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yuhuan","middleName":"","lastName":"Qin","suffix":""},{"id":414626658,"identity":"e4cbc3a8-84c1-4bc4-ad98-91f4407ef052","order_by":1,"name":"Xian Liu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Liu","suffix":""},{"id":414626659,"identity":"25706640-f777-4108-afa5-6437b7544dc2","order_by":2,"name":"Wei Luo","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Luo","suffix":""},{"id":414626660,"identity":"0814695e-40f6-47f8-a9eb-ee296edb5336","order_by":3,"name":"Xia Li","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Li","suffix":""},{"id":414626661,"identity":"b2d88a28-12dd-482d-916a-9751601862de","order_by":4,"name":"Yong Meng","email":"","orcid":"","institution":"Luzhou Laojiao Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Meng","suffix":""},{"id":414626662,"identity":"6b3a0be5-e660-4d8a-bb34-d61f315e806e","order_by":5,"name":"Hui Qin","email":"","orcid":"","institution":"Luzhou Laojiao Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Qin","suffix":""},{"id":414626663,"identity":"966a7764-8bd0-4fca-aa44-2f839f5b71ac","order_by":6,"name":"Xuepin Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYFAC5oYDDAw2EDYPcVoYQVrSSNQCJA6ToMXgdmPjgR8V5+35ZyQwPnjbxiBvTkiL5JyDDQd7ztxOnHEjgdlwbhuD4c4GAlr4JRIbDvC23U4wkEhgk+ZtY0gwOEBACxtQy8G/befsgVrYfxOlBWTLYd62A4wbgLYwE6UF5JfDMmeSE2ecedgsOeechOEGQloMbjcf/vimws6evz354Ic3ZTbyBG1hkICzwBEkgVMhNi2jYBSMglEwCnAAABHAQWUSzSEfAAAAAElFTkSuQmCC","orcid":"","institution":"Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Xuepin","middleName":"","lastName":"Liao","suffix":""},{"id":414626664,"identity":"e93c5b2e-16b8-434c-8835-ef9e1a8474dd","order_by":7,"name":"Bi Shi","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Bi","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-02-06 05:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5970012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5970012/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76255750,"identity":"1081fa6c-fd25-4bec-82c5-6770a85a8776","added_by":"auto","created_at":"2025-02-14 04:58:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275901,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the preparation of Ga-OA\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/4868e0cb102b3afed6b519be.png"},{"id":76255752,"identity":"e5a1630f-df56-4cad-8e22-836d559cacf0","added_by":"auto","created_at":"2025-02-14 04:58:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279546,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of OA and Ga-OA:(a-c) FT-IR spectroscopy, (d-f) O1s XPS spectroscopy, (g-i) Raman spectroscopy.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/7b43bf1faab31a287fc376ff.png"},{"id":76255856,"identity":"e830f969-0ea7-4a25-b402-bbea58fa2fde","added_by":"auto","created_at":"2025-02-14 05:06:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea-c:\u003c/strong\u003e Antibacterial activity (inhibition zone) of OA，Ga\u003csup\u003e3+ \u003c/sup\u003eand Ga-OA against \u003cem\u003eE. coli,\u003c/em\u003e \u003cem\u003eS. aueeus, Salmonella \u003c/em\u003e(OA concentrations were all 40 mmol/L, Ga\u003csup\u003e3+\u003c/sup\u003e concentrations were all 20 mmol/L); d: Growth kinetic curve of \u003cem\u003eS. aureus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/5c2d2edbfa04ddbf186debc7.png"},{"id":76257793,"identity":"31f5d47c-687b-4bb1-a945-9e06bdbdd9ed","added_by":"auto","created_at":"2025-02-14 05:30:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":323462,"visible":true,"origin":"","legend":"\u003cp\u003e(a): Observation of Ultrastructure of \u003cem\u003eS. aureus \u003c/em\u003eby SEM, incubated at 37 ℃ for 6 h. (b): Observation of Ultrastructure of \u003cem\u003eS. aureus \u003c/em\u003eby TEM, incubated at 37 ℃ for 6 h.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/f03dea56a809ebf4722b0b27.png"},{"id":76255759,"identity":"7f9b785d-2ec6-45ae-8daf-aa35e8b6eeb4","added_by":"auto","created_at":"2025-02-14 04:58:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174221,"visible":true,"origin":"","legend":"\u003cp\u003e(a-b) Volcano plots of gene expression in Ga\u003csup\u003e3+\u003c/sup\u003e vs Con, and Ga-Lac vs Con; (c) Heatmap of inter-sample correlation.\u003c/p\u003e\n\u003cp\u003eNote: Red points (up-regulated), blue points (down-regulated), and Gray points (non-significant differentially).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/80a559486c7b89647ad89962.png"},{"id":76255757,"identity":"ba1f88a2-4c7a-4255-aa7c-b590b8e96786","added_by":"auto","created_at":"2025-02-14 04:58:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":210999,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Distribution of DEGs in major functions; (b and c) KEGG annotation and enrichment analysis of down-regulated DEGs in the Ga-Lac vs Con group; (d-f): Expression profiles of down-regulated DEGs associated with Carbohydrate transport and metabolism, Coenzyme transport and metabolism and Amino acid transport and metabolism in \u003cem\u003eS. aureus\u003c/em\u003e in Ga-Lac vs Con.\u003c/p\u003e\n\u003cp\u003eNote: CTAM: Carbohydrate transport and metabolism; AATAM: Amino acid transport and metabolism; CW/B/EB: Cell wall/membrane/envelope biogenesis; CC-P: Cellular community - prokaryotes; F, SAD: Folding, sorting and degradation; PTS:Phosphotransferase system; BOVOSM:Biosynthesis of various other secondary metabolites; FAMM:Fructose and mannose metabolism; PACB: Pantothenate and CoA biosynthesis; CAMM: Cysteine and methionine metabolism; V, LAID/B: Valine, leucine and isoleucine degradation/biosynthesis\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/b78bfc886359d3c3a7b149ca.png"},{"id":76674842,"identity":"1ab71d3d-187f-4a89-9847-ddb652fa67f3","added_by":"auto","created_at":"2025-02-19 14:17:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2649758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/28438ea8-75f4-4ea3-8c53-4153bc553eff.pdf"},{"id":76255763,"identity":"97c35a35-2436-4291-bad1-e592139d0060","added_by":"auto","created_at":"2025-02-14 04:58:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":534159,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5970012/v1/6d3626c2c49e5d8042deb9b8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced antibacterial activity of organic acids via gallium chelation: a promising antibiotic alternative","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eThe widespread use of antibiotics as growth promoters in livestock feed\u003csup\u003e[1]\u003c/sup\u003e has resulted in antibiotic overuse, contributing to food safety concerns and the emergenceof antibiotic-resistant bacteria in animals\u003csup\u003e[2][3][4]\u003c/sup\u003e. This situation poses a significant threat to global human health\u003csup\u003e[5][6]\u003c/sup\u003e. Consequently, the livestock industry is undergoing a global shift towards \"antibiotic-free farming\", demanding the urgent development of novel alternatives to antibiotics.\u003c/p\u003e \u003cp\u003eCurrently recognized novel and safe antibacterial agents that can serve as alternatives to antibiotics include acidifiers, oligosaccharides, plant extracts, antimicrobial peptides, enzyme preparations, etc\u003csup\u003e[7]\u003c/sup\u003e. Among these, acidifiers are particularly notable for their diverse benefits. They create an acidic environment in the livestock gastrointestinal tract, reducing inflammation, inhibits pathogen growth and reproduction, preventing excessive gastrointestinal dysfunction, and enhancing livestock resilience\u003csup\u003e[8][9]\u003c/sup\u003e. Furthermore, acidifiers improve palatability and digestibility, increase digestive enzyme activity and antioxidant properties, and optimize the gastrointestinal microbiota and nutrient absorption, leading to improved digestion and absorption\u003csup\u003e[10]\u003c/sup\u003e. Acidifiers also participate in the biosynthesis and metabolism of substances, promoting the absorption of minerals and other nutrients. These compounds can be broadly classified into three primary classes : inorganic acids, organic acids and composite acidifiers. Numerous studies have consistently shown that organic acidifiers exhibit greater effectiveness than their inorganic counterparts\u003csup\u003e[11][12][13]\u003c/sup\u003e. Specifically, inorganic acids undergo rapid dissociation leading to a marked decrease in pH values within the animal's esophagus and stomach, potentially causing tissue damage and developmental problems; Conversely, organic acids display a more controlled release of protons within the stomach. Organic acids serve as effective feed growth promoters in livestock and poultry by inhibiting the proliferation of pathogenic microorganisms. These benefits include antimicrobial activity, immune modulation, and enhanced mineral and vitamin absorption\u003csup\u003e[14]\u003c/sup\u003e. Research has shown that broiler chickens treated with organic acidifiers gained weight and exhibited lower levels of low-density lipoprotein and higher levels of triglycerides, indicating that acidifiers can serve as effective alternatives to antibiotics\u003csup\u003e[15]\u003c/sup\u003e. Qiu \u003cem\u003eet al.\u003c/em\u003e found that incorporating 0.8% acidifier into feed can enhance the immune system, antioxidant capacity, and liver lipid metabolism in broiler chickens, ultimately promoting their growth and production\u003csup\u003e[16]\u003c/sup\u003e. Consequently, organic acids contribute to improve the live weight and growth performance of livestock while maintaining high animal safety. However, acidifiers often suffered the following problems, including limited specificity, poor efficacy against certain pathogens and inability to replace all antibiotics. Accordingly, a significant focus of future research and development on acidifiers should be to broaden their antimicrobial spectrum and enhance their antibacterial efficacy.\u003c/p\u003e \u003cp\u003eIn recent years, Ga(III) has emerged as a promising antibacterial agent, exhibiting both bacteriostatic and bactericidal effects. Study by Harrington \u003cem\u003eet al\u003c/em\u003e. demonstrated that gallium nitrate can inhibit and kill \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, while oral administration of gallium maltophenol in mice effectively cures infections caused by this bacterium\u003csup\u003e[17]\u003c/sup\u003e. Olakanmi \u003cem\u003eet al\u003c/em\u003e. found that gallium nitrate can inhibit the activity of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e ribonucleotide reductase, a key enzyme for DNA replication, in a mouse pulmonary tuberculosis model, thereby inhibiting the growth of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e\u003csup\u003e[18]\u003c/sup\u003e. These findings highlight the potential of Ga(III) as a therapeutic agent against various bacterial pathogens. It was suggested the significant chemical resemblance between Ga(III) and Fe(III) makes it difficult for bacteria to effectively discriminate between the two metals\u003csup\u003e[19][20]\u003c/sup\u003e. Upon cellular uptake, Ga(III) disrupts bacterial iron homeostasis by interfering with the iron transport system, resulting in iron deficiency and ultimately bacterial death\u003csup\u003e[21][22][23][24]\u003c/sup\u003e. This process can also lead to decreased or inactivated iron-dependent enzyme activities\u003csup\u003e[25]\u003c/sup\u003e, disruption of the bacterial electron transport chain\u003csup\u003e[26]\u003c/sup\u003e, and the induction of oxidative stress\u003csup\u003e[27]\u003c/sup\u003e. However, the high aqueous solubility of gallium salts results in weak cellular affinity, which limits their efficacy as monotherapies due to the challenge of intracellular delivery required for their antibacterial effects\u003csup\u003e[28]\u003c/sup\u003e. Nevertheless, the coordination chemistry of Ga(III) presents a promising avenue to overcome this limitation.\u003c/p\u003e \u003cp\u003eHerein, gallium-organic acid complexes (Ga-OA) with enhanced antibacterial effects were synthesized through autonomous assembly and coordination. Organic acids, capable of associating with phospholipids and proteins in bacterial cell walls, act as both antibacterial agents and efficient carriers for Ga(III) entry into cells. This dual role amplifies the inhibitory effects of both Ga(III) and the organic acid, leading to enhanced antibacterial activity in the Ga(III)- OA complexes. We investigated the antibacterial activity of Ga-OA against three common pathogenic bacteria in livestock and poultry: \u003cem\u003eEscherichia coli, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003eSalmonella spp\u003c/em\u003e. Furthermore, transcriptomic analysis was used to explore the antibacterial mechanisms of these complexes.\u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eGallium nitrate (Ga (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), analytical pure, purchased from Aladdin. L-lactic acid (Lac), acetic acid (Ac), and butyric acid (BA) are all analytical pure and purchased from Aladdin. NaCl, Agar powder, glutaraldehyde, methanol and other chemicals used in the study were all of analytical grade.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli ATCC\u003c/em\u003e25922, \u003cem\u003eStaphylococcus aureus ATCC\u003c/em\u003e23656, and \u003cem\u003eSalmonella SIIA\u003c/em\u003e235 were provided by the Tanning Biotechnology Laboratory of Sichuan University. Medium: The liquid medium is Mueller Hinton (MH) broth medium, while the solid agar plate and test tube slanted medium are both supplemented with 1.5% agar in nutrient broth (NB) medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of Gallium-Organic Acid Complex (Ga-OA)\u003c/h2\u003e \u003cp\u003eA series of gallium-organic acid complex were synthesized via a liquid-phase method. Optimally, precise quantities of organic acids (OA) and gallium nitrate Ga(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e were weighed based on the molar ratios of 0.5:1 and 0.25:1, respectively. Each compound was individually dissolved in purified water. Subsequently, the Ga(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e solution was added dropwise to the OA solution. The mixture was stirred at room temperature for 8 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) to facilitate complex formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization\u003c/h2\u003e \u003cp\u003eThe chemical structure of Ga-OA complexes were characterized using FT-IR spectroscopy in the wavenumber range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The surface elemental composition and binding energy of the prepared samples were determined through X-ray photoelectron spectroscopy (XPS) with AI Kα X-rays source. The chemical structure of the Ga-OA was further analyzed by Raman spectroscopy (Roman), and the sample was scanned by 532 nm laser in the range of 200\u0026ndash;2000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of bacterial suspension.\u003c/h2\u003e \u003cp\u003eTo prepare bacterial suspensions, each test strain was initially activated and cultured on nutrient broth (NB) slant medium for 24 hours, undergoing two generations of activation. Subsequently, several loops of well-growing colonies were scraped and inoculated into MH liquid medium for expansion culture. The cultures were incubated in a shaker at 37\u0026deg;C and 180 r/min for 24 hours. Finally, an appropriate volume of the expanded culture broth was collected, washed with sterile physiological saline, and diluted to a concentration of 10\u003csup\u003e7\u003c/sup\u003e CFU/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Antibacterial activity test of Ga-OA\u003c/h2\u003e \u003cp\u003eInhibition circle diameter: The diameter of the zone of inhibition of the Ga-OA against three common poultry and livestock pathogens was determined using the horizontal diffusion method (Oxford cup method). ConFig.d plates were incubated at 37 ℃ for 24 h, and the diameter of the zone of inhibition on each plate was measured by vernier caliper (mm).\u003c/p\u003e \u003cp\u003eMIC and MBC determination. The minimum inhibitory concentration (MIC) of Ga-OA against the pathogenic bacteria was determined using the twofold dilution method. Samples were incubated at 37℃ for 24 h, and the growth of bacteria was visually observed. The lowest concentration at which the solution became clear and transparent was defined as the minimum inhibitory concentration (MIC). To determine the minimum bactericidal concentration (MBC), the lowest inhibitory concentration (MIC) and a concentration higher than the MIC from the first three groups were inoculated onto sterile agar plates and incubated at 37 \u003csup\u003eo\u003c/sup\u003eC for 24 hours. The lowest concentration that resulted in no bacterial growth on the agar plate was defined as the minimum bactericidal concentration (MBC).\u003c/p\u003e \u003cp\u003eGrowth dynamics: \u003cem\u003eS. aureus\u003c/em\u003e was selected to determine its growth kinetic curve. Following 8 hours of expansion culture, \u003cem\u003eS. aureus\u003c/em\u003e cells were centrifuged and diluted to prepare a bacterial suspension at a concentration of 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. The suspension was then combined with the corresponding MIC concentration of Ga-OA and MH medium mixture. Samples were collected every 3 h, and measure the OD\u003csub\u003e600\u003c/sub\u003e. A curve was plotted according to the results to observe the effect of the complex on the growth of bacteria during action times.\u003c/p\u003e \u003cp\u003eThe content of Ga\u003csup\u003e3+\u003c/sup\u003e in cells: To determine the content of Ga\u003csup\u003e3+\u003c/sup\u003e in the cells, inductively coupled plasma spectrometry (ICP-OES) was used. \u003cem\u003eS. aureus\u003c/em\u003e after extended activation culture was prepared into a suspension of 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. It was added into the MH medium with Ga-OA and Ga\u003csup\u003e3+\u003c/sup\u003e at the concentration of MIC and cultured for 24 h. Bacteria were sampled and collected every 3 h and digested with concentrated nitric acid and hydrogen peroxide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Antibacterial mechanism of Ga-OA\u003c/h2\u003e \u003cp\u003eField Emission Scanning Electron Microscopy (FESEM) Analysis: SEM was employed to examine the microscopic morphology of \u003cem\u003eS. aureus\u003c/em\u003e cells treated with the Ga-OA complexes. Following treatment by Ga-OA, \u003cem\u003eS. aureus\u003c/em\u003e cells were fixed with 2.5% glutaraldehyde, centrifugation with ethanol gradient, and finally dried at room temperature.\u003c/p\u003e \u003cp\u003eTEM transmission electron microscopy (TEM) analysis: In order to investigate the effect of Ga-OA complexes on the internal structure of \u003cem\u003eS. aureus\u003c/em\u003e cells, the bacteria were treated with Ga-OA and equimolar concentrations of Ga\u003csup\u003e3+\u003c/sup\u003e and OA, respectively. The organisms were the collected, fixed, dehydrated, embedded, and stained to make ultrathin sections. Ultrastructural changes of the cells were subsequently observed using a Hitachi-HT7700 transmission electron microscope.\u003c/p\u003e \u003cp\u003eTranscriptome sequencing analysis: To further investigate the genetic effects of the Ga-OA complex on \u003cem\u003eS. aureus\u003c/em\u003e, RNA sequencing was conducted. Accordingly, \u003cem\u003eS. aureus\u003c/em\u003e cells in the logarithmic growth phase were inoculated in MH medium containing Ga\u003csup\u003e3+\u003c/sup\u003e or Ga-Acsolution and incubated at 37 ℃ for 4 h. Then, bacteria were collected and stored frozen, and the total RNA was extracted from the bacterial samples, and sequencing libraries were prepared using the NovaSeqXPlus platform at Shanghai Meiji Biotechnology Co. High-quality reads were aligned to the genome of \u003cem\u003eS. aureus\u003c/em\u003e GCF_000756205.1 using Bowtie software. Transcripts per million (TPM) values were used to quantify gene expression levels in the samples. Differential expression analysis was performed using DEGSeq2 software to identify differentially expressed genes (DEGs) based on the criteria of |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026ge; 1 and p-adjusted value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional enrichment analysis was then conducted on the DEGs to elucidate the inhibitory mechanisms of the Ga-OA complexes against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result and discuss","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Chemical Structural Characterization of Ga-OA.\u003c/h2\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 FT-IR analysis\u003c/h2\u003e\n \u003cp\u003eThe chemical structures of the organic acids and their gallium complexes was characterized by FT-IR. The FT-IR spectra of the Ga-OA complexes showed substantial differences from those of the individual organic acids (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). All three OAs exhibit a pronounced broad absorption peak around 3450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which corresponds to the stretching vibration of \u0026upsilon; O-H in the carboxylic acid moiety of OA molecule\u003csup\u003e[29]\u003c/sup\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(a), lactic acid (Lac) exhibited a distinct single peak near 1652 cm⁻\u0026sup1;, characteristic of the C\u0026thinsp;=\u0026thinsp;O bond in the carboxyl group\u003csup\u003e[30]\u003c/sup\u003e. Additionally, a stretching vibration peak of the C-O bond was observed at 1243 cm\u003csup\u003e\u0026minus;\u0026thinsp;1[31]\u003c/sup\u003e. In the Ga-Lac complex, these two peaks were significantly weakened, suggesting the involvement of the carbonyl group participates in the coordination reaction. This observation was further supported by the decrease in peak intensity upon complex formation. Furthermore, the principles of organic acid-metal ion interactions indicate that complexation leads to alterations in the C-O bond energy within the carboxylic acid\u003csup\u003e[32][33][34][35]\u003c/sup\u003e. Similarly, in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(b), acetic acid (Ac) exhibits a characteristic C-O stretching vibration peak at 1278 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was significantly weakened in the complexes, and the peaks at 1652 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the complexes are shifted to higher wavenumbers. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(c) shows that butyric acid (BA) displayed a characteristic C-O stretching vibration peak at 1203 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which also diminished upon complexation. These findings indicate that the gallium ion coordinates with the oxygen atom of the carbonyl group in the organic acid to form the organic acid-gallium ion complexes.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2 XPS spectral analysis\u003c/h2\u003e\n \u003cp\u003eX-ray photoelectron spectroscopy (XPS) analysis of the O1s spectra of OA and Ga-OA (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed-f) provided further evidence of complex formation. Compared to OA, the characteristic peak positions of the Ga-OA complex exhibited notable shifts. The O1s spectrum of Lac displays characteristic peaks at binding energies of 531.93 eV and 532.67 eV, corresponding to C\u0026thinsp;=\u0026thinsp;O and C-O (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively\u003csup\u003e[36][37]\u003c/sup\u003e. In contrast, the O1s spectrum of the Ga-Lac complex Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(d) revealed a new characteristic peak at a binding energy of 533.18 eV, attributed to the bond of Ga-O. This shift arises from the coordination reaction between the carbonyl group of Lac and Ga\u003csup\u003e3+\u003c/sup\u003e. Specifically, the electrons on the carbonyl oxygen shift to the empty orbitals under the influence of the central Ga\u003csup\u003e3+\u003c/sup\u003e ion\u003csup\u003e[38]\u003c/sup\u003e, resulting in a decrease in the electron cloud density of the ligand Lac. Therefore, the bond energy of the carbonyl oxygen shifted towards higher binding energies, forming a new Ga-O coordination bond. Similarly, upfield shifts in the O1s spectra of acetic acid (Ac) (Fig. S2) and butyric acid (BA) (Fig. S3) and their respective Ga\u003csup\u003e3+\u003c/sup\u003e complexes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef) confirmed the coordination reaction leading to the upfield shift of the carbonyl groups in OA. In general, these XPS findings provide further evidence of the successful coordination and bonding of Ga\u003csup\u003e3+\u003c/sup\u003e with the carbonyl groups of OA.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.3 Raman spectral analysis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(g-i) shows the Raman spectra of OA and Ga-OA. Compared to OA, the Raman spectra of Ga-OA complexes in the range of 200\u0026ndash;2000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are significantly different. The Raman spectrum of OA lacks discernible characteristic absorption peaks, whereas the Ga-OA spectrum exhibits a prominent novel characteristic peak near 1050 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, attributable to the bending vibration of the carboxyl group C-O\u003csup\u003e[39]\u003c/sup\u003e. This peak is absent in the spectrum of OA, definitively indicating a coordination reaction between OA and Ga\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Antimicrobial activity of the Ga-OA\u003c/h2\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 Inhibitory circle diameter\u003c/h2\u003e\n \u003cp\u003eThe diameter of the inhibition zone serves as a preliminary indicator of antibacterial activity, exhibiting a positive correlation with antibacterial potency within a defined range. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (a-c) depicts the diameters of inhibition zones generated by organic acids, gallium, and organic acid-gallium complexes with varying molar ratios against three common livestock pathogens: \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (a-c), the three organic acids (lactic acid, acetic acid, and butyric acid) exhibit moderate inhibitory effects against these pathogens, with inhibition zone diameters ranging from approximately 14 to 17 mm. Gallium ions also demonstrate some inhibitory activity, with inhibition zone diameters of approximately 8 to 13 mm. Significantly, the antibacterial activity of the Ga-OA surpasses that of both the individual organic acids and gallium ions. At a coordination molar ratio of 0.5:1 (Ga/OA), Ga-OA exhibits inhibition zone diameters of approximately 16\u0026ndash;20 mm against \u003cem\u003eE. coli\u003c/em\u003e, 20\u0026ndash;24 mm against \u003cem\u003eS. aureus\u003c/em\u003e, and 15\u0026ndash;18 mm against \u003cem\u003eSalmonella\u003c/em\u003e. Even at lower coordination molar ratios (as shown in Fig. S4-S6), the Ga-OA demonstrate higher antibacterial activity compared to their individual components.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2 MIC and MBC\u003c/h2\u003e\n \u003cp\u003eWe further validated the enhanced antibacterial efficacy of Ga-OA by assessing the MIC and MBC values against pathogenic bacteria using both the monomer and its complex. As presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the MIC values of Ga-OA ( molar ratio of Ga:OA was 0.5:1) were significantly lower than those of Ga\u0026sup3;⁺ and OA alone, indicating a substantial enhancement in its antibacterial performance. Similarly, the MBC values of Ga-OA ( molar ratio of Ga:OA was 0.5:1), as shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, were also reduced compared to the individual use of Ga\u0026sup3;⁺ and OA, further supporting the notion of a synergistic antibacterial effect. A comparison of the MIC and MBC values (Tables \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) reveals that Ga-OA exhibited the most potent inhibitory effect against \u003cem\u003eS. aureus\u003c/em\u003e, which aligns with the findings from the inhibition zone assays. The lower MBC values for Ga-OA, compared to Ga\u0026sup3;⁺ and OA alone, suggest an improved bactericidal efficacy of the complex, thereby highlighting its potential as an effective antimicrobial agent. The enhanced antibacterial activity of Ga-OA complexes arises from a muti-faceted mechanism. Upon coordination with OA, a portion of the positive charge on the Ga\u003csup\u003e3+\u003c/sup\u003e ion is transferred to the OA molecule, inducing electron delocalization within the resulting complex. This electron delocalization effect reduces the polarity of Ga\u003csup\u003e3+\u003c/sup\u003e while enhancing the its ability to permeate the lipid bilayer of cell membranes\u003csup\u003e[40][41][42]\u003c/sup\u003e. Consequently, the complex readily penetrates the cell interior, disrupting cellular processes and leading to enhanced antibacterial potency. Notably, Ga-OA exhibits varying degrees of inhibitory activities against different pathogenic bacteria, with \u003cem\u003eS. aureus\u003c/em\u003e exhibiting the most pronounced inhibitory effect. This variation in susceptibility could be attributed to differences in the cell wall structure of different microorganisms. Ga-OA complexes are more likely to penetrate the cell wall and accumulate intracellularly, potentially affecting the synthesis of intracellular DNA replication-related enzymes, hindering base replication, and ultimately inhibiting DNA synthesis, thereby inhibiting bacterial growth\u003csup\u003e[40]\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3 Growth kinetics of S. aureus\u003c/h2\u003e\n \u003cp\u003eTo further investigate the inhibitory effects of Ga-OA against \u003cem\u003eS. aureus\u003c/em\u003e, the Ga-Lac complex was selected to study its growth inhibition. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(c) presents the growth kinetic curves of \u003cem\u003eS. aureus\u003c/em\u003e after treatment with Ga-Lac and Lac, respectively. Compared to the blank control group, \u003cem\u003eS. aureus\u003c/em\u003e in the experimental groups (Ga-Lac, Lac) exhibited a degree of growth, however, their growth rates were lower than that of the blank control. The order of inhibitory strength was Ga-Lac\u0026thinsp;\u0026gt;\u0026thinsp;Lac\u0026thinsp;\u0026gt;\u0026thinsp;blank control, indicating that the gallium organic acid complex effectively suppressed the growth of \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMIC of OA, Ga\u003csup\u003e3+\u003c/sup\u003e and Ga-OA against three poultry pathogenic bacteria.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMIC (mmol/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eButyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Lactic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Acetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Butyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMBC of OA, Ga\u003csup\u003e3+\u003c/sup\u003e and Ga-OA against three poultry pathogenic bacteria.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMBC (mmol/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eButyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Lactic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Acetic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGa-Butyric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Antimicrobial mechanism\u003c/h2\u003e\n \u003cp\u003eAntibacterial activity investigations revealed that Lac, Ga\u003csup\u003e3+\u003c/sup\u003e, and Ga-OA all exhibited inhibitory effects against three bacterial species, leading to alterations in bacterial cell morphology. To assess these morphological alterations, SEM was employed to observe the changes in \u003cem\u003eS. aureus\u003c/em\u003e cells following treatment with Lac, Ga\u003csup\u003e3+\u003c/sup\u003e, and Ga-Lac, respectively. The results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(a-d). Compared to the control group (sterile saline), significant alterations in cellular morphology were observed in the experimental groups treated with Lac, Ga\u003csup\u003e3+\u003c/sup\u003e and Ga-Lac with varying degrees of impact among Lac, Ga\u003csup\u003e3+\u003c/sup\u003e, and Ga-Lac. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, typically \u003cem\u003eS. aureus\u003c/em\u003e cells exhibit a spherical shape with a smooth surface. Following Lac treatment, cells displayed slight deformation with partial inward concavity on the surface. This could be attributed to Lac\u0026apos;s small molecular size and lipophilic nature, allowing it to traverse the cell wall and membrane\u003csup\u003e[41]\u003c/sup\u003e, interacting with intracellular active substances and causing morphological changes\u003csup\u003e[43]\u003c/sup\u003e. Cells treated with Ga\u003csup\u003e3+\u003c/sup\u003e also underwent mild deformation and displayed a rougher surface (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, comparing Lac and Ga\u003csup\u003e3+\u003c/sup\u003e, cells exposed to Ga-OA demonstrated more pronounced concavity. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec, the cell was severely deformed, the cell wall structure collapsed, and the cell membrane ruptured, indicating Ga-OA showed stronger synergistic bacterial inhibition. This heightened efficacy arises from the Ga-Lac complex inheriting the liposolubility of Lac, facilitating its penetration through the cell wall and membrane into the cell interior to exert its antibacterial function. Further, TEM was employed to investigate alterations in the intracellular ultrastructure of bacteria, with the results presented in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee-h. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eh, S. \u003cem\u003eaureus\u003c/em\u003e bacterium in the blank control group maintained a smooth spherical shape, featuring relatively intact cell walls, cell membranes, a plump and evenly distributed cytoplasm, and abundant organelles. In contrast, while the cell walls and membranes remained relatively intact in Lac-treated cells, the cytoplasm exhibited slight coagulation, and weaker plasmolysis was observed at the cell termini (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). Integrating the SEM findings, it can be inferred that Lac displays good affinity towards cells but exerts limited effects. Similarly, in Ga\u003csup\u003e3+\u003c/sup\u003e-treated cells, the cell walls and membranes remained intact, while slight surface deformation was observed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef), accompanied by cytoplasmic condensation and vacuolation, indicating that the primary target of Ga\u003csup\u003e3+\u003c/sup\u003e lies within the cytoplasm. However, due to its weak affinity with cells, only a small amount of Ga\u003csup\u003e3+\u003c/sup\u003e entered the cytoplasm to exert its effects. Notably, the most pronounced morphological changes were observed in Ga-OA treated cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eg), characterized by a roughened cell surface, distinct ruptures in the cell wall and membrane, cytoplasmic leakage, uneven cytoplasmic distribution, severe coagulation and vacuolation, and incomplete cell morphology. These findings further suggest that the active site of the complex primarily resides in the cytoplasm. Combined with the SEM results, it can be postulated that upon forming a complex with OA, Ga\u003csup\u003e3+\u003c/sup\u003e utilizes OA as a carrier to enhance its liposolubility, facilitating easier penetration into cells and subsequently causing cellular damage or even apoptosis, thereby synergistically potentiating the antibacterial efficacy.\u003c/p\u003e\n \u003cp\u003eTo verify whether gallium ions in the complex enter cells to exert their effects, the accumulation of gallium ions in \u003cem\u003eS. aureus\u003c/em\u003e cells at different time points was analyzed. As shown in Fig. S7, the experimental group was Ga-Lac, while the control group was Lac. Within 0 to 12 hours, the content of Ga in cells from the control group remained essentially zero, whereas in the experimental group, the Ga content initially increased and then decreased with prolonged treatment time. This indicates that Ga-Lac in its complex form more readily penetrates the cell membrane, entering the interior. As the treatment duration lengthened, the inhibitory effects of both Ga-Lac and Ga\u0026sup3;⁺ prompted bacterial cells to secrete lysozyme, leading to autolysis, membrane rupture, and solute leakage, thereby decreasing the Ga content. These findings suggest that Ga-Lacexhibits a higher affinity for cells, manifesting in a stronger inhibitory capacity, consistent with observations from SEM images and TEM images.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Transcriptome Analysis\u003c/h2\u003e\n \u003cp\u003eRNA-seq technique was used to analyze the gene expressions of three groups (Con, Ga\u003csup\u003e3+\u003c/sup\u003e and Ga-Lac) to further illustrate the molecular mechanism of complex inhibition. The transcriptome sequencing results revealed 1565 and 1576 DEGs expressed by \u003cem\u003eS. aureus\u003c/em\u003e that treated with Ga\u003csup\u003e3+\u003c/sup\u003e vs Con and Ga-Lac vs Con groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea-b), with 761 genes up-regulated and 815 genes down-regulated in Ga-Lac vs Con. The differences between the groups were significant, especially the Ga-Lac vs Con group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003eTo elucidate the functional implications of these DEGs, a Clusters of Orthologous Groups (COG) analysis was performed, categorizing the DEGs into eight predicted functional categories in \u003cem\u003eS. aureus\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). The results suggested that the repression of genes associated with metabolism had a greater impact on \u003cem\u003eS. aureus\u003c/em\u003e growth. To further explore these metabolic pathways, KEGG annotations analysis was conducted on DEGs in the Ga-Lac vs Con group. Downregulated genes were primarily concentrated in metabolic and environmental information processing pathways (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). KEGG enrichment analysis indicated that the phosphotransferase system and galactose metabolism pathways were significantly enriched (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec), suggesting that metabolism-related and transport system-related genes were significantly affected by Ga-Lac treatment.\u003c/p\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1 Carbohydrate transport and metabolism\u003c/h2\u003e\n \u003cp\u003eThe down-regulated DEGs in Ga-Lac vs Con involved in carbohydrate transport and metabolism were shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed. Gene \u003cem\u003eCEP64_RS14610\u003c/em\u003e encoding proteins involved in pentose and gluconate interconversion pathway as well as the synthesis of the PTS fructose transporter subunit was down-regulated. Genes (\u003cem\u003efurA, furB, pfkA\u003c/em\u003e) involved in fructose and mannose metabolism were down-regulated. The expression of galactose metabolism-related genes (\u003cem\u003ebgaB, lacE, CEP64_RS14625, CEP64_RS14615, lacA, CEP64_RS14710, CEP64_RS1470\u003c/em\u003e) was distinctively repressed. Furthermore, the down-regulation of pentose phosphate pathway-related genes (\u003cem\u003efbaA, CEP64_RS15195, CEP64_RS18360, hxlA, CEP64_RS2514\u003c/em\u003e) were significant. Additionally, genes involved in glycolysis/gluconeogenesis, including \u003cem\u003eCEP64_RS24405, CEP64_RS8920, CEP64_RS15110\u003c/em\u003e were also remarkably down-regulated. The above results indicated that Ga-Lac inhibits the growth of \u003cem\u003eS. aureus\u003c/em\u003e by disrupting its carbohydrate transport and metabolism.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2 Coenzyme transport and metabolism\u003c/h2\u003e\n \u003cp\u003eThe down-regulated DEGs in Ga-Lac vs Con involved in coenzyme transport and metabolism were shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee. Cofactors synthesis are essential for a range of intracellular chemical reactivity and specificity[43]. Down-regulation of NAD-dependent aldehyde dehydrogenase-related genes (\u003cem\u003eCEP64_RS15110\u003c/em\u003e) inhibited intracellular redox reactions. The gene (\u003cem\u003eCEP64_RS14395\u003c/em\u003e) encoding the NAD (P)-binding protein in siroheme synthase, which was involved in porphyrin metabolism, was down-regulated, as were \u003cem\u003eilvD, panC\u003c/em\u003e, genes related to the biosynthesis pathway of pantothenate and coenzyme A. Synthesis inhibition of these coenzymes in anabolic and catabolic pathways leads to bacterial metabolic disruption.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.3 Amino acid transport and metabolism\u003c/h2\u003e\n \u003cp\u003eThe down-regulated DEGs in Ga-Lac vs Con involved in amino acid transport and metabolism were shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef. These genes are mainly involved in the biosynthesisof alanine, aspartic acid, and glutamic acid (\u003cem\u003egabT, CEP64_RS12660\u003c/em\u003e), lysine (\u003cem\u003epepV\u003c/em\u003e), cysteine (\u003cem\u003emetC, CEP64_RS26510\u003c/em\u003e), and tryptophan (\u003cem\u003eCEP64_RS20535\u003c/em\u003e). This down-regulation resulted in the inhibition of cellular components synthesis and intracellular processes of \u003cem\u003eS. aureus\u003c/em\u003e, including signal transduction and molecular recognition. These results suggest that Ga-Lac may induce \u003cem\u003eS. aureus\u003c/em\u003e apoptosis by deterring amino acid metabolism, potentially leading to apoptosis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.4 Environmental information processing\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e\n \u003ch2\u003e3.4.4.1 ABC transporter system\u003c/h2\u003e\n \u003cp\u003eDown-regulated DEGs involved in membrane transport in Ga-Lac vs Con were mainly concentrated in the ABC transporter system and PTS transporter system (Fig S8). The expression of genes involved in the ABC-type transporter system is shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The genes (\u003cem\u003eCEP64_RS16540\u003c/em\u003e) encoding ABC transporter proteases were down-regulated, which inhibited periplasmic protein synthesis and galactose transport. Similarly, down-regulation of \u003cem\u003eCEP64_RS24125\u003c/em\u003e, encoding the amino acid ABC transporter protein permease, inhibited the ABC-type amino acid transport system. Additionally, the down-regulated DEGs (\u003cem\u003eCEP64_RS24130, CEP64_RS25245\u003c/em\u003e) inhibited the substrate-binding structural domain proteins of transporter. These findings suggest that inhibition of the ABC-type iron transport system disrupts the bacterial protein transport system as well as the para-energetic conversion system, thereby blocking intracellular protein transport as well as energy conversion in \u003cem\u003eS. aureus.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section4\"\u003e\n \u003ch2\u003e3.4.4.2 PTS transport system\u003c/h2\u003e\n \u003cp\u003eThe carbohydrate-phosphotransferase system (PTS) is a key carbohydrate transport system that recognizes both intracellular and extracellular signals, transferring the high-energy phosphate of phosphoenolpyruvate (PEP) to other enzymes and proteins\u003csup\u003e[44]\u003c/sup\u003e. Down-regulation DEGs (\u003cem\u003eCEP64_RS24390\u003c/em\u003e) involved in the synthesis of cellulose-specific component related genes were observed, Galactitol-specific component related genes (\u003cem\u003eCEP64_RS14615, CEP64_RS24780\u003c/em\u003e) and fructose-specific component related genes (\u003cem\u003eCEP64_RS25080, CEP64_RS2509\u003c/em\u003e) were significantly down-regulated, which deterred phosphotransferase system (PTS). These results suggest that Ga-Lac inhibits \u003cem\u003eS. aureus\u003c/em\u003e by disrupting the ABC transporter system and PTS transporter systems, which are essential for disrupts the bacterial protein transport system and energy conversion system.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eExpression of DEGs related to Environmental information processing to \u003cem\u003eS. aureus\u003c/em\u003e in Ga-Lac vs Con\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eABC transporter system and PTS transporter system\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGene ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\n \u003cp\u003e(Lac vs Con)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS16540\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS16540\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eextracellular solute-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.3711\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS16545\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS16545\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esugar ABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.5588\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24125\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24125\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eamino acid ABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.7373\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24130\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24130\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etransporter substrate-binding domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.3453\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24135\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24135\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eamino acid ABC transporter ATP-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.7349\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25245\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25245\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etransporter substrate-binding domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.3968\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25230\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25230\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eamino acid ABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.7213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS22820\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eOpp3b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eoligopeptide ABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.2234\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS14515\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS14515\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFe(3+) dicitrate ABC transporter substrate-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.5025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24390\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24390\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS cellobiose transporter subunit IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.2356\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS14615\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS14615\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS galactitol transporter subunit IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.1063\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24780\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS24780\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS sugar transporter subunit IIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.7316\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25080\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25080\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS fructose transporter subunit IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.2642\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS25090\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCEP64_RS2509\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS fructose transporter subunit IIB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.0612\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study reports the novel synthesis and characterization of Ga-OA, an antimicrobial agent formed by complexing organic acids with gallium ion (Ga\u003csup\u003e3+\u003c/sup\u003e). While the organic acids exhibit broad-spectrum antibacterial activity, but they are often suffered the problems of limited specificity, poor efficacy against certain pathogens and inability to replace all antibiotics. In contrast, Ga\u003csup\u003e3+\u003c/sup\u003e exhibit strong antibacterial activity, but the high aqueous solubility has limited its application. These problems were effectively addressed in this study by the chelation of organic acids with Ga\u003csup\u003e3+\u003c/sup\u003e. Ga-OA significantly enhances the lipid solubility of Ga\u003csup\u003e3+\u003c/sup\u003e, facilitating greater cell penetration and disruption of cellular metabolism. Transcriptome sequencing revealed downregulation of genes involved in the glycolysis pathway in \u003cem\u003eS. aureus\u003c/em\u003e treated with Ga-OA, highlighting its potential to target critical metabolic pathways. Ga-OA holds promising potential as a novel antimicrobial agent due to its superior antibacterial activity and low toxicity profile. It presents a viable alternative to traditional antibiotics for controlling bacterial infections in poultry and livestock farming.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFounding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emianynag habio bioengineering co., Ltd, and Horizontal Science and Technology Project（No．NJGS-2023000594）, Luzhou Laojiao Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this study was conducted in accordance with ethical standards and guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe participant has consented to the submission of the case report to the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuhuan Qin: Supervision, Conceptualization.\u0026nbsp;Xian Liu: Investigation.\u0026nbsp;Wei Luo: Supervision, Conceptualization.\u0026nbsp;Xia Li:\u0026nbsp;Investigation. Mengyang Huang: Investigation.\u0026nbsp;Hui Qin: Investigation. Xuepin Liao: Supervision.\u0026nbsp;Bi Shi:\u0026nbsp;Investigation. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the financial supports provided by the mianynag habio bioengineering co., Ltd, and Horizontal Science and Technology Project（No．NJGS-2023000594）, Luzhou Laojiao Co., Ltd.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQYH completed the experimental content and wrote the article, All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n \u003cli\u003eT. Pulingam, T. Parumasivam, A. M. Gazzali, A. M. Sulaiman, J. Y. Chee, M. j Lakshmanan, C. F. Chin, K. Sudesh.\u0026nbsp;(2022)Antimicrobial resistance: prevalence, economic burden, mechanisms of resistance and strategies to overcome, Eur J Pharm. 170106103.\u003c/li\u003e\n \u003cli\u003eDibner J J, Richards J D, Antibiotic growth promoters in agriculture: history and mode of action, Poultry Science. 84(2005) 634-643.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRonquillo M G, Hernandez J C A, Antibiotic and synthetic growth promoters in animal diets: review of impact and an alytical methods, Food Control. 72(2017)255-267.\u003c/li\u003e\n \u003cli\u003eBrown K, Uwiera RRE Uwiera, ML Kalmokoff, SPJ Brooks, GD Inglis, Antimicrobial growth promoter use in livestock: a requirement to unde rstand their modes of action to develop effective alternatives, International Journal of Antimicrobial Agents. 49(2017)12-24.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eA. Pormohammad , R. J Turner, Silver Antibacterial Synergism Activities with Eight Other Metal(loid)-Based Antimicrobials against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eAntibiotics\u003c/em\u003e. 9(2020)853.\u003c/li\u003e\n \u003cli\u003eV. M. Dcosta, C. E. King, L. Kalan, M. Morar, W. W. L. Sung, C. Schwarz, D. Froese, G. Zazula, F. Calmels, R. Debruyne, G. B. Golding, H. N. Poinar \u0026amp; G. D. Wright, Antibiotic resistance is ancient, Nature. 477(2011)457\u0026ndash;461.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eH. Ayalew, H. Zhang, J. Wang, S. Wu, K. Qiu,G. Qi, Potential Feed Additives as Antibiotic Alternatives in Broiler Production, Frontiers in Veterinary Science. 17(2022)9164732022.\u003c/li\u003e\n \u003cli\u003eE. Kowalczyk, E. Patyra, K. Kwiatek, Organic acids and their importance in animal husbandry, Medycyna Weterynaryjna-Veterinary Medicine-Science and Practice. 69(2013)269-273.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eY. Sun, A. Ni, Y. Jiang, Y. l. Li, Z. Huang, L. Shi, H. Xu, C. Chen, D. l. Li, Y. Han, J. Chen, Effects of replacing in-feed antibiotics with synergistic organic acids on growth performance, health, carcass, and immune and oxidative statuses of broiler chickens under clostridium perfringens type a challenge,\u0026nbsp;\u003cem\u003eAvian Dis.\u0026nbsp;\u003c/em\u003e64(2020)393-400.\u003c/li\u003e\n \u003cli\u003eW. Y. Bing, W. Y, L. X. Jing, F. Qiu, Potential effects of acidifier and amylase as substitutes for antibiotic on the growth performance, nutrient digestion and gut microbiota in yellow-feathered broilers,\u0026nbsp;\u003cem\u003eAnimals.\u0026nbsp;\u003c/em\u003e10(2020) 1858.\u003c/li\u003e\n \u003cli\u003eD. Li, X. Huang, F. Hao,Y. Lv, H. Chen, W. Xiong, P. Liu, H Luo,S. Wu, Preparation of organic-inorganic composites with high antibacterial activity based on sepiolite, chitosan and zinc: The study of the active antibacterial sites of chitosan‑zinc oxide structure, Applied Clay Science. 216(2022)106325.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eS. Brul, P. Coote, Preservative agents in foods: Mode of action and microbial resistance mechanisms, International Journal of Food Microbiology. 50(1999)1-17.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eK. S. Agrawal, A. V. Sarda, R. Shrotriya,\u003cem\u003e\u0026nbsp;\u003c/em\u003eM. Bachhav, G. Nataraj, Acetic acid dressings: finding the Holy Grail for infected wound management Indian, Plast Surg. 50( 2017)273-280.\u003c/li\u003e\n \u003cli\u003eC. Q. Gao, H. Q. Shi, W. Y. Xie, L. \u0026nbsp;Zhao, J. Zhang, C. Ji, Q. G. Ma, Dietary supplementation with acidifiers improves the growth performance, meat quality and intestinal health of broiler chickens,\u0026nbsp;\u003cem\u003eAnim Nutr.\u0026nbsp;\u003c/em\u003e7(2021)762\u0026ndash;769.\u003c/li\u003e\n \u003cli\u003eS. K. Nath , M. T. Hossain, M. Ferdous, A. Siddika, A. Hossain, A. A. Maruf, A. \u0026nbsp;T. \u0026nbsp;Chowdhory, T. C. Nath, Effects of antibiotic, acidifier, and probiotic supplementation on mortality rates, lipoprotein profile, and carcass traits of broiler chickens, Veterinary and Animal Science. 22(2023) 22100325.\u003c/li\u003e\n \u003cli\u003eW. Pelyuntha, A. Yafa, B. Charoenwong, K. Vongkamjan, Effectiveness of the Organic Acid-Based Antimicrobial Agent to Prevent Bacterial Contamination in Fish Meal,\u0026nbsp;\u003cem\u003eAnimals\u003c/em\u003e. 12(2022)3367.\u003c/li\u003e\n \u003cli\u003eJ. R. Harrington, R. J. Martens, D. CohenN, L. R. Bernstein, Antimicrobial activity of gallium against virulent Rhodococcus equiin vitro an in vivo, J Vet Pharmacol Ther. 29(2006)121-7.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eO. Olakanmi, B. Kesavalu, R. Pasula,\u003cem\u003e\u0026nbsp;\u003c/em\u003eY. Maher, L. Abdalla, B. Schlesinger, B. Britigan, The transition metal gallium nitrate is efficacious in murine models of tuberculousis and inhibits key bacterial Fe-dependent enzymes, Antimicrob Agents Chemother. 57(2013)6074-80.\u003c/li\u003e\n \u003cli\u003eR. Garc\u0026iacute;a-Contreras, B. P\u0026eacute;rez-Eretza, E. Lira-Silva, R. Jasso-Ch\u0026aacute;vez, R. Coria-Jim\u0026eacute;nez, A. Rangel-Vega, T. Maeda, T. K Wood, Gallium induces the production of virulence factors in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003ePathogens and Disease.\u0026nbsp;\u003c/em\u003e70(2014)95\u0026ndash;98.\u003c/li\u003e\n \u003cli\u003eG. M. Rodriguez, R. Gardner, N. Kaur, O. Phanstiel, Utilization of Fe3+ -acinetoferrin analogs as an iron source by Mycobacterium tuberculosis, Bio Metals. 21(2008)93-103.\u003c/li\u003e\n \u003cli\u003eK. Nikoleta, D. Todor, Gallium as an antibacterial agent: a DFT/ SMD study of the Ga3+/Fe3+ competition for binding bacterial siderophores, Inorg Chem. 59(2020)6242-6254.\u003c/li\u003e\n \u003cli\u003eT. Olczak, D. Maszczak-S, J. W. Smalley, M. Olczak, Gallium (III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells, Arch Microbiol. 194(2012)719-724.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eM. Piatek, D. M. Griffith, K. Kavanagh, Quantitative proteomic reveals gallium maltolate induces an iron-limited stress response and reduced quorum-sensing in Pseudomonas aeruginosa, J Biol Inorg Chem.\u003c/li\u003e\n \u003cli\u003eCH Goss，Y Kaneko，L Khuu，GD Anderson，S Ravishankar，ML Aitken，N Lechtzin，G Zhou，DM Czyz，K Mclean, Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections, Sci Transl Med. 10(2018)7520.\u003c/li\u003e\n \u003cli\u003eS. R. Choi, B. E. Britigan, P. Narayanasamy, Iron/Heme metabolism-targeted gallium(iii) nanoparticles are active against extracellular and intracellular pseudomonas aeruginosa and acinetobacter baumannii, Antimicrob Agents Chemother. 63(2019)e02643-18.\u003c/li\u003e\n \u003cli\u003eL. Lei, C. Hao, Y. Nie, M. Li, Y. Hou, W. Rao, Superior antibacterial activity of gallium based liquid metals due to Ga3+ induced intracellular ROS generation, J Mater Chem B. 9(2020)85-93.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eI. Stojiljkovic, B D. Evavold, V. Kumar, Antimicrobial properties of porphyrins, Expert Opin Investig Drugs. 10(2001)309-320.\u003c/li\u003e\n \u003cli\u003e\u0026Eacute;. A Enyedy, O. D\u0026ouml;m\u0026ouml;t\u0026ouml;r, B. Krisztina, A. Het\u0026eacute;nyi, T. Tuccinardi, B. K Keppler, Interaction of the anticancer gallium(III) complexes of 8-hydroxyquinoline and maltol with human serum proteins, Journal of biological inorganic chemistry. 20(2015)77-88.\u003c/li\u003e\n \u003cli\u003eNI. Gorshkov, А Y Murko, II Gavrilova, М А Bezrukova 1, А I Kipper 1, S V Shatik 2, А V Tokarev 2, V D Krasikov 1, Е F Panarin 1, Metal-Polymer Complexes of Gallium/Gallium-68 with Copolymers of N-Vinylpyrrolidonewith N-Vinylformamideand N-Vinyliminodiacetic Acid: A Hint for Radiolabeling of Water-Soluble Synthetic Flexible Chain Macromolecules, Polymers. 12(2020)2889.\u003c/li\u003e\n \u003cli\u003eC. Zhu, H X Yang, Y Z Wang, Y Z Wanng,D Q Zhang.Y Chen, L X Gao, Synergistic effect between glutamic acid and rare earth cerium (III) as corrosion inhibitors on AA5052 aluminum alloy in neutral chloride medium, Ionics. 25(2019)1395-1406.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eB Kabak, E. Kend\u0026uuml;zler, Europium metal-organic frameworks: Synthesis, characterization, and application as fluorescence sensors for the detection of Cu\u003csup\u003e2+\u003c/sup\u003e, Ni2+ cations and T3, T4 hormones, Talanta. 266(2024)124944.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eL. Todorov, L. Saso, K. Benarous, M. Traykova, Synthesis, Structure and Impact of 5-Aminoorotic Acid and Its Complexes with Lanthanum(III) and Gallium(III) on the Activity of Xanthine Oxidase, Molecules. 26(2021)4503.\u003c/li\u003e\n \u003cli\u003eEl Habeeb Abeer A, Novel Gallium(III), Germanium(IV), and Hafnium(IV) Folate Complexes and Their Spectroscopic, Thermal Decomposition, Morphological, and Biological Characteristics, Bioinorganic Chemistry and Applications. 2020, 2020: 6678688.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eM. A. Akhtar, Z. Hadzhieva, I, Dlouh\u0026yacute;, A. Boccaccini, Electrophoretic Deposition and Characterization of Functional Coatings Based on an Antibacterial Gallium (III)-Chitosan Complex, Coatings. 10(2020)483. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eS. P. Valappil, E. A. Abou Neel, K. M. Zakir Hossain, W. Paul, Durgadas Cherukaraveedu, B. Wade, Tahera I. Ansari, Christopher K. Hope, S. M. Higham, Chandra P, Sharma f Novel lactoferrin-conjugated gallium complex to treat Pseudomonas aeruginosa wound infection, International Journal of Biological Macromolecules, 258(2024)128838.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eS. Saini, Haritha Sree Yaddanapudi, K. Tian, Yinong Yin, D. Magginetti, A. Tiwari, Terbium Ion Doping in Ca3Co4O9: A Step towards High-Performance Thermoelectric Materials, Scientific Reports. 7(2017)44621.\u003c/li\u003e\n \u003cli\u003eM Jovan\u0026iacute;, A Sanz, H Beltr\u0026aacute;n-Mir, E. Cordoncillo, New red-shade environmental-friendly multifunctional pigment based on Tb and Fe doped Y2Zr2O7 for ceramic applications and cool roof coatings, Dyes Pigments. 133(2016)33-40.\u003c/li\u003e\n \u003cli\u003eR. Lapuente, C. Quijada, F. Huerta, F. Cases \u0026amp; Jos\u0026eacute; L V\u0026aacute;zquez, X-ray photoelectron spectroscopy study of the composition of polyphenol films formed on Pt by electropolymerisation of phenol in the presence of sulphide in carbonate medium, Polymer Journal. 35(2003)911- 916.\u003c/li\u003e\n \u003cli\u003eŚciana B, W. Dawidowski, D. Radziewicz, J. Jadczak, M. C L\u0026oacute;pez-Escalante 3, V. Gonz\u0026aacute;lez de la Cruz, M. Gab\u0026aacute;s, Influence of As-N Interstitial Complexes on Strain Generated in GaAsN Epilayers Grown by AP-MOVPE, Energies. 15(2022)3036.\u003c/li\u003e\n \u003cli\u003eS R Choi, B. Switzer, B E Britigan, P. Narayanasamy,\u003cem\u003e\u0026nbsp;\u003c/em\u003eGallium Porphyrin and Gallium Nitrate Synergistically Inhibit Mycobacterial Species by Targeting Different Aspects of Iron/Heme Metabolism[J]. \u003cem\u003eACS Infect Dis\u003c/em\u003e, 26(2020)2582-2591.\u003c/li\u003e\n \u003cli\u003eC. Auger, J. Lemire, V. Appanna, V. Appanna, Gallium in bacteria, metabolic and medical implications, Springer New York. (2013)800-807.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eA Stinzi, C Barnes, J Xu, K N Raymond, Raymond, Microbial iron transport via a siderophore shuttle: a membrane iron transport paradigm, Proc Natl Acad Sci. 97(2000)10691-10696.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eG. Rhys, G. Chris, Cofactor F420: an expanded view of its distribution, biosynthesis, and roles in bacteria and archaea, FEMS microbiology reviews. 45(2021)465-478.\u003c/li\u003e\n \u003cli\u003ePfl\u0026uuml;ger-Grau K, Lorenzo V, From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida, FEMS Microbiology Letters. 356(2014)144-153.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"organic acids (OA), gallium (Ga³⁺), complexes, antibacterial activity, transcriptome analysis","lastPublishedDoi":"10.21203/rs.3.rs-5970012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5970012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs an alternative to antibiotics, acidifiers have gained widespread application in the feed industry. However, current acidifier products often suffer from limited antibacterial efficacy. To tackle this issue, we synthesized a series of organic acid - gallium complexes (Ga-OA) using organic acids (OA) and Ga\u003csup\u003e3+\u003c/sup\u003e as precursors, via a liquid-phase synthesis method. The antimicrobial activity of Ga-OA against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eSalmonella spp.\u003c/em\u003e was assessed using the Oxford cup and agar dilution methods to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. It was found that the Ga-OA complexes showed markedly higher antibacterial activity than each individually, and the antibacterial activity of Ga-OA complexes followed the order: Ga-Lac (lactic acid)\u0026thinsp;\u0026gt;\u0026thinsp;Ga-Ac (acetic acid)\u0026thinsp;\u0026gt;\u0026thinsp;Ga-BA (butyric acid). Furthermore, The MIC values of Ga-Lac against \u003cem\u003eEscherichia coli, Staphylococcus aureus, and Salmonella spp\u003c/em\u003e were 2.84, 0.18, and 2.84 mmol/L, respectively, meanwhile, the MBC values of Ga-Lac against these three bacteria were 5.68, 1.42, and 5.68 mmol/L, respectively. Transcriptome analysis revealed that the antibacterial mechanism of Ga-OA is initiated by organic acid (OA) binding to bacterial membranes, which promotes Ga\u003csup\u003e3+\u003c/sup\u003e entry into the cell. This intracellular Ga\u0026sup3;⁺ then disrupts iron transport, ultimately resulting in bacterial death. These results suggest that Ga-OA complexes have the potential to be a promising, safe, and effective antibacterial agent in animal husbandry, providing a solution to antibiotic resistance concerns.\u003c/p\u003e","manuscriptTitle":"Enhanced antibacterial activity of organic acids via gallium chelation: a promising antibiotic alternative","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-14 04:58:19","doi":"10.21203/rs.3.rs-5970012/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50adbf2d-c4c7-492e-bda6-36740077753b","owner":[],"postedDate":"February 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-12T10:25:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-14 04:58:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5970012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5970012","identity":"rs-5970012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}