Metabolomics study of the inhibitory effects of tubuloside A on Streptococcosis suis biofilm

Metabolomics study of the inhibitory effects of tubuloside A on Streptococcosis suis biofilm | 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 Metabolomics study of the inhibitory effects of tubuloside A on Streptococcosis suis biofilm Ruixiang Che, Yiyang Sun, Jianjun Zhao, Dongbo Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9345583/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Streptococcosis suis ( S. suis ) is an important zoonotic pathogen. After forming biofilms, it can cause persistent and chronic infections in the host, which increase the risk of zoonotic infections, and pose a threat to public health. Clinically, it is mainly treated with antibacterial drugs. However, the drug resistance of S. suis that forms biofilms is enhanced, and conventional drugs cannot eradicate it. At present, screening traditional Chinese medicine monomer drugs to interfere with the formation of biofilms has become one of the common methods for treating S. suis . In this study, the mechanism of tubuloside A intervention on S. suis ATCC700794 biofilm formation was explored by metabolomics. The minimum inhibitory concentration (MIC) of tubuloside A against the ATCC700794 was determined by two-fold serial dilution of the microbroth. The effects of tubuloside A were studied using crystal violet staining. The morphology of tubuloside A treated ATCC700794 cells was observed by scanning electron microscopy. Differentially expressed metabolites were screened using metabolomics and biological information analyses. The MIC of tubuloside A was 64ug/mL, whereas 1/2 MIC (32ug/mL) of tubuloside A significantly inhibited biofilm formation without affecting the bacterial growth and prevented the formation of biofilm structure. Using 1/2 MIC of tubuloside A, 65 metabolites were identified, of which 42 were upregulated and 23 were downregulated. Bioinformatic analysis showed that the changes in the ATCC700794 strains after tubuloside A intervention primarily involved glycine, serine and threonine metabolism, purine metabolism, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, citrate cycle (TCA), arginine and proline metabolism, pyruvate metabolism. This study elucidated the mechanism which tubuloside A inhibited from a metabolomics perspective, providing novel insights for biofilm control strategies. Tubuloside A Streptococcosis suis biofilm metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights 1.This study is the first to show that tubuloside A inhibits biofilm formation in Streptococcus suis at sub-inhibitory concentrations. 2.This study reveals the metabolic changes associated with the antibiofilm effect of tubuloside A using untargeted metabolomics. 3.This study identifies amino acid metabolism and central carbon metabolism as potential pathways involved in tubuloside A-mediated inhibition of S. suis biofilm formation. 1 Introduction Streptococcus suis ( S. suis ) is a widely prevalent zoonotic pathogen worldwide. It is the most important causes of bacterial infection and mortality in post-weaning piglets, causing huge economic losses to the swine industry. Its infection can cause meningitis and sepsis in pigs, while infected pigs or contaminated raw pork products can also transmit the pathogen to humans via mucous membranes, wounds and ingestion, resulting in septic shock and meningitis[ 1 , 2 ]. In 1960, De Moor first isolated this bacterium from outbreaks and sporadic cases of septicemia in pigs[ 3 ]. At present, based on the differences in the antigenic characteristics of the bacterial capsule, Streptococcus suis is currently classified into at least 29 serotypes[ 4 ]. Among them, serotype 2 is the most frequently isolated serotype and also the one most frequently associated with diseases. The bacteria exists in planktonic and biofilm states in nature[ 5 ]. Bacterial biofilm (BF) refers to a large number of aggregated, membrane-like bacterial aggregates formed by bacteria adhering to a contact surface and enclosing themselves by secreting polysaccharide matrices, fibrin, lipoproteins[ 6 ]. The polysaccharide matrix usually refers to a polysaccharide-protein complex, as well as organic and inorganic substances precipitated from the periphery[ 7 ]. The formation of bacterial biofilm is a life phenomenon that helps bacteria adapt to the natural environment and survive better. Once formed, biofilms possess innate resistance to antibiotics and host immunity[ 8 , 9 ]. It is difficult to completely eliminate them through antibiotics. Instead, only the free bacteria on the surface of the biofilm or in the blood that cause infection can be killed[ 10 ]. Therefore, the formation of biofilms enables bacteria to resist various adverse factors in the environment and acquire adaptive traits, and it is closely related to the pathogenicity and drug resistance of pathogenic microorganisms. Tubuloside A is a major phenylethanoid glycoside isolated from Cistanche tubulosa (Orobanchaceae), one of the most important bioactive constituents responsible for the pharmacological effects of Cistanche species[ 11 ]. It exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, neuroprotective, anti-aging, immunomodulatory, hepatoprotective, and memory-improving effects[ 12 ]. Due to its natural origin, favorable safety profile, and multiple pharmacological targets, tubuloside A has become a promising candidate for research in natural medicines, functional foods, and the prevention and adjuvant treatment of neurodegenerative and inflammation-related diseases. Previous studies have reported the mechanism of biofilm formation in S. suis through metabolomics[ 1 ]. Furthermore, tubuloside A has been shown to be an effective candidate for biofilm eradication. The antibacterial mechanisms of tubuloside A against methicillin-resistant Staphylococcus aureus (MRSA)[ 13 ]. Tubuloside A significantly impedes Staphylococcus aureus ( S.aureus )’s adherence to fibrinogen, notably disrupting biofilm development and the integration of staphylococcal protein A (SpA) into the cell wall[ 13 ]. However, whether tubuloside A can inhibit the biofilm formation of S. suis and the mechanism by which it inhibits S. suis biofilm formation remains unclear. Therefore, the present study aimed to identify and analyze the differentially expressed metabolites during tubuloside A inhibition of S. suis biofilms using untargeted metabolomics. This study is expected to provide a reliable basis for elucidating the mechanisms by which Tubuloside A disrupts S. suis biofilms, and offer insights for the development of drugs that inhibit S. suis biofilms. 2 Materials and methods 2.1 Chemicals and reagents Tubuloside A was purchased from Dalian Meilun Biotechnology Co., Ltd.(CAS# 112516-05-9, Dalian, China). Crystal violet, methanol, ethanol, glacial acetic acid, absolute ethanol, glutaraldehyde, tert-butyl alcohol, phosphate buffered saline (PBS), and other reagents were purchased from Tianjin KOMIO Chemical Reagents Company, Ltd. (Tianjin, China). Todd-Hewitt broth (THB) (THB: HB0311-3, Qingdao, China). 2.2 Bacterial Strains and Culture Conditions Streptococcus suis ATCC 700794 used in this study was kindly provided by the laboratory of Prof. Yanhua Li, Northeast Agricultural University. S.suis was grown in Todd-Hewitt broth (THB) with 5% (v/v) fetal bovine serum (Sijiqing Ltd, Hangzhou, Zhejiang, China). S.suis were incubated at 37°C in aconstant-temperature incubator at a rotational speed of 180–200 rpm. 2.3 Minimum inhibitory concentrations and growth rates The MIC of S. suis to tubuloside A was determined by microtitre broth dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). Cultures were diluted to 1 × 10 6 colony-forming units (CFU)/mL using THB. Finally, 100 µL of cell suspensions were inoculated into the wells of a 96-well plates (Corning Costar® 3599 Corning, NY, USA) containing serial dilutions of tubuloside A culture medium as previously reported[ 14 ]. At the same time, negative control was set up as outlined in a previous study[ 15 ]. The inoculated microplates were incubated at 37°C for 24 h. The growth rates of S. suis ATCC 700794 treated with and without 1/2 MIC (32 µg/mL) tubuloside A were analyzed[ 16 ], which were incubated at 37℃ for 12 h. The growth inhibition test was performed according to a previously reported method[ 17 ]. The culture medium was added with 1/2 MIC (32ug/mL) of tubuloside A and incubated at 37◦C for 12h. Control cells were also incubated in the absence of tubuloside A. Samples were taken every hour for measuring OD 600 nm for experimental group and control group. 2.4 Crystal violet staining assays Inoculate S. suis ATCC700794 into THB liquid medium and incubate in a 37°C constant temperature incubator for 18–24 hours. Dilute the culture with THB liquid medium to achieve a bacterial concentration of 1×10⁶ CFU/mL. 100 µL of the diluted S. suis suspension were dispensed into 96-well microplates. To these wells, 100 µL of tubuloside A solution at various sub‑MIC concentrations 1/2MIC (32ug/mL), 1/4MIC (16ug/mL) ,1/8 MIC (8ug/mL) were added, negative control wells were included. Subsequently, the plates were incubated statically at 37°C in a humidified incubator for 72 h to allow biofilm formation. After 72 h, remove the tissue culture plate to assess biofilm formation ability. Wells containing only THB liquid medium serve as the negative control group, and the edge wells of the tissue culture plate filled with physiological saline serve as the blank control. Wash the wells three times with PBS, dried and fixed with 200 µLof 99% methanol for 5 min, left to dry and stained with 200 µL of 2% crystal violet for another 5 min[ 16 ]. Excess stain was rinsed off gently by water and solubilized with 200 µL of 33% glacial acetic acid. The absorbance of the crystal violet-stained ATCC 700794 biofilms was measured at 595 nm[ 16 ]. 2.5 Scanning electron microscopy (SEM) Mid-exponential growth phase cultures of S. suis ATCC 700794 were adjusted to an optical density of 0.1 at 600 nm (OD600). Then, 2 mL cultures were transferred to the wells of a 6-well microplate containing an 11 × 11 mm sterilized rough glass slide (Mosutech Co., Ltd., Shanghai, China) on the bottom. After culturing for 72 h at 37℃without shaking, the glass slide was removed with tweezers, and the biofilms on the rough glass slide were washed with sterile PBS. The remaining biofilms were fixed with fixative solution [4% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 2 mM CaCl 2 in 0.2 M cacodylate buffer, pH 7.2] for 6 h and washed three times with 0.1 M PBS 10 min each, then fixed in 2% osmium tetroxide containing 2 mM potassium ferrocyanide and 6% (w/v) sucrose in cacodylate buffer. The samples were dried, gold sputtered with an ion sputtering instrument (current 15 mA, 2 min) and observed using SEM (FEI Quanta, Netherland)[ 16 ]. 2.6 Metabolomics assay The culture medium from the cultured S. suis ATCC 700794 was removed using pipette. Then the cells were washed with PBS under 37°C and the PBS was removed. 800 µL of cold methanol/acetonitrile (1:1, v/v) to remove the protein and extract the metabolites. The mixture was collected into a new centrifuge tube, and centrifuged at 14000g for 20 min to collect the supernatant. The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 µL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14000 g at 4 ℃ for 15 min, then the supernatant was injected. To monitor the stability and repeatability of instrument analysis, quality control (QC) samples were prepared by pooling 10 µL of each sample and analyzed together with the other samples. The QC samples were inserted regularly and analyzed in every 5 samples [ 18 ] . Analysis was performed using an UHPLC (Vanquish UHPLC, Thermo) coupled to a Orbitrap. Exploris™ 480 in Shanghai Applied Protein Technology Co., Ltd. For HILIC separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH Amide 1.7 µm column (waters, Ireland). In both ESI positive and negative modes, the mobile phase contained A = 25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. The gradient was 95% B for 0.5 min and was linearly reduced to 65% in 6.5 min, then reduced to 40% in 1 min and kept for 1min, then increased to 95% in 0.1 min and kept for 2.9min. The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 50, Ion Source Gas2(Gas2) as 2, source temperature: 350℃, IonSpray Voltage Floating (ISVF) :+3500 V/-2800V. In MS only acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000 and the accumulation time was set at 100ms. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000 and the accumulation time was set at 100ms, exclude time within 4s. The raw MS data were converted to MzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z = 10 ppm, peakwidth = c (10, 60), prefilter = c (10, 100). For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was sued for annotation of isotopes and adducts. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. Compound identification of metabolites was performed by comparing of accuracy m/z value (< 10 ppm), and MS/MS spectra with an in-house database established with available authentic standards. After sum-normalization, the processed data were analyzed by R package (ropls), where it was subjected to multivariate data analysis, including Pareto-scaled principal component analysis (PCA)and orthogonal partial least-squares discriminant analysis (OPLS-DA). The 7-fold cross-validation and response permutation testing were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Student’s t test was applied to determine the significance of differences between two groups of independent samples. VIP > 1 and p value < 0.05 were used to screen significant changed metabolites. Pearson’s correlation analysis was performed to determine the correlation between two variables. 2.7 Statistical analysis The significance of differences was determined by t-test. P < 0.05 was considered to be significant. 2.8 Clinical trial registration number Not applicable. 2.9 Ethics statement, informed consent from subjects, and informed consent for publication Not applicable. 3 Results 3.1 Tubuloside A affects the growth of S. suis The MIC of tubuloside A against S. suis ATCC700794 was 64ug/mL. The culture medium in which 1/2 MIC of tubuloside A was added to the S. suis group as the experimental group, and the group of S. suis without adding tubuloside A was designated as the control group. The results show that the growth curves were no significantly different between experimental group and control group in 12h (Fig. 1 ). It suggested that the growth of S. suis was unaffected by the addition of 1/2 MIC (32ug/mL) tubuloside A. 3.2 Tubuloside A affects the biofilm of S. suis We used crystal violet staining to study the effects of tubuloside A on the number of S. suis ATCC700794 biofilms. Compared with the control group, the OD 595nm value of 1/2 MIC of TA was significantly lower ( P < 0.01, Fig. 2 A), the OD 595nm value of 1/4 MIC of TA was significantly lower ( P < 0.01, Fig. 2 A), the OD 595nm value of 1/8 MIC of TA was significantly lower ( P < 0.05, Fig. 2 B). The inhibition rates of 60.60%, 53.62% and 28.55%, respectively. We used SEM to observe the effects of tubuloside A on the morphology of S. suis ATCC700794 biofilms. Without 1/2 MIC tubuloside A, S. suis ATCC700794 can form a three-level structure of biofilm on a sterile slide (Fig. 3 A). However, when 1/2 MIC tubuloside A is present, only a small number of bacteria adhere (Fig. 3 B). This results suggested that tubuloside A at 1/2MIC significantly inhibited the biofilm formation of S. suis in vitro. 3.3 Metabolomics analysis In order to investigate the effect of 1/2MIC tubuloside A on S. suis , we conducted an analysis based on untargeted metabolomics using the LC-MS/MS instrument. The principal component analysis (PCA) scoring plot revealed that principal component 1 (PC1) accounted for 48.2%, while PC2 explained 15%(Figure 4 A). Compared to the strain ATCC700794, 65 metabolites showed differences in their levels, with 42 metabolites increasing and 23 metabolites decreasing ( p < 0.05) ༈Figure 4 B༉. Stratified cluster analysis of metabolites showed that numerous differentially abundant metabolites were significantly upregulated in S. suis following treatment with 1/2 MIC tubuloside A༈Figure 4 C༉. The top20 of KEGG pathways enrichment in nucleotide metabolism, metabolic pathways, biosynthesis of amino acids, glycine, serine and threonine metabolism, purine metabolism, ABC transporters, cysteine and methionine metabolism, central carbon metabolism in cancer, carbon metabolism, glucagon signaling pathway, pyrimidine metabolism, alanine, aspartate and glutamate metabolism, citrate cycle (TCA cycle), biosynthesis of cofactors, carbon fixation by calvin cycle, other carbon fixation pathways, prostate cancer, arginine and proline metabolism, pyruvate metabolism, nicotinate and nicotinamide metabolism༈Figure 4 D༉. 4 Discussion Biofilms have been proven to be one of the key enablers of bacterial drug resistance and have also become an important intervention target for reducing bacterial drug resistance[ 19 ]. Studies have found that biofilms significantly reduce the penetration efficiency and bactericidal efficacy of antibiotics through their unique extracellular matrix barrier, changes in bacterial metabolic status, and specific expression of drug-resistant genes, enabling bacteria to survive and reproduce continuously in high-concentration antibiotic environments, further exacerbating the spread and dissemination of multidrug-resistant strains[ 19 ]. Biofilm formation poses a great challenge to its therapeutic aspects[ 20 ]. As a prevalent pathogen in swine, S. suis is capable of forming biofilms, which influences the development of antimicrobial resistance and virulence, thereby inflicting substantial economic losses on the pig farming industry[ 21 ]. Therefore, elucidating the mechanism(s) underlying Streptococcus suis biofilm formation and exploring strategies to interfere with this process are essential for the prevention and treatment of S. suis disease[ 21 ]. Tubuloside A is a phenylethanoid glycoside natural active ingredient isolated from Cistanche tubulosa (Orobanchaceae), which has attracted extensive attention in pharmacological and anti-drug resistance research in recent years[ 22 , 23 ]. In this study, we investigated the antibacterial activity and related mechanisms of action of tubuloside A against S. suis . In our study, the 1/2 MIC (32ug/mL) of tubuloside A did not inhibit the growth of S. suis . The 1/2 MIC, 1/4 MIC, and 1/8 MIC of tubuloside A significantly inhibited S. suis biofilm in the crystal violet staining experiments. Meanwhile, treatment with 1/2 MIC of tubuloside A was also found to significantly alter the morphology and structure of S. suis biofilms. These results show that 1/2 MIC of tubuloside A significantly inhibited S. suis biofilm formation. Studies have shown that as the concentration of tubuloside A increased, the inhibition of Staphylococcus aureus USA300 adhesion to fibrinogen became more significant[ 13 ]. In the murine pneumonia infection model, tubuloside A effectively reduced the bacterial load, demonstrating a significant capacity to mitigate MRSA pathogenicity[ 13 ]. The study have shown that tubuloside A inhibits the formation of MRSA biofilm, based on our experimental findings, tubuloside A may be a potential candidate for interfering with bacterial biofilm formation. To further explore the mechanism by which tubuloside A interferes with biofilm formation in S. suis ATCC 700794, we used untargeted metabolomics to investigate metabolic alterations in this strain following treatment with 1/2 MIC of TnA. The result that 65 metabolites showed differences in their levels, with 42 metabolites increasing and 23 metabolites decreasing ( p < 0.05). These metabolites are mainly enriched in nitrogen metabolism and carbon metabolism pathways, which included glycine, serine and threonine metabolism; cysteine and methionine metabolism; alanine, aspartate and glutamate metabolism; arginine and proline metabolism; pyruvate metabolism; citrate cycle (TCA cycle). Most of nitrogen metabolism are amino acid metabolic pathways. It is revealed that these pathways may be associated with bacterial biofilm formation. Studies have shown that L-serine can regulate the biosynthesis of glycine and cysteine. L-serine had a significantly decreased ability to form biofilms in the macrolide-resistant S. suis [ 12 ]. In our study, serine expression decreased significantly. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of glycine, serine, and threonine metabolism by L-serine. Cysteine has been shown to be involved in the biofilm formation of S. suis [ 24 ]. Under the action of cystathionine γ-synthase, cystathionine is formed from cysteine, which is then converted to homocysteine by cystathionine β-lyase and enters the methionine cycle. The content of cysteine can affect homocysteine in the methionine pathway, thereby further influencing biofilm formation[ 25 ]. In our study, the L-cystathionine and S-adenosyl-L-methionine significantly decreased. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of cysteine and methionine metabolism. Alanine, aspartate and glutamate metabolism has been demonstrated to be involved in biofilm formation. Aspartate produced by this pathway can be converted to arginine via the urea cycle. Studies have shown that L-arginine can been regulated the formation of biofilms in Pseudomonas [ 26 ]. In Staphylococcus aureus , exogenous L-arginine of argGH can also led to decreased biofilm formation in parental strains[ 27 ]. In our study, aspartate and citrulline expression decreased significantly. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of alanine, aspartate and glutamate metabolism or arginine and proline metabolism. Pyruvate cycle and TCA cycle are related to carbon metabolism. Our results suggest that carbon metabolism is an important regulatory factor in tubuloside A intervention of S. suis biofilm formation. In Streptococcus mutans , the pyruvate metabolism directs carbon flow into various pathways that contribute to its biofilm formation. In our study, phosphoenolpyruvate and pyruvate in pyruvate metabolism were significantly down-regulated. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of TCA cycle. TCA cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG in methicillin-sensitive Staphylococcus aureus (MSSA)[ 28 ]. Citrate as a carbon source enhanced biofilm formation in Pseudomonas fluorescens [ 29 ]. In our study, citrate expression decreased significantly, the expression level of succinate was up-regulated. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of TCA cycle. The tubuloside A exerts antibacterial effects mainly by regulating nitrogen metabolism, carbon metabolism, and other metabolic pathways. It may be an effective candidate for the development of novel strategies to treat S. suis infections in the future. 5 Conclusion The present study shows that tubuloside A has good antibacterial activity against S. suis ATCC700794. Tubuloside A can be used as a foundation for further research on the inhibitory mechanisms of S. suis biofilm formation and drug development. Abbreviations S. suis Streptococcus suis MIC Minimum inhibitory concentration Declarations Author contributions S-DB designed the whole experiment. R-XC directed the completion of the experiment. S-YYwere supportive during the experiment. Acknowledgments The authors appreciate applied ppotein technology for their support during the metabolites database searching and data analysis. Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by Heilongjiang Postdoctoral Financial Assistance (LBH-Z21192); Natural Science Foundation of Heilongjiang Province of China (JJ2023LH1529); Heilongjiang Bayi Agricultural University Talent Research Start-up Fund (XYB202018). References Wang H, Fan Q, Wang Y, Yi L, Wang Y. 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Advancing treatment strategies against MRSA: unveiling the potency of tubuloside A in targeting sortase A and mitigating pathogenicity. World J Microbiol Biotechnol. 2025;41(2):29; doi: 10.1007/s11274-024-04185-7. Zhou YH, Yu F, Chen M, Zhang YF, Qu QW, Wei YR, et al. Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM. Frontiers in Veterinary Science. 2022;8; doi: 10.3389/fvets.2021.829899. Dong CL, Wu T, Dong Y, Qu QW, Chen XY, Li YH. Exogenous methionine contributes to reversing the resistance of Streptococcus suis to macrolides. Microbiology Spectrum. 2024;12(2); doi: 10.1128/spectrum.02803-23. Rossi CS, Barrientos-Moreno L, Paone A, Cutruzzolà F, Paiardini A, Espinosa-Urgel M, et al. Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas. International Journal of Molecular Sciences. 2022;23(8); doi: 10.3390/ijms23084386. Manna AC, Leo S, Girel S, González-Ruiz V, Rudaz S, Francois P, et al. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 16 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviews received at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 17 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 07 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9345583","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":632000096,"identity":"096ee1eb-d3e9-4397-bcc3-48e9f5756330","order_by":0,"name":"Ruixiang Che","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ruixiang","middleName":"","lastName":"Che","suffix":""},{"id":632000097,"identity":"a6fe62bf-ca7a-4cc5-85d5-1837a220aa8c","order_by":1,"name":"Yiyang Sun","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yiyang","middleName":"","lastName":"Sun","suffix":""},{"id":632000098,"identity":"e9dbde0e-2967-4140-9508-fac1c037a909","order_by":2,"name":"Jianjun Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACxmYgIQHjfTCwsSNNC+OMgrRk0qxk5vlwiLGBoKp25mcPLNvu2Mv3H3/82cbgADMD++GjG/A7jM3cQLLtGTNjw4EE4xyDO3wMPGlpN/BrYTCTkGw7zAbSk5xj8IyZQYLHjIAW9m8gLTwgPYctDA4zNhDWwgO2RQKkp5mBSC1lEhLnDhuA9fQYpCWzEfKLYf/xbdISZYfBIfbhxx8bO372w8fwa2kABrQEsggbPuUgIA9y3AdCqkbBKBgFo2BkAwBbMEEIq0RYbgAAAABJRU5ErkJggg==","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jianjun","middleName":"","lastName":"Zhao","suffix":""},{"id":632000099,"identity":"b0505311-ec7f-425e-b606-65961c95fef2","order_by":3,"name":"Dongbo Sun","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dongbo","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-04-07 13:09:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9345583/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9345583/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108589838,"identity":"a1cafcaa-6060-4b6d-a6e4-17fc7338aec5","added_by":"auto","created_at":"2026-05-06 09:32:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth curve of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. suis\u003c/em\u003ein the absence of tubuloside A and in the presence of tubuloside A at 1/2 MIC (32ug/mL). The data are expressed as the mean ± stand.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9345583/v1/5346f1ef03bd0749c65801b4.png"},{"id":108805969,"identity":"c5cdc98c-cf3a-4e0f-9f13-2076b8c563bb","added_by":"auto","created_at":"2026-05-08 15:27:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of tubuloside A on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. suis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm formation in vitro\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eOD 595nm values of \u003cem\u003eS. suis\u003c/em\u003e ATCC799794 biofilms after treatments with 1/2 MIC, 1/4 MIC, and 1/8 MIC of tubuloside A(*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eInhibition rates of\u003cem\u003e S. suis\u003c/em\u003e ATCC799794 biofilms after treatments with 1/2 MIC, 1/4 MIC, and 1/8 MIC of tubuloside A.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9345583/v1/84aaea5706c00ebc8c85845d.png"},{"id":108589840,"identity":"38a2e713-b1e5-4b8a-825d-c4cbd1357280","added_by":"auto","created_at":"2026-05-06 09:32:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":333478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of tubuloside A on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSuis\u003c/em\u003ebiofilm by scanning electron microscope. \u003cstrong\u003e(A)\u003c/strong\u003e Control group; \u003cstrong\u003e(B) \u003c/strong\u003eAdd with 1/2MIC tubuloside A.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9345583/v1/768516869497130e154dcc90.png"},{"id":108589841,"identity":"f11bbdce-b557-45bd-b3d0-a5ec014456d8","added_by":"auto","created_at":"2026-05-06 09:32:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUntargeted metabolomics analysis of differentially expressed metabolites in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. suis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ATCC700794 exposed to 1/2 MIC of tubuloside A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eprincipal component analysis (PCA) score plot of metabolites in \u003cem\u003eS.suis\u003c/em\u003e ATCC799794 and \u003cem\u003eS. suis\u003c/em\u003e ATCC799794 with 1/2 MIC of tubuloside A .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eA volcano plot demonstrated metabolites altered significantly are from comparisons of \u003cem\u003eS.suis \u003c/em\u003eATCC799794 and \u003cem\u003eS. suis\u003c/em\u003e ATCC799794 with 1/2 MIC of tubuloside A .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eCluster analysis plot of metabolites of \u003cem\u003eS.suis \u003c/em\u003eATCC799794 and \u003cem\u003eS. suis\u003c/em\u003eATCC799794 with 1/2 MIC of tubuloside A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003eKyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differential metabolites of S.suis ATCC799794 and S. suis ATCC799794 with 1/2 MIC of tubuloside A.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9345583/v1/ab7f89fb1aa2f872748f26eb.png"},{"id":108814694,"identity":"150c7311-3cd3-45ea-80b2-843834971037","added_by":"auto","created_at":"2026-05-08 16:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":735661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9345583/v1/d8afc26d-5ad1-4fee-9a1e-286aa813e4fe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolomics study of the inhibitory effects of tubuloside A on Streptococcosis suis biofilm","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1.This study is the first to show that tubuloside A inhibits biofilm formation in Streptococcus suis at sub-inhibitory concentrations.\u003c/p\u003e\u003cp\u003e2.This study reveals the metabolic changes associated with the antibiofilm effect of tubuloside A using untargeted metabolomics.\u003c/p\u003e\u003cp\u003e3.This study identifies amino acid metabolism and central carbon metabolism as potential pathways involved in tubuloside A-mediated inhibition of S. suis biofilm formation.\u003c/p\u003e"},{"header":"1 Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus suis\u003c/em\u003e (\u003cem\u003eS. suis\u003c/em\u003e) is a widely prevalent zoonotic pathogen worldwide. It is the most important causes of bacterial infection and mortality in post-weaning piglets, causing huge economic losses to the swine industry. Its infection can cause meningitis and sepsis in pigs, while infected pigs or contaminated raw pork products can also transmit the pathogen to humans via mucous membranes, wounds and ingestion, resulting in septic shock and meningitis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 1960, De Moor first isolated this bacterium from outbreaks and sporadic cases of septicemia in pigs[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. At present, based on the differences in the antigenic characteristics of the bacterial capsule, \u003cem\u003eStreptococcus suis\u003c/em\u003e is currently classified into at least 29 serotypes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among them, serotype 2 is the most frequently isolated serotype and also the one most frequently associated with diseases.\u003c/p\u003e \u003cp\u003eThe bacteria exists in planktonic and biofilm states in nature[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bacterial biofilm (BF) refers to a large number of aggregated, membrane-like bacterial aggregates formed by bacteria adhering to a contact surface and enclosing themselves by secreting polysaccharide matrices, fibrin, lipoproteins[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The polysaccharide matrix usually refers to a polysaccharide-protein complex, as well as organic and inorganic substances precipitated from the periphery[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The formation of bacterial biofilm is a life phenomenon that helps bacteria adapt to the natural environment and survive better. Once formed, biofilms possess innate resistance to antibiotics and host immunity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is difficult to completely eliminate them through antibiotics. Instead, only the free bacteria on the surface of the biofilm or in the blood that cause infection can be killed[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, the formation of biofilms enables bacteria to resist various adverse factors in the environment and acquire adaptive traits, and it is closely related to the pathogenicity and drug resistance of pathogenic microorganisms.\u003c/p\u003e \u003cp\u003eTubuloside A is a major phenylethanoid glycoside isolated from Cistanche tubulosa (Orobanchaceae), one of the most important bioactive constituents responsible for the pharmacological effects of Cistanche species[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, neuroprotective, anti-aging, immunomodulatory, hepatoprotective, and memory-improving effects[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Due to its natural origin, favorable safety profile, and multiple pharmacological targets, tubuloside A has become a promising candidate for research in natural medicines, functional foods, and the prevention and adjuvant treatment of neurodegenerative and inflammation-related diseases.\u003c/p\u003e \u003cp\u003ePrevious studies have reported the mechanism of biofilm formation in \u003cem\u003eS. suis\u003c/em\u003e through metabolomics[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Furthermore, tubuloside A has been shown to be an effective candidate for biofilm eradication. The antibacterial mechanisms of tubuloside A against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Tubuloside A significantly impedes \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS.aureus\u003c/em\u003e)\u0026rsquo;s adherence to fibrinogen, notably disrupting biofilm development and the integration of staphylococcal protein A (SpA) into the cell wall[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, whether tubuloside A can inhibit the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e and the mechanism by which it inhibits \u003cem\u003eS. suis\u003c/em\u003e biofilm formation remains unclear. Therefore, the present study aimed to identify and analyze the differentially expressed metabolites during tubuloside A inhibition of \u003cem\u003eS. suis\u003c/em\u003e biofilms using untargeted metabolomics. This study is expected to provide a reliable basis for elucidating the mechanisms by which Tubuloside A disrupts \u003cem\u003eS. suis\u003c/em\u003e biofilms, and offer insights for the development of drugs that inhibit \u003cem\u003eS. suis\u003c/em\u003e biofilms.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and reagents\u003c/h2\u003e \u003cp\u003eTubuloside A was purchased from Dalian Meilun Biotechnology Co., Ltd.(CAS# 112516-05-9, Dalian, China). Crystal violet, methanol, ethanol, glacial acetic acid, absolute ethanol, glutaraldehyde, tert-butyl alcohol, phosphate buffered saline (PBS), and other reagents were purchased from Tianjin KOMIO Chemical Reagents Company, Ltd. (Tianjin, China). Todd-Hewitt broth (THB) (THB: HB0311-3, Qingdao, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Bacterial Strains and Culture Conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eStreptococcus suis\u003c/em\u003e ATCC 700794 used in this study was kindly provided by the laboratory of Prof. Yanhua Li, Northeast Agricultural University. \u003cem\u003eS.suis\u003c/em\u003e was grown in Todd-Hewitt broth (THB) with 5% (v/v) fetal bovine serum (Sijiqing Ltd, Hangzhou, Zhejiang, China). \u003cem\u003eS.suis\u003c/em\u003e were incubated at 37\u0026deg;C in aconstant-temperature incubator at a rotational speed of 180\u0026ndash;200 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Minimum inhibitory concentrations and growth rates\u003c/h2\u003e \u003cp\u003eThe MIC of \u003cem\u003eS. suis\u003c/em\u003e to tubuloside A was determined by microtitre broth dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). Cultures were diluted to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e colony-forming units (CFU)/mL using THB. Finally, 100 \u0026micro;L of cell suspensions were inoculated into the wells of a 96-well plates (Corning Costar\u0026reg; 3599 Corning, NY, USA) containing serial dilutions of tubuloside A culture medium as previously reported[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. At the same time, negative control was set up as outlined in a previous study[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The inoculated microplates were incubated at 37\u0026deg;C for 24 h.\u003c/p\u003e \u003cp\u003eThe growth rates of \u003cem\u003eS. suis\u003c/em\u003e ATCC 700794 treated with and without 1/2 MIC (32 \u0026micro;g/mL) tubuloside A were analyzed[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which were incubated at 37℃ for 12 h. The growth inhibition test was performed according to a previously reported method[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The culture medium was added with 1/2 MIC (32ug/mL) of tubuloside A and incubated at 37◦C for 12h. Control cells were also incubated in the absence of tubuloside A. Samples were taken every hour for measuring OD 600 nm for experimental group and control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Crystal violet staining assays\u003c/h2\u003e \u003cp\u003eInoculate \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 into THB liquid medium and incubate in a 37\u0026deg;C constant temperature incubator for 18\u0026ndash;24 hours. Dilute the culture with THB liquid medium to achieve a bacterial concentration of 1\u0026times;10⁶ CFU/mL. 100 \u0026micro;L of the diluted \u003cem\u003eS. suis\u003c/em\u003e suspension were dispensed into 96-well microplates. To these wells, 100 \u0026micro;L of tubuloside A solution at various sub‑MIC concentrations 1/2MIC (32ug/mL), 1/4MIC (16ug/mL) ,1/8 MIC (8ug/mL) were added, negative control wells were included. Subsequently, the plates were incubated statically at 37\u0026deg;C in a humidified incubator for 72 h to allow biofilm formation. After 72 h, remove the tissue culture plate to assess biofilm formation ability. Wells containing only THB liquid medium serve as the negative control group, and the edge wells of the tissue culture plate filled with physiological saline serve as the blank control. Wash the wells three times with PBS, dried and fixed with 200 \u0026micro;Lof 99% methanol for 5 min, left to dry and stained with 200 \u0026micro;L of 2% crystal violet for another 5 min[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Excess stain was rinsed off gently by water and solubilized with 200 \u0026micro;L of 33% glacial acetic acid. The absorbance of the crystal violet-stained ATCC 700794 biofilms was measured at 595 nm[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eMid-exponential growth phase cultures of \u003cem\u003eS. suis\u003c/em\u003e ATCC 700794 were adjusted to an optical density of 0.1 at 600 nm (OD600). Then, 2 mL cultures were transferred to the wells of a 6-well\u003c/p\u003e \u003cp\u003emicroplate containing an 11 \u0026times; 11 mm sterilized rough glass slide (Mosutech Co., Ltd., Shanghai, China) on the bottom. After culturing for 72 h at 37℃without shaking, the glass slide was removed with tweezers, and the biofilms on the rough glass slide were washed with sterile PBS. The remaining biofilms were fixed with fixative solution [4% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e in 0.2 M cacodylate buffer, pH 7.2] for 6 h and washed three times with 0.1 M PBS 10 min each, then fixed in 2% osmium tetroxide containing 2 mM potassium ferrocyanide and 6% (w/v) sucrose in cacodylate buffer. The samples were dried, gold sputtered with an ion sputtering instrument (current 15 mA, 2 min) and observed using SEM (FEI Quanta, Netherland)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Metabolomics assay\u003c/h2\u003e \u003cp\u003eThe culture medium from the cultured \u003cem\u003eS. suis\u003c/em\u003e ATCC 700794 was removed using pipette. Then the cells were washed with PBS under 37\u0026deg;C and the PBS was removed. 800 \u0026micro;L of cold methanol/acetonitrile (1:1, v/v) to remove the protein and extract the metabolites. The mixture was collected into a new centrifuge tube, and centrifuged at 14000g for 20 min to collect the supernatant. The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 \u0026micro;L acetonitrile/water (1:1, v/v) solvent and centrifuged at 14000 g at 4 ℃ for 15 min, then the supernatant was injected. To monitor the stability and repeatability of instrument analysis, quality control (QC) samples were prepared by pooling 10 \u0026micro;L of each sample and analyzed together with the other samples. The QC samples were inserted regularly and analyzed in every 5 samples [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eAnalysis was performed using an UHPLC (Vanquish UHPLC, Thermo) coupled to a Orbitrap. Exploris\u0026trade; 480 in Shanghai Applied Protein Technology Co., Ltd. For HILIC separation, samples were analyzed using a 2.1 mm \u0026times; 100 mm ACQUIY UPLC BEH Amide 1.7 \u0026micro;m column (waters, Ireland). In both ESI positive and negative modes, the mobile phase contained A\u0026thinsp;=\u0026thinsp;25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B= acetonitrile. The gradient was 95% B for 0.5 min and was linearly reduced to 65% in 6.5 min, then reduced to 40% in 1 min and kept for 1min, then increased to 95% in 0.1 min and kept for 2.9min.\u003c/p\u003e \u003cp\u003eThe ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 50, Ion Source Gas2(Gas2) as 2, source temperature: 350℃, IonSpray Voltage Floating (ISVF) :+3500 V/-2800V. In MS only acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000 and the accumulation time was set at 100ms. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 70-1200 Da, the resolution was set at 60000 and the accumulation time was set at 100ms, exclude time within 4s. The raw MS data were converted to MzXML files using ProteoWizard MSConvert before importing into freely available XCMS software. For peak picking, the following parameters were used: centWave m/z\u0026thinsp;=\u0026thinsp;10 ppm, peakwidth\u0026thinsp;=\u0026thinsp;c (10, 60), prefilter\u0026thinsp;=\u0026thinsp;c (10, 100). For peak grouping, bw\u0026thinsp;=\u0026thinsp;5, mzwid\u0026thinsp;=\u0026thinsp;0.025, minfrac\u0026thinsp;=\u0026thinsp;0.5 were used. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was sued for annotation of isotopes and adducts. In the extracted ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept. Compound identification of metabolites was performed by comparing of accuracy m/z value (\u0026lt;\u0026thinsp;10 ppm), and MS/MS spectra with an in-house database established with available authentic standards.\u003c/p\u003e \u003cp\u003eAfter sum-normalization, the processed data were analyzed by R package (ropls), where it was subjected to multivariate data analysis, including Pareto-scaled principal component analysis (PCA)and orthogonal partial least-squares discriminant analysis (OPLS-DA). The 7-fold cross-validation and response permutation testing were used to evaluate the robustness of the model. The variable importance in the projection (VIP) value of each variable in the OPLS-DA model was calculated to indicate its contribution to the classification. Student\u0026rsquo;s t test was applied to determine the significance of differences between two groups of independent samples. VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 and p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were used to screen significant changed metabolites. Pearson\u0026rsquo;s correlation analysis was performed to determine the correlation between two variables.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe significance of differences was determined by t-test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Clinical trial registration number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Ethics statement, informed consent from subjects, and informed consent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Tubuloside A affects the growth of \u003cem\u003eS. suis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe MIC of tubuloside A against \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 was 64ug/mL. The culture medium in which 1/2 MIC of tubuloside A was added to the \u003cem\u003eS. suis\u003c/em\u003e group as the experimental group, and the group of \u003cem\u003eS. suis\u003c/em\u003e without adding tubuloside A was designated as the control group. The results show that the growth curves were no significantly different between experimental group and control group in 12h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It suggested that the growth of \u003cem\u003eS. suis\u003c/em\u003e was unaffected by the addition of 1/2 MIC (32ug/mL) tubuloside A.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Tubuloside A affects the biofilm of \u003cem\u003eS. suis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eWe used crystal violet staining to study the effects of tubuloside A on the number of \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 biofilms. Compared with the control group, the OD 595nm value of 1/2 MIC of TA was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the OD 595nm value of 1/4 MIC of TA was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the OD 595nm value of 1/8 MIC of TA was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The inhibition rates of 60.60%, 53.62% and 28.55%, respectively. We used SEM to observe the effects of tubuloside A on the morphology of \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 biofilms. Without 1/2 MIC tubuloside A, \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 can form a three-level structure of biofilm on a sterile slide (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, when 1/2 MIC tubuloside A is present, only a small number of bacteria adhere (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This results suggested that tubuloside A at 1/2MIC significantly inhibited the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e in vitro.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Metabolomics analysis\u003c/h2\u003e \u003cp\u003eIn order to investigate the effect of 1/2MIC tubuloside A on \u003cem\u003eS. suis\u003c/em\u003e, we conducted an analysis based on untargeted metabolomics using the LC-MS/MS instrument. The principal component analysis (PCA) scoring plot revealed that principal component 1 (PC1) accounted for 48.2%, while PC2 explained 15%(Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to the strain ATCC700794, 65 metabolites showed differences in their levels, with 42 metabolites increasing and 23 metabolites decreasing (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ༈Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB༉. Stratified cluster analysis of metabolites showed that numerous differentially abundant metabolites were significantly upregulated in \u003cem\u003eS. suis\u003c/em\u003e following treatment with 1/2 MIC tubuloside A༈Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC༉. The top20 of KEGG pathways enrichment in nucleotide metabolism, metabolic pathways, biosynthesis of amino acids, glycine, serine and threonine metabolism, purine metabolism, ABC transporters, cysteine and methionine metabolism, central carbon metabolism in cancer, carbon metabolism, glucagon signaling pathway, pyrimidine metabolism, alanine, aspartate and glutamate metabolism, citrate cycle (TCA cycle), biosynthesis of cofactors, carbon fixation by calvin cycle, other carbon fixation pathways, prostate cancer, arginine and proline metabolism, pyruvate metabolism, nicotinate and nicotinamide metabolism༈Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD༉.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eBiofilms have been proven to be one of the key enablers of bacterial drug resistance and have also become an important intervention target for reducing bacterial drug resistance[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Studies have found that biofilms significantly reduce the penetration efficiency and bactericidal efficacy of antibiotics through their unique extracellular matrix barrier, changes in bacterial metabolic status, and specific expression of drug-resistant genes, enabling bacteria to survive and reproduce continuously in high-concentration antibiotic environments, further exacerbating the spread and dissemination of multidrug-resistant strains[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Biofilm formation poses a great challenge to its therapeutic aspects[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a prevalent pathogen in swine, \u003cem\u003eS. suis\u003c/em\u003e is capable of forming biofilms, which influences the development of antimicrobial resistance and virulence, thereby inflicting substantial economic losses on the pig farming industry[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, elucidating the mechanism(s) underlying Streptococcus suis biofilm formation and exploring strategies to interfere with this process are essential for the prevention and treatment of \u003cem\u003eS. suis\u003c/em\u003e disease[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Tubuloside A is a phenylethanoid glycoside natural active ingredient isolated from Cistanche tubulosa (Orobanchaceae), which has attracted extensive attention in pharmacological and anti-drug resistance research in recent years[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, we investigated the antibacterial activity and related mechanisms of action of tubuloside A against \u003cem\u003eS. suis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn our study, the 1/2 MIC (32ug/mL) of tubuloside A did not inhibit the growth of \u003cem\u003eS. suis\u003c/em\u003e. The 1/2 MIC, 1/4 MIC, and 1/8 MIC of tubuloside A significantly inhibited \u003cem\u003eS. suis\u003c/em\u003e biofilm in the crystal violet staining experiments. Meanwhile, treatment with 1/2 MIC of tubuloside A was also found to significantly alter the morphology and structure of \u003cem\u003eS. suis\u003c/em\u003e biofilms. These results show that 1/2 MIC of tubuloside A significantly inhibited \u003cem\u003eS. suis\u003c/em\u003e biofilm formation. Studies have shown that as the concentration of tubuloside A increased, the inhibition of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e USA300 adhesion to fibrinogen became more significant[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the murine pneumonia infection model, tubuloside A effectively reduced the bacterial load, demonstrating a significant capacity to mitigate MRSA pathogenicity[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The study have shown that tubuloside A inhibits the formation of MRSA biofilm, based on our experimental findings, tubuloside A may be a potential candidate for interfering with bacterial biofilm formation.\u003c/p\u003e \u003cp\u003eTo further explore the mechanism by which tubuloside A interferes with biofilm formation in \u003cem\u003eS. suis\u003c/em\u003e ATCC 700794, we used untargeted metabolomics to investigate metabolic alterations in this strain following treatment with 1/2 MIC of TnA. The result that 65 metabolites showed differences in their levels, with 42 metabolites increasing and 23 metabolites decreasing (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These metabolites are mainly enriched in nitrogen metabolism and carbon metabolism pathways, which included glycine, serine and threonine metabolism; cysteine and methionine metabolism; alanine, aspartate and glutamate metabolism; arginine and proline metabolism; pyruvate metabolism; citrate cycle (TCA cycle). Most of nitrogen metabolism are amino acid metabolic pathways. It is revealed that these pathways may be associated with bacterial biofilm formation.\u003c/p\u003e \u003cp\u003eStudies have shown that L-serine can regulate the biosynthesis of glycine and cysteine. L-serine had a significantly decreased ability to form biofilms in the macrolide-resistant \u003cem\u003eS. suis\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our study, serine expression decreased significantly. These results indicate that tubuloside A may influence the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e through the regulation of glycine, serine, and threonine metabolism by L-serine.\u003c/p\u003e \u003cp\u003eCysteine has been shown to be involved in the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Under the action of cystathionine γ-synthase, cystathionine is formed from cysteine, which is then converted to homocysteine by cystathionine β-lyase and enters the methionine cycle. The content of cysteine can affect homocysteine in the methionine pathway, thereby further influencing biofilm formation[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our study, the L-cystathionine and S-adenosyl-L-methionine significantly decreased. These results indicate that tubuloside A may influence the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e through the regulation of cysteine and methionine metabolism.\u003c/p\u003e \u003cp\u003eAlanine, aspartate and glutamate metabolism has been demonstrated to be involved in biofilm formation. Aspartate produced by this pathway can be converted to arginine via the urea cycle. Studies have shown that L-arginine can been regulated the formation of biofilms in \u003cem\u003ePseudomonas\u003c/em\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, exogenous L-arginine of argGH can also led to decreased biofilm formation in parental strains[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In our study, aspartate and citrulline expression decreased significantly. These results indicate that tubuloside A may influence the biofilm formation of S. suis through the regulation of alanine, aspartate and glutamate metabolism or arginine and proline metabolism.\u003c/p\u003e \u003cp\u003ePyruvate cycle and TCA cycle are related to carbon metabolism. Our results suggest that carbon metabolism is an important regulatory factor in tubuloside A intervention of \u003cem\u003eS. suis\u003c/em\u003e biofilm formation. In \u003cem\u003eStreptococcus mutans\u003c/em\u003e, the pyruvate metabolism directs carbon flow into various pathways that contribute to its biofilm formation. In our study, phosphoenolpyruvate and pyruvate in pyruvate metabolism were significantly down-regulated. These results indicate that tubuloside A may influence the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e through the regulation of TCA cycle.\u003c/p\u003e \u003cp\u003eTCA cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG in methicillin-sensitive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MSSA)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Citrate as a carbon source enhanced biofilm formation in \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In our study, citrate expression decreased significantly, the expression level of succinate was up-regulated. These results indicate that tubuloside A may influence the biofilm formation of \u003cem\u003eS. suis\u003c/em\u003e through the regulation of TCA cycle.\u003c/p\u003e \u003cp\u003eThe tubuloside A exerts antibacterial effects mainly by regulating nitrogen metabolism, carbon metabolism, and other metabolic pathways. It may be an effective candidate for the development of novel strategies to treat \u003cem\u003eS. suis\u003c/em\u003e infections in the future.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe present study shows that tubuloside A has good antibacterial activity against \u003cem\u003eS. suis\u003c/em\u003e ATCC700794. Tubuloside A can be used as a foundation for further research on the inhibitory mechanisms of \u003cem\u003eS. suis\u003c/em\u003e biofilm formation and drug development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eS. suis Streptococcus suis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMIC Minimum inhibitory concentration\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS-DB designed the whole experiment. R-XC directed the completion of the experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS-YYwere supportive during the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors appreciate applied ppotein technology for their support during the metabolites database searching and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Heilongjiang Postdoctoral Financial Assistance (LBH-Z21192); Natural Science Foundation of Heilongjiang Province of China (JJ2023LH1529); Heilongjiang Bayi Agricultural University Talent Research Start-up Fund (XYB202018).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWang H, Fan Q, Wang Y, Yi L, Wang Y. 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World J Microbiol Biotechnol. 2025;41(2):29; doi: 10.1007/s11274-024-04185-7.\u003c/li\u003e\n \u003cli\u003eZhou YH, Yu F, Chen M, Zhang YF, Qu QW, Wei YR, et al. Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM. Frontiers in Veterinary Science. 2022;8; doi: 10.3389/fvets.2021.829899.\u003c/li\u003e\n \u003cli\u003eDong CL, Wu T, Dong Y, Qu QW, Chen XY, Li YH. Exogenous methionine contributes to reversing the resistance of Streptococcus suis to macrolides. Microbiology Spectrum. 2024;12(2); doi: 10.1128/spectrum.02803-23.\u003c/li\u003e\n \u003cli\u003eRossi CS, Barrientos-Moreno L, Paone A, Cutruzzol\u0026agrave; F, Paiardini A, Espinosa-Urgel M, et al. Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas. International Journal of Molecular Sciences. 2022;23(8); doi: 10.3390/ijms23084386.\u003c/li\u003e\n \u003cli\u003eManna AC, Leo S, Girel S, Gonz\u0026aacute;lez-Ruiz V, Rudaz S, Francois P, et al. Teg58, a small regulatory RNA, is involved in regulating arginine biosynthesis and biofilm formation in Staphylococcus aureus. Scientific Reports. 2022;12(1); doi: 10.1038/s41598-022-18815-3.\u003c/li\u003e\n \u003cli\u003eDe Backer S, Sabirova J, De Pauw I, De Greve H, Hernalsteens JP, Goossens H, et al. Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300. Microorganisms. 2018;6(4); doi: 10.3390/microorganisms6040113.\u003c/li\u003e\n \u003cli\u003eAswathanarayan JB, Vittal RR. Attachment and biofilm formation of Pseudomonas fluorescens PSD4 isolated from a dairy processing line. Food Science and Biotechnology. 2014;23(6):1903-10; doi: 10.1007/s10068-014-0260-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tubuloside A, Streptococcosis suis, biofilm, metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-9345583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9345583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStreptococcosis suis\u003c/em\u003e (\u003cem\u003eS. suis\u003c/em\u003e) is an important zoonotic pathogen. After forming biofilms, it can cause persistent and chronic infections in the host, which increase the risk of zoonotic infections, and pose a threat to public health. Clinically, it is mainly treated with antibacterial drugs. However, the drug resistance of \u003cem\u003eS. suis\u003c/em\u003e that forms biofilms is enhanced, and conventional drugs cannot eradicate it. At present, screening traditional Chinese medicine monomer drugs to interfere with the formation of biofilms has become one of the common methods for treating \u003cem\u003eS. suis\u003c/em\u003e. In this study, the mechanism of tubuloside A intervention on \u003cem\u003eS. suis\u003c/em\u003e ATCC700794 biofilm formation was explored by metabolomics. The minimum inhibitory concentration (MIC) of tubuloside A against the ATCC700794 was determined by two-fold serial dilution of the microbroth. The effects of tubuloside A were studied using crystal violet staining. The morphology of tubuloside A treated ATCC700794 cells was observed by scanning electron microscopy. Differentially expressed metabolites were screened using metabolomics and biological information analyses. The MIC of tubuloside A was 64ug/mL, whereas 1/2 MIC (32ug/mL) of tubuloside A significantly inhibited biofilm formation without affecting the bacterial growth and prevented the formation of biofilm structure. Using 1/2 MIC of tubuloside A, 65 metabolites were identified, of which 42 were upregulated and 23 were downregulated. Bioinformatic analysis showed that the changes in the ATCC700794 strains after tubuloside A intervention primarily involved glycine, serine and threonine metabolism, purine metabolism, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, citrate cycle (TCA), arginine and proline metabolism, pyruvate metabolism. This study elucidated the mechanism which tubuloside A inhibited from a metabolomics perspective, providing novel insights for biofilm control strategies.\u003c/p\u003e","manuscriptTitle":"Metabolomics study of the inhibitory effects of tubuloside A on Streptococcosis suis biofilm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 09:32:52","doi":"10.21203/rs.3.rs-9345583/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-16T06:27:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180877284689302079911735475222430050557","date":"2026-05-08T11:13:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308316080587409257831372014805784629273","date":"2026-05-08T10:53:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T14:05:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127011640391195561527502171271482667720","date":"2026-05-07T10:47:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T14:53:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T12:54:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-09T00:04:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2026-04-07T13:05:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e8a7fea7-3a3c-4506-9477-53b72d8ea084","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-16T06:27:02+00:00","index":36,"fulltext":""},{"type":"reviewerAgreed","content":"180877284689302079911735475222430050557","date":"2026-05-08T11:13:05+00:00","index":32,"fulltext":""},{"type":"reviewerAgreed","content":"308316080587409257831372014805784629273","date":"2026-05-08T10:53:16+00:00","index":31,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T14:05:58+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"127011640391195561527502171271482667720","date":"2026-05-07T10:47:52+00:00","index":19,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T09:32:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 09:32:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9345583","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9345583","identity":"rs-9345583","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}