Antibacterial activity and mechanism analysis of deoxycholic acid against Clostridium perfringens

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However, its precise mechanistic action remains unclear. Objective In this study, we examined the inhibitory mechanism of DCA against C. perfringens through in vitro growth inhibition assays coupled with transcriptomic analysis. Result The study results indicate that DCA can effectively inhibit the formation of C. perfringens biofilms, disrupt their cell walls, increase cell membrane permeability, and cause nucleic acid leakage. Transcriptome analysis revealed that DCA can mediate its oxidative damage by up-regulating the oxidative phosphorylation pathway in C. perfringens and down-regulating antioxidant activity, peroxidase activity, and oxidoreductase activity. This study provides insights into the antimicrobial activity of DCA and its mechanisms, laying a theoretical foundation for its development as a novel antimicrobial agent or feed additive. Clostridium perfringens Deoxycholic acid Antimicrobial activity Transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction C. perfringens , a gram-positive anaerobic bacterium, is widely found in the intestinal microbiota of humans and animals, in water bodies, and in soil. It significantly impairs animal health and production performance, resulting in substantial annual economic losses to the global livestock industry[1–3]. Furthermore, C. perfringens is an important zoonotic pathogen. It not only endangers the health of animals but can also be transmitted to humans through contaminated food, leading to public health issues such as food poisoning. The Centers for Disease Control and Prevention, USA, reported that approximately 48 million Americans fall ill each year due to food poisoning caused by C. botulinum infection. In some developing and relatively poor countries, the public health risk posed by C. perfringens is even more serious[4, 5]. Treatment for C. perfringens infections typically involves antibiotics; however, C. perfringens has developed resistance to multiple antimicrobial agents, including chloramphenicol, lincosamides, tetracyclines, aminoglycosides, and macrolides[6–8]. Notably, β-lactams and oxazolamides are the drugs of choice for treating C. perfringens , but strains resistant to these drugs have now emerged. [8, 9]. Plasmids have mediated the evolution and rapid adaptation of these drug-resistant strains, posing a major threat to veterinary health, food safety, and public health[10].Consequently, there is an urgent need to develop novel antimicrobial agents or antibacterial strategies to escalating issue of antimicrobial resistance in C. perfringens . In recent years, the significant role of gut microbiota-derived metabolites in enhancing host health and regulating immune function has been widely confirmed. These metabolites, known as postbiotics, include exopolysaccharides (EPS), short chain fatty acids (SCFAs), bile acids (BAs), and other[11]. DCA is a secondary bile acid derived from the conversion of hepatocyte-synthesized primary bile acid by intestinal flora upon entering the intestine[12, 13]. While previous research on DCA primarily centered on its involvement in liver and gastrointestinal diseases[14, 15], recent studies have revealed its broad-spectrum antibacterial activity. DCA exhibits antimicrobial activity against pathogens such as Klebsiella pneumoniae, Streptococcus pneumoniae , Staphylococcus aureus , and C. perfringens . It can regulate the intestinal microbiota and alleviate the inflammatory response triggered by these pathogens, thereby making it a potential antimicrobial drug[16–19]. Studies have shown that deoxycholic acid has a strong specific antibacterial effect on C.perfringens , and can alleviate necrotic enteritis in chickens caused by C. perfringens through multiple pathways, including attenuating inflammatory cyclooxygenase signaling and enhancing host defense peptide synthesis[19, 20]. However, its antibacterial mechanism of DCA remains incompletely understood. Considering the broad-spectrum antimicrobial properties of DCA, this study aims to systematically evaluate its in vitro antibacterial activity against C. perfringens and investigate the underlying inhibitory mechanisms. The research results are expected to provide theoretical reference for DCA as a new antibacterial agent against C. perfringens or as a feed additive in animal husbandry. Materials and methods Bacterial and antimicrobial agents C. perfringens (CVCC2015) type A as an experimental strain. Bacteria were cultured using Tryptic Sulfite Cycloserine (TSC) agar and Fluid Thioglycollate Medium (FTG) under anaerobic conditions. DCA was purchased from Beijing Solarbio Technology Co., Ltd, purity > 99%. Minimum inhibitory concentration (MIC) and Minimum bacterial concentration (MBC) of DCA against C. perfringens The minimum inhibitory concentration (MIC) of DCA against C. perfringens was determined using the broth microdilution method in a 96-well plate. The MIC value was defined as the lowest concentration required to inhibit bacterial growth in the 96-well plate. At the MIC concentration, bacterial growth is inhibited but the bacteria are not killed in the culture medium. Subsequently, we spread the culture medium from the 96-well plate at concentrations above the MIC onto tryptic sulfate cycloserine (TSC) agar medium, and the concentration at which no bacterial growth was observed on the agar was defined as the MBC [21]. Effect of DCA on the growth curve of C. perfringens C. perfringens was inoculated in FTG broth supplemented with DCA at concentrations of 1/4, 1/2 and 1 MIC, and the same volume of PBS was added to the control group. Bacteria were collected every hour to measure OD600 nm . Each concentration treatment group was set up in three times. Assessment of cell wall integrity and cell membrane permeability Currently, alkaline phosphatase (AKP) and β-galactosidase (β-gal) have become important indicators for determining the integrity of bacterial cell walls and cell membranes[22, 23]. AKP is located between the bacterial cell wall and the cell membrane. Upon disruption of the cell wall, AKP is released into the extracellular environment[5]. Similarly, alterations in cell membrane permeability can be assessed by measuring the intracellular release of β-gal in C. perfringens . After 6 hours of cultivation, the bacterial concentration was adjusted to an optical density (OD) of 0.5 at 600 nm. Subsequently, C. perfringens was exposed to different concentrations of DCA, with PBS serving as the negative control. After treatment, the supernatant was collected by centrifugation, and the levels of AKP and β-gal were measured to assess the extent of damage to the bacterial cell wall and membrane. At the same time, the concentration of dsDNA in the supernatant was measured using an ultra-micro ultraviolet spectrophotometer to observe the level of intracellular nucleic acid leakage. Effect of DCA on the biofilm of C. perfringens The impact of DCA on the biofilm formation of C. perfringens was assessed utilizing the crystal violet assay[24]. FTG broth, supplemented with varying concentrations of DCA, was aliquoted into 96-well microtiter plates, with each concentration being triplicated across three rows. Subsequently, C. perfringens was then inoculated into the wells and incubated overnight to facilitate biofilm formation. Following incubation, the bacterial suspension in the wells was removed, and the plates were gently washed twice with PBS to remove non-adherent bacteria and residual culture medium. After drying, 100 µL of anhydrous methanol was added to each well to fix the biofilm for 15 minutes. Subsequently, 200 µL of 1% crystal violet solution was applied to stain the wells for 30 minutes. Then wash the plate with PBS and dissolve the biofilm with 33% glacial acetic acid for 10 minutes. Finally, the absorbance at OD570 nm was measured. The absorbance value was directly proportional to the biofilm mass, thereby reflecting the effect of DCA on the formation of C. perfringens biofilm. Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) inspection The concentration of C. perfringens cultured for 6 hours was adjusted to OD600 nm of 0.5. The bacterial precipitate was subsequently inoculated with DCA solution at concentrations of 0, 1 MIC and 8 MIC, and co-cultured for 2 hours at 37°C. Following incubation, the bacteria were collected by centrifugation, gently rinsed with PBS, and the PBS was discarded. The 2.5% glutaraldehyde electron microscopy fixative was added, and the bacteria were resuspended in the fixative for two hours at room temperature. The samples were processed according to a previously published protocol and subsequently examined using TEM and SEM[25, 26]. Total RNA extraction and RNA sequencing Total RNA extraction and RNA sequencing Four independent samples of the DCA-treated group (1 MIC) and the control group of C. perfringens were cultured for 6 h at 37°C. Total RNA was extracted from bacterial precipitates, and the extracted RNA was quality assessed to ensure its integrity, concentration, and purity. Libraries were constructed using the TruSeq™ Stranded Total RNA Library Prep Kit with rRNA depletion. NovaSeqXPlus sequencing platform (ShangHai majorbio Bio-pharm technology Co.,ltd) was used for mRNA sequencing. Analysis of differentially expressed genes The EBSeq algorithm was used to screen the differentially expressed genes, and the criteria for screening were Fold Change (FC) greater than 1.5 and FDR < 0.05. The differentially expressed genes were annotated in the GO (Gene Ontology) database and KEGG (Kyoto Encyclopedia of Genes and Genomes) database, and the enrichment analysis was carried out; the significance levels of the GO and KEGG Pathways enriched with differentially expressed genes were analysed by the Fisher exact test based on the hypergeometric distribution. KEGG Pathway significance levels, and screened out the GO items and metabolic pathways that were significantly enriched for differentially expressed genes. qPCR for Detection of Gene Expression For validation of transcriptional profiling from RNA-Seq, quantitative real-time PCR (qRT-PCR) was performed. Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan). qPCR was performed with ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme, Nanjing, China). Primers for qPCR were obtained by querying the gene database at NCBI and were synthesized by Bioengineering Biotechnology Co. (Bioengineering Biotechnology, Shanghai, China). The relative expression level of genes was represented by 2^(-∆∆Ct), and 16sRNA was used as a reference gene[22]. The primer details are shown in supplementary table S1 . Statistical analysis All the experimental data were replicated three times or more, statistically analyzed by SPSS Statistics 26.0, and plotted by GraphPad Prism 9.0. The results of the experiments were expressed as the ‘mean ± standard deviation’, and the t-test was used to compare the differences between the groups, with * representing a significant difference ( p < 0.05) and ** representing a highly significant difference ( p < 0.01). Result Effect of DCA on the growth curve and biofilm of C. perfringens The results showed that DCA had a strong inhibitory effect on C. perfringens with a MIC value of 0.125 mg/mL and MBC value of 1 mg/mL (8 MIC). As is shown in Fig. 1 A, when the DCA concentration is 1/4 MIC and 1/2 MIC, it can significantly inhibit the growth of C. perfringens . When the concentration is 1 MIC, DCA can completely inhibit the growth of C. perfringens . As is shown in Fig. 1 B, the results of the crystal violet assay showed that DCA at concentrations ranging from 1/4 MIC to 1 MIC significantly inhibited the formation of C. perfringens biofilm in a concentration-dependent manner compared with the control group. Effect of DCA on the membrane integrity of C. perfringens cell walls From the detection of AKP and β-gal content (Fig. 2 A, Fig. 2 C), DCA can destroy the integrity of cell walls and the permeability of cell membranes, and this destructive ability is proportional to the concentration of DCA. As shown in Fig. 2 B, the concentration of extracellular dsDNA significantly increased after DCA treatment, indicating that DCA can alter cell membrane permeability, leading to nucleic acid leakage, which may have a negative impact on maintaining normal bacterial physiological functions. Effect of DCA on C. perfringens morphology TEM and SEM further showed the disruptive effect of DCA on the structure of C. perfringens. As can be seen in Fig. 3 and Fig. 4 , the untreated cells (control) showed a typical C. perfringens ultrastructure, with the cell membrane, cell wall and no cellular damage was observed. After treatment with MIC concentration of DCA for 2 hours, some of the cell edges were blurred and the surface of the cell wall appeared wrinkled. Under the treatment with MBC concentration of DCA, the cell wall membrane was completely blurred and the bacterial surface showed a large number of breaks and was no longer intact. Scanning electron microscopy and projection results showed that low concentrations of DCA had a certain destructive effect on the cell membrane and cell wall of C. perfringens , while at the concentration of MBC, DCA could directly destroy the cell structure of C. perfringens leading to the death of the bacteria. Analysis of differentially expressed genes (DEGs) Transcriptomics analysis was used to understand the gene expression changes between DCA-treated and untreated groups. A total of 659 DEGs were identified compared to the control group of these, 355 were up-regulated and 304 were down-regulated (Fig. 5 A). Details of the DEGs are shown in supplementary table S2 . To further explore the effect of DCA on C. perfringens , the differentially expressed genes were subjected to GO functional gene enrichment analysis. As seen in Fig. 6 A, GO enrichment results showed that 202 GO terms were up-regulation, mainly involving protein-containing complex, ribonucleoprotein complex, structural constituent of ribosome and structural molecule activity. The down-regulated GO terms are enriched in molecular function, specifically enriched in four GO terms: antioxidant activity, peroxidase activity, oxidoreductase activity and protein serine/threonine kinase activity(Fig. 6 B). While Details of GO enrichment of DEGs are shown in supplementary table S3 . The differentially expressed genes of C. perfringens after DCA were subjected to KEGG enrichment analysis, and as shown in Fig. 6 C, after DCA treatment, up-regulated genes were enriched in four pathways: Ribosome, oxidative phosphorylation, pyruvate metabolism and taurine and hypotaurine metabolism. Down-regulated genes were enriched in pathways that did not show significant differences (Fig. 6 D). See supplementary table S4 for details. Discussion The spores of C. perfringens exhibit strong environmental resistance, capable of withstanding extreme conditions such as high temperatures and dryness, and demonstrate significant resistance to conventional chemical disinfectants[27]. Additionally, with the widespread use of antibiotics, this strain has gradually evolved to develop multidrug resistance. To reduce antibiotic use and mitigate the threat this bacterium poses to public health, the development of novel antimicrobial agents to address C. perfringens infections has become an urgent research priority. Currently, there is very little research on non-antibiotic antimicrobial drugs for C. perfringens . Zheng et al. studied the antibacterial activity of natural propolis and identified caffeic acid as its primary component against C. perfringens , concluding that propolis could serve as an alternative to antibiotics[5]. DCA was proposed in 2019 as a potential treatment for necrotic enteritis (NE) caused by C. perfringens in chickens[20]. Subsequently, Kim et al. conducted further research and found that DCA can induce the production of host defense peptides to alleviate NE, further revealing the potential of DCA as an antibiotic alternative[19]. However, these two studies focused on the effects of DCA on the host, and there have been no studies on how DCA acts on C. perfringens . Therefore, the aim of our study was to reveal the antibacterial activity and mechanism of DCA against C. perfringens . By detecting AKP and β-gal activity, we can observe that DCA can disrupt the cell wall and cell membrane structure of C. perfringens . To validate these results, we directly observed the cellular structure using SEM and TEM. The control group of C. perfringens exhibited a thick, mature, and dense three-dimensional structure. At a DCA concentration of 1 MIC, the cell wall already showed significant wrinkling and deformation, and at an 8 MIC concentration, the cells began to rupture, with intracellular material leaking out, indicating that DCA can effectively disrupt the cellular structure of C. perfringens . The virulence and drug resistance of C.perfringens is closely related to the formation of biofilms, which can prevent or delay the penetration of antibiotics and allow them to escape the killing effect of antimicrobial drugs[28]. As can be seen from Fig. 1 D, DCA can significantly inhibit the formation of C. perfringens biofilms , and transcriptomic results indicated that DCA treatment significantly down-regulated the expression of the spoVG gene in C. perfringens (log 2 FC=-3.00). The spovG gene is widely present in bacteria and has been confirmed to be a key regulatory factor in biofilm formation in bacteria such as Bacillus subtilis , Listeria monocytogenes , Staphylococcus aureus , Bacillus cereus , and Staphylococcus epidermidis [28–30]. However, whether the spoVG gene can regulate the formation of C. perfringens biofilms has not yet been confirmed and requires further research. Among the DEGs, def gene was significantly down-regulated(log2FC=-3.52), the down-regulation of this gene was also verified by qPCR(RS09240). The def gene encodes peptide deformylase (PDF), an enzyme whose core function is to catalyze the removal of the formyl group (-CHO) from the N-terminal formylmethionine of newly synthesized polypeptide chains, thereby forming methionine. This is an important step in post-translational modification of bacterial proteins. If PDF is inhibited, it leads to the accumulation of un-deformylated proteins, resulting in bacterial growth arrest or death[31–33]. This enzyme, which is widely present in bacteria, has become an important target for the development of new antimicrobial drugs[34, 35]. This indicates that DCA exerts its antibacterial effect against C. perfringens by suppressing bacterial protein synthesis through down-regulation of the def gene. Transcriptomics is important for understanding the antibacterial mechanism of DCA. KEGG enrichment results showed that the oxidative phosphorylation pathway was significantly enriched. Oxidative phosphorylation is the main pathway for ATP production in bacteria, and up-regulation of this pathway promotes the activity of the bacterial electron transport chain; however, the high activity of the ETC may lead to electron leakage, which reacts with oxygen to generate large amounts of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide[36]. GO enrichment analysis showed that antioxidant activity, peroxidase activity and oxidoreductase activity were down-regulated in C. perfringens . These enzymes are normally responsible for scavenging ROS from the cell, and if the activity of these enzymes is reduced, the ability of the bacteria to process ROS is diminished, leading to ROS accumulation[37, 38]. This indicates that DCA can induce ROS by upregulating the oxidative phosphorylation pathway. At the same time, the bacterial antioxidant system is inhibited and unable to effectively clear these ROS, resulting in an increase in intracellular ROS levels and triggering oxidative stress. Excessive ROS can damage bacterial DNA, proteins, and lipids, leading to the destruction of cellular structures and ultimately causing cell death[39, 40]. Conclusions In conclusion, DCA exhibits potent antibacterial activity against C. perfringens , effectively inhibiting biofilm formation, disrupting bacterial cell wall integrity, and increasing membrane permeability. Transcriptome results indicate that DCA mediates oxidative damage by upregulating the oxidative phosphorylation pathway in C. perfringens and downregulating antioxidant activity, peroxidase activity, and oxidoreductase activity. Additionally, DCA suppresses the expression of the def gene, thereby impairing bacterial protein synthesis. This multi-targeted mechanism underscores DCA’s broad-spectrum antibacterial efficacy, making it a promising candidate for combating C. perfringens infections. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by the Financial Program of BJAST (25CA005) Author Contribution X.-L.S. conducted the experiment and wrote the main manuscript. Z.-Y.Z., J.-D.B., and Q.-Y.G. carried out the analysis and reviewed the final manuscript, M.-H.W. contributed to reviewing the final manuscript, C.-S.Y. conducted the laboratory analysis. Q.-X.Z. designed the experiment and revised the manuscript. Acknowledgement We sincerely thank the Beijing Milu Ecological research Center, China Agricultural University, and Anhui Agricultural University for providing the experimental conditions. We would like to express our gratitude to all personnel involved in this experiment. All authors participated in the critical review of the manuscript; gave final approval for the version to be published; agreed to submit the manuscript to the journal; and agreed to take responsibility for all aspects of the work. Data Availability The datasets generated and analyzed during the present study can be obtained from the first author upon reasonable request. The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1314926.（https://dataview.ncbi.nlm.nih.gov/?search=SUB15589106&archive=bioproject）. Other data are provided in the supplementary information file. References Uzal FA, Navarro MA, Asin J, Boix O, Ballarà-Rodriguez I, Gibert X. Clostridial diarrheas in piglets: A review. Vet Microbiol. 2023;280:109691. https://doi.org/10.1016/j.vetmic.2023.109691. Alizadeh M, Shojadoost B, Boodhoo N, Astill J, Taha-Abdelaziz K, Hodgins DC, et al. Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination. Anim Health Res Rev. 2021;22:147–62. https://doi.org/10.1017/S146625232100013X. Mwangi S, Timmons J, Fitz-coy S, Parveen S. Characterization of Clostridium perfringens recovered from broiler chicken affected by necrotic enteritis. Poult Sci. 2019;98:128–35. https://doi.org/10.3382/ps/pey332. Johnson S, Skinner AM, Lostutter C, Duke T, Posthaus H. Enteritis necroticans and Clostridium perfringens type C; Epidemiological and pathological findings over the past 20 years. PLoS Negl Trop Dis. 2025;19:e0012836. https://doi.org/10.1371/journal.pntd.0012836. Zheng X, Al Naggar Y, Wu Y, Liu D, Hu Y, Wang K, et al. Untargeted metabolomics description of propolis’s in vitro antibacterial mechanisms against Clostridium perfringens . Food Chem. 2023;406:135061. https://doi.org/10.1016/j.foodchem.2022.135061. Adams V, Han X, Lyras D, Rood JI. Antibiotic resistance plasmids and mobile genetic elements of Clostridium perfringens . Plasmid. 2018;99:32–9. https://doi.org/10.1016/j.plasmid.2018.07.002. Fayez M, Elsohaby I, Al-Marri T, Zidan K, Aldoweriej A, El-Sergany E, et al. Genotyping and antimicrobial susceptibility of Clostridium perfringens isolated from dromedary camels, pastures and herders. Comp Immunol Microbiol Infect Dis. 2020;70. https://doi.org/10.1016/j.cimid.2020.101460. Wu K, Li Z, Fang M, Yuan Y, Fox EM, Liu Y, et al. Genome characteristics of the optrA-positive Clostridium perfringens strain QHY-2 carrying a novel plasmid type. mSystems. 2023. https://doi.org/10.1128/msystems.00535-23. Gulliver EL, Adams V, Marcelino VR, Gould J, Rutten EL, Powell DR, et al. Extensive genome analysis identifies novel plasmid families in Clostridium perfringens . Microb Genom. 2023;9. https://doi.org/10.1099/mgen.0.000995. Feng Y, Fan X, Zhu L, Yang X, Liu Y, Gao S, et al. Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of Clostridium perfringens . Microb Genom. 2020;6:1–15. https://doi.org/10.1099/mgen.0.000441. Liu C, Ma N, Feng Y, Zhou M, Li H, Zhang X, et al. From probiotics to postbiotics: Concepts and applications. Animal Research and One Health. 2023;1:92–114. https://doi.org/10.1002/aro2.7. Jin D, Huang K, Xu M, Hua H, Ye F, Yan J, et al. Deoxycholic acid induces gastric intestinal metaplasia by activating STAT3 signaling and disturbing gastric bile acids metabolism and microbiota. Gut Microbes. 2022;14. https://doi.org/10.1080/19490976.2022.2120744. Wahlström A, Brumbaugh A, Sjöland W, Olsson L, Wu H, Henricsson M, et al. Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism. Nat Commun. 2024;15:4276. https://doi.org/10.1038/s41467-024-48543-3. Sato Y, Kobayashi M, Ohira M, Funayama R, Maekawa M, Karasawa H, et al. Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells. Cancer Sci. 2024;115:1778–90. https://doi.org/10.1111/cas.16132. Tanaka M, Nakura H, Tateishi T, Watanabe M, Nakaya S, Kumai T, et al. Ursodeoxycholic acid prevents hepatic cytochrome P450 isozyme reduction in rats with deoxycholic acid-induced liver injury. J Hepatol. 1999;31:263–70. https://doi.org/10.1016/S0168-8278(99)80223-2. Zheng Y, Yue C, Zhang H, Chen H, Liu Y, Li J. Deoxycholic Acid and Lithocholic Acid Alleviate Liver Injury and Inflammation in Mice with Klebsiella pneumoniae -Induced Liver Abscess and Bacteremia. J Inflamm Res. 2021;Volume 14:777–89. https://doi.org/10.2147/JIR.S298495. Vidal JE, Wier MN, A. Angulo-Zamudio U, McDevitt E, Jop Vidal AG, Alibayov B, et al. Prophylactic Inhibition of Colonization by Streptococcus pneumoniae with the Secondary Bile Acid Metabolite Deoxycholic Acid. Infect Immun. 2021;89. https://doi.org/10.1128/IAI.00463-21. Zhao W, Wang J, Li X, Li Y, Ye C. Deoxycholic acid inhibits Staphylococcus aureus -induced endometritis through regulating TGR5/PKA/NF-κB signaling pathway. Int Immunopharmacol. 2023;118:110004. https://doi.org/10.1016/j.intimp.2023.110004. Kim DM, Liu J, Whitmore MA, Tobin I, Zhao Z, Zhang G. Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis. J Anim Sci Biotechnol. 2024;15:29. https://doi.org/10.1186/s40104-024-00995-9. Wang H, Latorre JD, Bansal M, Abraha M, Al-Rubaye B, Tellez-Isaias G, et al. Microbial metabolite deoxycholic acid controls Clostridium perfringens -induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling. Sci Rep. 2019;9:14541. https://doi.org/10.1038/s41598-019-51104-0. Li J, Li C, Shi C, Aliakbarlu J, Cui H, Lin L. Antibacterial mechanisms of clove essential oil against Staphylococcus aureus and its application in pork. Int J Food Microbiol. 2022;380:109864. https://doi.org/10.1016/j.ijfoodmicro.2022.109864. Xin J, Pu Q, Wang R, Gu Y, He L, Du X, et al. Antibacterial activity and mechanism of chelerythrine against Streptococcus agalactiae . Front Vet Sci. 2024;11. https://doi.org/10.3389/fvets.2024.1408376. Yu Y, Dong J, Wang Y, Gong X. RNA-seq analysis of antibacterial mechanism of Cinnamomum camphora essential oil against Escherichia coli . PeerJ. 2021;9:e11081. https://doi.org/10.7717/peerj.11081. Bai B, Wang M, Zhang Z, Guo Q, Yao J. Mechanistic Investigation of the Pyrolysis Temperature of Reed Wood Vinegar for Maximising the Antibacterial Activity of Escherichia coli and Its Inhibitory Activity. Biology (Basel). 2024;13:912. https://doi.org/10.3390/biology13110912. Liu X, Shu Q, Chen Q, Pang X, Wu Y, Zhou W, et al. Antibacterial Efficacy and Mechanism of Mannosylerythritol Lipids-A on Listeria monocytogenes . Molecules. 2020;25:4857. https://doi.org/10.3390/molecules25204857. Rao W, Fang Z, Chen Z, Wu J, Fang X. Antibacterial mechanism of metabolites of Leuconostoc mesenteroides against Serratia liquefaciens . LWT. 2023;187:115335. https://doi.org/10.1016/j.lwt.2023.115335. Lindström M, Heikinheimo A, Lahti P, Korkeala H. Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiol. 2011;28:192–8. https://doi.org/10.1016/j.fm.2010.03.020. Hu WS, Woo DU, Kang YJ, Koo OK. Biofilm and Spore Formation of Clostridium perfringens and Its Resistance to Disinfectant and Oxidative Stress. Antibiotics. 2021;10:396. https://doi.org/10.3390/antibiotics10040396. Shi C, Zheng L, Lu Z, Zhang X, Bie X. The global regulator SpoVG regulates Listeria monocytogenes biofilm formation. Microb Pathog. 2023;180:106144. https://doi.org/10.1016/j.micpath.2023.106144. Huang Q, Zhang Z, Liu Q, Liu F, Liu Y, Zhang J, et al. SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus . BMC Microbiol. 2021;21:172. https://doi.org/10.1186/s12866-021-02239-6. Sangshetti J, Khan FA, Shinde D. Peptide Deformylase: A New Target in Antibacterial, Antimalarial and Anticancer Drug Discovery. Curr Med Chem. 2014;22:214–36. https://doi.org/10.2174/0929867321666140826115734. Merzoug A, Chikhi A, Bensegueni A, Boucherit H, Okay S. Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics. Mol Inform. 2018;37. https://doi.org/10.1002/minf.201700087. Gokhale KM, Telvekar VN. Novel peptidomimetic peptide deformylase (PDF) inhibitors of Mycobacterium tuberculosis . Chem Biol Drug Des. 2021;97:148–56. https://doi.org/10.1111/cbdd.13769. Singhal A, Fang FC. Direct and Indirect Inhibition of Salmonella Peptide Deformylase by Nitric Oxide. mBio. 2020;11. https://doi.org/10.1128/mBio.01383-20. Merzoug A, Chikhi A, Bensegueni A, Boucherit H, Okay S. Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics. Mol Inform. 2018;37. https://doi.org/10.1002/minf.201700087. Imlay JA. Pathways of Oxidative Damage. Annu Rev Microbiol. 2003;57:395–418. https://doi.org/10.1146/annurev.micro.57.030502.090938. Imlay JA. Cellular Defenses against Superoxide and Hydrogen Peroxide. Annu Rev Biochem. 2008;77:755–76. https://doi.org/10.1146/annurev.biochem.77.061606.161055. Ezraty B, Gennaris A, Barras F, Collet J-F. Oxidative stress, protein damage and repair in bacteria. Nat Rev Microbiol. 2017;15:385–96. https://doi.org/10.1038/nrmicro.2017.26. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell. 2007;130:797–810. https://doi.org/10.1016/j.cell.2007.06.049. Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N, et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proceedings of the National Academy of Sciences. 2014;111. https://doi.org/10.1073/pnas.1401876111. Additional Declarations No competing interests reported. Supplementary Files supplementarytableS3.csv supplementarytableS4.csv supplementarytableS1.csv supplementarytableS2.csv Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 24 Nov, 2025 Reviews received at journal 21 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviews received at journal 04 Nov, 2025 Reviewers agreed at journal 26 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers invited by journal 23 Oct, 2025 Editor invited by journal 23 Oct, 2025 Editor assigned by journal 04 Sep, 2025 Submission checks completed at journal 03 Sep, 2025 First submitted to journal 03 Sep, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":150375,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibitory effect of DCA on the growth and biofilm formation of \u003cem\u003eC. perfringens\u003c/em\u003e.\u003cstrong\u003e A\u003c/strong\u003e Effect of different concentrations of DCA on the growth curve of \u003cem\u003eC. perfringens.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eB\u003c/strong\u003e Effect of DCA on biofilm formation of \u003cem\u003eC. perfringens\u003c/em\u003e, OD570\u003csub\u003enm\u003c/sub\u003e indicates biofilm content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/c0fd9cfadec8e4f5cd22f9a7.png"},{"id":95215717,"identity":"c0ae3c61-fd61-4ce4-a743-d3749d8e4317","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":263736,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of DCA on the cell membrane permeability and cell wall integrity of \u003cem\u003eC. perfringens\u003c/em\u003e. \u003cstrong\u003eA \u003c/strong\u003eEffect of different concentrations of DCA treatment on cell membrane integrity of \u003cem\u003eC. perfringens\u003c/em\u003e, OD415 nm represents extracellular β-Gal content, and higher values indicate more severe cell membrane disruption. \u003cstrong\u003eB\u003c/strong\u003e Extracellular AKP content in \u003cem\u003eC. perfringens\u003c/em\u003e after treatment with different concentrations of DCA, a value that indirectly reflects cell wall integrity. \u003cstrong\u003eC\u003c/strong\u003eExtracellular DNA concentration of \u003cem\u003eC. perfringens\u003c/em\u003e after treatment with different concentrations of DCA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/a09884f70d4a79033b43e17d.png"},{"id":95215746,"identity":"62da24e5-4b39-4a8d-b457-2c54d0a9917d","added_by":"auto","created_at":"2025-11-05 15:04:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":528869,"visible":true,"origin":"","legend":"\u003cp\u003eTEM micrographs of \u003cem\u003eC. perfringens\u003c/em\u003etreated with DCA.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/38cd0a4d61f5c6338b633015.png"},{"id":95215724,"identity":"88ccb720-baa4-48bf-aabf-c0dfc152d3ad","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":792637,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of \u003cem\u003eC. perfringens\u003c/em\u003etreated with DCA at the MIC/MBC (8MIC) and control cells without treatment.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/2c66ccb105dc11d80541bdc3.png"},{"id":95215722,"identity":"3865e520-03dd-435a-b806-980c6e2a7fdc","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":306434,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the gene expression analysis. \u003cstrong\u003eA\u003c/strong\u003eVolcano mapping of differentially expressed genes (DEGs) based on RNA-seq analysis of \u003cem\u003eC. perfringens\u003c/em\u003e after untreated and DCA treatment. \u003cstrong\u003eB\u003c/strong\u003ePrincipal component analysis (PCA) of \u003cem\u003eC. perfringens\u003c/em\u003e gene expression in the DCA treated and control groups. \u003cstrong\u003eC\u003c/strong\u003e Hierarchical clustering heat map of different gene expressions in different experimental conditions. Red represents high gene expression and blue represents low gene expression. \u003cstrong\u003eD\u003c/strong\u003eComparison of fold changes in DEGs between RNA-seq and RT-PCR methods.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/e613257a068a96263ab6e2ed.png"},{"id":95215720,"identity":"4ba9657b-bf1e-4c42-be72-5798616fce5a","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":435060,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs KEGG and GO enrichment ranking results. \u003cstrong\u003eA\u003c/strong\u003eGO enrichment analysis of up-regulated genes in \u003cem\u003eC. perfringens\u003c/em\u003e after DCA treatment. \u003cstrong\u003eB\u003c/strong\u003e GO enrichment analysis of down-regulated genes in \u003cem\u003eC. perfringens\u003c/em\u003e after DCA treatment. \u003cstrong\u003eC\u003c/strong\u003e KEGG enrichment analysis of up-regulated genes in \u003cem\u003eC. perfringens\u003c/em\u003e after DCA treatment. \u003cstrong\u003eD\u003c/strong\u003e KEGG enrichment analysis of down-regulated genes in \u003cem\u003eC. perfringens\u003c/em\u003e after DCA treatment.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/2f55aca2aaddf3fe7131d13b.png"},{"id":100614889,"identity":"989c87da-35b1-4803-8e54-3c08e0a7b69d","added_by":"auto","created_at":"2026-01-19 17:27:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3377944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/570f4783-0003-4f68-805c-988831a2af6f.pdf"},{"id":95215715,"identity":"c0c46594-b342-4a61-8058-85491b5a9fdc","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"csv","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15120,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytableS3.csv","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/50ed9f0ab4494b4ee40a9acc.csv"},{"id":95215718,"identity":"955189d9-ab1e-4d0f-a048-725d7f8b0f0a","added_by":"auto","created_at":"2025-11-05 15:04:00","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10560,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytableS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/bc36c810728c936f81d77bcd.csv"},{"id":95215743,"identity":"7981540e-7392-4a41-8dfe-ca8d0a2dbfec","added_by":"auto","created_at":"2025-11-05 15:04:03","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24064,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytableS1.csv","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/71faef5adedbf29ffcdeb54c.csv"},{"id":95215745,"identity":"c23e178c-2111-465a-8ff6-c7582fe25fe6","added_by":"auto","created_at":"2025-11-05 15:04:10","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":853719,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytableS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-7454314/v1/c4e5659c1f2c31c87ffc7e20.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antibacterial activity and mechanism analysis of deoxycholic acid against Clostridium perfringens","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eC. perfringens\u003c/em\u003e, a gram-positive anaerobic bacterium, is widely found in the intestinal microbiota of humans and animals, in water bodies, and in soil. It significantly impairs animal health and production performance, resulting in substantial annual economic losses to the global livestock industry[1\u0026ndash;3]. Furthermore, \u003cem\u003eC. perfringens\u003c/em\u003e is an important zoonotic pathogen. It not only endangers the health of animals but can also be transmitted to humans through contaminated food, leading to public health issues such as food poisoning. The Centers for Disease Control and Prevention, USA, reported that approximately 48\u0026nbsp;million Americans fall ill each year due to food poisoning caused by \u003cem\u003eC. botulinum\u003c/em\u003e infection. In some developing and relatively poor countries, the public health risk posed by \u003cem\u003eC. perfringens\u003c/em\u003e is even more serious[4, 5].\u003c/p\u003e\u003cp\u003eTreatment for \u003cem\u003eC. perfringens\u003c/em\u003e infections typically involves antibiotics; however, \u003cem\u003eC. perfringens\u003c/em\u003e has developed resistance to multiple antimicrobial agents, including chloramphenicol, lincosamides, tetracyclines, aminoglycosides, and macrolides[6\u0026ndash;8]. Notably, β-lactams and oxazolamides are the drugs of choice for treating \u003cem\u003eC. perfringens\u003c/em\u003e, but strains resistant to these drugs have now emerged. [8, 9]. Plasmids have mediated the evolution and rapid adaptation of these drug-resistant strains, posing a major threat to veterinary health, food safety, and public health[10].Consequently, there is an urgent need to develop novel antimicrobial agents or antibacterial strategies to escalating issue of antimicrobial resistance in \u003cem\u003eC. perfringens\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn recent years, the significant role of gut microbiota-derived metabolites in enhancing host health and regulating immune function has been widely confirmed. These metabolites, known as postbiotics, include exopolysaccharides (EPS), short chain fatty acids (SCFAs), bile acids (BAs), and other[11]. DCA is a secondary bile acid derived from the conversion of hepatocyte-synthesized primary bile acid by intestinal flora upon entering the intestine[12, 13]. While previous research on DCA primarily centered on its involvement in liver and gastrointestinal diseases[14, 15], recent studies have revealed its broad-spectrum antibacterial activity. DCA exhibits antimicrobial activity against pathogens such as \u003cem\u003eKlebsiella pneumoniae, Streptococcus pneumoniae\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eC. perfringens\u003c/em\u003e. It can regulate the intestinal microbiota and alleviate the inflammatory response triggered by these pathogens, thereby making it a potential antimicrobial drug[16\u0026ndash;19].\u003c/p\u003e\u003cp\u003eStudies have shown that deoxycholic acid has a strong specific antibacterial effect on \u003cem\u003eC.perfringens\u003c/em\u003e, and can alleviate necrotic enteritis in chickens caused by \u003cem\u003eC. perfringens\u003c/em\u003e through multiple pathways, including attenuating inflammatory cyclooxygenase signaling and enhancing host defense peptide synthesis[19, 20]. However, its antibacterial mechanism of DCA remains incompletely understood. Considering the broad-spectrum antimicrobial properties of DCA, this study aims to systematically evaluate its in vitro antibacterial activity against \u003cem\u003eC. perfringens\u003c/em\u003e and investigate the underlying inhibitory mechanisms. The research results are expected to provide theoretical reference for DCA as a new antibacterial agent against \u003cem\u003eC. perfringens\u003c/em\u003e or as a feed additive in animal husbandry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial and antimicrobial agents\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. perfringens\u003c/em\u003e (CVCC2015) type A as an experimental strain. Bacteria were cultured using Tryptic Sulfite Cycloserine (TSC) agar and Fluid Thioglycollate Medium (FTG) under anaerobic conditions. DCA was purchased from Beijing Solarbio Technology Co., Ltd, purity\u0026thinsp;\u0026gt;\u0026thinsp;99%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMinimum inhibitory concentration (MIC) and Minimum bacterial concentration (MBC) of DCA against\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe minimum inhibitory concentration (MIC) of DCA against \u003cem\u003eC. perfringens\u003c/em\u003e was determined using the broth microdilution method in a 96-well plate. The MIC value was defined as the lowest concentration required to inhibit bacterial growth in the 96-well plate. At the MIC concentration, bacterial growth is inhibited but the bacteria are not killed in the culture medium. Subsequently, we spread the culture medium from the 96-well plate at concentrations above the MIC onto tryptic sulfate cycloserine (TSC) agar medium, and the concentration at which no bacterial growth was observed on the agar was defined as the MBC [21].\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of DCA on the growth curve of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. perfringens\u003c/em\u003e was inoculated in FTG broth supplemented with DCA at concentrations of 1/4, 1/2 and 1 MIC, and the same volume of PBS was added to the control group. Bacteria were collected every hour to measure OD600\u003csub\u003enm\u003c/sub\u003e. Each concentration treatment group was set up in three times.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAssessment of cell wall integrity and cell membrane permeability\u003c/h3\u003e\n\u003cp\u003eCurrently, alkaline phosphatase (AKP) and β-galactosidase (β-gal) have become important indicators for determining the integrity of bacterial cell walls and cell membranes[22, 23]. AKP is located between the bacterial cell wall and the cell membrane. Upon disruption of the cell wall, AKP is released into the extracellular environment[5]. Similarly, alterations in cell membrane permeability can be assessed by measuring the intracellular release of β-gal in \u003cem\u003eC. perfringens\u003c/em\u003e. After 6 hours of cultivation, the bacterial concentration was adjusted to an optical density (OD) of 0.5 at 600 nm. Subsequently, \u003cem\u003eC. perfringens\u003c/em\u003e was exposed to different concentrations of DCA, with PBS serving as the negative control. After treatment, the supernatant was collected by centrifugation, and the levels of AKP and β-gal were measured to assess the extent of damage to the bacterial cell wall and membrane. At the same time, the concentration of dsDNA in the supernatant was measured using an ultra-micro ultraviolet spectrophotometer to observe the level of intracellular nucleic acid leakage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of DCA on the biofilm of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe impact of DCA on the biofilm formation of \u003cem\u003eC. perfringens\u003c/em\u003e was assessed utilizing the crystal violet assay[24]. FTG broth, supplemented with varying concentrations of DCA, was aliquoted into 96-well microtiter plates, with each concentration being triplicated across three rows. Subsequently, \u003cem\u003eC. perfringens\u003c/em\u003e was then inoculated into the wells and incubated overnight to facilitate biofilm formation. Following incubation, the bacterial suspension in the wells was removed, and the plates were gently washed twice with PBS to remove non-adherent bacteria and residual culture medium. After drying, 100 \u0026micro;L of anhydrous methanol was added to each well to fix the biofilm for 15 minutes. Subsequently, 200 \u0026micro;L of 1% crystal violet solution was applied to stain the wells for 30 minutes. Then wash the plate with PBS and dissolve the biofilm with 33% glacial acetic acid for 10 minutes. Finally, the absorbance at OD570\u003csub\u003enm\u003c/sub\u003e was measured. The absorbance value was directly proportional to the biofilm mass, thereby reflecting the effect of DCA on the formation of \u003cem\u003eC. perfringens\u003c/em\u003e biofilm.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM) and Scanning electron microscopy (SEM) inspection\u003c/h3\u003e\n\u003cp\u003eThe concentration of \u003cem\u003eC. perfringens\u003c/em\u003e cultured for 6 hours was adjusted to OD600\u003csub\u003enm\u003c/sub\u003e of 0.5. The bacterial precipitate was subsequently inoculated with DCA solution at concentrations of 0, 1 MIC and 8 MIC, and co-cultured for 2 hours at 37\u0026deg;C. Following incubation, the bacteria were collected by centrifugation, gently rinsed with PBS, and the PBS was discarded. The 2.5% glutaraldehyde electron microscopy fixative was added, and the bacteria were resuspended in the fixative for two hours at room temperature. The samples were processed according to a previously published protocol and subsequently examined using TEM and SEM[25, 26].\u003c/p\u003e\n\u003ch3\u003eTotal RNA extraction and RNA sequencing\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eTotal RNA extraction and RNA sequencing\u003c/div\u003e\u003cp\u003eFour independent samples of the DCA-treated group (1 MIC) and the control group of \u003cem\u003eC. perfringens\u003c/em\u003e were cultured for 6 h at 37\u0026deg;C. Total RNA was extracted from bacterial precipitates, and the extracted RNA was quality assessed to ensure its integrity, concentration, and purity. Libraries were constructed using the TruSeq\u0026trade; Stranded Total RNA Library Prep Kit with rRNA depletion. NovaSeqXPlus sequencing platform (ShangHai majorbio Bio-pharm technology Co.,ltd) was used for mRNA sequencing.\u003c/p\u003e\n\u003ch3\u003eAnalysis of differentially expressed genes\u003c/h3\u003e\n\u003cp\u003eThe EBSeq algorithm was used to screen the differentially expressed genes, and the criteria for screening were Fold Change (FC) greater than 1.5 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The differentially expressed genes were annotated in the GO (Gene Ontology) database and KEGG (Kyoto Encyclopedia of Genes and Genomes) database, and the enrichment analysis was carried out; the significance levels of the GO and KEGG Pathways enriched with differentially expressed genes were analysed by the Fisher exact test based on the hypergeometric distribution. KEGG Pathway significance levels, and screened out the GO items and metabolic pathways that were significantly enriched for differentially expressed genes.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eqPCR for Detection of Gene Expression\u003c/h2\u003e\u003cp\u003eFor validation of transcriptional profiling from RNA-Seq, quantitative real-time PCR (qRT-PCR) was performed. Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan). qPCR was performed with ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme, Nanjing, China). Primers for qPCR were obtained by querying the gene database at NCBI and were synthesized by Bioengineering Biotechnology Co. (Bioengineering Biotechnology, Shanghai, China). The relative expression level of genes was represented by 2^(-∆∆Ct), and 16sRNA was used as a reference gene[22]. The primer details are shown in \u003cb\u003esupplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll the experimental data were replicated three times or more, statistically analyzed by SPSS Statistics 26.0, and plotted by GraphPad Prism 9.0. The results of the experiments were expressed as the \u0026lsquo;mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation\u0026rsquo;, and the t-test was used to compare the differences between the groups, with * representing a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and ** representing a highly significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cb\u003eEffect of DCA on the growth curve and biofilm of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results showed that DCA had a strong inhibitory effect on \u003cem\u003eC. perfringens\u003c/em\u003e with a MIC value of 0.125 mg/mL and MBC value of 1 mg/mL (8 MIC). As is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, when the DCA concentration is 1/4 MIC and 1/2 MIC, it can significantly inhibit the growth of \u003cem\u003eC. perfringens\u003c/em\u003e. When the concentration is 1 MIC, DCA can completely inhibit the growth of \u003cem\u003eC. perfringens\u003c/em\u003e. As is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the results of the crystal violet assay showed that DCA at concentrations ranging from 1/4 MIC to 1 MIC significantly inhibited the formation of \u003cem\u003eC. perfringens\u003c/em\u003e biofilm in a concentration-dependent manner compared with the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of DCA on the membrane integrity of\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e \u003cb\u003ecell walls\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom the detection of AKP and β-gal content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), DCA can destroy the integrity of cell walls and the permeability of cell membranes, and this destructive ability is proportional to the concentration of DCA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the concentration of extracellular dsDNA significantly increased after DCA treatment, indicating that DCA can alter cell membrane permeability, leading to nucleic acid leakage, which may have a negative impact on maintaining normal bacterial physiological functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of DCA on\u003c/b\u003e \u003cb\u003eC. perfringens\u003c/b\u003e \u003cb\u003emorphology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTEM and SEM further showed the disruptive effect of DCA on the structure of \u003cem\u003eC. perfringens.\u003c/em\u003e As can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the untreated cells (control) showed a typical \u003cem\u003eC. perfringens\u003c/em\u003e ultrastructure, with the cell membrane, cell wall and no cellular damage was observed. After treatment with MIC concentration of DCA for 2 hours, some of the cell edges were blurred and the surface of the cell wall appeared wrinkled. Under the treatment with MBC concentration of DCA, the cell wall membrane was completely blurred and the bacterial surface showed a large number of breaks and was no longer intact. Scanning electron microscopy and projection results showed that low concentrations of DCA had a certain destructive effect on the cell membrane and cell wall of \u003cem\u003eC. perfringens\u003c/em\u003e, while at the concentration of MBC, DCA could directly destroy the cell structure of \u003cem\u003eC. perfringens\u003c/em\u003e leading to the death of the bacteria.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of differentially expressed genes (DEGs)\u003c/h2\u003e\u003cp\u003eTranscriptomics analysis was used to understand the gene expression changes between DCA-treated and untreated groups. A total of 659 DEGs were identified compared to the control group of these, 355 were up-regulated and 304 were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Details of the DEGs are shown in \u003cb\u003esupplementary table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the effect of DCA on \u003cem\u003eC. perfringens\u003c/em\u003e, the differentially expressed genes were subjected to GO functional gene enrichment analysis. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, GO enrichment results showed that 202 GO terms were up-regulation, mainly involving protein-containing complex, ribonucleoprotein complex, structural constituent of ribosome and structural molecule activity. The down-regulated GO terms are enriched in molecular function, specifically enriched in four GO terms: antioxidant activity, peroxidase activity, oxidoreductase activity and protein serine/threonine kinase activity(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). While Details of GO enrichment of DEGs are shown in \u003cb\u003esupplementary table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe differentially expressed genes of \u003cem\u003eC. perfringens\u003c/em\u003e after DCA were subjected to KEGG enrichment analysis, and as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, after DCA treatment, up-regulated genes were enriched in four pathways: Ribosome, oxidative phosphorylation, pyruvate metabolism and taurine and hypotaurine metabolism. Down-regulated genes were enriched in pathways that did not show significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). See \u003cb\u003esupplementary table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e for details.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe spores of \u003cem\u003eC. perfringens\u003c/em\u003e exhibit strong environmental resistance, capable of withstanding extreme conditions such as high temperatures and dryness, and demonstrate significant resistance to conventional chemical disinfectants[27]. Additionally, with the widespread use of antibiotics, this strain has gradually evolved to develop multidrug resistance. To reduce antibiotic use and mitigate the threat this bacterium poses to public health, the development of novel antimicrobial agents to address \u003cem\u003eC. perfringens\u003c/em\u003e infections has become an urgent research priority. Currently, there is very little research on non-antibiotic antimicrobial drugs for \u003cem\u003eC. perfringens\u003c/em\u003e. Zheng et al. studied the antibacterial activity of natural propolis and identified caffeic acid as its primary component against \u003cem\u003eC. perfringens\u003c/em\u003e, concluding that propolis could serve as an alternative to antibiotics[5]. DCA was proposed in 2019 as a potential treatment for necrotic enteritis (NE) caused by \u003cem\u003eC. perfringens\u003c/em\u003e in chickens[20]. Subsequently, Kim et al. conducted further research and found that DCA can induce the production of host defense peptides to alleviate NE, further revealing the potential of DCA as an antibiotic alternative[19]. However, these two studies focused on the effects of DCA on the host, and there have been no studies on how DCA acts on \u003cem\u003eC. perfringens\u003c/em\u003e. Therefore, the aim of our study was to reveal the antibacterial activity and mechanism of DCA against \u003cem\u003eC. perfringens\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eBy detecting AKP and β-gal activity, we can observe that DCA can disrupt the cell wall and cell membrane structure of \u003cem\u003eC. perfringens\u003c/em\u003e. To validate these results, we directly observed the cellular structure using SEM and TEM. The control group of \u003cem\u003eC. perfringens\u003c/em\u003e exhibited a thick, mature, and dense three-dimensional structure. At a DCA concentration of 1 MIC, the cell wall already showed significant wrinkling and deformation, and at an 8 MIC concentration, the cells began to rupture, with intracellular material leaking out, indicating that DCA can effectively disrupt the cellular structure of \u003cem\u003eC. perfringens\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe virulence and drug resistance of \u003cem\u003eC.perfringens\u003c/em\u003e is closely related to the formation of biofilms, which can prevent or delay the penetration of antibiotics and allow them to escape the killing effect of antimicrobial drugs[28]. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, DCA can significantly inhibit the formation of \u003cem\u003eC. perfringens biofilms\u003c/em\u003e, and transcriptomic results indicated that DCA treatment significantly down-regulated the expression of the \u003cem\u003espoVG\u003c/em\u003e gene in \u003cem\u003eC. perfringens\u003c/em\u003e (log\u003csub\u003e2\u003c/sub\u003eFC=-3.00). The \u003cem\u003espovG\u003c/em\u003e gene is widely present in bacteria and has been confirmed to be a key regulatory factor in biofilm formation in bacteria such as \u003cem\u003eBacillus subtilis\u003c/em\u003e, \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, and \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e[28\u0026ndash;30]. However, whether the \u003cem\u003espoVG\u003c/em\u003e gene can regulate the formation of \u003cem\u003eC. perfringens\u003c/em\u003e biofilms has not yet been confirmed and requires further research.\u003c/p\u003e\u003cp\u003eAmong the DEGs, \u003cem\u003edef\u003c/em\u003e gene was significantly down-regulated(log2FC=-3.52), the down-regulation of this gene was also verified by qPCR(RS09240). The \u003cem\u003edef\u003c/em\u003e gene encodes peptide deformylase (PDF), an enzyme whose core function is to catalyze the removal of the formyl group (-CHO) from the N-terminal formylmethionine of newly synthesized polypeptide chains, thereby forming methionine. This is an important step in post-translational modification of bacterial proteins. If PDF is inhibited, it leads to the accumulation of un-deformylated proteins, resulting in bacterial growth arrest or death[31\u0026ndash;33]. This enzyme, which is widely present in bacteria, has become an important target for the development of new antimicrobial drugs[34, 35]. This indicates that DCA exerts its antibacterial effect against \u003cem\u003eC. perfringens\u003c/em\u003e by suppressing bacterial protein synthesis through down-regulation of the \u003cem\u003edef\u003c/em\u003e gene.\u003c/p\u003e\u003cp\u003eTranscriptomics is important for understanding the antibacterial mechanism of DCA. KEGG enrichment results showed that the oxidative phosphorylation pathway was significantly enriched. Oxidative phosphorylation is the main pathway for ATP production in bacteria, and up-regulation of this pathway promotes the activity of the bacterial electron transport chain; however, the high activity of the ETC may lead to electron leakage, which reacts with oxygen to generate large amounts of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide[36]. GO enrichment analysis showed that antioxidant activity, peroxidase activity and oxidoreductase activity were down-regulated in \u003cem\u003eC. perfringens\u003c/em\u003e. These enzymes are normally responsible for scavenging ROS from the cell, and if the activity of these enzymes is reduced, the ability of the bacteria to process ROS is diminished, leading to ROS accumulation[37, 38]. This indicates that DCA can induce ROS by upregulating the oxidative phosphorylation pathway. At the same time, the bacterial antioxidant system is inhibited and unable to effectively clear these ROS, resulting in an increase in intracellular ROS levels and triggering oxidative stress. Excessive ROS can damage bacterial DNA, proteins, and lipids, leading to the destruction of cellular structures and ultimately causing cell death[39, 40].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, DCA exhibits potent antibacterial activity against \u003cem\u003eC. perfringens\u003c/em\u003e, effectively inhibiting biofilm formation, disrupting bacterial cell wall integrity, and increasing membrane permeability. Transcriptome results indicate that DCA mediates oxidative damage by upregulating the oxidative phosphorylation pathway in \u003cem\u003eC. perfringens\u003c/em\u003e and downregulating antioxidant activity, peroxidase activity, and oxidoreductase activity. Additionally, DCA suppresses the expression of the \u003cem\u003edef\u003c/em\u003e gene, thereby impairing bacterial protein synthesis. This multi-targeted mechanism underscores DCA\u0026rsquo;s broad-spectrum antibacterial efficacy, making it a promising candidate for combating \u003cem\u003eC. perfringens\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Financial Program of BJAST (25CA005)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.-L.S. conducted the experiment and wrote the main manuscript. Z.-Y.Z., J.-D.B., and Q.-Y.G. carried out the analysis and reviewed the final manuscript, M.-H.W. contributed to reviewing the final manuscript, C.-S.Y. conducted the laboratory analysis. Q.-X.Z. designed the experiment and revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe sincerely thank the Beijing Milu Ecological research Center, China Agricultural University, and Anhui Agricultural University for providing the experimental conditions. We would like to express our gratitude to all personnel involved in this experiment. All authors participated in the critical review of the manuscript; gave final approval for the version to be published; agreed to submit the manuscript to the journal; and agreed to take responsibility for all aspects of the work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the present study can be obtained from the first author upon reasonable request. The raw sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1314926.（https://dataview.ncbi.nlm.nih.gov/?search=SUB15589106\u0026amp;amp;archive=bioproject）. Other data are provided in the supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eUzal FA, Navarro MA, Asin J, Boix O, Ballarà-Rodriguez I, Gibert X. Clostridial diarrheas in piglets: A review. Vet Microbiol. 2023;280:109691. https://doi.org/10.1016/j.vetmic.2023.109691.\u003c/li\u003e\n\u003cli\u003eAlizadeh M, Shojadoost B, Boodhoo N, Astill J, Taha-Abdelaziz K, Hodgins DC, et al. Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination. Anim Health Res Rev. 2021;22:147–62. https://doi.org/10.1017/S146625232100013X.\u003c/li\u003e\n\u003cli\u003eMwangi S, Timmons J, Fitz-coy S, Parveen S. Characterization of \u003cem\u003eClostridium perfringens\u003c/em\u003e recovered from broiler chicken affected by necrotic enteritis. Poult Sci. 2019;98:128–35. https://doi.org/10.3382/ps/pey332.\u003c/li\u003e\n\u003cli\u003eJohnson S, Skinner AM, Lostutter C, Duke T, Posthaus H. Enteritis necroticans and \u003cem\u003eClostridium perfringens\u003c/em\u003e type C; Epidemiological and pathological findings over the past 20 years. PLoS Negl Trop Dis. 2025;19:e0012836. https://doi.org/10.1371/journal.pntd.0012836.\u003c/li\u003e\n\u003cli\u003eZheng X, Al Naggar Y, Wu Y, Liu D, Hu Y, Wang K, et al. Untargeted metabolomics description of propolis’s in vitro antibacterial mechanisms against \u003cem\u003eClostridium perfringens\u003c/em\u003e. Food Chem. 2023;406:135061. https://doi.org/10.1016/j.foodchem.2022.135061.\u003c/li\u003e\n\u003cli\u003eAdams V, Han X, Lyras D, Rood JI. Antibiotic resistance plasmids and mobile genetic elements of \u003cem\u003eClostridium perfringens\u003c/em\u003e. Plasmid. 2018;99:32–9. https://doi.org/10.1016/j.plasmid.2018.07.002.\u003c/li\u003e\n\u003cli\u003eFayez M, Elsohaby I, Al-Marri T, Zidan K, Aldoweriej A, El-Sergany E, et al. Genotyping and antimicrobial susceptibility of \u003cem\u003eClostridium perfringens\u003c/em\u003e isolated from dromedary camels, pastures and herders. Comp Immunol Microbiol Infect Dis. 2020;70. https://doi.org/10.1016/j.cimid.2020.101460.\u003c/li\u003e\n\u003cli\u003eWu K, Li Z, Fang M, Yuan Y, Fox EM, Liu Y, et al. Genome characteristics of the optrA-positive \u003cem\u003eClostridium perfringens\u003c/em\u003e strain QHY-2 carrying a novel plasmid type. mSystems. 2023. https://doi.org/10.1128/msystems.00535-23.\u003c/li\u003e\n\u003cli\u003eGulliver EL, Adams V, Marcelino VR, Gould J, Rutten EL, Powell DR, et al. Extensive genome analysis identifies novel plasmid families in \u003cem\u003eClostridium perfringens\u003c/em\u003e. Microb Genom. 2023;9. https://doi.org/10.1099/mgen.0.000995.\u003c/li\u003e\n\u003cli\u003eFeng Y, Fan X, Zhu L, Yang X, Liu Y, Gao S, et al. Phylogenetic and genomic analysis reveals high genomic openness and genetic diversity of \u003cem\u003eClostridium perfringens\u003c/em\u003e. Microb Genom. 2020;6:1–15. https://doi.org/10.1099/mgen.0.000441.\u003c/li\u003e\n\u003cli\u003eLiu C, Ma N, Feng Y, Zhou M, Li H, Zhang X, et al. From probiotics to postbiotics: Concepts and applications. Animal Research and One Health. 2023;1:92–114. https://doi.org/10.1002/aro2.7.\u003c/li\u003e\n\u003cli\u003eJin D, Huang K, Xu M, Hua H, Ye F, Yan J, et al. Deoxycholic acid induces gastric intestinal metaplasia by activating STAT3 signaling and disturbing gastric bile acids metabolism and microbiota. Gut Microbes. 2022;14. https://doi.org/10.1080/19490976.2022.2120744.\u003c/li\u003e\n\u003cli\u003eWahlström A, Brumbaugh A, Sjöland W, Olsson L, Wu H, Henricsson M, et al. Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism. Nat Commun. 2024;15:4276. https://doi.org/10.1038/s41467-024-48543-3.\u003c/li\u003e\n\u003cli\u003eSato Y, Kobayashi M, Ohira M, Funayama R, Maekawa M, Karasawa H, et al. Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells. Cancer Sci. 2024;115:1778–90. https://doi.org/10.1111/cas.16132.\u003c/li\u003e\n\u003cli\u003eTanaka M, Nakura H, Tateishi T, Watanabe M, Nakaya S, Kumai T, et al. Ursodeoxycholic acid prevents hepatic cytochrome P450 isozyme reduction in rats with deoxycholic acid-induced liver injury. J Hepatol. 1999;31:263–70. https://doi.org/10.1016/S0168-8278(99)80223-2.\u003c/li\u003e\n\u003cli\u003eZheng Y, Yue C, Zhang H, Chen H, Liu Y, Li J. Deoxycholic Acid and Lithocholic Acid Alleviate Liver Injury and Inflammation in Mice with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e-Induced Liver Abscess and Bacteremia. J Inflamm Res. 2021;Volume 14:777–89. https://doi.org/10.2147/JIR.S298495.\u003c/li\u003e\n\u003cli\u003eVidal JE, Wier MN, A. Angulo-Zamudio U, McDevitt E, Jop Vidal AG, Alibayov B, et al. Prophylactic Inhibition of Colonization by \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e with the Secondary Bile Acid Metabolite Deoxycholic Acid. Infect Immun. 2021;89. https://doi.org/10.1128/IAI.00463-21.\u003c/li\u003e\n\u003cli\u003eZhao W, Wang J, Li X, Li Y, Ye C. Deoxycholic acid inhibits \u003cem\u003eStaphylococcus aureus\u003c/em\u003e-induced endometritis through regulating TGR5/PKA/NF-κB signaling pathway. Int Immunopharmacol. 2023;118:110004. https://doi.org/10.1016/j.intimp.2023.110004.\u003c/li\u003e\n\u003cli\u003eKim DM, Liu J, Whitmore MA, Tobin I, Zhao Z, Zhang G. Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis. J Anim Sci Biotechnol. 2024;15:29. https://doi.org/10.1186/s40104-024-00995-9.\u003c/li\u003e\n\u003cli\u003eWang H, Latorre JD, Bansal M, Abraha M, Al-Rubaye B, Tellez-Isaias G, et al. Microbial metabolite deoxycholic acid controls \u003cem\u003eClostridium perfringens\u003c/em\u003e-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling. Sci Rep. 2019;9:14541. https://doi.org/10.1038/s41598-019-51104-0.\u003c/li\u003e\n\u003cli\u003eLi J, Li C, Shi C, Aliakbarlu J, Cui H, Lin L. Antibacterial mechanisms of clove essential oil against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and its application in pork. Int J Food Microbiol. 2022;380:109864. https://doi.org/10.1016/j.ijfoodmicro.2022.109864.\u003c/li\u003e\n\u003cli\u003eXin J, Pu Q, Wang R, Gu Y, He L, Du X, et al. Antibacterial activity and mechanism of chelerythrine against \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e. Front Vet Sci. 2024;11. https://doi.org/10.3389/fvets.2024.1408376.\u003c/li\u003e\n\u003cli\u003eYu Y, Dong J, Wang Y, Gong X. RNA-seq analysis of antibacterial mechanism of Cinnamomum camphora essential oil against \u003cem\u003eEscherichia coli\u003c/em\u003e. PeerJ. 2021;9:e11081. https://doi.org/10.7717/peerj.11081.\u003c/li\u003e\n\u003cli\u003eBai B, Wang M, Zhang Z, Guo Q, Yao J. Mechanistic Investigation of the Pyrolysis Temperature of Reed Wood Vinegar for Maximising the Antibacterial Activity of \u003cem\u003eEscherichia coli\u003c/em\u003e and Its Inhibitory Activity. Biology (Basel). 2024;13:912. https://doi.org/10.3390/biology13110912.\u003c/li\u003e\n\u003cli\u003eLiu X, Shu Q, Chen Q, Pang X, Wu Y, Zhou W, et al. Antibacterial Efficacy and Mechanism of Mannosylerythritol Lipids-A on \u003cem\u003eListeria monocytogenes\u003c/em\u003e. Molecules. 2020;25:4857. https://doi.org/10.3390/molecules25204857.\u003c/li\u003e\n\u003cli\u003eRao W, Fang Z, Chen Z, Wu J, Fang X. Antibacterial mechanism of metabolites of Leuconostoc mesenteroides against \u003cem\u003eSerratia liquefaciens\u003c/em\u003e. LWT. 2023;187:115335. https://doi.org/10.1016/j.lwt.2023.115335.\u003c/li\u003e\n\u003cli\u003eLindström M, Heikinheimo A, Lahti P, Korkeala H. Novel insights into the epidemiology of \u003cem\u003eClostridium perfringens\u003c/em\u003e type A food poisoning. Food Microbiol. 2011;28:192–8. https://doi.org/10.1016/j.fm.2010.03.020.\u003c/li\u003e\n\u003cli\u003eHu WS, Woo DU, Kang YJ, Koo OK. Biofilm and Spore Formation of \u003cem\u003eClostridium perfringens\u003c/em\u003e and Its Resistance to Disinfectant and Oxidative Stress. Antibiotics. 2021;10:396. https://doi.org/10.3390/antibiotics10040396.\u003c/li\u003e\n\u003cli\u003eShi C, Zheng L, Lu Z, Zhang X, Bie X. The global regulator \u003cem\u003eSpoVG\u003c/em\u003e regulates \u003cem\u003eListeria monocytogenes\u003c/em\u003e biofilm formation. Microb Pathog. 2023;180:106144. https://doi.org/10.1016/j.micpath.2023.106144.\u003c/li\u003e\n\u003cli\u003eHuang Q, Zhang Z, Liu Q, Liu F, Liu Y, Zhang J, et al. \u003cem\u003eSpoVG\u003c/em\u003e is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in \u003cem\u003eBacillus cereus\u003c/em\u003e. BMC Microbiol. 2021;21:172. https://doi.org/10.1186/s12866-021-02239-6.\u003c/li\u003e\n\u003cli\u003eSangshetti J, Khan FA, Shinde D. Peptide Deformylase: A New Target in Antibacterial, Antimalarial and Anticancer Drug Discovery. Curr Med Chem. 2014;22:214–36. https://doi.org/10.2174/0929867321666140826115734.\u003c/li\u003e\n\u003cli\u003eMerzoug A, Chikhi A, Bensegueni A, Boucherit H, Okay S. Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics. Mol Inform. 2018;37. https://doi.org/10.1002/minf.201700087.\u003c/li\u003e\n\u003cli\u003eGokhale KM, Telvekar VN. Novel peptidomimetic peptide deformylase (PDF) inhibitors of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. Chem Biol Drug Des. 2021;97:148–56. https://doi.org/10.1111/cbdd.13769.\u003c/li\u003e\n\u003cli\u003eSinghal A, Fang FC. Direct and Indirect Inhibition of \u003cem\u003eSalmonella\u003c/em\u003e Peptide Deformylase by Nitric Oxide. mBio. 2020;11. https://doi.org/10.1128/mBio.01383-20.\u003c/li\u003e\n\u003cli\u003eMerzoug A, Chikhi A, Bensegueni A, Boucherit H, Okay S. Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics. Mol Inform. 2018;37. https://doi.org/10.1002/minf.201700087.\u003c/li\u003e\n\u003cli\u003eImlay JA. Pathways of Oxidative Damage. Annu Rev Microbiol. 2003;57:395–418. https://doi.org/10.1146/annurev.micro.57.030502.090938.\u003c/li\u003e\n\u003cli\u003eImlay JA. Cellular Defenses against Superoxide and Hydrogen Peroxide. Annu Rev Biochem. 2008;77:755–76. https://doi.org/10.1146/annurev.biochem.77.061606.161055.\u003c/li\u003e\n\u003cli\u003eEzraty B, Gennaris A, Barras F, Collet J-F. Oxidative stress, protein damage and repair in bacteria. Nat Rev Microbiol. 2017;15:385–96. https://doi.org/10.1038/nrmicro.2017.26.\u003c/li\u003e\n\u003cli\u003eKohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell. 2007;130:797–810. https://doi.org/10.1016/j.cell.2007.06.049.\u003c/li\u003e\n\u003cli\u003eDwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD, Takahashi N, et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proceedings of the National Academy of Sciences. 2014;111. https://doi.org/10.1073/pnas.1401876111.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Clostridium perfringens, Deoxycholic acid, Antimicrobial activity, Transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-7454314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7454314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDeoxycholic acid (DCA), a gut microbiota-derived secondary metabolite, exhibits broad-spectrum antimicrobial activity, particularly against \u003cem\u003eClostridium perfringens\u003c/em\u003e(\u003cem\u003eC. perfringens\u003c/em\u003e). However, its precise mechanistic action remains unclear.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eIn this study, we examined the inhibitory mechanism of DCA against \u003cem\u003eC. perfringens\u003c/em\u003e through in vitro growth inhibition assays coupled with transcriptomic analysis.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eThe study results indicate that DCA can effectively inhibit the formation of \u003cem\u003eC. perfringens\u003c/em\u003e biofilms, disrupt their cell walls, increase cell membrane permeability, and cause nucleic acid leakage. Transcriptome analysis revealed that DCA can mediate its oxidative damage by up-regulating the oxidative phosphorylation pathway in \u003cem\u003eC. perfringens\u003c/em\u003e and down-regulating antioxidant activity, peroxidase activity, and oxidoreductase activity. This study provides insights into the antimicrobial activity of DCA and its mechanisms, laying a theoretical foundation for its development as a novel antimicrobial agent or feed additive.\u003c/p\u003e","manuscriptTitle":"Antibacterial activity and mechanism analysis of deoxycholic acid against Clostridium perfringens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 15:03:55","doi":"10.21203/rs.3.rs-7454314/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T05:49:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T12:12:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T08:09:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58025968512609703516530715355051787445","date":"2025-11-21T07:50:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"324503566348243821679393198080137151834","date":"2025-11-21T06:03:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211992741721123329163225092275627588058","date":"2025-11-20T09:34:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86239159500476569979313041241235591449","date":"2025-11-20T02:59:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-04T07:29:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50158670613366299845060090274633362808","date":"2025-10-26T14:59:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124427845996239903903507581564612484600","date":"2025-10-26T01:42:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169614901178839773011358158484880485656","date":"2025-10-24T07:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-24T01:39:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-23T11:58:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T06:29:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T03:47:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-09-04T03:44:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"392abc7e-9d0f-4f5e-83f7-0d86f2787bc3","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:50:36+00:00","versionOfRecord":{"articleIdentity":"rs-7454314","link":"https://doi.org/10.1186/s12866-025-04676-z","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-16 16:31:09","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-11-05 15:03:55","video":"","vorDoi":"10.1186/s12866-025-04676-z","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04676-z","workflowStages":[]},"version":"v1","identity":"rs-7454314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7454314","identity":"rs-7454314","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}