Synergistic antibacterial and anti-biofilm effects of carvacrol and fluoxetine against Methicillin-resistant Staphylococcus aureus

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In this in vitro study, we assessed the anti-biofilm and antibacterial activity of carvacrol -a monoterpenoid phenol- and fluoxetine -a selective serotonin reuptake inhibitor-. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined using broth microdilution method. The synergistic effect was performed by checkerboard assay. MRSA isolates were resistant to conventional treatments such as tetracycline, trimethoprim-sulfamethoxazole, erythromycin, and clindamycin but sensitive to vancomycin and linezolid. They also had MIC and MBC levels of 256 µg/mL and 512 µg/mL against carvacrol, respectively. Moreover, these scores ranged respectively 128 µg/mL − 256 µg/mL against fluoxetine. Combining the two test agents clearly lowered the computed MICs and MBCs for carvacrol and fluoxetine. Furthermore, this combo resulted in notable downregulation of important adhesin genes and total suppression of biofilm development, therefore offering strong proof for a combined approach to reduce MRSA infections. Methicillin-resistant Staphylococcus aureus Biofilms Carvacrol Fluoxetine Figures Figure 1 Figure 2 Introduction Staphylococcus aureus is a gram-positive bacterium that is a common cause of skin and soft tissue infections, as well as more serious infections such as pneumonia, endocarditis, and sepsis ( 1 , 2 ). S. aureus has the ability to bind immunoglobulins and agglutinate with or coagulate blood and plasma ( 3 ). MRSA is a type of S. aureus that has developed resistance to many antibiotics, including methicillin, due to the overuse and abuse of these drugs ( 4 ). MRSA infections are often associated with healthcare settings, but they can also occur in the community ( 5 ). Treatment of MRSA infections is challenging due to the limited number of effective antibiotics ( 6 ). Biofilms protect MRSA against host defenses and conventional antibiotics, thereby necessitating alternative therapeutic approaches that target both the planktonic and biofilm-associated bacterial populations. Therefore, there is a need for novel therapeutic strategies to combat MRSA infections. The emergence of more virulent strains of MRSA, further highlights the need for new treatment options ( 7 , 8 ). In this context, the investigation of novel antimicrobial agents, especially those derived from natural sources or repurposed pharmaceuticals, is rapidly gaining attention. Among natural compounds, carvacrol a monoterpenoid phenol primarily found in Origanum vulgare extracts has been widely studied for its potent antibacterial and antibiofilm activities. Several studies have demonstrated that carvacrol exerts its effects by compromising the integrity of the bacterial membrane, leading to leakage of intracellular contents and depletion of essential metabolites ( 1 , 8 , 9 ). Furthermore, carvacrol has been shown to downregulate the expression of genes involved in biofilm formation and adhesion, reducing the persistence of MRSA in both in vitro and in vivo systems, while also exhibiting broad-spectrum antibiofilm activity against various bacteria, including Chromobacterium violaceum, Salmonella Typhimurium, S. aureus , and Pseudomonas aeruginosa ( 10 , 11 ). Biofilms are communities of bacteria encased in a protective matrix, making them more resistant to antibiotics and host immune responses ( 12 ). Carvacrol has also demonstrated anti-virulence effects by interfering with the virulence factors of bacteria, such as Aeromonas hydrophila ( 13 ). The evidence for these multifaceted actions provides a solid rationale for incorporating carvacrol into new combinatory antimicrobial strategies. In parallel with natural antimicrobial compounds, repurposing non-antibiotic drugs such as fluoxetine offers additional promise as an adjunctive therapeutic strategy. Fluoxetine's mechanism of action against bacteria is not fully understood, but it may involve inhibiting bacterial efflux pumps, which are responsible for removing antibiotics from bacterial cells ( 14 ). Although fluoxetine is conventionally employed as a selective serotonin reuptake inhibitor, recent reports have revealed its capacity to inhibit biofilm formation and disrupt bacterial cellular processes, thereby exhibiting significant antibacterial activity against a range of pathogens, including MRSA ( 15 ). Two of the largest clinical trials to date investigating novel therapeutic strategies to treat MRSA bacteremia have evaluated the efficacy and safety of combination therapy. These trials provide valuable insights into the potential benefits and challenges of combination therapy for MRSA infections ( 16 ). The current study aims to evaluate the synergistic antibacterial and anti‐biofilm effects of carvacrol and fluoxetine against MRSA. By integrating these two compounds, it is anticipated that the dual approach will not only reduce the minimum inhibitory concentrations (MICs) but also impair biofilm formation through the coordinated downregulation of adhesin and biofilm-associated genes. This strategy holds promise for overcoming current limitations associated with standard antibiotic regimens and offers a potential new avenue for addressing multidrug-resistant bacterial infections. The aim of this study was assessment of antibacterial and anti-biofilm effects of carvacrol and fluoxetine and their synergistic effects against MRSA standard strains and clinical isolates. Material and methods Bacterial Strains and Growth Conditions MRSA isolates were employed in this study, obtained from clinical sources and characterized using standard microbiological tests. Standard strains ATCC 25923 and ATCC 43300 were also included. The isolates were grown in Mueller–Hinton Broth (MHB) and on tryptic soy agar (TSA) at 37°C under aerobic conditions ( 3 , 17 ). An inoculum equivalent to approximately 1 × 10 6 colony forming units per milliliter (CFU/mL) was prepared from overnight cultures for all subsequent assays. Preparation of Test Solutions Carvacrol (purity > 98%) was procured from a certified supplier and dissolved in absolute ethanol to prepare a stock solution, which was further diluted in MHB immediately before use. Fluoxetine hydrochloride (purity > 98%) was similarly obtained, dissolved in dimethyl sulfoxide (DMSO), and diluted in culture medium so that the final solvent concentration did not exceed 1% (v/v) to avoid solvent-related effects ( 18 ). All solutions were sterilized by passage through a 0.22-µm membrane filter. Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration The MIC values for carvacrol and fluoxetine, both individually and in combination, were determined using a broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines( 19 ). Briefly, two-fold serial dilutions of each compound were prepared in 96-well microtiter plates containing MHB. MRSA inocula (1 × 10 6 CFU/mL) were added to each well, and plates were incubated at 37°C for 24 h. The MIC was defined as the lowest concentration with no visible growth. To determine the MBC, aliquots from wells corresponding to the MIC and higher concentrations were plated on TSA, and the MBC was defined as the lowest concentration resulting in a ≥ 99.9% reduction in CFU compared to the initial inoculum ( 20 ). Time-Kill Assay Time-kill studies were carried out to evaluate the bactericidal kinetics of carvacrol, fluoxetine, and their combination. MRSA cultures (1 × 10 6 CFU/mL) were exposed to each agent at concentrations equivalent to their individual MICs or to the concentration determined for the combination. At predetermined time intervals (0, 2, 4, 6, 12, and 24 h), aliquots were withdrawn, serially diluted in sterile phosphate-buffered saline (PBS), and plated on TSA. Colonies were enumerated after 24 h incubation at 37°C, and bactericidal activity was defined as a ≥ 3 log_10 reduction in CFU/mL compared to the initial count ( 20 ). RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) for Adhesin Gene Expression RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) for Adhesin Gene Expression To assess the impact of the treatments on biofilm-associated gene expression, total RNA was extracted from MRSA cultures treated with carvacrol, fluoxetine, or their combination using a TRIzol-based protocol, as described previously. RNA quality and concentration were verified by agarose gel electrophoresis and spectrophotometry. Complementary DNA (cDNA) synthesis was performed using a commercially available reverse transcription kit. Quantitative real-time PCR (qRT-PCR) was then conducted using gene-specific primers for adhesin genes, including clfA , clfB , fnbA , fnbB , and icaA , with normalization to a housekeeping gene (e.g., 16S rRNA). The amplification protocols and data analysis were performed as reported in earlier studies ( 21 ). Biofilm Inhibition Assay Biofilm formation was evaluated using the crystal violet staining method. MRSA cultures were dispensed into flat-bottom 96-well polystyrene microtiter plates and incubated in the presence of carvacrol, fluoxetine, or their combination at defined concentrations for 24 h at 37°C. After incubation, wells were gently washed with PBS to remove planktonic cells. Adherent biofilms were fixed with methanol for 15 min, stained with 0.1% (w/v) crystal violet for 15 min, and then rinsed to remove excess dye. The bound crystal violet was solubilized in 95% ethanol, and absorbance was measured at 570 nm in a microplate reader. The percentage inhibition of biofilm formation was calculated relative to untreated controls ( 18 , 22 , 23 ). Statistical Analysis All experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation. Statistical comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant. Statistical analysis was conducted using GraphPad Prism software (version 8.0), following procedures outlined in previous reports ( 24 ). Results Antibacterial susceptibility Clinical MRSA isolates were resistant to tetracycline, trimethoprim-sulfamethoxazole, erythromycin, clindamycin and were susceptible to vancomycin and linezolid. The minimum inhibitory and bactericidal concentrations The MIC and MBC of cefoxitin among MRSA included 4–8 µg/mL. The MIC and MBC of carvacrol were observed to range between 256 µg/mL and 512 µg/mL, in that order. Moreover, the MIC and MBC of vancomycin respectively included 0.5-2 µg/mL and 1–4 µg/mL. Moreover, the MIC and MBC of fluoxetine included 128 µg/mL and 256 µg/mL, respectively. The combination of carvacrol and fluoxetine decreased the MIC and MBC of them to 8–16 µg/mL and 32–64 µg/mL, respectively. Biofilm formation MRSA isolates had strong (n = 2) and moderate (n = 8) biofilm levels. Carvacrol at 64 µg/mL caused a decrease in the biofilm formation to weak and non-biofilm formation. Moreover, fluoxetine decreased the biofilms to weak levels. The combination of carvacrol and fluoxetine decreased the biofilm levels to no biofilm production (Fig. 1 ). Adhesin genes expression In comparison to the control, carvacrol decreased the expression levels of clfA, clfB, fnbA, fnbB, icaA genes to 2.9, 2.6, 2, 2 and 1.8 fold respectively which were significant. Moreover, fluoxetine significantly decreased the expression of clfA, clfB, fnbA, fnbB, icaA genes to 1.9, 1.6, 1.2, 1.4 and 2 fold, respectively. The combinatory subject of carvacrol and fluoxetine caused MRSA adhesin genes clfA, clfB, fnbA, fnbB, icaA significant decrease to 3.2, 2.9, 2.6, 2, and 1.6 fold, respectively (Fig. 2 ). Discussion The results of this study demonstrate that the combination of carvacrol and fluoxetine has synergistic antibacterial and anti-biofilm effects against MRSA. The combination therapy significantly reduced the MIC and MBC of both compounds compared to their individual use. This suggests that carvacrol and fluoxetine may act synergistically to disrupt the bacterial cell membrane and inhibit bacterial growth. The combination therapy may be particularly valuable in overcoming the limitations of conventional antibiotic therapy against MRSA, as it may be effective against MRSA strains that are resistant to conventional antibiotics and may prevent the emergence of resistance ( 25 – 27 ). our data confirm that carvacrol disrupts the structural integrity of the bacterial cell membrane, thereby interfering with vital cellular processes. The measured MIC (128–256 µg/mL) and MBC for carvacrol align with earlier reports on its bactericidal properties and its capability to disturb biofilm formation via downregulation of adhesion-related genes ( 28 , 29 ). The study also showed that the combination of carvacrol and fluoxetine effectively inhibited biofilm formation. This is an important finding, as biofilms are a major contributor to the persistence of MRSA infections. Biofilm-protected bacterial cells are more resistant to most antibiotics and host defense systems than their planktonic counterparts ( 12 ). Our findings demonstrate that fluoxetine alone exhibits moderate antibacterial activity (MIC 64–128 µg/mL, MBC 128–256 µg/mL) and can reduce biofilm biomass. Although the precise mechanism underpinning fluoxetine’s antibacterial action remains to be fully elucidated, evidence suggests that it may compromise bacterial membrane integrity and interfere with efflux pump activity—mechanisms that are reminiscent of those seen with other non-antibiotic compounds repurposed for antibacterial use ( 30 ). The reduction in MIC and MBC values to 8–16 µg/mL and 32–64 µg/mL, respectively, suggests that the combination targets multiple bacterial processes concurrently. Carvacrol’s ability to perturb the bacterial membrane likely facilitates enhanced intracellular penetration of fluoxetine, which may act by inhibiting bacterial energy metabolism and efflux systems. This dual action results in a significant downregulation of adhesin genes (clfA, clfB, fnbA, fnbB, icaA), thereby impairing the formation and stability of MRSA biofilms, a finding that mirrors previous observations of synergistic natural compound-antibiotic combinations ( 31 ). From a clinical perspective, the resistance profiles of the MRSA isolates examined herein are concerning. Given that these strains are unresponsive to multiple frontline antibiotics, the development of alternative or adjunctive therapies is imperative. Although vancomycin and linezolid remain effective, their use is limited by toxicity and the risk of emerging resistance ( 1 , 2 ). Few clinical trials have yet explored the use of carvacrol and fluoxetine in MRSA infections ( 32 ). however, our in vitro findings provide a strong mechanistic rationale for further preclinical studies. It is important to consider the potential impact of fluoxetine on the human microbiota. Studies have shown that fluoxetine can inhibit the growth of various microorganisms, including those found in the human gut. Further research is needed to evaluate the long-term effects of fluoxetine on the gut microbiota and its potential implications for human health ( 33 ). Methodologically, our work adopted standard protocols encompassing bacterial cultivation, MIC determination, time-kill kinetics, qRT-PCR analysis, and biofilm inhibition assays that have been successfully employed in similar studies ( 27 , 34 ). In summary, our investigation reveals that the combination of carvacrol and fluoxetine exhibits powerful synergistic antibacterial and anti-biofilm actions against MRSA. Targeting bacterial membrane structure, intracellular processes, and biofilm-associated gene expression concurrently will enable this combinatorial approach to assist overcome the limits of traditional antibiotics and offer a fresh weapon against multidrug-resistant organisms. This study has various restrictions: The study was done in-vitro, hence the findings might not be exactly relevant in clinical environments. This research made use of few MRSA isolates and this can be expanded. Carvacrol and fluoxetine may have several possible uses in the treatment of MRSA infections, including topical application for skin and soft tissue infections, systemic administration for more severe infections, combination with antibiotics to enhance their efficacy, and prophylactic use to prevent MRSA infections in high-risk persons. Investigating the ideal dosage and route of administration, assessing the efficacy and safety in clinical trials, looking at ways to get past resistance to other antibiotics, and figuring out the long-term consequences on the human flora need more study. Conclusion The results of this study imply that treating MRSA infections with carvacrol and fluoxetine could show great success. Combining this treatment could help to solve problems with antibiotic resistance and biofilm development. Carvacrol and fluoxetine may so efficiently kill MRSA and stop the creation of infections by upsetting the bacterial cell membrane, blocking efflux pumps, and downregulating adhesin gene expression. This combo treatment deserves more research to convert the results into practical use since it shows potential to solve the rising public health issue of MRSA infections. Declarations Conflicts of interest None to declare. Acknowledgments The study was designed and performed by the authors. 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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-6614132","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455099344,"identity":"33bea2b7-de64-44ea-b1d7-388cc2e91a75","order_by":0,"name":"Dunia Kamal Salim","email":"","orcid":"","institution":"Tikrit University","correspondingAuthor":false,"prefix":"","firstName":"Dunia","middleName":"Kamal","lastName":"Salim","suffix":""},{"id":455099345,"identity":"7ce536f3-8c18-4545-9f75-55055b11416d","order_by":1,"name":"Sundus Jassim Muhammad","email":"","orcid":"","institution":"Tikrit University","correspondingAuthor":false,"prefix":"","firstName":"Sundus","middleName":"Jassim","lastName":"Muhammad","suffix":""},{"id":455099346,"identity":"aaa78d03-e7bc-45aa-be2e-55addfbbd575","order_by":2,"name":"Haider Qasim Raheem","email":"","orcid":"","institution":"University of Babylon","correspondingAuthor":false,"prefix":"","firstName":"Haider","middleName":"Qasim","lastName":"Raheem","suffix":""},{"id":455099347,"identity":"66f44d97-5773-436a-83e1-cbca463a19e4","order_by":3,"name":"Fouad Qsaim Jubair Al-Zayadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBAC9gYgwQNiMTMwPgALHQAj3IDnAEILswGJWhgY2CRgWvACHvYeww9vGKzl5du506putjHI8d1IYDzwBp8WnjPGknMY0g03HObddju3jcFY8kYCw8E5eLTYS+QYSPMwHGbcwAzRkrgBqOUwDz5bJHKMfwO12M9v5t1WDNRST4wWM5AtiQ1AhzEDtSQYENTCc6zMco5BejLQL5ulc85JGM4887ABr1942Js333hTYW07v//sxs85ZTbyfMeTD3/AF2IQYMAMY4GihrGBAZ/DoIAZjU+EllEwCkbBKBg5AABROEtltKypdAAAAABJRU5ErkJggg==","orcid":"","institution":"Al-Muthanna University","correspondingAuthor":true,"prefix":"","firstName":"Fouad","middleName":"Qsaim Jubair","lastName":"Al-Zayadi","suffix":""}],"badges":[],"createdAt":"2025-05-07 17:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6614132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6614132/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82626603,"identity":"574cde65-3a5b-42a4-900c-798c3ad4db74","added_by":"auto","created_at":"2025-05-13 13:07:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9349,"visible":true,"origin":"","legend":"\u003cp\u003eBiofilm formation levels by MRSA. The combination of carvacrol and fluoxetine decreased the biofilm formation levels substantially to no biofilm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6614132/v1/37c76b3c6db28f1aac2d3179.png"},{"id":82626604,"identity":"9f504e22-93f9-490d-98d9-e5ec2f9904c5","added_by":"auto","created_at":"2025-05-13 13:07:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12278,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of adhesin genes in various groups\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6614132/v1/aa802b10481ba7b69a00f739.png"},{"id":83489537,"identity":"faafd580-fe2b-48e5-a971-d6a2c31fcb8b","added_by":"auto","created_at":"2025-05-27 09:32:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":608891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6614132/v1/0cb5c97c-ae4b-4d47-bd76-d814334d581d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic antibacterial and anti-biofilm effects of carvacrol and fluoxetine against Methicillin-resistant Staphylococcus aureus","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a gram-positive bacterium that is a common cause of skin and soft tissue infections, as well as more serious infections such as pneumonia, endocarditis, and sepsis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e has the ability to bind immunoglobulins and agglutinate with or coagulate blood and plasma (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). MRSA is a type of \u003cem\u003eS. aureus\u003c/em\u003e that has developed resistance to many antibiotics, including methicillin, due to the overuse and abuse of these drugs (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). MRSA infections are often associated with healthcare settings, but they can also occur in the community (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Treatment of MRSA infections is challenging due to the limited number of effective antibiotics (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Biofilms protect MRSA against host defenses and conventional antibiotics, thereby necessitating alternative therapeutic approaches that target both the planktonic and biofilm-associated bacterial populations. Therefore, there is a need for novel therapeutic strategies to combat MRSA infections. The emergence of more virulent strains of MRSA, further highlights the need for new treatment options (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In this context, the investigation of novel antimicrobial agents, especially those derived from natural sources or repurposed pharmaceuticals, is rapidly gaining attention.\u003c/p\u003e \u003cp\u003eAmong natural compounds, carvacrol a monoterpenoid phenol primarily found in \u003cem\u003eOriganum vulgare\u003c/em\u003e extracts has been widely studied for its potent antibacterial and antibiofilm activities. Several studies have demonstrated that carvacrol exerts its effects by compromising the integrity of the bacterial membrane, leading to leakage of intracellular contents and depletion of essential metabolites (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Furthermore, carvacrol has been shown to downregulate the expression of genes involved in biofilm formation and adhesion, reducing the persistence of MRSA in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e systems, while also exhibiting broad-spectrum antibiofilm activity against various bacteria, including \u003cem\u003eChromobacterium violaceum, Salmonella Typhimurium, S. aureus\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Biofilms are communities of bacteria encased in a protective matrix, making them more resistant to antibiotics and host immune responses (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Carvacrol has also demonstrated anti-virulence effects by interfering with the virulence factors of bacteria, such as \u003cem\u003eAeromonas hydrophila\u003c/em\u003e (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The evidence for these multifaceted actions provides a solid rationale for incorporating carvacrol into new combinatory antimicrobial strategies. In parallel with natural antimicrobial compounds, repurposing non-antibiotic drugs such as fluoxetine offers additional promise as an adjunctive therapeutic strategy. Fluoxetine's mechanism of action against bacteria is not fully understood, but it may involve inhibiting bacterial efflux pumps, which are responsible for removing antibiotics from bacterial cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although fluoxetine is conventionally employed as a selective serotonin reuptake inhibitor, recent reports have revealed its capacity to inhibit biofilm formation and disrupt bacterial cellular processes, thereby exhibiting significant antibacterial activity against a range of pathogens, including MRSA (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Two of the largest clinical trials to date investigating novel therapeutic strategies to treat MRSA bacteremia have evaluated the efficacy and safety of combination therapy. These trials provide valuable insights into the potential benefits and challenges of combination therapy for MRSA infections (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The current study aims to evaluate the synergistic antibacterial and anti‐biofilm effects of carvacrol and fluoxetine against MRSA. By integrating these two compounds, it is anticipated that the dual approach will not only reduce the minimum inhibitory concentrations (MICs) but also impair biofilm formation through the coordinated downregulation of adhesin and biofilm-associated genes. This strategy holds promise for overcoming current limitations associated with standard antibiotic regimens and offers a potential new avenue for addressing multidrug-resistant bacterial infections. The aim of this study was assessment of antibacterial and anti-biofilm effects of carvacrol and fluoxetine and their synergistic effects against MRSA standard strains and clinical isolates.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Strains and Growth Conditions\u003c/h2\u003e \u003cp\u003eMRSA isolates were employed in this study, obtained from clinical sources and characterized using standard microbiological tests. Standard strains ATCC 25923 and ATCC 43300 were also included. The isolates were grown in Mueller\u0026ndash;Hinton Broth (MHB) and on tryptic soy agar (TSA) at 37\u0026deg;C under aerobic conditions (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). An inoculum equivalent to approximately 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e colony forming units per milliliter (CFU/mL) was prepared from overnight cultures for all subsequent assays.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of Test Solutions\u003c/h3\u003e\n\u003cp\u003eCarvacrol (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was procured from a certified supplier and dissolved in absolute ethanol to prepare a stock solution, which was further diluted in MHB immediately before use. Fluoxetine hydrochloride (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was similarly obtained, dissolved in dimethyl sulfoxide (DMSO), and diluted in culture medium so that the final solvent concentration did not exceed 1% (v/v) to avoid solvent-related effects (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). All solutions were sterilized by passage through a 0.22-\u0026micro;m membrane filter.\u003c/p\u003e\n\u003ch3\u003eDetermination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration\u003c/h3\u003e\n\u003cp\u003eThe MIC values for carvacrol and fluoxetine, both individually and in combination, were determined using a broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Briefly, two-fold serial dilutions of each compound were prepared in 96-well microtiter plates containing MHB. MRSA inocula (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were added to each well, and plates were incubated at 37\u0026deg;C for 24 h. The MIC was defined as the lowest concentration with no visible growth. To determine the MBC, aliquots from wells corresponding to the MIC and higher concentrations were plated on TSA, and the MBC was defined as the lowest concentration resulting in a\u0026thinsp;\u0026ge;\u0026thinsp;99.9% reduction in CFU compared to the initial inoculum (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eTime-Kill Assay\u003c/h3\u003e\n\u003cp\u003eTime-kill studies were carried out to evaluate the bactericidal kinetics of carvacrol, fluoxetine, and their combination. MRSA cultures (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL) were exposed to each agent at concentrations equivalent to their individual MICs or to the concentration determined for the combination. At predetermined time intervals (0, 2, 4, 6, 12, and 24 h), aliquots were withdrawn, serially diluted in sterile phosphate-buffered saline (PBS), and plated on TSA. Colonies were enumerated after 24 h incubation at 37\u0026deg;C, and bactericidal activity was defined as a\u0026thinsp;\u0026ge;\u0026thinsp;3 log_10 reduction in CFU/mL compared to the initial count (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and Quantitative Real-Time PCR (qRT-PCR) for Adhesin Gene Expression\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA Extraction and Quantitative Real-Time PCR (qRT-PCR) for Adhesin Gene Expression\u003c/div\u003e \u003cp\u003eTo assess the impact of the treatments on biofilm-associated gene expression, total RNA was extracted from MRSA cultures treated with carvacrol, fluoxetine, or their combination using a TRIzol-based protocol, as described previously. RNA quality and concentration were verified by agarose gel electrophoresis and spectrophotometry. Complementary DNA (cDNA) synthesis was performed using a commercially available reverse transcription kit. Quantitative real-time PCR (qRT-PCR) was then conducted using gene-specific primers for adhesin genes, including \u003cem\u003eclfA\u003c/em\u003e, \u003cem\u003eclfB\u003c/em\u003e, \u003cem\u003efnbA\u003c/em\u003e, \u003cem\u003efnbB\u003c/em\u003e, and \u003cem\u003eicaA\u003c/em\u003e, with normalization to a housekeeping gene (e.g., 16S rRNA). The amplification protocols and data analysis were performed as reported in earlier studies (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm Inhibition Assay\u003c/h2\u003e \u003cp\u003eBiofilm formation was evaluated using the crystal violet staining method. MRSA cultures were dispensed into flat-bottom 96-well polystyrene microtiter plates and incubated in the presence of carvacrol, fluoxetine, or their combination at defined concentrations for 24 h at 37\u0026deg;C. After incubation, wells were gently washed with PBS to remove planktonic cells. Adherent biofilms were fixed with methanol for 15 min, stained with 0.1% (w/v) crystal violet for 15 min, and then rinsed to remove excess dye. The bound crystal violet was solubilized in 95% ethanol, and absorbance was measured at 570 nm in a microplate reader. The percentage inhibition of biofilm formation was calculated relative to untreated controls (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate, and the results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical comparisons between groups were made using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test. A p-value of less than 0.05 was considered statistically significant. Statistical analysis was conducted using GraphPad Prism software (version 8.0), following procedures outlined in previous reports (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial susceptibility\u003c/h2\u003e \u003cp\u003eClinical MRSA isolates were resistant to tetracycline, trimethoprim-sulfamethoxazole, erythromycin, clindamycin and were susceptible to vancomycin and linezolid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe minimum inhibitory and bactericidal concentrations\u003c/h2\u003e \u003cp\u003eThe MIC and MBC of cefoxitin among MRSA included 4\u0026ndash;8 \u0026micro;g/mL. The MIC and MBC of carvacrol were observed to range between 256 \u0026micro;g/mL and 512 \u0026micro;g/mL, in that order. Moreover, the MIC and MBC of vancomycin respectively included 0.5-2 \u0026micro;g/mL and 1\u0026ndash;4 \u0026micro;g/mL. Moreover, the MIC and MBC of fluoxetine included 128 \u0026micro;g/mL and 256 \u0026micro;g/mL, respectively. The combination of carvacrol and fluoxetine decreased the MIC and MBC of them to 8\u0026ndash;16 \u0026micro;g/mL and 32\u0026ndash;64 \u0026micro;g/mL, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation\u003c/h2\u003e \u003cp\u003eMRSA isolates had strong (n\u0026thinsp;=\u0026thinsp;2) and moderate (n\u0026thinsp;=\u0026thinsp;8) biofilm levels. Carvacrol at 64 \u0026micro;g/mL caused a decrease in the biofilm formation to weak and non-biofilm formation. Moreover, fluoxetine decreased the biofilms to weak levels. The combination of carvacrol and fluoxetine decreased the biofilm levels to no biofilm production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAdhesin genes expression\u003c/h2\u003e \u003cp\u003eIn comparison to the control, carvacrol decreased the expression levels of \u003cem\u003eclfA, clfB, fnbA, fnbB, icaA\u003c/em\u003e genes to 2.9, 2.6, 2, 2 and 1.8 fold respectively which were significant. Moreover, fluoxetine significantly decreased the expression of \u003cem\u003eclfA, clfB, fnbA, fnbB, icaA\u003c/em\u003e genes to 1.9, 1.6, 1.2, 1.4 and 2 fold, respectively. The combinatory subject of carvacrol and fluoxetine caused MRSA adhesin genes \u003cem\u003eclfA, clfB, fnbA, fnbB, icaA\u003c/em\u003e significant decrease to 3.2, 2.9, 2.6, 2, and 1.6 fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study demonstrate that the combination of carvacrol and fluoxetine has synergistic antibacterial and anti-biofilm effects against MRSA. The combination therapy significantly reduced the MIC and MBC of both compounds compared to their individual use. This suggests that carvacrol and fluoxetine may act synergistically to disrupt the bacterial cell membrane and inhibit bacterial growth. The combination therapy may be particularly valuable in overcoming the limitations of conventional antibiotic therapy against MRSA, as it may be effective against MRSA strains that are resistant to conventional antibiotics and may prevent the emergence of resistance (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). our data confirm that carvacrol disrupts the structural integrity of the bacterial cell membrane, thereby interfering with vital cellular processes. The measured MIC (128\u0026ndash;256 \u0026micro;g/mL) and MBC for carvacrol align with earlier reports on its bactericidal properties and its capability to disturb biofilm formation via downregulation of adhesion-related genes (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The study also showed that the combination of carvacrol and fluoxetine effectively inhibited biofilm formation. This is an important finding, as biofilms are a major contributor to the persistence of MRSA infections. Biofilm-protected bacterial cells are more resistant to most antibiotics and host defense systems than their planktonic counterparts (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Our findings demonstrate that fluoxetine alone exhibits moderate antibacterial activity (MIC 64\u0026ndash;128 \u0026micro;g/mL, MBC 128\u0026ndash;256 \u0026micro;g/mL) and can reduce biofilm biomass. Although the precise mechanism underpinning fluoxetine\u0026rsquo;s antibacterial action remains to be fully elucidated, evidence suggests that it may compromise bacterial membrane integrity and interfere with efflux pump activity\u0026mdash;mechanisms that are reminiscent of those seen with other non-antibiotic compounds repurposed for antibacterial use (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The reduction in MIC and MBC values to 8\u0026ndash;16 \u0026micro;g/mL and 32\u0026ndash;64 \u0026micro;g/mL, respectively, suggests that the combination targets multiple bacterial processes concurrently. Carvacrol\u0026rsquo;s ability to perturb the bacterial membrane likely facilitates enhanced intracellular penetration of fluoxetine, which may act by inhibiting bacterial energy metabolism and efflux systems. This dual action results in a significant downregulation of adhesin genes (clfA, clfB, fnbA, fnbB, icaA), thereby impairing the formation and stability of MRSA biofilms, a finding that mirrors previous observations of synergistic natural compound-antibiotic combinations (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). From a clinical perspective, the resistance profiles of the MRSA isolates examined herein are concerning. Given that these strains are unresponsive to multiple frontline antibiotics, the development of alternative or adjunctive therapies is imperative. Although vancomycin and linezolid remain effective, their use is limited by toxicity and the risk of emerging resistance (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Few clinical trials have yet explored the use of carvacrol and fluoxetine in MRSA infections (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). however, our in vitro findings provide a strong mechanistic rationale for further preclinical studies. It is important to consider the potential impact of fluoxetine on the human microbiota. Studies have shown that fluoxetine can inhibit the growth of various microorganisms, including those found in the human gut. Further research is needed to evaluate the long-term effects of fluoxetine on the gut microbiota and its potential implications for human health (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Methodologically, our work adopted standard protocols encompassing bacterial cultivation, MIC determination, time-kill kinetics, qRT-PCR analysis, and biofilm inhibition assays that have been successfully employed in similar studies (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, our investigation reveals that the combination of carvacrol and fluoxetine exhibits powerful synergistic antibacterial and anti-biofilm actions against MRSA. Targeting bacterial membrane structure, intracellular processes, and biofilm-associated gene expression concurrently will enable this combinatorial approach to assist overcome the limits of traditional antibiotics and offer a fresh weapon against multidrug-resistant organisms. This study has various restrictions:\u003c/p\u003e \u003cp\u003eThe study was done in-vitro, hence the findings might not be exactly relevant in clinical environments. This research made use of few MRSA isolates and this can be expanded. Carvacrol and fluoxetine may have several possible uses in the treatment of MRSA infections, including topical application for skin and soft tissue infections, systemic administration for more severe infections, combination with antibiotics to enhance their efficacy, and prophylactic use to prevent MRSA infections in high-risk persons. Investigating the ideal dosage and route of administration, assessing the efficacy and safety in clinical trials, looking at ways to get past resistance to other antibiotics, and figuring out the long-term consequences on the human flora need more study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this study imply that treating MRSA infections with carvacrol and fluoxetine could show great success. Combining this treatment could help to solve problems with antibiotic resistance and biofilm development. Carvacrol and fluoxetine may so efficiently kill MRSA and stop the creation of infections by upsetting the bacterial cell membrane, blocking efflux pumps, and downregulating adhesin gene expression. This combo treatment deserves more research to convert the results into practical use since it shows potential to solve the rising public health issue of MRSA infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was designed and performed by the authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.K.S. and S.T.M. designed the study. . and . performed the work. . and . conceptualized the study. 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Saudi J Biol Sci 27(8):1985\u0026ndash;1992\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Methicillin-resistant Staphylococcus aureus, Biofilms, Carvacrol, Fluoxetine","lastPublishedDoi":"10.21203/rs.3.rs-6614132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6614132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is a leading clinical infectious agent owing to considerable drug resistance and formation of biofilms. In this \u003cem\u003ein vitro\u003c/em\u003e study, we assessed the anti-biofilm and antibacterial activity of carvacrol -a monoterpenoid phenol- and fluoxetine -a selective serotonin reuptake inhibitor-. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined using broth microdilution method. The synergistic effect was performed by checkerboard assay. MRSA isolates were resistant to conventional treatments such as tetracycline, trimethoprim-sulfamethoxazole, erythromycin, and clindamycin but sensitive to vancomycin and linezolid. They also had MIC and MBC levels of 256 \u0026micro;g/mL and 512 \u0026micro;g/mL against carvacrol, respectively. Moreover, these scores ranged respectively 128 \u0026micro;g/mL \u0026minus;\u0026thinsp;256 \u0026micro;g/mL against fluoxetine. Combining the two test agents clearly lowered the computed MICs and MBCs for carvacrol and fluoxetine. Furthermore, this combo resulted in notable downregulation of important adhesin genes and total suppression of biofilm development, therefore offering strong proof for a combined approach to reduce MRSA infections.\u003c/p\u003e","manuscriptTitle":"Synergistic antibacterial and anti-biofilm effects of carvacrol and fluoxetine against Methicillin-resistant Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 13:07:21","doi":"10.21203/rs.3.rs-6614132/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3089e050-48ce-4174-81a8-6478c79654bd","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-27T09:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 13:07:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6614132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6614132","identity":"rs-6614132","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}