Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Therapeutic Evaluation of Reserpine against Biofilm-forming Staphylococcus aureus of Bovine Mastitis in a Murine Model

J
Jayant P. Hole1
0009-0009-4902-1519
K
Kalyani Putty2
0000-0002-9451-3047
S
Shivajyothi Jemmigumpula2
0000-0003-2528-6431
K
Krishna Satya Alapati1,*
0000-0001-6353-178X
1Department of Biotechnology, Acharya Nagarjuna University, Guntur-522 510, Andhra Pradesh, India.
2Department of Veterinary Biotechnology, College of Veterinary Science, Sri PV Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad-500 030, Telangana, India.

ABSTRACT

Background: Within the mammary gland, S. aureus establishes biofilm communities that contribute to treatment failure, recurrence and increased antimicrobial use in bovine mastitis. Strategies that disrupt biofilm-associated survival mechanisms may therefore improve therapeutic outcomes while supporting antimicrobial stewardship.

Methods: In this study, we integrated robust computational screening of seventeen biofilm-associated S. aureus proteins implicated in adhesion, extracellular matrix production and transcriptional regulation against selected plant-derived polyphenolic compounds, followed by in vitro and in vivo validation to identify adjunctive therapeutic strategies for mastitis management.

Result: Reserpine emerged as a multi-target candidate, with predicted interactions across key biofilm determinants, including adhesins and regulatory proteins. In vitro, reserpine significantly reduced biofilm biomass (68.6-71.5% inhibition) and decreased viable bacterial counts by >95%, indicating disruption of both biofilm matrix integrity and bacterial persistence. In a murine mastitis model, reserpine monotherapy modestly reduced intramammary bacterial load. However, when combined with ceftiofur, bacterial burden declined by approximately three log units relative to untreated controls, accompanied by reduced inflammatory infiltration and improved preservation of mammary architecture. Targeting intrinsic persistence pathways alongside conventional antibiotic therapy may present a clinically relevant approach for improving therapeutic outcomes in biofilm driven bovine mastitis while reducing reliance on escalating antimicrobial use in dairy systems.

INTRODUCTION

In addition to its substantial economic impact on dairy industry, bovine mastitis has also become a one health concern, as sustained antimicrobial use in dairy systems contributes to the selection of antimicrobial resistance, drug residues in milk and further spread of resistance determinants into the environment (Halasa et al., 2007; Oliver et al., 2011; Bhakat et al., 2025). Among mastitis pathogens, Staphylococcus aureus is particularly problematic; it causes both clinical and subclinical intramammary infections by its ability to persist within mammary tissue, evade host immunity and recur despite therapy (Brouillette et al., 2003; Archer et al., 2011). One of the key factors that contribute to this persistence is the biofilm formation (Sandhu et al., 2025). In the mammary gland, S. aureus develops structured, surface-associated communities wrapped in a self-produced extracellular matrix comprised of polysaccharides, proteins, extracellular DNA and host-derived factors. This matrix enables the generation of mechanical stability, as well as spatial gradients in oxygen, nutrients and antimicrobial exposure, which promote phenotypic heterogeneity and metabolic dormancy, both central determinants of antimicrobial tolerance (Otto, 2018; Flemming et al., 2023). A coordinated system of adhesins, autolysins and extracellular matrix-binding proteins, including icaADBC operon, which encode poly-β-1,6-N-acetylglucosamine synthesis, are involved in the development of biofilms (Cramton et al., 1999; Foster et al., 2014).
       
Notably, biofilm-associated survival isn’t completely regulated by classical antimicrobial resistance genes. Recent studies suggest that tolerance, a reversible physiological phenomenon, permits microbial survival at antibiotic concentrations exceeding planktonic MIC values in the absence of stable resistance mutations (Theuretzbacher et al., 2020). This tolerant phenotype is triggered by upregulation of efflux pumps, decreased metabolic rate, altered cell-cell wall behaviour and quorum-sensing modulation (Costa et al., 2013; Otto, 2018). Repeated and empirical antibiotic usage to address tolerance can inadvertently select for biofilm-adapted populations, complicating therapeutic interventions (Oliver et al., 2011). These challenges have led to a revival of antibiofilm approaches that aim at targeting pathogenic fitness (Clatworthy et al., 2007). These strategies are meant to destabilize adhesion, assembly of the matrix, or regulatory pathways and sensitize bacteria to host defences and standard antibiotics. Gram-positive pathogens have been shown to be susceptible to plant-derived polyphenols and small molecules, such as quercetin, resveratrol, ursolic acid and reserpine, exhibiting antibiofilm effects through the modulation of quorum sensing, efflux activity and extracellular matrix production (Borges et al., 2018; Shiva et al., 2018; Zhang et al., 2020; Greeshma et al., 2024).
       
Computational structural biology has made it possible to systematically prioritise biofilm-associated targets. Structural prediction can be done with reasonable accuracy in the absence of crystallographic data using homology modelling platforms (viz., Swiss-Model) (Waterhouse et al., 2018), whereas evaluation of either the stereochemical or structural reliability is done with validation programs (viz., PROCHECK and VERIFY3D) (Laskowski et al., 1993; Eisenberg et al., 1997). Sharp molecular docking systems, including HDOCK, can be used to perform high-throughput prediction of protein-ligand interactions to accelerate the rational screening of potential antibiofilm compounds (Yan et al., 2017; Yan et al., 2020). The combination of in silico modelling combined with in vitro and in vivo validation represents a potent, mechanistically guided therapeutic discovery pipeline. In this study, we adopted a multi-tiered approach where we (i) evaluated interactions between selected plant derived polyphenolic compounds and key biofilm-associated proteins of S. aureus using molecular docking and (ii) experimentally assessed the antibiofilm and antibiotic-potentiating activity of apparently potent polyphenol in vitro and in vivo in a murine mastitis model. The main objective of the study was to characterise potential adjunctive therapies to interrupt biofilm-mediated persistence and improve the antimicrobial efficacy.

MATERIALS AND METHODS

Ethical standards
 
All animal studies have been approved by the Institutional Animal Ethics Committee (IAEC/VL/09-2024-2025; dated 8/11/2024).
 
Bacterial strains used in the study
 
This study was conducted between August 2025 and January 2026. All experiments in the current study were conducted at Acharya Nagarjuna University, Guntur and PVNR Telangana Veterinary University, Hyderabad, India. The Staphylococcus aureus strain (S. aureus strain #C5) used in this study was previously isolated from a cow with clinical mastitis and was characterised as a strong biofilm-forming strain (data not shown). The bacterial strains were maintained on tryptic soy agar or broth and on mannitol salt agar (Himedia, India) and grown at 37°C for 12 h. Prior to experimentation, the cultures were revived from -80°C, phenotypically reconfirmed and prepared for subsequent analyses.
 
Target protein identification, ligand selection and molecular docking
 
Seventeen biofilm-associated proteins of S. aureus implicated in adhesion, extracellular polymeric substance production, quorum sensing and structural stability were selected as potential molecular targets based on their established or predicted roles in biofilm formation (based on a literature search on PubMed). Four plant derived polyphenolic compounds with reported antibiofilm and antimicrobial properties (based on PubMed literature search), viz., reserpine, quercetin, ursolic acid and resveratrol, were selected as ligands for in silico screening. Protein-ligand docking was performed using the HDOCK v 28.5.2 web server, which combines template-based and ab initio docking algorithms (Yan et al., 2017; Yan et al., 2020). Docking was performed using default global docking parameters without defined binding sites, to avoid bias. Binding affinities were estimated using HDOCK’s v 28.5.2 scoring function, integrating shape complementarity, electrostatic interactions, desolvation energy and statistical potentials. Complexes were ranked based on docking scores, with more negative values indicating stronger predicted binding affinity. Polyphenols demonstrating strong binding affinity with more than one biofilm associated proteins with stable interaction networks were considered promising therapeutic agents.
 
Antagonistic effect of reserpine on S. aureus biofilms in vitro
 
The antagonistic effect of biofilm formation by Reserpine was studied using different concentrations (500 μg/ml and 1000 μg/ml) and compared with the control group that did not receive any treatment. For this, an overnight culture of biofilm-forming S. aureus (strain #C5) was incubated for 12 h in tryptic soy broth supplemented with either 500 μg/ml or 1000 μg/ml of Reserpine (Sigma-Aldrich, USA CAS Number: 50-55-5). The inhibitory effect was studied by the MTP method as described before (Christensen et al., 1985; Stepanović et al., 2000). Briefly, 1:200 diluted aliquots (200 µL) of the prepared suspensions were inoculated in triplicate (in two sets) into sterile, flat-bottom 96-well microtiter plates, while sterile broth without inoculum served as a negative control. The plates were incubated at 37°C for 24 h. Following incubation, planktonic cells were removed and the wells were gently washed three times with sterile PBS to remove non-adherent bacteria. One set of triplicate wells was then plated on TSA plates for colony counting. Biofilm formation ability was determined in the other set of triplicate wells using the crystal violet staining method as follows. The adherent biofilm was fixed with methanol for 15 min at room temperature and stained with 0.5% crystal violet for 15 min. Excess stain was removed by washing under running water and the bound dye was solubilised using 95% ethanol. Optical density (OD) was measured at 590 nm using a BioTek microplate ELISA reader. The anti-biofilm effect was assessed by dividing the S. aureus isolates into three groups: control isolates, Reserpine-treated isolates at 500 µg/ml and Reserpine-treated isolates at 1000 μg/ml. The inhibitory rates were calculated using the formula:


Mice mastitis model
 
In vivo studies were carried out using a mouse mastitis model (Brouillette et al., 2004; Notebaert et al., 2006). Lactating C57BL6 mice of 12 to 14 days after offspring birth were used for the study. The pups were removed 1 h before challenge with S. aureus (strain #C5) (200 CFU/gland) via intramammary inoculation in both the left and right abdominal mammary glands. Animals were randomly assigned to five experimental groups (n = 8 per group) (Fig 2A). Group 1 served as the uninfected, untreated negative control. Group 2 comprised infected, PBS-treated animals and served as the disease control. Group 3 (infected) received reserpine at a dose of 500 µg/ml. Group 4 (infected) was treated with ceftiofur (1 mg/Kg b.wt) alone and served as the antibiotic control group. Group 5 (infected) was administered 500 µg/mg reserpine in combination with ceftiofur (1mg/Kg b.wt). All animals were observed periodically for clinical signs, behavioural changes and local inflammatory reactions. At 16 hours post-infection, animals were euthanised and mammary gland tissues were aseptically collected and homogenised. The homogenates were serially diluted in sterile PBS and plated on nutrient agar to quantify viable bacterial counts (CFU). Additionally, mammary tissues were collected for histopathological analysis.
 
Statistical analysis
 
All statistical analyses, unless otherwise stated, were conducted using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA). The error bars in the graphs show the standard error of the mean. Unpaired t-test and one-way ANOVA were used to compare the groups. Assays were performed in triplicate and the data were expressed as the mean of 3 independent experiments. The results were considered statistically significant at P<0.001.

RESULTS AND DISCUSSION

In silico evidence reveals that reserpine is a broad-spectrum inhibitor of S. aureus biofilm machinery
 
The development of biofilms in S. aureus involves a multi-factorial and tightly coordinated process that incorporates the adhesion of cells to the surface, the synthesis of the extracellular matrix, autolysis-driven remodelling and global transcriptional regulation (Otto, 2008; Archer et al., 2011). This phenotype is closely linked with chronic infection, antibiotic resistance and treatment failure in bovine mastitis (Melchior et al., 2006; Song et al., 2024). Since disrupting a single determinant is unlikely to destroy a strong system, we focused on identifying multiple functional nodes in the biofilm network. Based on existing literature sources in Pubmed, 17 experimentally validated S.aureus biofilm-associated proteins pertinent to bovine mastitis were shortlisted. These comprised important adhesins (ClfA, ClfB, Cna, FnbA), which mediate attaching to host extracellular matrix constituents (fibrinogen and fibronectin) (Foster et al., 2014); the polysaccharide intercellular adhesion operon proteins (IcaA-D), central to poly-b-1,6-N-acetylglucosamine (PNAG) matrix synthesis (Cramton et al., 1999); autolysins; and other surface-associated virulence factors (Eno, SdrG, Esp and SesA) (Melchior et al., 2009). To examine therapeutic vulnerabilities in this network, we analysed four FDA-approved bioactive plant derived polyphenols with reported antibiofilm activity, including quercetin, resveratrol, resperpin and ursolic acid (Lionta et al., 2014; Qin et al., 2014; Khameneh et al., 2015; Musini et al., 2023; He et al., 2025). Table 1 summarises binding affinities and interaction profiles of the selected polyphenols against the 17 target proteins. Reserpine exhibited a more pronounced predicted binding energy than the rest of the compounds tested across the target panel. Reserpine was also found to have a broad range of interaction with adhesion molecules, matrix biosynthetic enzymes and regulatory proteins as opposed to showing an isolated affinity to a restricted number of proteins. It is important to note that reserpine demonstrated a high predicted interaction with ClfA, FnbA and Cna, which are major adhesins needed to colonise host tissues (Houston et al., 2011; Foster et al., 2014). It also displayed high-affinity binding to IcaA-D complex that is the core in PNAG production and biofilm structural integrity (Cramton et al., 1999). Notably, reserpine also interacted with a transcriptional regulator, SarA whose inactivation has been found to severely affect biofilm formation and virulence (Beenken et al., 2003; Valle et al., 2003). Bap and EbhA, proteins that have been found to mediate biofilm maturation and persistence in mastitis isolates, were also predicted to interact with reserpine in a stable manner (Cucarella et al., 2001). Quercetin, resveratrol and ursolic acid, in turn, exhibited a weaker or target-restricted pattern of interaction, which is in line with previously reported mechanisms of action of the compounds (Ouyang et al., 2016; Borges et al., 2018). Together, this docking information indicates that reserpine exhibits multi-target interaction profile which concomitantly regulates adhesion, matrix biosynthesis and regulatory pathways needed to sustain and initiate biofilm in S. aureus. Since biofilm resilience is a multi network mechanism (Otto, 2018), the ability to disrupt the functions of several nodes can provide more translational potential in the case of bovine mastitis.

Table 1: Major biofilm-associated genes of S. aureus and their encoded proteins’ docking values with the selected polyphenols.


 
Reserpine interferes with S. aureus biofilm and decreases viable bacterial population in vitro
 
To experimentally validate (in vitro) the ability of reserpine to interfere with the biofilm phenotype, biofilm-forming S. aureus was exposed to two different concentrations: 1000 mg/mL (MIC) and 500 mg/mL (1/2 MIC) concentrations, both of which have been reported to inhibit gram-positive biofilms, including S. aureus (Kaatz et al., 2003). In the microtiter plate assay, biofilm biomass was significantly reduced by reserpine: at 500 mg/mL, reserpine inhibited biofilm growth by 68.6 per cent of the control and at 1000 mg/mL, it inhibited biofilm growth by 71.5 per cent of the control (p<0.001) (Fig 1A). The two doses caused significant suppression, however the small difference observed between concentrations is typical of a threshold or plateau effect commonly seen with regulatory pathways being targeted (Otto, 2018). Significantly, the disruption of biofilm was paralleled with a significant reduction in the viable bacterial counts after the treatment with reserpine. At 500 mg/mL, bacterial loads decreased from 7.32 log10 CFU/mL to 5.97 log10 CFU/mL (95.53% reduction) and at 1000 mg/mL from 7.32 log10 CFU/mL to 5.77 log10 CFU/mL (97.18% reduction) (Fig 1B). This simultaneous decrease in both biomass and CFU implies that reserpine destabilises both the form and viability of biomasses instead of merely dispersing the extracellular matrix material without affecting the confined cells, a drawback reported with a number of antibiofilm agents (Archer et al., 2011; Borges et al., 2018). These results are further consistent with the previously shown inhibition of NorA-mediated efflux by reserpine in S. aureus (Kaatz et al., 2003). Efflux systems play a role in creating antibiotic resistance as well as in biofilm maturation and stress adaptation which can be inhibited to disrupt the formation of biofilms and sensitise the embedded cells (Costa et al., 2013; Schindler and Kaatz, 2016). Combined with our in silico data of multi-target binding with adhesin and regulatory proteins, the current data is consistent with a model where reserpine disrupts biofilm stability at a variety of functional nodes. Since biofilm-related tolerance has the potential to raise the effective concentration of antibiotics by many orders of magnitude (Archer et al., 2011), the significant drop in viable counts in the present case is potentially translationally relevant.

Fig 1: Antibiofilm (A) and bactericidal (B) activities of reserpine (MIC and ½ MIC levels).


 
Reserpine has a significant therapeutic potential in the mouse mastitis model
 
To determine whether reserpine would translate its in vitro antibiofilm activities into therapeutic outcome in vivo we used a murine mastitis model. The untreated animals showed rapid growth in bacteria after 16 hours of S. aureus challenge with a maximum of 7.85 log10 CFU/g of mammary tissue. Such bacterial load is in line with the severity of acute mastitis at an early stage and demonstrates the capacity of S. aureus to form high-density populations in mammary alveoli, in which biofilm-like structures and deposition of the extracellular matrix increase survival (Brouillette et al., 2003; Brouillette et al., 2004; Archer et al., 2011). Monotherapy with reserpine (500 mg/mL) decreased the bacteria to 6.53 log10 CFU/g (P<0.001). Even though this was significant, this decrease was minor as compared to ceftiofur monotherapy, which reduced the burden to 5.62 log10 CFU/g. Ceftiofur is a third-generation cephalosporin, which binds to penicillin-binding proteins and prevents cross-linking of peptidoglycan, but its activity in biofilm-associated populations is compromised by limited penetration, changes in metabolic states and tolerance-based mechanisms (Archer et al., 2011; Otto, 2018). Strikingly, combination therapy (reserpine (500 μg/mL) + ceftiofur) further reduced the bacterial burden to 4.83 log10 CFU/g (P<0.0001), representing an approximate 3-log reduction relative to untreated controls (Fig 2B). This could be indicative of functional synergy that is mediated by the effect of interference with inherent tolerance mechanisms and not solely additive bactericidal action.

Fig 2: Therapeutic effects of reserpine on S. aureus induced acute mouse mastitis.


       
Mechanically, efflux pumps like NorA have functions other than those of classical multidrug resistance. In S. aureus, efflux activity induces quorum sensing, intracellular homeostasis, redox balance and stress adaptation, which are part of biofilm maturation and maintenance (Costa et al., 2013). NorA has been shown to be inhibited by reserpine, which has been found to decrease resistance to various classes of antibiotics through heightened retention of drugs in intracellular locations (Kaatz et al., 2003; Schindler and Kaatz, 2016). In the context of β-lactam therapy, enhanced intracellular stress combined with impaired cell wall synthesis may amplify bactericidal signalling cascades. Furthermore, efflux inhibition may reduce the capacity of biofilm-embedded cells to export toxic metabolic by-products, increasing susceptibility under nutrient-limited mammary conditions. This mechanistic explanation was corroborated by histopathologies (Fig 2C). Infected glands, which were untreated, exhibited significant alveolar wall thickening, congestion of the vascularity and intense neutrophil infiltration in the stroma and luminal regions, which are the signs of acute inflammatory mastitis (Rainard and Riollet, 2006). This type of inflammatory reactions, as crucial as it is in clearing bacteria, is also implicated in the destruction of tissues and disrupted glandular activity. Ceftiofur or reserpine monotherapy showed partial inflammatory pathology inhibition, which is in line with the decreased bacterial burden. Nevertheless, alveolar architecture and neutrophilic infiltration were most preserved with the combination treatment group (Fig 2C). The simultaneous decrease in CFU and histological damage suggests augmented bacterial clearance and limited persistent inflammatory signalling. Increased neutrophil recruitment and cytokine generation induced by persistent antigen exposure is known to be caused by biofilm-associated infections (Otto, 2018). Notably, reserpine itself failed to achieve complete bacteriological eradication, which supports its probable, though not exclusive, role as an adjuvant rather than a primary antimicrobial. This is consistent with the modern antibiotic-potentiation approaches, which are intended to sensitise pathogens to the already available antibiotics, but do not substitute them (Clatworthy et al., 2007; Theuretzbacher et al., 2020). Targeting tolerance pathways is a logical treatment in biofilm-related mastitis, where biofilm mediated tolerance instead of classical resistance can be the basis of therapeutic failure. Even though bovine mammary physiology cannot be completely recapitulated in murine models, they can give mechanistic understanding of the interaction of host-pathogen-drug interactions in vivo (Brouillette et al., 2004). The observed ~3-log reduction with combination therapy supports further pharmacodynamic and translational investigation in bovine systems.

CONCLUSION

Collectively, these findings demonstrate that reserpine can functionally disrupt biofilm-associated persistence and significantly potentiate β-lactam activity in experimental S. aureus mastitis. Targeting intrinsic tolerance mechanisms may therefore represent a viable strategy to enhance antimicrobial efficacy in biofilm-driven infections.

ACKNOWLEDGEMENT

No specific funding was received for this study. The authors would like to acknowledge PVNRTVU, Hyderabad and Acharya Nagarjuna University, Guntur, for providing resources to conduct the study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal studies have been approved by the Institutional Animal Ethics Committee (IAEC/VL/09-2024-2025; dated 8/11/2024).

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Archer, N.K., Mazaitis, M.J., Costerton, J.W., Leid, J.G., Powers, M.E. and Shirtliff, M.E. (2011). Staphylococcus aureus biofilms: Properties, regulation and roles in human disease. Virulence. 2: 445-459. doi: 10.4161/viru.2.5.17724.

  2. Beenken, K.E., Blevins, J.S. and Smeltzer, M.S. (2003). Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infection and Immunity. 71: 4206-4211. doi: 10.1128/ IAI.71.8.4206-4211.2003.

  3. Bhakat, C., Singh, A.K., Mandal, A., Karunakaran, M., Mohammad, A., Mandal, D.K., Rai, S., Chatterjee, A., Mondal, M. and Dutta, T.K. (2025). Udder health maintenance to augment milk production in dairy cattle: A review. Indian Journal of Animal Research. 59(7): 1085-1091. doi: 10.18805/IJAR.B-4816.

  4. Borges, A., Saavedra, M.J. and Simoes, M. (2018). Insights on antimicrobial resistance, biofilms and the use of phytochemicals as new antimicrobial agents. Current Medicinal Chemistry.  25: 1-21. doi: 10.2174/0929867324666170920151121.

  5. Brouillette, E., Talbot, B.G. and Malouin, F. (2003). The pathogenesis of Staphylococcus aureus mastitis. Journal of Mammary Gland Biology and Neoplasia. 8: 323-339. doi: 10.1023/ B:JOMG.0000010038.32054.16.

  6. Brouillette, E., Lacasse, P., Shkreta, L., Belanger, J., Grondin, G., Diarra, M.S., Fournier, S. and Talbot, B.G. (2004). DNA microarray analysis of the host response to Staphylococcus aureus infection in the mouse mammary gland. Infection and Immunity. 72: 3350-3360. doi: 10.1128/IAI.72.6.3350- 3360.2004.

  7. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M. and Beachey, E.H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology. 22: 996-1006. doi: 10.1128/ jcm.22.6.996-1006.1985.

  8. Clatworthy, A.E., Pierson, E. and Hung, D.T. (2007). Targeting virulence: A new paradigm for antimicrobial therapy. Nature Chemical Biology. 3: 541-548. doi: 10.1038/ nchembio.2007.24.

  9. Costa, S.S., Viveiros, M., Amaral, L. and Couto, I. (2013). Multidrug efflux pumps in Staphylococcus aureus: An update. Frontiers in Microbiology. 4: 178. doi: 10.3389/fmicb. 2013.00178.

  10. Cramton, S.E., Gerke, C., Schnell, N.F., Nichols, W.W. and Gotz, F. (1999). The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infection and Immunity. 67: 5427-5433. doi: 10.1128/IAI.67.10.5427-5433.1999.

  11. Cucarella, C., Solano, C., Valle, J., Amorena, B., Lasa, I. and Penadés, J.R. (2001). Bap, a Staphylococcus aureus surface protein involved in biofilm formation. Journal of Bacteriology. 183: 2888-2896. doi: 10.1128/JB.183.9. 2888-2896.2001.

  12. Eisenberg, D., Lüthy, R. and Bowie, J.U. (1997). VERIFY3D: Assessment of protein models with three dimensional profiles. Methods in Enzymology. 277: 396-404. doi: 10.1016/S0076- 6879(97)77022-8.

  13. Flemming, H.C. and Wuertz, S. (2023). Bacteria and archaea on earth and their abundance in biofilms. Nature Reviews Microbiology. 21: 513-528. doi: 10.1038/s41579-023- 00831-0.

  14. Foster, T.J., Geoghegan, J.A., Ganesh, V.K and Hook, M. (2014). Adhesion, invasion and evasion: The many functions of Staphylococcus aureus surface proteins. Nature Reviews Microbiology. 12: 49-62. doi: 10.1038/nrmicro3161.

  15. Greeshma, A.J., Pushpa Ramani, R.N., Lakshmi, K.K. and Rao, S.T. (2024). Efficacy of resveratrol and ursolic acid on biofilm inhibition and antimicrobial resistance of Streptococcus uberis. Indian Journal of Animal Research. 58(12): 2177- 2183. doi: 10.18805/IJAR.B-4697.

  16. Halasa, T., Huijps, K., Osteras, O. and Hogeveen, H. (2007). Economic effects of bovine mastitis. Journal of Dairy Science. 90: 4096-4108. doi: 10.3168/jds.2007-0132.

  17. He, J., Cui, Y., Liu, Y., Mao, J., Dong, Y., Yao, R., Yang, D., Fan, P. and Xue, J. (2025). Resveratrol inhibits the formation of Staphylococcus aureus biofilms by reducing PIA, eDNA release and ROS production. Frontiers in Veterinary Science. 12: 1594239. doi: 10.3389/fvets.2025.1594239.

  18. Houston, P., Rowe, S.E., Pozzi, C., Waters, E.M. and O’Gara, J.P. (2011). Essential role for the major autolysin in the fibronectin- binding protein-mediated Staphylococcus aureus biofilm phenotype. Infection and Immunity. 79: 1153-1165. doi: 10.1128/IAI.01158-10

  19. Kaatz, G.W., Moudgal, V.V., Seo, S.M. and Kristiansen, J.E. (2003). Inhibition of NorA-mediated efflux by reserpine in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 47: 329-333. doi: 10.1128/AAC.47.1.329-333.2003.

  20. Khameneh, B., Iranshahy, M., Ghandadi, M., Atashbeyk, D.G., Fazly Bazzaz, B.S. and Iranshahi, M. (2015). Investigation of the antibacterial activity and efflux pump inhibitory effect of plant alkaloid reserpine against Staphylococcus aureus. Drug Development and Industrial Pharmacy. 41: 989-994. doi: 10.3109/03639045.2014.920025.

  21. Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993). Procheck: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography.  26: 283-291. doi: 10.1107/S0021889892009944.

  22. Lionta, E., Spyrou, G., Vassilatis, D.K. and Cournia, Z. (2014). Structure-based virtual screening for drug discovery. Current Topics in Medicinal Chemistry. 14: 1923-1938. doi: 10.2174/1568026614666140923120832.

  23. Melchior, M.B., Vaarkamp, H. and Fink-Gremmels, J. (2006). Biofilms: A role in recurrent mastitis infection? Veterinary Journal. 171: 398-407. doi: 10.1016/j.tvjl.2005.01.006.

  24. Melchior, M.B., van Osch, M.H.J., Graat, R.M., van Duijkeren, E., Mevius, D.J., Nielen, M., Gaastra, W. and Fink-Gremmels, J. (2009). Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates. Veterinary Microbiology. 134: 206-213. doi: 10.1016/j.vetmic.2008.09.031.

  25. Musini, A., Ponnusamy, K. and Shanmugam, K. (2023). Antibiofilm activity of quercetin against drug-resistant Staphylococcus aureus. Journal of Applied Microbiology. 134: lxad123. doi: 10.1093/jambio/lxad123.

  26. Notebaert, S. and Meyer, E. (2006). Mouse models to study the pathogenesis and control of bovine mastitis. Veterinary Research. 37: 409-421. doi: 10.1051/vetres:2006003.

  27. Oliver, S.P., Murinda, S.E. and Jayarao, B.M. (2011). Impact of antibiotic use in adult dairy cows on antimicrobial resistance. Journal of Dairy Science. 94: 569-583. doi: 10.3168/ jds.2010-3505.

  28. Ouyang, J., Sun, F., Feng, W., Sun, Y., Qiu, X., Xiong, L., Liu, Y., Chen, Y. and Li, H. (2016). Quercetin is an effective inhibitor of Staphylococcus aureus biofilm formation. Journal of Medical Microbiology. 65: 1159-1167. doi: 10.1099/jmm.0.000338.

  29. Otto, M. (2008). Staphylococcal biofilms. Current Topics in Microbiology and Immunology. 322: 207-228. doi: 10.1007/978-3-540- 75418-3_10.

  30. Otto, M. (2018). Staphylococcal biofilms and their role in antibiotic resistance. Microbiology Spectrum. 6: 1-8. doi: 10.1128/ microbiolspec.GPP3-0023-2018.

  31. Qin, N., Tan, X., Jiao, Y., Liu, L., Zhao, W., Yang, S. and Jia, A. (2014). RNA-seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid. Scientific Reports. 4: 5467. doi: 10.1038/srep05467.

  32. Rainard, P. and Riollet, C. (2006). Innate immunity of the bovine mammary gland. Veterinary Research. 37: 369-400. doi: 10.1051/vetres:2006007.

  33. Sandhu, J., Chandra, M., Kaur, G., Narang, D. and Arora, A.K. (2025). Studies on the efficacy of phage in controlling Staphylococcus aureus biofilm phenotype associated with mastitis. Indian Journal of Animal Research. doi: 10.18805/IJAR.B-5371.

  34. Schindler, B.D. and Kaatz, G.W. (2016). Multidrug efflux pumps of gram-positive bacteria. Antimicrobial Agents and Chemotherapy. 60: 500-509. doi: 10.1128/AAC.01774-15.

  35. Shiva, J.J., Putty, K., Narasimha Reddy, Y., Dhanalakshmi, K. and Umair, M.A.H. (2018). Antagonistic effect of ursolic acid on Staphylococcus aureus biofilms and enhancement of antibiotic susceptibility. Veterinary World. 11: 1440- 1444. doi: 10.14202/vetworld.2018.1440-1444.

  36. Song, X., Zhang, D., Li, B., Zhou, P. and Huang, S.Y. (2024). Biofilm formation and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis. Journal of Animal Science and Biotechnology. 15: 45. doi: 10.1186/s40104- 024-00945-2.

  37. Stepanović, S., Vuković, D., Dakić, I., Savić, B. and Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods. 40: 175-179. doi: 10.1016/S0167-7012(00)00122-6.

  38. Theuretzbacher, U., Bush, K., Harbarth, S., Paul, M., Rex, J.H., Tacconelli, E., Thwaites, G.E., Klugman, K.P. and Carmeli, Y. (2020). Critical analysis of antibacterial agents in clinical development. Nature Reviews Microbiology. 18: 275-287. doi: 10.1038/s41579-019-0288-4.

  39. Valle, J., Toledo-Arana, A., Berasain, C., Ghigo, J.M., Amorena, B., Penadés, J.R. and Lasa, I. (2003). SarA and not óB is essential for biofilm development by Staphylococcus aureus. Molecular Microbiology. 48: 1075-1087. doi: 10.1046/j.1365-2958.2003.03493.x.

  40. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. and Schwede, T. (2018). Swiss- Model: Homology modelling of protein structures and complexes. Nucleic Acids Research. 46: W296-W303. doi: 10.1093/nar/gky427.

  41. Yan, Y., Zhang, D., Zhou, P., Li, B. and Huang, S.Y. (2017). HDOCK: A web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy. Nucleic Acids Research. 45: W365-W373. doi: 10.1093/nar/gkx407.

  42. Yan, Y., Tao, H., He, J. and Huang, S.Y. (2020). The HDOCK server for integrated protein-protein docking. Nature Protocols. 15: 1829-1852. doi: 10.1038/s41596-020-0312-x.

  43. Zhang, L., Liang, E., Cheng, Y., Mahmood, T., Ge, F., Zhou, K., Bao, M., Lv, L., Yi, J. and Wang, J. (2020). Is combined medication with natural medicine a promising therapy for bacterial biofilm infection? Biomedicine and Pharmacotherapy. 128: 110184. doi: 10.1016/j.biopha.2020.110184.
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
18
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Therapeutic Evaluation of Reserpine against Biofilm-forming Staphylococcus aureus of Bovine Mastitis in a Murine Model

    J
    Jayant P. Hole1
    0009-0009-4902-1519
    K
    Kalyani Putty2
    0000-0002-9451-3047
    S
    Shivajyothi Jemmigumpula2
    0000-0003-2528-6431
    K
    Krishna Satya Alapati1,*
    0000-0001-6353-178X
    1Department of Biotechnology, Acharya Nagarjuna University, Guntur-522 510, Andhra Pradesh, India.
    2Department of Veterinary Biotechnology, College of Veterinary Science, Sri PV Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad-500 030, Telangana, India.

    ABSTRACT

    Background: Within the mammary gland, S. aureus establishes biofilm communities that contribute to treatment failure, recurrence and increased antimicrobial use in bovine mastitis. Strategies that disrupt biofilm-associated survival mechanisms may therefore improve therapeutic outcomes while supporting antimicrobial stewardship.

    Methods: In this study, we integrated robust computational screening of seventeen biofilm-associated S. aureus proteins implicated in adhesion, extracellular matrix production and transcriptional regulation against selected plant-derived polyphenolic compounds, followed by in vitro and in vivo validation to identify adjunctive therapeutic strategies for mastitis management.

    Result: Reserpine emerged as a multi-target candidate, with predicted interactions across key biofilm determinants, including adhesins and regulatory proteins. In vitro, reserpine significantly reduced biofilm biomass (68.6-71.5% inhibition) and decreased viable bacterial counts by >95%, indicating disruption of both biofilm matrix integrity and bacterial persistence. In a murine mastitis model, reserpine monotherapy modestly reduced intramammary bacterial load. However, when combined with ceftiofur, bacterial burden declined by approximately three log units relative to untreated controls, accompanied by reduced inflammatory infiltration and improved preservation of mammary architecture. Targeting intrinsic persistence pathways alongside conventional antibiotic therapy may present a clinically relevant approach for improving therapeutic outcomes in biofilm driven bovine mastitis while reducing reliance on escalating antimicrobial use in dairy systems.

    INTRODUCTION

    In addition to its substantial economic impact on dairy industry, bovine mastitis has also become a one health concern, as sustained antimicrobial use in dairy systems contributes to the selection of antimicrobial resistance, drug residues in milk and further spread of resistance determinants into the environment (Halasa et al., 2007; Oliver et al., 2011; Bhakat et al., 2025). Among mastitis pathogens, Staphylococcus aureus is particularly problematic; it causes both clinical and subclinical intramammary infections by its ability to persist within mammary tissue, evade host immunity and recur despite therapy (Brouillette et al., 2003; Archer et al., 2011). One of the key factors that contribute to this persistence is the biofilm formation (Sandhu et al., 2025). In the mammary gland, S. aureus develops structured, surface-associated communities wrapped in a self-produced extracellular matrix comprised of polysaccharides, proteins, extracellular DNA and host-derived factors. This matrix enables the generation of mechanical stability, as well as spatial gradients in oxygen, nutrients and antimicrobial exposure, which promote phenotypic heterogeneity and metabolic dormancy, both central determinants of antimicrobial tolerance (Otto, 2018; Flemming et al., 2023). A coordinated system of adhesins, autolysins and extracellular matrix-binding proteins, including icaADBC operon, which encode poly-β-1,6-N-acetylglucosamine synthesis, are involved in the development of biofilms (Cramton et al., 1999; Foster et al., 2014).
           
    Notably, biofilm-associated survival isn’t completely regulated by classical antimicrobial resistance genes. Recent studies suggest that tolerance, a reversible physiological phenomenon, permits microbial survival at antibiotic concentrations exceeding planktonic MIC values in the absence of stable resistance mutations (Theuretzbacher et al., 2020). This tolerant phenotype is triggered by upregulation of efflux pumps, decreased metabolic rate, altered cell-cell wall behaviour and quorum-sensing modulation (Costa et al., 2013; Otto, 2018). Repeated and empirical antibiotic usage to address tolerance can inadvertently select for biofilm-adapted populations, complicating therapeutic interventions (Oliver et al., 2011). These challenges have led to a revival of antibiofilm approaches that aim at targeting pathogenic fitness (Clatworthy et al., 2007). These strategies are meant to destabilize adhesion, assembly of the matrix, or regulatory pathways and sensitize bacteria to host defences and standard antibiotics. Gram-positive pathogens have been shown to be susceptible to plant-derived polyphenols and small molecules, such as quercetin, resveratrol, ursolic acid and reserpine, exhibiting antibiofilm effects through the modulation of quorum sensing, efflux activity and extracellular matrix production (Borges et al., 2018; Shiva et al., 2018; Zhang et al., 2020; Greeshma et al., 2024).
           
    Computational structural biology has made it possible to systematically prioritise biofilm-associated targets. Structural prediction can be done with reasonable accuracy in the absence of crystallographic data using homology modelling platforms (viz., Swiss-Model) (Waterhouse et al., 2018), whereas evaluation of either the stereochemical or structural reliability is done with validation programs (viz., PROCHECK and VERIFY3D) (Laskowski et al., 1993; Eisenberg et al., 1997). Sharp molecular docking systems, including HDOCK, can be used to perform high-throughput prediction of protein-ligand interactions to accelerate the rational screening of potential antibiofilm compounds (Yan et al., 2017; Yan et al., 2020). The combination of in silico modelling combined with in vitro and in vivo validation represents a potent, mechanistically guided therapeutic discovery pipeline. In this study, we adopted a multi-tiered approach where we (i) evaluated interactions between selected plant derived polyphenolic compounds and key biofilm-associated proteins of S. aureus using molecular docking and (ii) experimentally assessed the antibiofilm and antibiotic-potentiating activity of apparently potent polyphenol in vitro and in vivo in a murine mastitis model. The main objective of the study was to characterise potential adjunctive therapies to interrupt biofilm-mediated persistence and improve the antimicrobial efficacy.

    MATERIALS AND METHODS

    Ethical standards
     
    All animal studies have been approved by the Institutional Animal Ethics Committee (IAEC/VL/09-2024-2025; dated 8/11/2024).
     
    Bacterial strains used in the study
     
    This study was conducted between August 2025 and January 2026. All experiments in the current study were conducted at Acharya Nagarjuna University, Guntur and PVNR Telangana Veterinary University, Hyderabad, India. The Staphylococcus aureus strain (S. aureus strain #C5) used in this study was previously isolated from a cow with clinical mastitis and was characterised as a strong biofilm-forming strain (data not shown). The bacterial strains were maintained on tryptic soy agar or broth and on mannitol salt agar (Himedia, India) and grown at 37°C for 12 h. Prior to experimentation, the cultures were revived from -80°C, phenotypically reconfirmed and prepared for subsequent analyses.
     
    Target protein identification, ligand selection and molecular docking
     
    Seventeen biofilm-associated proteins of S. aureus implicated in adhesion, extracellular polymeric substance production, quorum sensing and structural stability were selected as potential molecular targets based on their established or predicted roles in biofilm formation (based on a literature search on PubMed). Four plant derived polyphenolic compounds with reported antibiofilm and antimicrobial properties (based on PubMed literature search), viz., reserpine, quercetin, ursolic acid and resveratrol, were selected as ligands for in silico screening. Protein-ligand docking was performed using the HDOCK v 28.5.2 web server, which combines template-based and ab initio docking algorithms (Yan et al., 2017; Yan et al., 2020). Docking was performed using default global docking parameters without defined binding sites, to avoid bias. Binding affinities were estimated using HDOCK’s v 28.5.2 scoring function, integrating shape complementarity, electrostatic interactions, desolvation energy and statistical potentials. Complexes were ranked based on docking scores, with more negative values indicating stronger predicted binding affinity. Polyphenols demonstrating strong binding affinity with more than one biofilm associated proteins with stable interaction networks were considered promising therapeutic agents.
     
    Antagonistic effect of reserpine on S. aureus biofilms in vitro
     
    The antagonistic effect of biofilm formation by Reserpine was studied using different concentrations (500 μg/ml and 1000 μg/ml) and compared with the control group that did not receive any treatment. For this, an overnight culture of biofilm-forming S. aureus (strain #C5) was incubated for 12 h in tryptic soy broth supplemented with either 500 μg/ml or 1000 μg/ml of Reserpine (Sigma-Aldrich, USA CAS Number: 50-55-5). The inhibitory effect was studied by the MTP method as described before (Christensen et al., 1985; Stepanović et al., 2000). Briefly, 1:200 diluted aliquots (200 µL) of the prepared suspensions were inoculated in triplicate (in two sets) into sterile, flat-bottom 96-well microtiter plates, while sterile broth without inoculum served as a negative control. The plates were incubated at 37°C for 24 h. Following incubation, planktonic cells were removed and the wells were gently washed three times with sterile PBS to remove non-adherent bacteria. One set of triplicate wells was then plated on TSA plates for colony counting. Biofilm formation ability was determined in the other set of triplicate wells using the crystal violet staining method as follows. The adherent biofilm was fixed with methanol for 15 min at room temperature and stained with 0.5% crystal violet for 15 min. Excess stain was removed by washing under running water and the bound dye was solubilised using 95% ethanol. Optical density (OD) was measured at 590 nm using a BioTek microplate ELISA reader. The anti-biofilm effect was assessed by dividing the S. aureus isolates into three groups: control isolates, Reserpine-treated isolates at 500 µg/ml and Reserpine-treated isolates at 1000 μg/ml. The inhibitory rates were calculated using the formula:


    Mice mastitis model
     
    In vivo studies were carried out using a mouse mastitis model (Brouillette et al., 2004; Notebaert et al., 2006). Lactating C57BL6 mice of 12 to 14 days after offspring birth were used for the study. The pups were removed 1 h before challenge with S. aureus (strain #C5) (200 CFU/gland) via intramammary inoculation in both the left and right abdominal mammary glands. Animals were randomly assigned to five experimental groups (n = 8 per group) (Fig 2A). Group 1 served as the uninfected, untreated negative control. Group 2 comprised infected, PBS-treated animals and served as the disease control. Group 3 (infected) received reserpine at a dose of 500 µg/ml. Group 4 (infected) was treated with ceftiofur (1 mg/Kg b.wt) alone and served as the antibiotic control group. Group 5 (infected) was administered 500 µg/mg reserpine in combination with ceftiofur (1mg/Kg b.wt). All animals were observed periodically for clinical signs, behavioural changes and local inflammatory reactions. At 16 hours post-infection, animals were euthanised and mammary gland tissues were aseptically collected and homogenised. The homogenates were serially diluted in sterile PBS and plated on nutrient agar to quantify viable bacterial counts (CFU). Additionally, mammary tissues were collected for histopathological analysis.
     
    Statistical analysis
     
    All statistical analyses, unless otherwise stated, were conducted using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA). The error bars in the graphs show the standard error of the mean. Unpaired t-test and one-way ANOVA were used to compare the groups. Assays were performed in triplicate and the data were expressed as the mean of 3 independent experiments. The results were considered statistically significant at P<0.001.

    RESULTS AND DISCUSSION

    In silico evidence reveals that reserpine is a broad-spectrum inhibitor of S. aureus biofilm machinery
     
    The development of biofilms in S. aureus involves a multi-factorial and tightly coordinated process that incorporates the adhesion of cells to the surface, the synthesis of the extracellular matrix, autolysis-driven remodelling and global transcriptional regulation (Otto, 2008; Archer et al., 2011). This phenotype is closely linked with chronic infection, antibiotic resistance and treatment failure in bovine mastitis (Melchior et al., 2006; Song et al., 2024). Since disrupting a single determinant is unlikely to destroy a strong system, we focused on identifying multiple functional nodes in the biofilm network. Based on existing literature sources in Pubmed, 17 experimentally validated S.aureus biofilm-associated proteins pertinent to bovine mastitis were shortlisted. These comprised important adhesins (ClfA, ClfB, Cna, FnbA), which mediate attaching to host extracellular matrix constituents (fibrinogen and fibronectin) (Foster et al., 2014); the polysaccharide intercellular adhesion operon proteins (IcaA-D), central to poly-b-1,6-N-acetylglucosamine (PNAG) matrix synthesis (Cramton et al., 1999); autolysins; and other surface-associated virulence factors (Eno, SdrG, Esp and SesA) (Melchior et al., 2009). To examine therapeutic vulnerabilities in this network, we analysed four FDA-approved bioactive plant derived polyphenols with reported antibiofilm activity, including quercetin, resveratrol, resperpin and ursolic acid (Lionta et al., 2014; Qin et al., 2014; Khameneh et al., 2015; Musini et al., 2023; He et al., 2025). Table 1 summarises binding affinities and interaction profiles of the selected polyphenols against the 17 target proteins. Reserpine exhibited a more pronounced predicted binding energy than the rest of the compounds tested across the target panel. Reserpine was also found to have a broad range of interaction with adhesion molecules, matrix biosynthetic enzymes and regulatory proteins as opposed to showing an isolated affinity to a restricted number of proteins. It is important to note that reserpine demonstrated a high predicted interaction with ClfA, FnbA and Cna, which are major adhesins needed to colonise host tissues (Houston et al., 2011; Foster et al., 2014). It also displayed high-affinity binding to IcaA-D complex that is the core in PNAG production and biofilm structural integrity (Cramton et al., 1999). Notably, reserpine also interacted with a transcriptional regulator, SarA whose inactivation has been found to severely affect biofilm formation and virulence (Beenken et al., 2003; Valle et al., 2003). Bap and EbhA, proteins that have been found to mediate biofilm maturation and persistence in mastitis isolates, were also predicted to interact with reserpine in a stable manner (Cucarella et al., 2001). Quercetin, resveratrol and ursolic acid, in turn, exhibited a weaker or target-restricted pattern of interaction, which is in line with previously reported mechanisms of action of the compounds (Ouyang et al., 2016; Borges et al., 2018). Together, this docking information indicates that reserpine exhibits multi-target interaction profile which concomitantly regulates adhesion, matrix biosynthesis and regulatory pathways needed to sustain and initiate biofilm in S. aureus. Since biofilm resilience is a multi network mechanism (Otto, 2018), the ability to disrupt the functions of several nodes can provide more translational potential in the case of bovine mastitis.

    Table 1: Major biofilm-associated genes of S. aureus and their encoded proteins’ docking values with the selected polyphenols.


     
    Reserpine interferes with S. aureus biofilm and decreases viable bacterial population in vitro
     
    To experimentally validate (in vitro) the ability of reserpine to interfere with the biofilm phenotype, biofilm-forming S. aureus was exposed to two different concentrations: 1000 mg/mL (MIC) and 500 mg/mL (1/2 MIC) concentrations, both of which have been reported to inhibit gram-positive biofilms, including S. aureus (Kaatz et al., 2003). In the microtiter plate assay, biofilm biomass was significantly reduced by reserpine: at 500 mg/mL, reserpine inhibited biofilm growth by 68.6 per cent of the control and at 1000 mg/mL, it inhibited biofilm growth by 71.5 per cent of the control (p<0.001) (Fig 1A). The two doses caused significant suppression, however the small difference observed between concentrations is typical of a threshold or plateau effect commonly seen with regulatory pathways being targeted (Otto, 2018). Significantly, the disruption of biofilm was paralleled with a significant reduction in the viable bacterial counts after the treatment with reserpine. At 500 mg/mL, bacterial loads decreased from 7.32 log10 CFU/mL to 5.97 log10 CFU/mL (95.53% reduction) and at 1000 mg/mL from 7.32 log10 CFU/mL to 5.77 log10 CFU/mL (97.18% reduction) (Fig 1B). This simultaneous decrease in both biomass and CFU implies that reserpine destabilises both the form and viability of biomasses instead of merely dispersing the extracellular matrix material without affecting the confined cells, a drawback reported with a number of antibiofilm agents (Archer et al., 2011; Borges et al., 2018). These results are further consistent with the previously shown inhibition of NorA-mediated efflux by reserpine in S. aureus (Kaatz et al., 2003). Efflux systems play a role in creating antibiotic resistance as well as in biofilm maturation and stress adaptation which can be inhibited to disrupt the formation of biofilms and sensitise the embedded cells (Costa et al., 2013; Schindler and Kaatz, 2016). Combined with our in silico data of multi-target binding with adhesin and regulatory proteins, the current data is consistent with a model where reserpine disrupts biofilm stability at a variety of functional nodes. Since biofilm-related tolerance has the potential to raise the effective concentration of antibiotics by many orders of magnitude (Archer et al., 2011), the significant drop in viable counts in the present case is potentially translationally relevant.

    Fig 1: Antibiofilm (A) and bactericidal (B) activities of reserpine (MIC and ½ MIC levels).


     
    Reserpine has a significant therapeutic potential in the mouse mastitis model
     
    To determine whether reserpine would translate its in vitro antibiofilm activities into therapeutic outcome in vivo we used a murine mastitis model. The untreated animals showed rapid growth in bacteria after 16 hours of S. aureus challenge with a maximum of 7.85 log10 CFU/g of mammary tissue. Such bacterial load is in line with the severity of acute mastitis at an early stage and demonstrates the capacity of S. aureus to form high-density populations in mammary alveoli, in which biofilm-like structures and deposition of the extracellular matrix increase survival (Brouillette et al., 2003; Brouillette et al., 2004; Archer et al., 2011). Monotherapy with reserpine (500 mg/mL) decreased the bacteria to 6.53 log10 CFU/g (P<0.001). Even though this was significant, this decrease was minor as compared to ceftiofur monotherapy, which reduced the burden to 5.62 log10 CFU/g. Ceftiofur is a third-generation cephalosporin, which binds to penicillin-binding proteins and prevents cross-linking of peptidoglycan, but its activity in biofilm-associated populations is compromised by limited penetration, changes in metabolic states and tolerance-based mechanisms (Archer et al., 2011; Otto, 2018). Strikingly, combination therapy (reserpine (500 μg/mL) + ceftiofur) further reduced the bacterial burden to 4.83 log10 CFU/g (P<0.0001), representing an approximate 3-log reduction relative to untreated controls (Fig 2B). This could be indicative of functional synergy that is mediated by the effect of interference with inherent tolerance mechanisms and not solely additive bactericidal action.

    Fig 2: Therapeutic effects of reserpine on S. aureus induced acute mouse mastitis.


           
    Mechanically, efflux pumps like NorA have functions other than those of classical multidrug resistance. In S. aureus, efflux activity induces quorum sensing, intracellular homeostasis, redox balance and stress adaptation, which are part of biofilm maturation and maintenance (Costa et al., 2013). NorA has been shown to be inhibited by reserpine, which has been found to decrease resistance to various classes of antibiotics through heightened retention of drugs in intracellular locations (Kaatz et al., 2003; Schindler and Kaatz, 2016). In the context of β-lactam therapy, enhanced intracellular stress combined with impaired cell wall synthesis may amplify bactericidal signalling cascades. Furthermore, efflux inhibition may reduce the capacity of biofilm-embedded cells to export toxic metabolic by-products, increasing susceptibility under nutrient-limited mammary conditions. This mechanistic explanation was corroborated by histopathologies (Fig 2C). Infected glands, which were untreated, exhibited significant alveolar wall thickening, congestion of the vascularity and intense neutrophil infiltration in the stroma and luminal regions, which are the signs of acute inflammatory mastitis (Rainard and Riollet, 2006). This type of inflammatory reactions, as crucial as it is in clearing bacteria, is also implicated in the destruction of tissues and disrupted glandular activity. Ceftiofur or reserpine monotherapy showed partial inflammatory pathology inhibition, which is in line with the decreased bacterial burden. Nevertheless, alveolar architecture and neutrophilic infiltration were most preserved with the combination treatment group (Fig 2C). The simultaneous decrease in CFU and histological damage suggests augmented bacterial clearance and limited persistent inflammatory signalling. Increased neutrophil recruitment and cytokine generation induced by persistent antigen exposure is known to be caused by biofilm-associated infections (Otto, 2018). Notably, reserpine itself failed to achieve complete bacteriological eradication, which supports its probable, though not exclusive, role as an adjuvant rather than a primary antimicrobial. This is consistent with the modern antibiotic-potentiation approaches, which are intended to sensitise pathogens to the already available antibiotics, but do not substitute them (Clatworthy et al., 2007; Theuretzbacher et al., 2020). Targeting tolerance pathways is a logical treatment in biofilm-related mastitis, where biofilm mediated tolerance instead of classical resistance can be the basis of therapeutic failure. Even though bovine mammary physiology cannot be completely recapitulated in murine models, they can give mechanistic understanding of the interaction of host-pathogen-drug interactions in vivo (Brouillette et al., 2004). The observed ~3-log reduction with combination therapy supports further pharmacodynamic and translational investigation in bovine systems.

    CONCLUSION

    Collectively, these findings demonstrate that reserpine can functionally disrupt biofilm-associated persistence and significantly potentiate β-lactam activity in experimental S. aureus mastitis. Targeting intrinsic tolerance mechanisms may therefore represent a viable strategy to enhance antimicrobial efficacy in biofilm-driven infections.

    ACKNOWLEDGEMENT

    No specific funding was received for this study. The authors would like to acknowledge PVNRTVU, Hyderabad and Acharya Nagarjuna University, Guntur, for providing resources to conduct the study.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal studies have been approved by the Institutional Animal Ethics Committee (IAEC/VL/09-2024-2025; dated 8/11/2024).

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Archer, N.K., Mazaitis, M.J., Costerton, J.W., Leid, J.G., Powers, M.E. and Shirtliff, M.E. (2011). Staphylococcus aureus biofilms: Properties, regulation and roles in human disease. Virulence. 2: 445-459. doi: 10.4161/viru.2.5.17724.

    2. Beenken, K.E., Blevins, J.S. and Smeltzer, M.S. (2003). Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infection and Immunity. 71: 4206-4211. doi: 10.1128/ IAI.71.8.4206-4211.2003.

    3. Bhakat, C., Singh, A.K., Mandal, A., Karunakaran, M., Mohammad, A., Mandal, D.K., Rai, S., Chatterjee, A., Mondal, M. and Dutta, T.K. (2025). Udder health maintenance to augment milk production in dairy cattle: A review. Indian Journal of Animal Research. 59(7): 1085-1091. doi: 10.18805/IJAR.B-4816.

    4. Borges, A., Saavedra, M.J. and Simoes, M. (2018). Insights on antimicrobial resistance, biofilms and the use of phytochemicals as new antimicrobial agents. Current Medicinal Chemistry.  25: 1-21. doi: 10.2174/0929867324666170920151121.

    5. Brouillette, E., Talbot, B.G. and Malouin, F. (2003). The pathogenesis of Staphylococcus aureus mastitis. Journal of Mammary Gland Biology and Neoplasia. 8: 323-339. doi: 10.1023/ B:JOMG.0000010038.32054.16.

    6. Brouillette, E., Lacasse, P., Shkreta, L., Belanger, J., Grondin, G., Diarra, M.S., Fournier, S. and Talbot, B.G. (2004). DNA microarray analysis of the host response to Staphylococcus aureus infection in the mouse mammary gland. Infection and Immunity. 72: 3350-3360. doi: 10.1128/IAI.72.6.3350- 3360.2004.

    7. Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F., Melton, D.M. and Beachey, E.H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology. 22: 996-1006. doi: 10.1128/ jcm.22.6.996-1006.1985.

    8. Clatworthy, A.E., Pierson, E. and Hung, D.T. (2007). Targeting virulence: A new paradigm for antimicrobial therapy. Nature Chemical Biology. 3: 541-548. doi: 10.1038/ nchembio.2007.24.

    9. Costa, S.S., Viveiros, M., Amaral, L. and Couto, I. (2013). Multidrug efflux pumps in Staphylococcus aureus: An update. Frontiers in Microbiology. 4: 178. doi: 10.3389/fmicb. 2013.00178.

    10. Cramton, S.E., Gerke, C., Schnell, N.F., Nichols, W.W. and Gotz, F. (1999). The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infection and Immunity. 67: 5427-5433. doi: 10.1128/IAI.67.10.5427-5433.1999.

    11. Cucarella, C., Solano, C., Valle, J., Amorena, B., Lasa, I. and Penadés, J.R. (2001). Bap, a Staphylococcus aureus surface protein involved in biofilm formation. Journal of Bacteriology. 183: 2888-2896. doi: 10.1128/JB.183.9. 2888-2896.2001.

    12. Eisenberg, D., Lüthy, R. and Bowie, J.U. (1997). VERIFY3D: Assessment of protein models with three dimensional profiles. Methods in Enzymology. 277: 396-404. doi: 10.1016/S0076- 6879(97)77022-8.

    13. Flemming, H.C. and Wuertz, S. (2023). Bacteria and archaea on earth and their abundance in biofilms. Nature Reviews Microbiology. 21: 513-528. doi: 10.1038/s41579-023- 00831-0.

    14. Foster, T.J., Geoghegan, J.A., Ganesh, V.K and Hook, M. (2014). Adhesion, invasion and evasion: The many functions of Staphylococcus aureus surface proteins. Nature Reviews Microbiology. 12: 49-62. doi: 10.1038/nrmicro3161.

    15. Greeshma, A.J., Pushpa Ramani, R.N., Lakshmi, K.K. and Rao, S.T. (2024). Efficacy of resveratrol and ursolic acid on biofilm inhibition and antimicrobial resistance of Streptococcus uberis. Indian Journal of Animal Research. 58(12): 2177- 2183. doi: 10.18805/IJAR.B-4697.

    16. Halasa, T., Huijps, K., Osteras, O. and Hogeveen, H. (2007). Economic effects of bovine mastitis. Journal of Dairy Science. 90: 4096-4108. doi: 10.3168/jds.2007-0132.

    17. He, J., Cui, Y., Liu, Y., Mao, J., Dong, Y., Yao, R., Yang, D., Fan, P. and Xue, J. (2025). Resveratrol inhibits the formation of Staphylococcus aureus biofilms by reducing PIA, eDNA release and ROS production. Frontiers in Veterinary Science. 12: 1594239. doi: 10.3389/fvets.2025.1594239.

    18. Houston, P., Rowe, S.E., Pozzi, C., Waters, E.M. and O’Gara, J.P. (2011). Essential role for the major autolysin in the fibronectin- binding protein-mediated Staphylococcus aureus biofilm phenotype. Infection and Immunity. 79: 1153-1165. doi: 10.1128/IAI.01158-10

    19. Kaatz, G.W., Moudgal, V.V., Seo, S.M. and Kristiansen, J.E. (2003). Inhibition of NorA-mediated efflux by reserpine in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 47: 329-333. doi: 10.1128/AAC.47.1.329-333.2003.

    20. Khameneh, B., Iranshahy, M., Ghandadi, M., Atashbeyk, D.G., Fazly Bazzaz, B.S. and Iranshahi, M. (2015). Investigation of the antibacterial activity and efflux pump inhibitory effect of plant alkaloid reserpine against Staphylococcus aureus. Drug Development and Industrial Pharmacy. 41: 989-994. doi: 10.3109/03639045.2014.920025.

    21. Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993). Procheck: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography.  26: 283-291. doi: 10.1107/S0021889892009944.

    22. Lionta, E., Spyrou, G., Vassilatis, D.K. and Cournia, Z. (2014). Structure-based virtual screening for drug discovery. Current Topics in Medicinal Chemistry. 14: 1923-1938. doi: 10.2174/1568026614666140923120832.

    23. Melchior, M.B., Vaarkamp, H. and Fink-Gremmels, J. (2006). Biofilms: A role in recurrent mastitis infection? Veterinary Journal. 171: 398-407. doi: 10.1016/j.tvjl.2005.01.006.

    24. Melchior, M.B., van Osch, M.H.J., Graat, R.M., van Duijkeren, E., Mevius, D.J., Nielen, M., Gaastra, W. and Fink-Gremmels, J. (2009). Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates. Veterinary Microbiology. 134: 206-213. doi: 10.1016/j.vetmic.2008.09.031.

    25. Musini, A., Ponnusamy, K. and Shanmugam, K. (2023). Antibiofilm activity of quercetin against drug-resistant Staphylococcus aureus. Journal of Applied Microbiology. 134: lxad123. doi: 10.1093/jambio/lxad123.

    26. Notebaert, S. and Meyer, E. (2006). Mouse models to study the pathogenesis and control of bovine mastitis. Veterinary Research. 37: 409-421. doi: 10.1051/vetres:2006003.

    27. Oliver, S.P., Murinda, S.E. and Jayarao, B.M. (2011). Impact of antibiotic use in adult dairy cows on antimicrobial resistance. Journal of Dairy Science. 94: 569-583. doi: 10.3168/ jds.2010-3505.

    28. Ouyang, J., Sun, F., Feng, W., Sun, Y., Qiu, X., Xiong, L., Liu, Y., Chen, Y. and Li, H. (2016). Quercetin is an effective inhibitor of Staphylococcus aureus biofilm formation. Journal of Medical Microbiology. 65: 1159-1167. doi: 10.1099/jmm.0.000338.

    29. Otto, M. (2008). Staphylococcal biofilms. Current Topics in Microbiology and Immunology. 322: 207-228. doi: 10.1007/978-3-540- 75418-3_10.

    30. Otto, M. (2018). Staphylococcal biofilms and their role in antibiotic resistance. Microbiology Spectrum. 6: 1-8. doi: 10.1128/ microbiolspec.GPP3-0023-2018.

    31. Qin, N., Tan, X., Jiao, Y., Liu, L., Zhao, W., Yang, S. and Jia, A. (2014). RNA-seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid. Scientific Reports. 4: 5467. doi: 10.1038/srep05467.

    32. Rainard, P. and Riollet, C. (2006). Innate immunity of the bovine mammary gland. Veterinary Research. 37: 369-400. doi: 10.1051/vetres:2006007.

    33. Sandhu, J., Chandra, M., Kaur, G., Narang, D. and Arora, A.K. (2025). Studies on the efficacy of phage in controlling Staphylococcus aureus biofilm phenotype associated with mastitis. Indian Journal of Animal Research. doi: 10.18805/IJAR.B-5371.

    34. Schindler, B.D. and Kaatz, G.W. (2016). Multidrug efflux pumps of gram-positive bacteria. Antimicrobial Agents and Chemotherapy. 60: 500-509. doi: 10.1128/AAC.01774-15.

    35. Shiva, J.J., Putty, K., Narasimha Reddy, Y., Dhanalakshmi, K. and Umair, M.A.H. (2018). Antagonistic effect of ursolic acid on Staphylococcus aureus biofilms and enhancement of antibiotic susceptibility. Veterinary World. 11: 1440- 1444. doi: 10.14202/vetworld.2018.1440-1444.

    36. Song, X., Zhang, D., Li, B., Zhou, P. and Huang, S.Y. (2024). Biofilm formation and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis. Journal of Animal Science and Biotechnology. 15: 45. doi: 10.1186/s40104- 024-00945-2.

    37. Stepanović, S., Vuković, D., Dakić, I., Savić, B. and Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods. 40: 175-179. doi: 10.1016/S0167-7012(00)00122-6.

    38. Theuretzbacher, U., Bush, K., Harbarth, S., Paul, M., Rex, J.H., Tacconelli, E., Thwaites, G.E., Klugman, K.P. and Carmeli, Y. (2020). Critical analysis of antibacterial agents in clinical development. Nature Reviews Microbiology. 18: 275-287. doi: 10.1038/s41579-019-0288-4.

    39. Valle, J., Toledo-Arana, A., Berasain, C., Ghigo, J.M., Amorena, B., Penadés, J.R. and Lasa, I. (2003). SarA and not óB is essential for biofilm development by Staphylococcus aureus. Molecular Microbiology. 48: 1075-1087. doi: 10.1046/j.1365-2958.2003.03493.x.

    40. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. and Schwede, T. (2018). Swiss- Model: Homology modelling of protein structures and complexes. Nucleic Acids Research. 46: W296-W303. doi: 10.1093/nar/gky427.

    41. Yan, Y., Zhang, D., Zhou, P., Li, B. and Huang, S.Y. (2017). HDOCK: A web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy. Nucleic Acids Research. 45: W365-W373. doi: 10.1093/nar/gkx407.

    42. Yan, Y., Tao, H., He, J. and Huang, S.Y. (2020). The HDOCK server for integrated protein-protein docking. Nature Protocols. 15: 1829-1852. doi: 10.1038/s41596-020-0312-x.

    43. Zhang, L., Liang, E., Cheng, Y., Mahmood, T., Ge, F., Zhou, K., Bao, M., Lv, L., Yi, J. and Wang, J. (2020). Is combined medication with natural medicine a promising therapy for bacterial biofilm infection? Biomedicine and Pharmacotherapy. 128: 110184. doi: 10.1016/j.biopha.2020.110184.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)