Medicinal plants have long been recognized as valuable sources of therapeutic agents, playing a vital role in traditional medicine and contributing to the improvement of human health and quality of life. Many plant species have been scientifically reported to possess medicinal properties, largely due to their bioactive compounds that participate in biological processes essential for disease treatment and prevention [1]. For many communities, particularly in rural or indigenous settings, medicinal plants often serve as the primary—sometimes the only—source of healthcare [2]. Their use is especially critical in developing countries, where access to conventional medicine may be limited and reliance on traditional remedies forms a key component of primary healthcare [3,4]. Despite advances in pharmaceutical research and the development of numerous antimicrobial agents in recent decades, infectious diseases remain a major cause of morbidity and mortality, particularly in underresourced regions [4]. Compounding this challenge is the rising problem of antimicrobial resistance. Microorganisms, especially bacteria, possess the genetic capacity to acquire and transmit resistance to drugs, diminishing the efficacy of many conventional treatments [5]. In vitro studies focusing on plants traditionally used in medicine have been widely conducted within microbiology, particularly regarding their effects on pathogenic bacterial growth. However, investigations into the synergistic potential between plant extracts and conventional antimicrobial agents remain limited. In this study, we assessed the in vitro synergistic interactions between extracts of Neem (Azadirachta indica) [6], Guava (Psidium guajava) [7], Clove (Syzygium aromaticum) [8], Garlic (Allium sativum) [9], Ear-pod Wattle (Acacia auriculiformis) [10], Ginger (Zingiber officinale) [11], Papaya (Carica papaya) [12], and Mint (Mentha piperita) [13], and selected antimicrobial drugs. The assays were performed against the Staphylococcus aureus RN6390 strain using the Kirby-Bauer disk diffusion method.

Plant samples

Samples of A. indica, P. guajava, C. papaya, M. piperita, and A. auriculiformis were collected in July 2025 from Janta Inter College, Behat, Saharanpur-247121. Voucher specimens were prepared and deposited at the designated herbarium for future reference. The collected leaves were dried at 40°C and ground into fine powder using a mechanical mill [14,15]. In the same time, A. sativum, S. aromaticum, and Z. officinale were procured fresh from the local market and used in their natural form for extract preparation.

Preparation of plant extracts

Plant materials—dried (Azadirachta indica, Psidium guajava, Carica papaya, Mentha piperita, Acacia auriculiformis) and fresh (Allium sativum, Syzygium aromaticum, Zingiber officinale)— were finely ground and extracted with 70% methanol. The initial extraction process lasted 48 hours, after which the mixtures were filtered [15]. The residual plant matter was re-extracted with fresh 70% methanol for an additional 24 hours, followed by a second filtration [15,16]. The combined methanolic extracts were then concentrated using a rotary evaporator at 45°C to remove the solvent. The crude extracts were stored in sterile bottles under refrigerated conditions until use. To determine the extract concentrations (mg/ml), the dry weight was calculated by complete evaporation of the remaining solvent [16].

Bacterial strains

Staphylococcus aureus RN6390, a commonly used laboratory strain that was developed from NCTC8325, was utilized in this study. The NCTC8325 strain was obtained from the Department of Microbiology at NYU Grossman School of Medicine, located at 540 First Avenue, 2nd Floor, Lab 1-2, New York, NY 10016.

Media used

To maintain bacterial broth cultures, a nutrient broth was prepared using 0.5 g of NaCl, 0.5 g of peptone, and 0.3 g of beef extract per 100 ml of distilled water. For solid media, nutrient agar was formulated by adding 1.5 g of agar to the broth base [17].

Preparation of working slant

The S. aureus NCTC8325 stock culture was maintained at 4°C on semi-solid agar slants composed of 0.5% peptone, 0.3% beef extract, and 1.5% agar. For experimental use, a loopful of the culture was aseptically transferred from the stock to initiate active working cultures [17]. These slants were then incubated at 36 ± 1.0°C for 24 hours.

Broth preparation

Under sterile conditions, isolated colonies from the cultured S. aureus NCTC8325 slant were picked using a sterile inoculating loop and transferred into a cooled, autoclaved liquid broth medium containing 0.5% peptone and 0.3% beef extract [17,18]. The inoculated broth was then incubated at 36 ± 1.0°C for 24 hours, until visible turbidity indicated active bacterial growth.

Antimicrobial tests

Prior to evaluating the synergistic effects between plant extracts and antimicrobial agents, the Minimum Inhibitory Concentration (MIC) of each extract was determined against the Staphylococcus aureus RN6390 strain. This was done by incorporating serial dilutions of the extracts into Mueller Hinton Agar (MHA) media NCCLS (2004a,b). Petri dishes containing various concentrations of the plant extracts (mg/ml), along with appropriate controls, were inoculated with approximately 10⁴ CFU of S. aureus RN6390 using a Steer’s replicator and incubated at 37°C for 24 hours. The lowest concentration of each plant extract that visibly inhibited bacterial growth was recorded as the MIC, and the MIC₉₀ (the concentration inhibiting 90% of isolates) was subsequently calculated. For synergism assays, onefourth of the MIC₉₀ was used as the sub-inhibitory concentration, as described previously [19]. These assays were performed on S. aureus RN6390 and NCTC8325 strains using the disk diffusion method (Kirby-Bauer), following NCCLS (2004) guidelines on Mueller Hinton Agar (MHA). A total of thirteen antimicrobial agents were tested: cefuroxime (CEO; 10 IU), cefazolin (CEF; 1 µg), cefixime (CEI; 30 µg), imipenem (IMP; 10 µg), cephalexin (CFL; 30 µg), ertapenem (EPN; 30 µg), clarithromycin (CLM; 30 µg), linezolid (LNZ; 10 µg), streptomycin (STM; 30 µg), tigecycline (TIC; 30 µg), amikacin (AMC; 15 µg), sulfadiazine (SUZ; 25 µg), and ciprofloxacin (CPF; 5 µg). For each S. aureus RN6390 strain, two sets of antibiograms were performed in duplicate: one on control plates containing plain Mueller Hinton Agar (MHA), and the other on MHA supplemented with one-fourth the MIC₉₀ of the respective plant extract. After incubation at 37°C for 18 hours, the diameters (in mm) of the inhibition zones were measured and recorded [19,20].

Statistical analysis

Data obtained from the synergism assays were analysed using the Wilcoxon nonparametric test to compare the inhibition zone diameters (mm) generated by the disk diffusion method. Statistical analysis was performed using Minitab Statistical Software, version 13.32 [20,21]. A p-value of less than 0.05 was considered statistically significant.

Characteristics, MIC 90% (mg/ml) against S. aureus RN6390 strain, and one-fourth the MIC 90% values obtained in the synergism assays for the plants and their respective extracts are presented in Table 1.

Anti-S. aureus activity was verified for all the plants. S. aromaticum showed the highest activity, followed by P. guajava; the lowest activity was recorded for A. auriculiformis. The MIC 90% range was 0.37 mg/ml for clove and 18.01 mg/ml for A. auriculiformis and it is not surprising the differences in the antimicrobial activity of plants tested, due to phytochemical properties and differences among species. Although the antimicrobial activities of A. auriculiformis, C. papaya, and Z. afficinale, have not been relatively high, synergism assays were carried out for them and the synergism rate of A. auriculiformis was as high as that of S. aromaticum Table 2.

(-): For non-dried (fresh) plant materials, the extract efficacy was considered as 100% of the original sample.

X: synergism when p<= 0.05, (-) no synergism.

Antimicrobial mechanisms of the drugs used here were variable and the protein synthesis inhibitors were those that presented strongest synergistic effect (5.1 extracts/drug) together with folic acid (4 extracts/drug) and bacterial cell wall synthesis (3.9 extracts/drug) inhibitors. Inhibitors of the nucleic acid synthesis (2 extracts/drug) showed weak synergism with plant extracts. Among the protein synthesis inhibitors, tigecycline showed synergism with all the extracts, followed by clarithromycin and streptomycin. The synergistic capacity was promising for the extracts of some plants such as S. aromaticum, A. auriculiformis, and P. guajava, which presented synergism with 11, 11, and 9 drugs, respectively; while garlic and ginger showed synergism with only 4 and 3 drugs, respectively.

The synergism recorded here to plant extracts with weak action on S. aureus RN6390 growth, such as A. auriculiformis, is an important data since it showed a synergism profile similar to that of the clove extract, considered the most efficient S. aureus RN6390 growth inhibitor in this study. Thus, the researchers should investigate the synergistic capacity of plant extracts or other natural products, independent of the antimicrobial activity they have. Therefore, the results of the present study seem to be promising and may enhance the natural products uses, showing the potential of these plants in the treatment of infectious diseases caused by S. aureus

Future studies on the chemical characteristics of extracts and active components should be carried out for each plant and antimicrobial property, since only crude extracts and their dry weight have been used in MIC determination (expressed in mg/ml) and synergism assays. In the current study, the plant extract antimicrobial activity against S. aureus RN690 strain was confirmed and synergism was possible with all the antimicrobial drugs tested. tigecycline showed synergism with all plant extracts; and the A. auriculiformis extract, although with the lowest antimicrobial activity, presented a synergism profile similar to that of S. aromaticum, whose extract showed a relatively high inhibitory capacity on S. aureus RN6390 growth. The possible activities of substances found in plant extracts on ribosome structure and bacterial enzymes inhibition appear to be related with synergism profile between plant extracts and inhibitors of protein synthesis, however, the understanding of synergism mechanism is fundamental to development of pharmacological agents to treat diseases by S. aureus using medicinal plants.

Author contributions

All authors have reviewed the final version to be published and agreed to be accountable for all aspects of the work.

Disclosures human subjects

All authors have confirmed that this study did not involve human participants or tissue.

Conflicts of interest

No conflicts of interest.

Payment/services info

All authors have declared that no financial support was received from any organization for the submitted work.

Financial relationships

All authors have declared that they have no financial relationships at present with any organizations that might have an interest in the submitted work.

Other relationships

All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

Acknowledgments

To professor Richard P. Novick, MD, Principal Investigator, Research Professor in the Department of Microbiology and Professor Emeritus of Microbiology and Medicine, NYU Grossman School of Medicine’s Department of Microbiology at 540 First Avenue, 2nd floor, Lab 1-2, New York, NY 10016 for providing the S. aureus RN6390 strain.