Abstract
Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml−1 with 86% mortality. At a sub-MIC dosage of 0.125 mg ml−1, the biofilm formation was reduced by 71%. The two major cannabinoids detected were cannabidiol and delta-9-tetrahydrocannabinol (Δ9-THC), which were majorly attributed to substantial inhibitory action against MRSA. The time-kill kinetics demonstrated a bactericidal action at 4 MIC over an 8–20-h time window with a 90% reduction in growth rate. The results from SEM, and light microscopy Giemsa staining revealed a reduction in cells in the treated group with increased AKP activity, indicating bacterial cell membrane breakdown. These findings suggested cannabinoids may be a promising alternative to antibiotic therapy for bovine biofilm-associated MRSA.
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Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information: Imperative criteria for the zone of inhibition diameter are provided in Supplementary Table 1. Primers used for DNA amplification of MRSA genes are provided in Supplementary Table 2. The antibacterial activity of plant crude ethanolic leaf extract using disc diffusion method against MRSA isolated from bovine mastitis by disc diffusion method is provided in Supplementary Table 3. The chemical composition (%) of the ethanol extract of Cannabis sativa L. leaf is determined by GC–MS, provided in Supplementary Table 4. The antibacterial activity of Cannabis extract is provided in Supplementary Table 5. The staphylococcal bacterial species was confirmed through 16 s rRNA gene sequencing under accession number Accession no: OP418348 and is available at the following URL: https://www.ncbi.nlm.nih.gov/nuccore/OP418348.1/.
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Acknowledgements
We would like to express our gratitude to Dr. Dheer Singh, Director, ICAR-National Dairy Research Institute, Karnal—132001, Haryana, India, for the necessary support and assistance during the study. We are grateful to the sampling staff of Referral Veterinary Diagnostic and Extension Centre, Uchani, Karnal (Haryana) India, for their contributions in sample collection. We gracefully thank SEM laboratory, Dept of Metallurgical & Materials Engg, IIT Roorkee, Roorkee – 247667, Uttarakhand, India, for providing SEM facility. We are also thankful to Jawaharlal Nehru University’s University Science Instrumentation Centre, AIRF, for providing us GC-MS Instrument facility, New Mehrauli Road, New Delhi, 110067.
Funding
This work was supported by the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, for funding the project (Grant number: EMR/2017/ 004602) and DBT-SRF fellowship from the Department of Biotechnology, GOI, India, during this project to Mayank Roshan (Fellow no: DBT/2018/NDRI/1003).
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M.R: Conceptualization, Visualization, Methodology, Formal analysis, Writing – original draft, I.S: Formal analysis, M.S: Data curation, Formal analysis, A.V: Conceptualization, review & editing, D.P.S: Conceptualization, Resources, D.G: Methodology, Formal analysis; S.R: Writing – review & editing,J.T: Conceptualization, Resources, S.P: Resources, Supervision, S.De: Conceptualization, Supervision, review & Funding acquisition
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The mastitis milk sample was collected from LUVAS Uchani in Karnal, Haryana, India, as part of the SERB-funded project “Mastitis related antibiotic resistance pattern mapping in three districts of Haryana (FILENO. EMR/2017/004602)”, which detailed the research aims and methodology. Approval for the study and its protocols, materials, and methods was granted by the Institutional Biosafety Committee (IBSC) of the National Dairy Research Institute (NDRI) in Karnal, India.
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Roshan, M., Singh, I., Vats, A. et al. Antimicrobial and antibiofilm effect of cannabinoids from Cannabis sativa against methicillin-resistant Staphylococcus aureus (MRSA) causing bovine mastitis. Int Microbiol (2024). https://doi.org/10.1007/s10123-024-00505-x
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DOI: https://doi.org/10.1007/s10123-024-00505-x