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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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/.
References
Ajose DJ et al (2022) Combating bovine mastitis in the dairy sector in an era of antimicrobial resistance: ethno-veterinary medicinal option as a viable alternative approach. Front Vet Sci 9:800322. https://doi.org/10.3389/fvets.2022.800322
Akinduti PA et al (2022) Antibacterial activities of plant leaf extracts against multi-antibiotic resistant Staphylococcus aureus associated with skin and soft tissue infections. BMC Complement Med Ther 22(1):1–11. https://doi.org/10.1186/s12906-022-03527-y
Alaiya MA, Odeniyi MA (2023) Utilisation of mangifera indica plant extracts and parts in antimicrobial formulations and as a pharmaceutical excipient: a review. Future J Pharm Sci 9(1):1–19. https://doi.org/10.1186/s43094-023-00479-z
Anywar GU et al (2022) Cytotoxicity of medicinal plant species used by traditional healers in treating people suffering from HIV/AIDS in Uganda. Front Toxicol 4:2022. https://doi.org/10.3389/ftox.2022.832780
Arsene MM et al (2022) Phytochemical analysis, antibacterial and antibiofilm activities of aloe vera aqueous extract against selected resistant gram-negative bacteria involved in urinary tract infections. Fermentation 8(11):626 (https://www.mdpi.com/2311-5637/8/11/626/htm)
Asghar N et al (2016) Compositional difference in antioxidant and antibacterial activity of all parts of the Carica papaya using different solvents. Chem Cent J 10:5. https://doi.org/10.1186/s13065-016-0149-0
Bandyopadhyay S et al (2015) Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant Staphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis – three cases reported from India. Veterinary Quarterly 35(1):56–61
Bazargani MM, Rohloff J (2016) Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms. Food Control 61:156–164
Behera M et al (2023) Novel AadA5 and DfrA17 variants of class 1 integron in multidrug-resistant Escherichia Coli CAUSING bovine mastitis. Appl Microbiol Biotechnol 107(1):433–446
Blaskovich MA et al (2021) The antimicrobial potential of cannabidiol. Commun Biol 4(1):1–18. https://doi.org/10.1038/s42003-020-01530-y
Bonini SA et al (2018) Cannabis sativa: a comprehensive ethnopharmacological review of a medicinal plant with a long history. J Ethnopharmacol 227(300–315):2023. https://doi.org/10.1016/j.jep.2018.09.004
Caneschi A, Bardhi A, Barbarossa A, Zaghini A (2023) Plant essential oils as a tool in the control of bovine mastitis: an update. Molecules 28(8):3425 (https://www.mdpi.com/1420-3049/28/8/3425/htm)
CLSI. 2020. 40 Clsi CLSI (Clinical and Laboratory Standards Institute )M100-ED29: 2021 Performance Standards for Antimicrobial Susceptibility Testing, 30th edn.
de Andrade B, Neto J et al (2021) Anti-MRSA activity of curcumin in planktonic cells and biofilms and determination of possible action mechanisms. Microb Pathog 155:104892
Frassinetti S et al (2020) Antimicrobial and antibiofilm activity of Cannabis sativa L. seeds extract against Staphylococcus aureus and growth effects on probiotic Lactobacillus spp. LWT 124:109149
Gomes F et al (2019) Anti-biofilm activity of hydroethanolic plant extracts against Staphylococcus aureus isolates from bovine mastitis. Heliyon 1 5(5). https://doi.org/10.1016/j.heliyon.2019.e01728
Gonelimali Faraja D et al (2018) Antimicrobial properties and mechanism of action of some plant extracts against food pathogens and spoilage microorganisms. Front Microbiol 9:389–103 (www.frontiersin.org )
Grover M et al (2021) In vitro phytochemical screening, cytotoxicity studies of Curcuma longa extracts with isolation and characterisation of their isolated compounds. Molecules 26(24):7509. https://doi.org/10.3390/molecules26247509
Gupta K et al (2019) Inhibition of Staphylococcus aureus and Pseudomonas aeruginosa biofilm and virulence by active fraction of Syzygium cumini (L.) skeels leaf extract: in-vitro and in silico studies. Indian Journal of Microbiology 59(1):13–21
Jain VK et al (2022) Virulence and antimicrobial resistance gene profiles of Staphylococcus aureus associated with clinical mastitis in cattle. PLoS ONE 17(5):e0264762. https://doi.org/10.1371/journal.pone.0264762
Khameneh B, Iranshahy M, Soheili V, Fazly Bazzaz BS (2019) Review on plant antimicrobials: a mechanistic viewpoint. Antimicrob Resist Infect Control 8(1):1–28. https://doi.org/10.1186/s13756-019-0559-6
Krishnamoorthy P, Goudar AL, Suresh KP, Roy P (2021) Global and countrywide prevalence of subclinical and clinical mastitis in dairy cattle and buffaloes by systematic review and meta-analysis. Res Vet Sci 136(6450):561–586
Kumar DV et al (2017) Tinospora cordifolia: the antimicrobial property of the leaves of amruthaballi. J Bacteriol Mycol Open Access 5(5):00147. https://doi.org/10.15406/jbmoa.2017.05.00147
Kumar M et al (2021) Futuristic non-antibiotic therapies to combat antibiotic resistance: a review. Front Microbiol 12:609459. https://doi.org/10.3389/fmicb.2021.609459
Lakshmana Prabu S, Umamaheswari A, Grace Felciya G (2020) Investigation on the biofilm eradication potential of selected medicinal plants against methicillin-resistant Staphylococcus aureus. Biotechnol Repo 28:e00523. https://doi.org/10.1016/j.btre.2020.e00523
Liang M, et al. et al (2022) Phytochemicals with activity against methicillin-resistant Staphylococcus aureus. Phytomedicine 100:154073. https://doi.org/10.1016/j.phymed.2022.154073
Liu YX et al (2022) Antibacterial and Antibiofilm properties of dichloromethane fraction of extracts from adventitious roots of Eurycoma longifolia against Staphylococcus aureus. LWT 162:113438
Monistero V et al (2018) Staphylococcus aureus isolates from bovine mastitis in eight countries: genotypes, detection of genes encoding different toxins and other virulence genes. Toxins 10(6). https://pubmed.ncbi.nlm.nih.gov/29914197/
Mordmuang A et al (2019) Evaluation of a Rhodomyrtus tomentosa ethanolic extract for its therapeutic potential on Staphylococcus aureus infections using in vitro and in vivo models of mastitis. Vet Res 50:1–11. https://doi.org/10.1186/s13567-019-0664-9
Omidi Mitra et al (2020) Ability of biofilm production and molecular analysis of spa and ica genes among clinical isolates of methicillin-resistant Staphylococcus aureus. BMC Research Notes 13(1):1–7. https://doi.org/10.1186/s13104-020-4885-9
Requena R et al (2019) Study of the potential synergistic antibacterial activity of essential oil components using the thiazolyl blue tetrazolium bromide (MTT) assay. Lwt 101:183–190. https://doi.org/10.1016/j.lwt.2018.10.093
Romano L, Hazekamp A (2018) An overview of galenic preparation methods for medicinal cannabis. Curr Bioact Compd 15(2):174–195
Roshan M et al (2022) Virulence and enterotoxin gene profile of methicillin-resistant Staphylococcus aureus isolates from bovine mastitis. CIMID 80: 101724. https://doi.org/10.1016/j.cimid.2021.101724. https://pubmed.ncbi.nlm.nih.gov/34826723/
Sánchez E et al (2016) Antibacterial and antibiofilm activity of methanolic plant extracts against nosocomial microorganisms. eCAM 1572697:8. https://doi.org/10.1155/2016/1572697
Schilcher K, Horswill AR (2020) Staphylococcal biofilm development: structure, regulation, and treatment strategies. MMBR 84(3):10–1128. https://doi.org/10.1128/mmbr.00026-19
Schofs L et al (2021) The antimicrobial effect behind Cannabis sativa. PRP 9(2):e00761. https://doi.org/10.1002/prp2.761
Selvaraj A et al (2020) Carvacrol targets SarA and CrtM of methicillin-resistant Staphylococcus aureus to mitigate biofilm formation and staphyloxanthin synthesis: an in vitro and in vivo approach. ACS Omega 5(48):31100–31114
Selvaraj A, Valliammai A, Premika M, Priya A, Bhaskar JP, Krishnan V, Pandian SK (2021) Sapindus mukorossi Gaertn. and its bioactive metabolite oleic acid impedes methicillin-resistant Staphylococcus aureus biofilm formation by down regulating adhesion genes expression. Microbiol Res 242:126601
Shalaby MA, Dokla EM, Serya RA, Abouzid KA (2020) Penicillin binding protein 2a: an overview and a medicinal chemistry perspective. Eur J Med Chem 199:112312
Singh Ila et al (2023) Evaluation of virulence, antimicrobial resistance and biofilm forming potential of methicillin-resistant Staphylococcus aureus (MRSA) isolates from bovine suspected with mastitis. Curr Microbiol 80(6):1–12. https://doi.org/10.1007/s00284-023-03303-2
Sionov RV, Steinberg D (2022) Anti-microbial activity of phytocannabinoids and endocannabinoids in the light of their physiological and pathophysiological roles. Biomedicines 10(3):631. https://doi.org/10.3390/biomedicines10030631
Subbiah M et al (2020) Antimicrobial resistant enteric bacteria are widely distributed amongst people, animals and the environment in Tanzania. Nat Commun 11(1):228. https://doi.org/10.1038/s41467-019-13995-5
Tajik T et al (2022) Extracellular vesicles of cannabis with high CBD content induce anticancer signalling in human hepatocellular carcinoma. Biomed Pharmacother 152:113209. https://doi.org/10.1016/j.biopha.2022.113209
Turner Nicholas A et al (2019) Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17(4):203–18 (www.nature.com/nrmicro )
Vats A et al (2020) Poly I: C Stimulation in-vitro as a marker for an antiviral response in different cell types generated from buffalo (Bubalus bubalis). Mol Immunol 121:136–143
Zengin G et al (2018) Chromatographic analyses, in vitro biological activities, and cytotoxicity of Cannabis sativa L. essential oil: A multidisciplinary study. Molecules 23(12):3266. https://doi.org/10.3390/molecules23123266
Zhang Ke Xin et al (2020) Extract from Oenothera biennis L. stalks efficiently inhibits Staphylococcus aureus activity by affecting cell permeability and respiratory metabolism. Ind Crops Prod 158:112961
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).
Author information
Authors and Affiliations
Contributions
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
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethical approval
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.
Consent to participate
No consent was required as no experiments related to humans were performed.
Competing interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s10123-024-00505-x