Assessment of antimicrobial activity and genetic diversity of Cannabis Sativa at University of Limpopo

Abstract

Background: Antimicrobial resistance remains a significant challenge worldwide, making it difficult to manage microbial infections caused by multidrug-resistant strains. This necessitates the development of new antimicrobial drugs. Cannabis sativa is a medicinal plant with promising antimicrobial properties, and humans have been utilising it for thousands of years as both food and for medicinal purposes. Since research on Cannabis sativa has only been conducted in recent years following its legalisation, there is limited understanding of Cannabis sativa’s antimicrobial activity and genetic diversity. This study aimed to assess the antimicrobial activity of Cannabis sativa against multidrug-resistant microorganisms and explore its genetic diversity for the potential development of alternative antimicrobial agents, as well as to improve knowledge on genetic variation and genetic improvement of Cannabis sativa found in the Limpopo Province. Objectives: The study aimed to assess the antimicrobial activity and genetic diversity of Cannabis sativa at the University of Limpopo. Methods: The study was experimental and quantitative, conducted at the University of Limpopo. Two Cannabis sativa strains were donated by Heavenly Favour Farm, followed by extraction using methanol and distilled water as solvents separately. The antimicrobial activity of methanol and distilled water extracts of the two Cannabis sativa strains was assessed against five human multidrug-resistant strains using disk diffusion and serial dilution methods. Antimicrobial activity of Cannabis sativa strains against MRSA was compared using a non-parametric Kruskal-Wallis test. A p-value of ≤ 0.05 was considered statistically significant. Genetic analysis involved PCR amplification using genetic markers to identify the Cannabidiolic Acid (CBDA) and Tetrahydrocannabinolic acid (THCA) synthase genes in Cannabis sativa, which are crucial for cannabinoid production. Furthermore, genetic diversity was assessed by using simple sequence repeat (SSR) markers to identify genetic variations between the two strains.Results: The four Cannabis sativa extracts exhibited an antimicrobial activity against Methicillin-resistant Staphylococcus aureus only, and no inhibition of growth was observed on Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. The methanol extracts exhibited higher antimicrobial activity by consistently showing larger inhibition zones against MRSA compared to distilled water extracts. No significant difference was observed in antimicrobial activity against MRSA between the two strains in both methanol and distilled water extracts. The Minimum Inhibitory Concentrations (MICs) of the extracts ranged from 3.13 mg/ml to 12.5 mg/ml, while the Minimum Bactericidal Concentrations (MBCs) values indicated bactericidal activity at concentrations above 6.25 mg/ml. For genetic diversity analysis, two out of six SSR markers (B01-CANN1 and CAN0093) were detected on the Futura 75 strain of Cannabis sativa only. Due to the limited detection of markers, assessing the genetic relationship between the two Cannabis sativa strains was not possible using genetic markers. Conclusion: The findings suggest that Cannabis sativa extracts exhibited observable antimicrobial activity against MRSA. This is due to the diverse bioactive compounds found in the extracts, indicating a new and promising alternative treatment against the increasing multidrug-resistant strains. Furthermore, findings revealed different genetic variations by detecting only two SSR markers on one Cannabis sativa strain. This provides knowledge for maintaining and characterising the genetic variations of Cannabis sativa strains. However, the limited number of Cannabis sativa strains and genetic markers used has limited the study’s ability to completely represent the genetic diversity within the species.