Despite the prevalence of literature characterizing staphylococcal pathogenesis in humans, Staphylococcus aureus is a major cause of infections and diseases in a plethora of animal hosts, leading to a considerable impact on public health and agriculture¹. As a highly adaptive pathogen, this bacterium is responsible for a wide range of clinical problems, from minor skin infections to life-threatening bacteremia and meningitis². Serious infections associated with S. aureus cause approximately 20,000 human deaths annually in the US and 5000 in the EU, costing an estimated 380 million € in health costs³. While penicillins were historically effective against S. aureus⁴, the use of conventional antibiotics as a standard treatment of staphylococcal infections has become problematic due to their serious side effects⁵ and the emergence of drug-resistant S. aureus strains¹. Currently, the global spread of methicillin-resistant S. aureus (MRSA) is one of the most serious public health challenges because MRSA strains have emerged with concomitant resistance to other groups of antibiotics, such as aminoglycosides, macrolides, and tetracyclines as well as the beta-lactam antibiotics⁶. The rise of antibiotic resistance poses a growing threat to bacterial infection treatment⁷, with MRSA strains significantly contributing to morbidity and mortality compared to methicillin-sensitive S. aureus (MSSA) (15.6% vs. 6.2%)⁸. The development of new anti-staphylococcal agents, encompassing the use of novel molecules and natural extracts as innovative therapeutics, is an effective strategy to address growing bacterial resistance^9,10, and innovative approaches to medicinal plant cultivation play a crucial role in this endeavor¹¹.

Recognizing the escalating threat of antibiotic-resistant strains, including those of S. aureus, cultivators of medicinal plants for pharmaceutical and healthcare purposes are turning to controlled indoor facilities for cultivation. These environments mitigate risks associated with unpredictable outdoor conditions and pests, allowing for the production of homogeneous plant material^12,13. The choice of cultivation substrate is critical, involving soil-based or soilless media (hydroponics)¹⁴. Soilless culture systems, defined as growing plants without using soil as a rooting medium while supplying inorganic nutrients via irrigation nutrient solution^15,16, offer cost-effectiveness, higher yields, and prompt harvests^17,18. Soilless systems also generally have higher water and nutrient use efficiencies^19,20. A proper supply of mineral nutrients is essential for efficient and sustainable cultivation²¹. Notably, essential nutrients such as nitrogen (N), phosphorus (P), and potassium (K) are pivotal in regulating the profile of secondary metabolites in plants^22,23. Although emphasis is placed on the availability of sufficient amounts of these major plant nutrients, the potential effects of micronutrients²⁴ and plant biostimulants²⁵ must be considered. Iron (Fe), for instance, is indispensable for fundamental plant processes such as photosynthesis and respiration²⁶. Furthermore, the integration of plant biostimulants, particularly amino acids, is gaining recognition for their role in modulating nitrogen absorption and assimilation by regulating the enzymes and structural proteins involved in these processes²⁷. While the effects of essential nutrients and biostimulants on plant metabolism are a significant area of many studies^28–30, further research is needed to fully understand their impact on the formation of secondary plant metabolites.

Among the various crop species grown in soilless systems, such as spices³¹ and culinary aromatic herbs³², medical cannabis (Cannabis sativa L.) is commonly cultivated to control nutrient delivery and environmental conditions for optimal bioactive compound production³³. The acceptability, utilization, and subsequent medicinal use of C. sativa continue to expand, as shown by the growing number of countries that allow its use for specific therapeutic purposes^34–36. The beneficial potential of cannabis can be attributed to its rich metabolite profile, with more than 480 compounds detected³⁷, particularly to cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), and Δ⁹-tetrahydrocannabinol (Δ⁹-THC)³⁸. The diverse pharmacological profile of cannabis includes anticancer or anti-inflammatory effects³⁹ and, notably, antimicrobial activity against various pathogenic bacteria such as S. aureus (including MRSA), Streptococcus mutans, or Listeria monocytogenes^40,41. The ability of crude cannabis extracts and phytocannabinoids to curtail the growth and spread of S. aureus has been the subject of many studies^1,37 since the pioneering work by Van Klingeren and Ten Ham in 1976⁴². Their study highlighted the anti-staphylococcal effects of phytocannabinoids, with CBD and Δ⁹-THC significantly affecting the growth of S. aureus. Furthermore, extracts from C. sativa and phytocannabinoids have demonstrated the ability to inhibit the synthesis of bacterial virulence factors and the formation of biofilms⁴³, providing a potential solution to the challenges posed by limited sensitivity to antibiotics^44,45. However, the antibacterial activity of these compounds depends on various factors, including the specific phytocannabinoid compound being used and its concentration⁴⁶. Considering the strong antibacterial potential of cannabis extracts, this study evaluated the growth-inhibitory effect of ethanolic extracts of C. sativa L. against two S. aureus strains, including the antibiotic-resistant form, across the vegetation cycle to identify cannabinoids with the highest antimicrobial activity and to determine the optimal harvest time in the context of soilless cultivation.