Efficacy of Cannabidiol in Reducing Virulence of Listeria monocytogenes

Source / Quelle: https://www.mdpi.com/1422-0067/27/6/2682

Abstract

Listeria monocytogenes (LM) is a major foodborne pathogen causing illnesses ranging from gastroenteritis to severe systemic infections. The key virulence factors include bacterial motility, hemolysin and lecithinase production, and invasion of host tissues. This study investigated the anti-virulence effects of cannabidiol (CBD), the main non-psychoactive compound in Cannabis sativa, against LM. The minimum inhibitory concentration (MIC, 2289 μM; 719.8 µg/mL) and sub-inhibitory concentration (SIC, 11.92 μM; 3.75 µg/mL) of CBD were determined for LM strains Scott A and ATCC 19115. Cultures were treated with SIC, 6× SIC, 1/4× MIC, and MIC to assess effects on motility, hemolysin and lecithinase production, and adhesion and invasion of human intestinal (Caco-2) and brain endothelial (HBMEC) cells, alongside virulence gene expression by RT-qPCR. Cannabidiol’s efficacy was also determined using a Galleria mellonella larval infection model at SIC and 6× SIC. Cannabidiol at 6× SIC significantly reduced motility, toxin production, and host cell adhesion and invasion (p < 0.05). RT-qPCR revealed downregulation of key virulence genes, including prfA, hly, plcA, plcB, iap, motA, motB, actA, inlA, and inlB. In vivo, CBD enhanced larval survival in a dose-dependent manner and cytotoxicity was observed at concentrations above 33.75 µg/mL. These results indicate that CBD, at non-bactericidal levels, effectively suppresses multiple virulence mechanisms in LM, highlighting its potential as a novel anti-virulence agent for food safety and therapeutic applications.

1. Introduction

Listeria monocytogenes is a Gram-positive, facultative intracellular rod bacteria that causes listeriosis in humans and animals [1]. It is a significant food-borne pathogen that causes the highest number of hospitalizations (>90%) [2] and a mortality rate of around 20% to 30% [3], especially in susceptible individuals, ranking as the third leading cause of food-borne related deaths. Each year, LM causes approximately 1600 illnesses, 1500 hospitalizations, and 260 deaths in the U.S., primarily affecting older adults, pregnant women, newborns, and individuals with weakened immune systems [4,5]. However, listeriosis is a mild and self-resolving disease for those with strong immune systems, typically manifesting as mild gastroenteritis [6]. The psychrotrophic ability of LM coupled with its resistance to both elevated salinity and acidic environments presents a formidable challenge in controlling this pathogen in foods [7,8].

The pathogenesis of LM occurs upon ingesting contaminated food, resulting in gastroenteritis, septicemia, meningitis, and abortion in pregnant women [9,10,11]. Foods such as ready-to-eat meat products [12], soft cheese, unpasteurized milk [13], and fresh produce [14] are high-risk products contributing to listeriosis. Approximately 83% of listeriosis cases are linked to deli meat that is cut and packaged at retail [12,15,16]. The infective dose of listeriosis is estimated to be 104 to 107 cells in immunocompromised people but it is more than 107 in healthy individuals [17,18].

The major virulence factors in LM include its motility, lecithinase activity, hemolysis of RBCs due to the production of listeriolysin O (LLO), and its ability to attach and colonize the intestinal and brain cells. Internalins [19,20], LLO [21,22], phospholipases [23,24], and actin polymerization protein (ActA) [25,26] help in the attachment, vacuolar escape, intracellular proliferation and cell–cell spread of the bacterium. Antibiotics are the drug of choice for treating LM infection, but LM has gained resistance to a wide range of antibiotics commonly used to treat listeriosis [27,28,29,30].

The treatment of listeriosis is primarily guided by antimicrobial susceptibility profiles of LM, with β-lactam antibiotics forming the cornerstone of therapy. Agents such as penicillin, ampicillin, and amoxicillin are widely used, either as monotherapy or in combination with an aminoglycoside, most commonly gentamicin, to enhance bactericidal activity [31,32,33,34,35]. In severe clinical presentations, including listerial meningitis, adults with normal renal function are typically treated with high-dose ampicillin administered at 2 g intravenously every 4–6 h, or penicillin G at 4 million units intravenously every 4 h, in combination with gentamicin at 1.7 mg/kg intravenously every 8 h for a minimum duration of three weeks [35]. When β-lactam antibiotics are contraindicated due to reduced susceptibility or patient intolerance, alternative agents with activity against Gram-positive bacteria may be considered. These include tetracyclines, erythromycin, chloramphenicol, vancomycin, and trimethoprim–sulfamethoxazole (TMP/SMX) [32]. TMP/SMX is frequently used in patients unable to tolerate ampicillin and is typically administered at 3–5 mg/kg (trimethoprim component) intravenously every 6 h for at least three weeks [32,35,36,37]. Vancomycin is often reserved for cases of LM bacteremia, while erythromycin provides an additional option for individuals who cannot receive ampicillin and/or gentamicin [32,38]. More recently, fluoroquinolones such as levofloxacin have demonstrated activity against LM in vitro and in animal infection models, suggesting potential utility under specific clinical circumstances [34,35]. Treatment considerations become particularly complex during pregnancy, where both maternal outcomes and fetal safety must be carefully balanced. In pregnant patients with listeriosis, ampicillin or erythromycin administered intravenously, or oral amoxicillin, is commonly prescribed for a minimum of 14 days and may be continued until delivery if clinically indicated [18,39]. In cases of penicillin intolerance, TMP/SMX is generally recommended; however, because trimethoprim may exert teratogenic effects during early gestation, erythromycin is preferred in pregnant patients due to its established safety profile for the fetus [18,40].

While LM is traditionally regarded as susceptible to several antimicrobials commonly used to treat Gram-positive infections, including β-lactams, gentamicin, erythromycin, tetracycline, rifampicin, and vancomycin, effective clinical management is increasingly challenged by both intrinsic and acquired resistance mechanisms [18,41,42]. The organism is inherently resistant to cephalosporins, nalidixic acid, and polymyxin E, and many isolates exhibit reduced susceptibility to fluoroquinolones, third- and fourth-generation cephalosporins, fosfomycin, oxacillin, and lincosamides, thereby limiting therapeutic options [18,41,43]. In addition, elevated levels of resistance to tetracyclines have been reported in certain strains, further complicating treatment strategies [41,44]. Importantly, antimicrobial susceptibility profiles of LM are highly heterogeneous and influenced by geographic origin, source of isolation, and temporal factors, reflecting the dynamic and evolving nature of resistance patterns [18]. The growing clinical relevance of antimicrobial resistance is further highlighted by the emergence of multidrug-resistant (MDR) LM strains. The first MDR strain of human origin was identified in France in 1988 and demonstrated resistance to multiple antibiotic classes, including chloramphenicol, erythromycin, streptomycin, and tetracycline, with resistance genes located on a plasmid [45]. Since this initial report, additional MDR isolates have been recovered from clinical, food, and environmental sources across diverse geographic regions, underscoring the pathogen’s ability to acquire and disseminate resistance determinants via mobile genetic elements [33,44,46,47,48,49,50,51]. Taken together, the increasing prevalence of antimicrobial resistance, combined with the severe clinical consequences of listeriosis, underscores the urgent need to pursue novel antibiotics as well as complementary therapeutic strategies beyond conventional bactericidal approaches.

The growing challenge of antimicrobial resistance in LM has prompted increased interest in non-conventional strategies to manage infection beyond standard antibiotic therapy [18,52]. Consequently, increasing attention has been directed toward plant-derived antimicrobial compounds as alternative or adjunct agents. These natural products act through multiple mechanisms, such as compromising bacterial membrane structure, altering permeability, or interfering with efflux systems essential for bacterial survival [18,53]. Several plant-derived chemical compounds have been extensively documented for their inhibitory activity against LM, providing strong justification for exploring natural alternatives to conventional antimicrobials [54]. Compounds such as trans-cinnamaldehyde from cinnamon, eugenol from clove, thymol and carvacrol from thyme and oregano, citral from lemongrass, and oleuropein from olive have been shown to effectively suppress LM growth and survival in vitro [55,56,57,58]. Terpenoid compounds, including limonene and carvacrol, have shown notable activity against LM [59,60]. Beyond growth inhibition, sub-inhibitory concentrations of several phytochemicals significantly downregulated virulence-associated genes involved in motility, toxin production, adhesion, and host cell invasion, resulting in reduced hemolytic activity, impaired motility, and diminished epithelial cell adhesion and invasion [22,58,61,62]. Importantly, attenuation of virulence by these compounds has been validated in biologically relevant infection models, including human intestinal and endothelial cell lines, where treatment with trans-cinnamaldehyde, carvacrol, thymol, or eugenol markedly improved host survival outcomes [58,61,62,63]. Moreover, studies using the Galleria mellonella infection model have revealed that phytochemicals such as trans-cinnamaldehyde, carvacrol, and thymol can significantly attenuate LM virulence rather than solely inhibiting growth [63]. Although these findings highlight the therapeutic potential of natural antimicrobial compounds, their clinical application for the treatment of listeriosis remains dependent on further validation in mammalian models and human studies [53]. From a translational perspective, numerous studies have demonstrated that plant-derived compounds can be successfully incorporated into food-relevant delivery systems, such as active packaging films, edible coatings, marinades, and encapsulated formulations, leading to substantial reductions of LM in meat, dairy, produce, and ready-to-eat food models during storage [64,65,66,67,68]. The demonstrated feasibility of plant-derived antimicrobials and antivirulence compounds provides a strong conceptual basis for evaluating CBD as a natural approach to reduce LM pathogenicity. While plant-based compounds have been explored for food-related applications, the present study does not merely address the incorporation of CBD into food delivery systems; rather, it also explores on elucidating its potential role in controlling LM infection in the context of clinical regimen.

Historically, plants have contributed to the development of novel drugs and served as active components in a number of herbal and traditional medicines [69]. Many plant-derived chemicals have previously been shown to possess significant antibacterial properties against Gram-positive and Gram-negative bacteria [61,70,71,72,73,74,75,76]. Cannabidiol (C21H30O2) (CBD) is a non-psychoactive plant-derived compound obtained from the plant Cannabis sativa. It is reported to possess antimicrobial properties against a wide range of microorganisms, especially Gram-positive bacteria [77,78,79,80,81,82]. Wassmann et al. (2020) reported that CBD is an effective adjuvant in combination with bacitracin for killing Gram-positive bacteria [78]. Cannabidiol has neuroprotective properties [83], including the blood–brain barrier (BBB) [84,85]. It is also reported to exert protective effects on the intestinal barrier [86,87]. The FDA in 2018 and the European Medicines Agency (EMA) in 2019 approved a pure oil-based liquid formulation of CBD, known as Epidiolex® and Epidyolex, respectively for the oral management of two epilepsy conditions, namely Dravet syndrome and Lennox–Gastaut syndrome [80]. The application of CBD as an ingredient in food and health supplements has been increasing in recent years, especially driven by increasing consumer interest in natural wellness products [88].

Cannabidiol has been reported to exert antibacterial activity through multiple, primarily membrane-associated mechanisms. Evidence from radiolabeled macromolecular synthesis assays demonstrates that CBD rapidly inhibits protein, DNA, RNA, and peptidoglycan synthesis at concentrations near the minimum inhibitory concentration, consistent with a membrane-targeted bactericidal effect rather than inhibition of a single biosynthetic pathway [80]. This membrane-disruptive activity is further supported by observations of membrane depolarization, bacterial cytological profiling characteristic of membrane-permeabilizing agents, and rapid uptake of the normally membrane-impermeable SYTOX™ Green dye, collectively indicating loss of cytoplasmic membrane integrity and function [80]. In addition to direct membrane disruption, CBD has been shown to modulate bacterial pathogenicity by inhibiting membrane vesicle release and altering vesicle protein composition in Gram-negative bacteria, thereby enhancing the bactericidal activity of selected antibiotics and functioning as a potential antibiotic adjuvant [89]. Moreover, studies in Gram-positive bacteria have demonstrated that CBD damages bacterial cell walls and membranes, leading to widespread disruption of metabolic and biosynthetic pathways and global alterations in proteomic and metabolomic profiles, further contributing to its antibacterial activity [90].

The objective of this study was to investigate the efficacy of CBD in attenuating the major virulence factors in LM in vitro and controlling listeriosis in the invertebrate model, G. mellonella.

2. Results

2.1. Effect of CBD on the Growth of LM

To evaluate the effect of CBD on the growth of LM, bacterial cultures were exposed to increasing concentrations of CBD in tryptic soy broth and incubated at 37 °C for 24 h, after which bacterial growth was quantified by CFU enumeration to determine the sub-inhibitory concentration (SIC) and minimum inhibitory concentration (MIC). In this study, the SIC of CBD was defined as the highest concentration that did not result in a statistically significant reduction in LM growth relative to the control but had an effect on the transcription of genes while MIC was defined as the lowest concentration of CBD that completely inhibited bacterial growth, yielding CFU counts comparable to the initial inoculum at 24 h.

Using these criteria, the SIC and MIC of CBD against LM Scott A and ATCC 19115 were found to be the same. The SIC, 6× SIC, 1/4× MIC and MIC against LM were 11.92 μM (0.000375% w/v; 3.75 µg/mL), 71.52 μM (0.00225%; 22.5 µg/mL), 572.16 μM (0.018%; 180.0 µg/mL) and 2289.56 μM (0.072%; 719.8 µg/mL), respectively. The effect of various concentrations of CBD on LM growth is depicted in Figure 1. Cannabidiol demonstrated a concentration-dependent inhibitory effect LM with no significant reductions in bacterial counts (p < 0.05) at SIC and 6× SIC. A minimal reduction of less than 0.5 log CFU/mL in LM population was observed at 6× SIC, whereas a 3 log CFU/mL reduction was noted at 1/4× MIC of CBD (p < 0.05). As expected, the MIC of CBD completely inhibited LM growth yielding counts similar to control (no CBD) at 0 h of incubation (inoculation level) (p < 0.05), indicating a bacteriostatic effect.

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