Antibacterial effects of sulforaphane – A phytonutrient derived from broccoli as promising candidate in the combat of bacterial infections

Abstract

Bacterial pathogens, particularly antibiotic-resistant strains may constitute major challenges for the successful treatment of infected patients. Therefore, novel antibiotics or alternative, antibiotics-independent compounds with antimicrobial properties such as phytonutrients are needed. Our systematic literature review summarizes current knowledge on antibacterial effects of sulforaphane (SFN) in vitro and in vivo, including human studies. The isothiocyanate SFN is abundant in plants from the Brassicaceae family including broccoli. The 28 reports reviewed herein revealed that SFN i.) exerted antimicrobial effects against a variety of Gram-positive and Gram-negative bacteria; ii.) counteracted distinct virulence factors such as biofilm formation and toxin production (e.g. Shiga toxin); iii.) enhanced antibacterial immune cell responses mounting in anti-oxidant and anti-inflammatory actions thereby supporting bacterial killing and dampening inflammatory cell and tissue damage; iv.) prevented from aspirin-induced small intestinal cell injury; and v.) alleviated Helicobacter pylori-induced gastritis. In conclusion, given its antibacterial, immune-modulatory, and disease-alleviating effects, SFN constitutes a promising alternative antibiotic-independent candidate for the treatment of bacterial infections, warranting further consideration in clinical trials.

1. Introduction

1.1. Increasing antibiotic resistance

The development of antimicrobial resistance by bacteria is one of the global challenges in modern medicine. Since the discovery of sulfonamide and penicillin, antibiotics have revolutionized the treatment of bacterial infections and contributed significantly to combat infectious diseases. According to the World Health Organization, the overuse of antibiotics in humans, animals, and plants is one of the main causes for increasing numbers of resistant bacterial strains [1]. The excessive and in many cases unnecessary application of antibiotics in medicine, farming, and agriculture leads to ongoing selective pressure on commensal as well as pathogenic bacteria. While susceptible bacteria are killed by the drugs, resistant strains survive and continue to replicate. In the long term, this mounts in the scenario that certain infections become increasingly difficult or even impossible to be treated by common antimicrobial agents. Particularly multidrug-resistant (MDR) Gram-negative bacterial species represent a growing problem leading to failure of standard therapeutic regimens [2]. This scenario does not only increase morbidity and mortality, but also healthcare costs [3]. According to estimates, 4.95 million deaths in 2019 were associated with antibiotic-resistant bacteria worldwide [4]. The rational and responsible use of existing antibiotics is essential, in order to slow down the fatal development of resistance. To counteract the progressive loss of effective antibiotics, alternative therapeutic approaches are needed. In addition to the development of new classes of antibiotics, alternative antimicrobial strategies are becoming increasingly important.

1.2. Plant-derived antibacterial compounds

Phytochemicals are chemicals of plant origin of which antimicrobial effects have been demonstrated for thousands of them worldwide [5]. Besides flavonoids, tannins, and quinones, isothiocyanates have gained great interest in recent years.

1.2.1. Isothiocyanates

Isothiocyanates result from hydrolysis reactions of glucosinolates by the enzyme myrosinase both of which can be found in different compartments of plant cells [6]. Tissue disruption due to chewing or cooking removes the physical barrier, and the chemical reaction can occur. Glucosinolates are mainly present in cabbage, mustard, and broccoli belonging to the Brassicaceae family, but also the gut microbiome can provide myrosinase activity. Isothiocyanates can be assigned to different groups depending on their side chains (i.e., aliphatic, aromatic, heterocyclic) with the (–N=C=S) group as common feature. The molecules may react with amines, thiols, and hydroxyls but their biological activities are mainly associated with reactions with sulfhydryl groups. Since cysteine ​​is involved in the arrangement and structure of proteins, the ability of isothiocyanates to react with sulfhydryl groups is fundamental for their pleiotropic effects [6].

1.2.2. Sulforaphane

In recent years several isothiocyanates including sulforaphane (SFN; 1-isothiocyanato-4-(methylsulfinyl)butan) have been extensively investigated regarding their antibacterial and health-beneficial effects both, in vitro and in vivo. SFN belongs to the aliphatic group of isothiocyanates and constitutes a hydrolysis product of the glucosinolate glucoraphanin, which is primarily found in broccoli [7]. After its first isolation from broccoli in 1992 [8], SFN has become the topic in diverse research areas given its anti-cancer, anti-inflammatory, but also anti-microbial properties [9] suggesting SFN as potential antibiotics-independent candidate molecule in the combat of infectious diseases caused by bacterial including MDR microorganisms.

1.3. Objective

The aim of this literature review is to compile the current knowledge on SFN as a potential antibacterial agent derived from both, in vitro and in vivo including clinical studies.

2. Methods

2.1. Inclusion and exclusion criteria

This review summarizes knowledge from studies in English or German addressing the antibacterial effects of SFN. Studies that evaluated other isothiocyanates than SFN or focussed on SFN's features beyond its antibacterial effects were excluded.

2.2. Search strategy

A structured literature survey was conducted from December 2, 2024 to January 16, 2025 applied on the meta database “PubMed” of the U.S. National Institute of Health. First, the key term “sulforaphane” was searched for. In order to exclude studies that might note the following search terms as additional effects, another search was carried out for the term “bacteria”. To avoid missing studies due to different wording, searches were conducted using both “antibiotic” and “anti bacteria”. The final search, following the Boolean logic, included all terms connected by “AND”, except for the latter two, indicated as alternatives by using “OR”, resulting in total of 57 hits (as indicated in Table 1). Six papers were excluded since they were reviews, one since the paper was written in Chinese. Additionally, two preprints and one clinical study, for which no results had been published yet, were excluded. After excluding all these studies, the abstracts of the remaining 47 were reviewed and further 19 were excluded based on the exclusion criteria. The remaining 28 studies were included in this research.

Table 1.

Search query

Search	Query	Results
#1	“sulforaphane”	3,232
#2	“bacteria”	1,736,738
#3	“antibiotic”	515,597
#4	“anti bacteria*”	527,084
#5	(#1) AND (#2) AND ((#3) OR (#4))	57

3. Results

3.1. Antibacterial effects of SFN

In 2008, Johansson et al. analyzed the antimicrobial effects of SFN against a broad variety of Gram-positive (including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enterococcus faecalis, Bacillus subtilis, and Bacillus cereus) and Gram-negative bacteria (such as Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Enterobacter cloacae, Salmonella Typhimurium, Shigella sonnii, Pasteurella multocida, Pseudomonas aeruginosa, and Neisseria sicca) [10]. Using ceftriaxone as a positive control the disk diffusion method was used to test susceptibility of both, bacterial references strains and clinical isolates. With the exceptions of three methicillin-resistant S. aureus (MRSA) isolates and one P. aeruginosa reference strain, SFN exhibited high levels of antimicrobial efficacies against the bacteria tested given overall minimum inhibitory concentrations (MICs) ranging from 1 to 4 mg L⁻¹ [10].

Another study addressed the bactericidal effects of L-SFN against defined respiratory pathogens such as Streptococcus pneumoniae, S. pyogenes, and Haemophilus influenzae [11]. Measurements of the optical density of the bacterial solutions to quantify the direct inhibitory effects of the applied SFN revealed a significant growth inhibition of H. influenzae, but neither of the S. pneumoniae nor S. pyogenes strains. Furthermore, bacterial killing assays applying THP-1 macrophages and HL-60 cells underscored that clearance of H. influenzae was enhanced upon L-SFN treatment while no bacterial clearance could be observed for the streptococci tested. The authors hypothesized that the pronounced cell wall thickness of the Gram-positive bacteria prevented from the bactericidal actions of L-SFN exhibiting its antibacterial effects against the Gram-negative H. influenzae bacteria [11].

In their study from 2009, Aires and coworkers tested the antimicrobial effects of intact glucosinolates as well as of enzymatic hydrolysis products and microbial catabolites of glucosinolates on facultative anaerobic bacteria [12]. Therefore, three Gram-positive (E. faecalis, S. aureus, and Staphylococcus saprophyticus) and 15 Gram-negative bacterial strains (including E. coli, K. pneumoniae, and Acinetobacter baumanii) that had been isolated from the human intestinal tract were subjected to different doses (ranging from 0.015 to 3.0 µmol) of respective compounds using the disk diffusion method. Dimethylsulfoxid (DMSO) was used as a negative control while vancomycin (30 µg), gentamicin (10 µg), and tobramycin (10 µg) were used as positive controls. Whereas the lowest dose did not exhibit any bacterial growth-inhibiting effects, the higher ones resulted in bacterial growth inhibition in a concentration-dependent manner. Three of the isothiocyanates, including SFN, consistently showed the most pronounced antibacterial activities against the Gram-positive bacteria. In fact, all three of them were even more susceptible to SFN if compared to the vancomycin control. Except for Salmonella Typhi, all tested Gram-negative bacteria were susceptible towards SFN which, remarkably, turned out to be even more effective than all synthetic antibiotic compounds used as positive controls [12].

Another study of Aires et al. published in 2013 aimed to evaluate the antimicrobial potential of eight different glucosinolate hydrolysis products against four Aeromonas strains (namely, Aeromonas allosaccharophila, Aeromonas hydrophila, Aeromonas media, and Aeromonas veronii) isolated from pig ileum segments [13]. All compounds were tested at different doses ranging from 0.015 to 3.0 µmol. DMSO was used as the negative control in all experiments while gentamicin served as positive control. A modification of the disk diffusion test was performed to explore the antibacterial activity of each glucosinolate hydrolysis product. Except for one compound, the glucosinolate hydrolysis products showed bacterial growth-inhibitory effects in a dose-dependent way, whereas the most effective ones were three of the isothiocyanates including SFN. Notably, higher doses of the isothiocyanates including SFN were even more effective than the gentamicin control. Furthermore, application of an isothiocyanate mixture resulted in the most pronounced growth inhibition of all four Aeromonas strains [13].

In 2020, Abukhabta and colleagues compared the antimicrobial activities of SFN extracts from raw or cooked broccoli and cooked broccoli with added mustard seeds both, in vitro and in vivo [14]. For their in vitro experiments, SFN and glucoraphanin were extracted from respective broccoli samples. In addition, a broccoli powder-based soup was prepared with or without additional mustard seed powder and the SFN extracted as well. When measuring the SFN contents it turned out that rather low SFN levels were detected in both raw and cooked broccoli, whereas the addition of mustard seeds, however, increased the SFN concentrations considerably. Then, the authors tested the antimicrobial effects of respective SFN compounds against both Gram-positive (i.e., S. aureus, Bacillus cereus, and Listeria monocytogenes) and Gram-negative bacterial isolates (including E. coli K12, E. coli O157:H7, S. Typhimurium, Salmonella enterica). Interestingly, the raw broccoli extracts showed virtually no antimicrobial activity except against B. cereus, while in tests with cooked broccoli prominent antibacterial effects could be assessed. The highest antibacterial activity, however, was observed upon combination of cooked broccoli and mustard seeds containing the enzyme myrosinase. Analysis of the soup proved that addition of 2% mustard seed powder elevated the SFN concentration by nearly four-fold. The MIC of the pure SFN when tested against E. coli was 5.65 mM [14].

In a study from 2010, multiple essences from fresh broccoli sprouts were examined for their antibacterial activity against Helicobacter pylori [15]. To obtain the essences, Moon et al. processed fresh broccoli sprouts with methanol and water and further extracted the solutions with either hexane, chloroform, ethyl acetate or n-butanol. Eventually, six samples (crude methanol extract, residual water fraction, hexane, chloroform, ethyl acetate, and butanol) were tested for their H. pylori growth-inhibiting activities. In addition, 18 SFN-related compounds were synthesized and included into the antibacterial tests. The disk diffusion method was conducted with either 10 mg of the obtained extracts or 5 mg of synthesized molecules. Except for the residual water fraction, all samples tested were able to inhibit H. pylori growth. The strongest antibacterial activity was found for the chloroform extract. To further determine the distinct molecules responsible for the observed antibacterial effects, the chloroform extract was analyzed by gas chromatography. Three isothiocyanates including SFN and two nitriles were identified in this extract. Among the 18 synthesized compounds, nine inhibited H. pylori growth. Of note, SFN and related compounds that were found in the essences in significant amounts were the major active molecules in broccoli sprouts responsible for the H. pylori growth inhibition [15].

In 2012, Saavedra et al. evaluated the antimicrobial effects of different glucosinolate hydrolysis products against distinct commensal bacteria isolated from the pig ileum [16]. Besides SFN, the authors investigated seven other glucosinolate hydrolysis products regarding their growth-inhibitory properties directed against both, Gram-negative (i.e., E. coli, K. pneumoniae, Enterobacter hormaechei) and Gram-positive (i.e., E. faecalis, Enterococcus flavescens, Enterococcus hirae) bacteria. All glucosinolate hydrolysis products were analyzed at concentrations ranging from 0.015 to 15.0 µmol, but SFN only up to 3.0 µmol. Remarkably, SFN at its highest tested dose turned out to be more effective against all Gram-positive bacteria than the positive control gentamicin, and also against the Gram-negative isolates with the exception of E. hormaechei and K. pneumoniae against which SFN showed rather moderate antimicrobial effects at this dose. Inhibitory effects were observed at the 0.15 µmol SFN dose for all the bacteria except for E. hormaechei [16].

Haristoy et al. (2005) evaluated the antimicrobial activity of twelve isothiocyanates including L-SFN, D-SFN, and a synthetic chiral mixture D,L-SFN against H. pylori [17]. Two reference and 23 clinical H. pylori strains were used to determine the MICs of respective molecules. The SFNs exhibited pronounced antibacterial activities with MIC ranging from 0.06 to 8 μg mL⁻¹. Further assessment of the compounds showing the lowest MICs regarding their bactericidal activity against extra- as well as intracellular bacteria (in cultured human epithelial cells) was conducted. Whereas four isothiocyanates displayed potent bactericidal activities against both extra- and intracellular H. pylori, SFNs did not [17].

In their studies Ko et al. (2016) demonstrated that ten isothiocyanates inhibited the growth of oral pathogens and the relationship between structure and function [18]. The experiments were assessed with different Gram-positive bacterial strains including S. aureus, E. faecalis, Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus casei. The MIC and minimum bactericidal concentration (MBC) for each isothiocyanate were determined using the broth microdilution method. The MICs and MBCs of SFN against the tested strains ranged from 0.25 to 1.0 mg mL⁻¹ and 0.25 - >1.0 mg mL⁻¹, respectively. Compared to the other isothiocyanates, SFN was among the rather less effective antibacterial compounds when tested against oral pathogens. The authors hypothesized that the effectiveness of the respective compound depended on its molecular size and chemical structure. SFN belongs to the aliphatic compounds with generally less antibacterial potency, whereas it exhibits a longer hydrocarbon chain if compared to other isothiocyanates [18].

For their study in 2020, Cierpial et al. synthesized several fluoroaryl analogues of SFN and compared their antibacterial (besides antifungal, antiviral, and anticancer) properties with SFN [19]. The MICs of all compounds were assessed by the broth microdilution method. The results showed that the included Gram-negative bacteria (i.e., E. coli, P. aeruginosa) were not susceptible to the SFN analogues. On the contrary, the Gram-positive bacteria (i.e., S. aureus, S. epidermidis, E. hirae, B. subtilis, B. cereus, and Micrococcus luteus) were inhibited by both, the SFN and the fluoroaryl analogues. Remarkably, two of these analogues exhibited potent antimicrobial activities directed against clinical MRSA isolates. Against probiotic Lactobacillus strains, however, the antimicrobial effects of SFN and the analogues were negligible or even absent [19].

In 2020, Nowicki et al. addressed the antibacterial activities and synergistic effects of several aliphatic isothiocyanates [20]. The authors determined the MICs and MBCs of the isothiocyanates and further, calculated the fractional inhibitory concentration (FIC) index to detect synergies of different compounds when tested in combination. Among the included isothiocyanates, SFN exhibited the lowest MIC of 87 mg L⁻¹. When combining distinct compounds with SFN, however, FIC indices could be determined indicative for additive or even synergistic effects. Additionally, the authors supposed, that the effectiveness was not only dependent on the applied isothiocyanate but also on the experimental conditions since the MIC values were up to ten-fold lower after changing the culture media (e.g., from MH broth to M9 medium). In addition, densitometric analyses were performed from lysed bacterial cells that had been treated with isothiocyanates or serine hydroxamate to determine (p)ppGpp alarmones that are integral part of the stringent response constituting a global bacterial stress regulatory system. The results showed that both, SFN alone and combinations of other isothiocyanates with SFN resulted in enhanced, but comparable accumulation of alarmones indicative for similar stringent responses [20].

In 2022, Zhao et al. tested the antibacterial effects of SFN directed against Mycobacterium tuberculosis, Mycobacterium smegmatis, and E. coli applying the broth microdilution method and revealed MICs of 5 μg mL⁻¹, 10 μg mL⁻¹, and 80 μg mL⁻¹, respectively [21]. A time-kill kinetic analysis showed synergistic effects of SFN and rifampicin upon combination. Further experiments with the membrane-impermanent reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) and catalase revealed no influence of the latter on the bactericidal activities of the SFN, whereas the antimicrobial effects of SFN could be antagonized to the level of NAC when they were tested in combination. Since catalase catalyses the decomposition of H₂O₂ only while NAC acts on superoxide radicals, the authors hypothesized that the effects of SFN were mainly related to superoxide radicals. Furthermore, time- and dose-dependent treatment of bacteria with SFN resulted in increased intracellular ROS levels that were associated with bacterial killing. To investigate intracellular effects, THP-1 macrophages were infected with M. smegmatis or M. tuberculosis and treated with SFN, catalyse or NAC. Whereas lower SFN concentrations (10 μg mL⁻¹) did not affect bacterial growth, higher concentrations exhibited potent intracellular bactericidal effects that were antagonised by both catalase and NAC. Additionally, the authors found significantly increased mitochondrial ROS levels after SFN treatment when applied in a concentration of 100 μg mL⁻¹ [21].

3.2. Impact on bacterial virulence factors and plasmid curing activity

Fahey et al. (2013) analyzed the mechanisms of the inhibitory effects of several isothiocyanates including SFN on H. pylori urease [22]. In fact, SFN could effectively inhibit urease derived from five H. pylori strains which was shown to be due to a covalent binding between enzyme and inhibitor. When testing H. pylori growth inhibition by SFN, MBCs ranging from 2.8 to 5.6 μg mL⁻¹ could be assessed which also held true when tested against an urease-negative H. pylori strain. The authors concluded that the anti-H. pylori effects of SFN were not primarily due to urease inactivation [22].

In 2016, Nowicki et al. compared different isothiocyanates including SFN regarding their antimicrobial effects against enterohemorrhagic E. coli (EHEC) producing Shiga toxin that is mainly responsible for the EHEC-induced kidney failure [23]. The authors found that SFN did not only inhibit bacterial growth of two clinical EHEC isolates with MBCs of 0.71 mg mL⁻¹, but also the development of Shiga toxin encoding prophages. The authors further found that the SFN-related effects were due to inhibition of alarmones as integral part of the stringent response. Hence, the direct inhibition of both, bacterial growth and Shiga toxin production by isothiocyanates including SFN might be considered as promising treatment options for EHEC infections [23].

In a very recent study from 2024, Bendary et al. addressed whether SFN either alone or in combination with standard synthetic antibiotics might be an effective target molecule to counteract P. aeruginosa infections [24]. When testing for growth-inhibitory effects, the broth microdilution analysis revealed a MIC of 10 μg mL⁻¹ for SFN against P. aeruginosa. Further analyses of the anti-virulence actions of SFN were conducted with sub-inhibitory concentrations of ¼ MIC either alone or in combination with other anti-Pseudomonas compounds. Interestingly, SFN alone could effectively reduce motility and biofilm formation of P. aeruginosa and interfered with the oxidative stress responses of the opportunistic pathogens. Furthermore, SFN incubation could diminish production of virulence factors such as pyocyanin and the extracellular enzymes elastase, proteases, and hemolysins. When applied in combination with anti-pseudomonal antibiotics, SFN could reduce the MICs of the former resulting in synergistic effects. Furthermore, SFN reduced the expression of genes involved in quorum sensing mechanisms thereby additionally reducing bacterial virulence. Remarkably, SFN could dampen the P. aeruginosa-induced immunopathology in mice further underscoring the potential of SFN as promising treatment option of P. aeruginosa infections [24].

In a study published in 2021, Krause et al. investigated the antimicrobial effects of two isothiocyanates including SFN against Vibrio cholerae, and their impact on virulence factors and biofilm formation [25]. The microdilution assay revealed a MIC of 0.5 mM for SFN when tested against the pathogens, whereas ½ and ¼ of the MIC could also effectively inhibit bacterial growth. Whereas the tested isothiocyanates did not affect bacterial oxidative stress responses, SFN could reduce already formed bacterial biofilm upon exposure at 1x and 5x MIC. In addition, SFN also prevented from biofilm formation when applied prophylactically. Transmission electron microscopy observation analysis revealed cell shrinkage and cytoplasm condensation after isothiocyanate treatment. What is more, cytosol leakage on distal parts of the cells could be observed after 120 min but no extensive damage of cells or cell membranes. The [³H]-radiolabeled nucleotide assay was assessed to compare effects on RNA or DNA synthesis. The results exhibited a significant decrease of stable RNA synthesis. [³H]-thymine incorporation revealed impaired DNA synthesis, whereas measurement of [³²P]-labeled nucleotides showed increased (p)ppGpp alarmones levels upon isothiocyanate treatment that were comparable to cultures under amino acid starvation induced by serine hydroxamate. Further, a real-time quantitative polymerase chain reaction (RT-qPCR) was performed to analyse virulence gene expression. Significant down-regulation of all tested genes (namely, toxT, toxR, toxS, ctxB, cpH, tcpP, and tcpA) was observed upon SFN treatment and also in the presence of serine hydroxamate. The activities of the isothiocyanates were examined in different human cell lines and resulted in significant decrease of the cytotoxic effects of the V. cholerae lysates, without exerting any toxic effects to the tested cell lines themselves [25].

Nowicki et al. studied the effects of SFN and another isothiocyanate on three Shiga toxin-producing Shigella dysenteriae strains [26]. At first, the broth microdilution method revealed that the MICs of SFN were in the range between 11 and 44 μg mL⁻¹. Two additionally included Shigella species (namely, Shigella sonnei and Shigella flexneri), however, were far less susceptible with MICs exceeding 100 μg mL⁻¹. The Formazan-assay was conducted to test Vero and HeLa cell viability after exposure to isothiocyanate-treated (¼ MIC) or untreated bacterial lysates. The results showed significantly attenuated cytotoxicity when the lysates were treated with isothiocyanates. To investigate the underlying mechanisms, (p)ppGpp alarmones were evaluated in bacterial cells treated with phenylethyl-isothiocyanate, SFN or serine hydroxamate compared to cells that were left untreated. The findings for isothiocyanate-treated bacteria revealed increased levels of (p)ppGpp that were comparable to those in cells treated with serine hydroxamate. Additional gene expression analyses exhibited significant down-regulation of stx expression after isothiocyanate treatment at the MIC or ¼ MIC. Collectively, the obtained results further underscore the potency of SFN in the treatment of Shigella infections [26].

In 2019, research on the inhibitory effects of several isothiocyanates on bacterial conjugation constituting the main mechanism of horizontal resistance genes transfer was published by Kwapong et al. [27]. For their experiments several S. aureus and E. coli strains were included and the antibacterial properties of the isothiocyanates determined applying the broth microdilution assay. Overall, the applied isothiocyanates revealed lower MICs against the Gram-positive if compared to the Gram-negative strains, whereas for SFN, MIC values between 32 and 64 mg L⁻¹ were assessed. Further, the authors tested the anti-conjugant activity for different plasmids. The SFN could inhibit conjugation with one of the plasmids (i.e., IncW plasmid R7K). At the same concentration (16 mg L⁻¹), plasmid curing could be observed and hence, SFN not considered as anti-conjugant molecule despite its antibacterial effects [27].

3.3. Antibacterial immune responses modulated by SFN

Yang et al. (2016) addressed whether SFN might be involved in the modulation of lipopolysaccharide (LPS)-induced pro-inflammatory responses in alveolar macrophages and investigated the epigenetic DNA methylation of CD14 by SFN treatment [28]. Therefore, the authors isolated alveolar macrophages from piglets and tested whether the pre-treatment with SFN improved cell viability following LPS challenge which in fact, occurred in a SFN concentration-dependent manner. Gene expression studies of CD14 and downstream genes in the TRIF pathway in SFN and LPS co-treated alveolar macrophages revealed that CD14 gene expression was down-regulated upon SFN pre-treatment which also held true for the expression of TRAM, TRIF, RIPK1, and TRAF3. Furthermore, SFN application was accompanied by decreased expression of tumor necrosis factor-alpha (TNF-α), interferon-beta (IFN-β), interleukin (IL)-1β, and IL-6 in LPS-stimulated alveolar macrophages, which could also be confirmed on protein level. Analyses of the DNA methyltransferase DNMT1 and DNMT3a expression showed an up-regulating effect of LPS on DNMT3a expression that was suppressed by SFN. Furthermore, bisulfite sequencing was applied to further unravel the molecular mechanisms of SFN-induced CD14 down-regulation. However, no methylation changes could be detected in the CD14 promoter region of any SFN-LPS-treated alveolar macrophages, whereas two alterations of the gene body methylation sites became evident. Furthermore, methylation was enhanced in LPS-treated cells and suppressed upon SFN challenge. The authors concluded that SFN mediated the epigenetic down-regulation of CD14 in the LPS-induced inflammatory responses within the TRIF pathway thereby limiting inflammation in pigs [28].

In their study from 2013, Koo et al. addressed whether SFN inhibited the binding of LPS to MD2, the co-receptor of the Toll-like receptor-4 (TLR-4) [29]. First, an in vitro assay was performed to verify inhibitory effects of SFN on the interaction of LPS with MD2 and revealed decreasing association of MD2 with biotinylated LPS. To confirm these results in cellular systems, Ba/F3 cells expressing TLR-4 and Flag-tagged MD2 were treated with LPS in the presence or absence of SFN. The immunoblot analysis demonstrated reduced association of LPS with MD2 in a SFN dose-dependent manner. In further experiments BMDMs were treated with fluorescent LPS. Analysis by confocal microscopy showed that SFN significantly blocked the co-localization of fluorescent LPS with MD2 in BMDMs. Immunoprecipitation and immunoblotting studies using Ba/F3 cells treated with LPS and SFN were performed in the presence or absence of NAC or dithiothreitol to determine whether the inhibitory effect of SFN was related to its reactivity with sulfhydryl groups. In fact, the suppressive effect of SFN could be reversed by supplementation with NAC and dithiothreitol, whereas the SFN-mediated inhibition was blocked by NAC. Furthermore, liquid chromatography-tandem mass spectrometry analysis and a docking simulation analysis showed a covalent binding of SFN to Cys133 exclusively in MD2 suggesting that this binding blocked the interaction between LPS and MD2. In consequence, SFN could suppress the LPS-dependent activation of TLR-4 [29].

In a study from 2020, Ali et al. compared the effects of four defined compounds including SFN on their anti-inflammatory properties on bacteria-infected macrophages [30]. At first, the applied MTT assay revealed that non-toxic concentrations of respective compounds did neither exert any cytotoxic effects when tested against THP-1-derived macrophages nor peripheral blood mononuclear cell (PBMC)-derived macrophages within 24 h post incubation. Furthermore, SFN treatment was found to increase nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of anti-inflammatory response pathways mounting in diminished pro-inflammatory and oxidative stress responses in LPS-stimulated macrophages as indicated by down-regulated gene expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Interestingly, SFN down-regulated M1 marker genes in LPS- and interferon-gamma (IFN-γ)-activated THP-1-derived macrophages, whereas the expression of M2 marker genes was up-regulated by SFN. Furthermore, both the THP-1-derived and PBMC-derived macrophages were pre-treated with respective compound and infected with either E. coli or S. aureus. Unexpectedly, bacterial counting revealed promoted E. coli survival upon SFN treatment in THP-1-derived macrophages while in PBMC-derived macrophages E. coli survival was significantly suppressed. In case of S. aureus infection, bacterial survival was decreased in both types of macrophages due to SFN intervention [30].

In 2022, Zhou et al. tested the effects of SFN on Nrf2 activation in M. tuberculosis-infected THP-1-derived macrophages [31]. At first, a cell viability assay revealed 20 µM as a suitable dose of SFN that was used for further experiments. Pre-treatment with SFN resulted in increased Nrf2 levels in both, uninfected and M. tuberculosis infected THP-1 macrophages that was accompanied by diminished bacteria-induced production of pro-inflammatory cytokines. Further, flow cytometry analyses revealed fewer infected THP-1 macrophages when cells were treated with SFN prior infection. While SFN treatment on uninfected cells did not induce apoptotic cell responses, pre-treatment significantly attenuated M. tuberculosis-induced apoptosis. Additionally, increased quantities of autophagy vesicles were observed through transmission electron microscopy when cells were pre-treated with SFN. Furthermore, the authors were able to show that SFN application could increase the expression of the autophagosome marker LC3 II due to its Nrf2-activating effect. In conclusion, SFN treatment resulted in enhanced mycobacterial killing effects through Nrf2 activation [31].

Deramaudt et al. tested the effects of SFN on the intracellular survival of S. aureus in macrophages [32]. Therefore, human THP1-derived macrophages, primary human PBMC-derived macrophages, and primary mouse bone marrow-derived macrophages (BMDMs) were treated with SFN prior S. aureus infection. The results revealed that prophylactic SFN application diminished intracellular S. aureus survival in macrophages via inhibition of the p38/JNK signaling pathway. In addition, the authors found that S. aureus-induced cell apoptosis was reduced upon SFN prophylaxis given lower cellular activated caspase-3/-7 levels. Even though the SFN-mediated inhibitory effects occurred Nrf2-independently, intracellular survival of S. aureus was enhanced in Nrf2^−/− BMDMs. Furthermore, SFN challenge suppressed S. aureus-induced up-regulation of the pro-inflammatory mediators TNF-α, IL-1β, and IL-6, which also held true for IL-23, inducible nitric oxide synthase (iNOS), and M1 marker genes. Collectively, the results suggest that activating effects of SFN on the bactericidal activity of macrophages was caused by decreased pro-inflammatory responses of JNK and p38 MAPK signalling [32].

Mazarakis et al. (2021) addressed the immune-modulatory effect of L-SFN and its metabolites on PBMCs [33]. Therefore, blood samples were collected from 14 healthy volunteers, then the PBMCs isolated, pre-treated with SFN or derived metabolites (in concentrations of 10 and 50 µM), and stimulated with either LPS or imiquimod. Flow cytometry analyses revealed that 10 μM L-SFN co-incubation resulted in higher proportions of CD3⁺ T cells and CD19⁺ B cells, whereas CD56⁺ natural killer (NK) cells and CD14⁺ monocytes levels decreased after treatment with 50 μM L-SFN. Chemokine and cytokine measurements exhibited significantly reduced levels of IL-6, monocyte chemotactic protein-1 (MCP-1), IL-1β, IL-10, and RANTES after pre-treatment with L-SFN and subsequent LPS stimulation, whereas TNF-α levels remained unaffected. Immune phenotyping of specific dendritic cell (DC) populations revealed significant increases in total DC numbers and dose-dependently elevated immature DCs but reduced CD14⁺ monocytes and plasmacytoid DCs. Furthermore, THP-1 cells that had been transfected with an anti-oxidant response element (ARE) construct were challenged with L-SFN and the Luciferase Reporter Assay applied to determine the Nrf2 activity in THP-1 cells. In fact, the results showed significantly increased Nrf2-ARE activity due to L-SFN treatment compared to the untreated control group [33].

Liang et al. (2020) addressed the potential effects of SFN in pneumococcal infections [34]. A combination of computational and in vitro methods revealed that L-SFN and distinct metabolites could affect adherence of pneumococci via modulating bacterial factors, host receptors, and inflammatory responses. In fact, pneumococcal adherence to Hep-2 cells could be diminished in the presence of L-SFN, L-sulforaphane-cysteine (L–SFN–CYS), L-sulforaphane N-acetyl-cysteine, and L-sulforaphane-glutathione (L–SFN–GSH). Furthermore, synthroton-Fourier transform infrared S-microspectroscopy revealed that treatment with L-SFN or metabolites caused direct changes in protein and lipid profiles in pulmonary epithelial cells. Furthermore, results from molecular docking and comprehensive network studies including a plethora of pneumococcal and host factors highlighted L–SFN–GSH as a potent candidate molecule interfering with plasminogen, fibronectin, and the pneumococcal pilus-associated antigen RrgA as key factors in the network of the pathogen and host in favor of the latter [34].

3.4. Application of SFN in animal studies

In 2013, a study was published by Yanaka et al. focussing on SFN as a potential preventive measure of non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal injuries [35]. Initial in vitro experiments revealed that prophylactic application of 5 µM SFN to rat-derived small intestinal mucosa cells could attenuate aspirin-induced cell injury that was accompanied by increased expression of the heme oxygenase-1 (HO-1) gene. When testing SFN in vivo, application of SFN glucosinolate constituting a biologically inactive SFN precursor that is converted to the active form in the lumen of the gastrointestinal tract could ameliorate indomethacin-induced small intestinal injury in mice and furthermore, prevent from mucosal overgrowth with commensal intestinal enterobacteria [35].

In the in vivo part of their work, Bendary et al. tested whether SFN could protect mice from P. aeruginosa infection [24]. Treatment of P. aeruginosa-infected mice with SFN at a concentration of ¼ MIC (i.e., 2.5 μg mL⁻¹) resulted in less pronounced mortality if compared to placebo. Whereas the latter showed irreversible tissue fibrosis and necrosis in ex vivo biopsies taken from the liver and kidney, mice from the SFN treated group exhibited only modest lesions and mild signs of inflammation [24].

After investigating the impact of SFN on biofilm formation, Krause et al. also performed in vivo experiments applying Vibrio cholerae-infected Galleria mellonella larvae [25]. Pre-treatment with SFN (25 mg kg⁻¹) 3 h before V. cholerae infection improved the survival of the larvae by 60% and 30% after 48 and 72 h, respectively, if compared to mock-treated counterparts. Moreover, both, hemocytes infiltration to larval hemolymph and phagocytosis rates by hemocytes were enhanced upon SFN treatment as assessed by fluorescence microscopy analysis [25].

Also in the G. mellonella model, Shiga toxin inhibition due to SFN treatment was tested in vivo by Nowicki et al. (2021) [26]. Therefore, G. mellonella larvae were infected with one of the S. dysenteriae strains. Without intervention, the 96 h-survival rate was 20%, whereas SFN treatment had similar effects like azithromycin used as positive control resulting in approximately 50% survival 96 h post infection. When applying SFN and the synthetic antibiotic together, additive effects could be achieved as indicated by an 80% survival rate at 96 h [26].

Yanaka and colleagues addressed the effects of SFN-rich broccoli sprouts on pathogenic colonization and induced gastric inflammation following H. pylori infection of mice [36]. Therefore, conventional wildtype and Nrf2-deficient mice were fed a salt-rich diet and orally infected with H. pylori Sydney strain 1. Following 8-week-feeding with broccoli sprout homogenates via the drinking water, wildtype mice displayed lower gastric H. pylori burdens, abrogated inflammatory responses in the corpus, and virtual absence of salt-induced gastric atrophy that was accompanied by down-regulated expression of pro-inflammatory cytokines such as TNF-α and IL-1β if compared to mock-treated counterparts. Conversely, oral treatment of Nrf2-deficient mice with SFN-rich broccoli sprouts did not exert any disease-alleviating effects following H. pylori infection indicating the pivotal role of the Nrf2-dependent anti-oxidant and anti-inflammatory effects exerted by SFN [36].

3.5. In vivo studies with SFN in humans

In order to investigate the disease-alleviating effects of a SFN-rich diet in H. pylori-positive humans, Yanaka et al. treated 25 subjects with 70 g of glucoraphanin-rich 3-day-old germinated broccoli sprouts per day for eight weeks, while 23 subjects received alfalfa as placebo [36]. Treatment with broccoli sprouts reduced gastric pathogen loads as indicated by less urea measured by the breath test, and less H. pylori antigen measured in stool samples that were accompanied by lowered pepsinogen I and II concentrations used as gastric inflammatory biomarkers. Like in mice, treatment of H. pylori-infected humans with SFN-rich broccoli sprouts points towards a promising antibiotics-independent intervention measure of pathogen-induced oxidative stress during gastritis but does not result in complete eradication of the infection [36].

In 2020, Abukhabta and colleagues conducted a human intervention trial where broccoli soup with and without mustard seeds was fed to 11 ileostoma patients (characterized by: age range 32–63 years, terminal ileostomies at least 1.5 years ago, non-smokers) [14]. Before and after soup intake, ileal fluids were collected and analyzed. The group that was fed broccoli soup without mustard seeds revealed mean SFN contents of 0.17 µmol in ileal fluids at 4 h post consumption that could be increased by adding mustard seeds to mean values of 1.05 µmol. When testing the SFN extracted from the ileal fluids regarding their antibacterial activity against E. coli, no such antibacterial effects could be observed, however. The authors concluded that SFN-rich broccoli soup might exert antimicrobial effects rather in the stomach and upper intestines, but not in more distal compartments such as the ileum and colon [14].

A study from 2019 by Chang et al. investigated the impact of SFN or a probiotic compound containing the yeast Saccharomyces boulardii on the H. pylori eradication rate when applied in combination with a standard first-line treatment regimen [37]. Therefore, 183 subjects were enrolled and included in three test groups. All patients were treated with the standard triple therapy consisting of 40 mg of pantoprazole, 1 g of amoxicillin, and 500 mg of clarithromycin taken twice a day for one week. Group A did not receive any other treatments while for groups B and C one capsule was provided additionally with either the probiotic (group B) or SFN (group C) three times daily for four weeks. Even though the eradication rate in group C turned out to be higher if compared to group A, none of the final eradication rates showed statistically significant differences in comparison to group A. Also, the frequency of adverse events did not differ between the groups. In conclusion, the authors could not observe increasing H. pylori eradication rates or decreasing adverse events due to supplementation of probiotics or SFN to a standard H. pylori eradication regimen [37].

4. Discussion

4.1. Main findings of the research

4.1.1. Antibacterial effects of SFN

Several studies comparing distinct isothiocyanates including SFN with each other revealed superior antibacterial effects of SFN [17, 18]. Overall, SFN and derived compounds were shown to exert potent antimicrobial properties against a broad variety of both, Gram-positive and Gram-negative commensal as well as (opportunistic) pathogenic bacteria [10–27] which also held true when testing for intracellular growth-inhibitory effects of respective compounds [11, 30–33]. The determination of the MICs and MBCs of SFN and derivatives revealed overt bacterial growth-inhibiting effects when testing against environmental and reference strains, but also clinical isolates. Remarkably, in some studies the bacterial growth-inhibiting properties were even more pronounced if compared to synthetic antibiotics used as positive controls [12, 13, 16]. However, individual studies reported discrepant results regarding SFN's antimicrobial effects when focussing on Gram-positive and Gram-negative bacterial classes. In one report, for instance, the tested L-SFN did not inhibit growth of the included Gram-positive respiratory pathogens such as S. pneumoniae and S. pyogenes but could clear extracellularly and even intracellularly growing Gram-negative H. influenzae bacteria [11]. Another study, however, revealed lower MICs of the isothiocyanates of interest including SFN when tested against Gram-positive S. aureus versus Gram-negative E. coli [27]. Besides the applied concentration and other defined experimental conditions, the antibacterial effects depended on the molecular size and the chemical structure including the isoform of the substance on one hand side and on the respective bacterial species and within the species on the isolate on the other, for instance [18]. Regarding its effects targeting MDR bacteria, one study reported that the tested SFN concentrations could not inhibit growth of three MRSA isolates [10]. Further investigations, including other MDR Gram-positive bacteria such as vancomycin-resistant enterococci (VRE) and particularly multi-resistant Gram-negative rods (3MRGN/4MRGN) might be of urgent need to further unravel to potential of isothiocyanates including SFN in the combat of infections caused by MDR bacteria. Interestingly research indicated that the concentration and the bioavailability of the contained SFN depended on the preparation and absorption methods. Comparing raw and cooked broccoli, for instance, revealed comparably low SFN concentrations, but far more pronounced antimicrobial effects of the latter versus the former when tested in vitro that could be enhanced by adding mustard seeds [14]. In vivo, however, the SFN-derived antimicrobial effects were rather minor in the distal intestinal tract (i.e., ileum, colon) as shown following feeding of SFN-rich broccoli soup with mustard seeds to ileostoma patients where the MICs against the tested bacteria were not low enough for sufficient bacterial growth inhibition [14].

4.1.2. Impact of SFN on bacterial virulence factors

In addition to the bacterial growth inhibition, SFN and metabolites were tested regarding their effects directed against defined virulence factors expressed by the pathogens. Studies on the expression of the stx gene encoding for the Shiga toxin produced by EHEC and Shigella species, for instance, revealed suppressed expression upon SFN treatment [23, 26]. Besides the Shiga toxin, other virulence factors involved for quorum sensing in Pseudomonas species were shown to be suppressed [24]. SFN also proved to be an inhibitor of urease produced by H. pylori [22]. This suppression was even shown to be irreversible contrary to another clinically used synthetic urease inhibitor.

Research also addressed whether isothiocyanates could affect bacterial plasmid conjugation thereby counter-acting resistance development and inter-bacterial exchange of toxin genes. Unfortunately, SFN did not prove to be an effective anti-conjugant agent even though minor effects had been observed [27]. Furthermore, two studies addressed the impact of SFN on bacterial biofilms. Remarkably, not only already existing biofilm mass decreased upon SFN application, but also new biofilm formation was attenuated [24, 25].

4.1.3. Putative molecular mechanisms underlying the antibacterial effects of SFN

4.1.3.1. SFN and the bacterial stringent response

In view of the molecular mechanisms that were responsible for the observed antibacterial properties of SFN, some studies revealed that the induction of the stringent response, a reaction mechanism of bacteria to environmental stress including nutrient deficiency, might be one explanation given increased levels of the alarmones (p)ppGpp upon SFN stimulation [20, 25, 26], but also the modulation of genes involved in the relA-mediated pathway by SFN was shown to play a role [20, 21, 23].

4.1.3.2. SFN and modulation of antibacterial immune responses

Other experimental approaches addressed the effects of isothiocyanates including SFN on the antibacterial immune responses on the host side and revealed, that macrophages and monocytes were stimulated upon SFN challenge to enhance killing of extracellular as well as intracellular pathogens by augmenting oxidative stress [21, 30–32]. Whereas SFN modulated LPS-induced pro-inflammatory immune responses and limited inflammation in pigs [28], it was also shown to suppress the LPS-dependent activation of TLR-4 [29]. Another study revealed that L-SFN application to human-derived PBMCs dampened pro-inflammatory mediator secretion, irrespective of a subsequent TLR stimulation, and resulted in shifts among distinct immune cell subsets further underscoring the immune-modulatory effects of the natural compound [33]. In support of its anti-oxidative capacities, SFN was proven as a promising preventive measure of NSAID-induced small intestinal injuries [35]. Furthermore, like in mice, treatment of H. pylori-infected humans with SFN-rich broccoli sprouts could dampen the oxidative stress responses and ameliorate pathogen-induced gastritis but failed to result in bacterial eradication [36].

4.1.4. Synergistic effects of SFN and other molecules of clinical impact

Another part of research addressed synergistic effects of SFN and other plant-derived compounds or antibiotics and found synergistic antibacterial effects in some cases [20, 21, 24], whereas however, SFN induced bacterial stringent responses were not further affected by combinatory regimens [20]. In one study the combination of SFN with rifampicin showed synergistic effects in killing of E. coli and mycobacteria in vitro [21], which was also the case when testing SFN in combination with different anti-pseudomonal antibiotics [24]. A trial with human subjects revealed no statistically significant increases in H. pylori eradication rates when SFN was used in addition to a standard antibiotic therapy [37].

4.2. Limitations

The presented extensive research on SFN's antibacterial effects provides a broad overview of current knowledge. However, it is difficult to summarize or compare results from the included studies, as heterogeneous groups/strains/isolates of bacteria were applied for testing, experiments under different conditions were conducted, and even when the same methods were applied, inter-individual variations were noticed which was even more pronounced when comparing results obtained from different reports. A limiting factor in the systematic literature review was also that the search and analysis of the individual sources were conducted by only one person. Therefore, despite the careful approach and precise work, mistakes cannot be excluded.

4.3. Conclusion and outlook

In summary, SFN has demonstrated a highly versatile effectiveness against a broad spectrum of bacterial (opportunistic) pathogens. Besides direct growth-inhibiting properties, down-regulatory effects on virulence factors have also been shown in many studies which further underscores SFN as a promising tool in the fight against rising antibiotic resistance, as it mitigates infections without increasing selective pressure for resistance development. Various immune-modulatory pathways have been identified through which infection processes are influenced as well. Previous work, for instance, highlights the importance of the triangle relationship between nutrients from the Brassicaceae family including broccoli, the commensal gut microbiota, and formation of pivotal components of the immune system orchestrating intestinal homeostasis and enhancing resistance to infections [38, 39]. However, many questions regarding the exact underlying molecular mechanism of (inter-)actions of SFN and potential synergies with common synthetic antibiotics or other phytochemicals, for instance, remain unanswered that need to be addressed in future research. In addition, studies focusing on potential adverse effects and interactions are required in order to safely apply SFN in the future in medicine to assure that individuals benefit from the multitude of proven health-promoting and disease-alleviating effects of the phytochemical.

List of abbreviations

ARE

antioxidant response element

BMDM

bone marrow-derived macrophage

DC

dendritic cell

DMSO

dimethylsulfoxid

EHEC

entero-hemorrhagic Escherichia coli

FIC

fractional inhibitory concentration

HO-1

heme oxygenase-1

IFN

interferon

IL

interleukin

LPS

lipopolysaccharide

L–SFN–CYS

L-sulforaphane-cysteine

L–SFN–GSH

L-sulforaphane-glutathione

MBC

minimum bactericidal concentration

MCP-1

monocyte chemotactic protein-1

MDR

multi-drug resistant

MIC

minimum inhibitory concentration

MRGN

multi-resistant Gram-negative rods

MRSA

methicillin-resistant Staphylococcus aureus

NAC

N-acetyl-L-cysteine

NK cell

natural killer cell

Nrf2

nuclear factor erythroid 2-related factor 2

NSAID

non-steroidal anti-inflammatory drugs

PBMC

peripheral blood mononuclear cell

ROS

reactive oxygen species

RT-qPCR

real-time quantitative polymerase chain reaction

SFN

sulforaphane

TLR-4

Toll-like receptor-4

TNF-α

tumor necrosis factor-alpha

VRE

vancomycin-resistant enterococci

Funding Statement

Funding: None.