**1. Introduction**

Historically, plants have provided a wide variety of active compounds, becoming a key element in the development of many cultures. The Celastraceae family, commonly known as the bittersweet family, comprises a group of plants distributed mainly in tropical and subtropical regions of the world including North Africa, South America, and East Asia. It has a long history in traditional medicine as a stimulant, diuretic, emmenagogue, antiinflammatory, antibacterial, anti-cancer, and for the treatment of gastrointestinal disorders among others [1]. In the Canary Islands, East Africa, and the Arabian Peninsula, the leaves of members of this family are chewed to combat fatigue [1,2]. In our search for antimicrobial compounds from plants, we isolated celastrol and its methylated derivative, pristimerin, which are the first and the most frequently reported celastroloids. The term celastroloid refers to methylene quinone nor-triterpenes with a 24-nor-*D: A*-friedo-oleanane skeleton, which, later on, was extended to related phenolic nor-triterpenes [3] and their dimer and trimer congeners. The two natural pentacyclic triterpenoids celastrol and pristimerin are commonly found in the roots and bark of Celastraceae species. Both compounds show a wide range of pharmacological activities, including anti-cancer, anti-inflammatory, antioxidant, hepatoprotective, or immunomodulatory, among others [4–8]. The anti-cancer properties of celastrol, one of the most studied methylene quinones, have been attributed

**Citation:** Padilla-Montaño, N.; de León Guerra, L.; Moujir, L. Antimicrobial Activity and Mode of Action of Celastrol, a Nortriterpen Quinone Isolated from Natural Sources. *Foods* **2021**, *10*, 591. https://doi.org/10.3390/foods10030591

Academic Editors: Ana Maria Loureiro da Seca and Antoaneta Trendafilova

Received: 12 February 2021 Accepted: 5 March 2021 Published: 11 March 2021

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to apoptosis and autophagy induction, cell cycle arrest, and anti-metastatic and antiangiogenic action [9,10]. Other works have evidenced the ability of celastrol in combating metabolic disorders such as obesity and type 2 diabetes [11]. Treatment with celastrol suppresses food intake, blocks reduction in energy consumption, and mediates weight loss by acting as a leptin sensitizer in mouse models [12,13].

In addition to these pharmacological activities, antimicrobial properties have also been demonstrated for these and other related triterpenoids such as tingenone, netzahualcoyone, or zeylasterone that exhibit inhibitory activity against Gram-positive bacteria [14–17]. Moreover, the anti-biofilm [18–20] and anti-fouling properties of celastrol and pristimerin have been reported on a wide variety of microorganisms [21], as well as anti-mycotic activity [22–27]. Besides, celastrol and its derivatives have also shown inhibitory activity against viruses producing hepatitis B and C [28,29], which makes them potentially useful compounds for the control of various diseases. This paper describes the antimicrobial activity and mechanism of action of celastrol and pristimerin using *Bacillus subtilis* as a model of spore-forming bacteria. Species of *Bacillus* have been associated with many food contaminations causing food-borne illness in humans [30], spoilage of processed food products [31,32], or certain pathologies such as pneumonia, bacteremia, and meningitis in immunosuppressed patients [33–35]. Regardless of variations in disease presentation, the etiologic agent is often the spore and the production of toxins that play a central role in the pathophysiology of the infection. Although it is generally accepted that the antimicrobial activity of terpenoid compounds involves damage on plasma membrane [36], little is known about how this affects other cellular processes essential to bacterial cell development. Thus, the action of these compounds on different metabolic pathways such as macromolecular synthesis, uptake of solutes and biosynthetic precursors, or the damage on membrane function was investigated.

#### **2. Materials and Methods**

#### *2.1. Microorganisms*

Strains used for determining antimicrobial activity included *Bacillus subtilis* ATCC 6051, *B. cereus* ATCC 21772, *B. megaterium* ATCC 25848, *B. pumilus* ATCC 7061, *Staphylococcus aureus* ATCC 6538, *S. epidermidis* ATCC 14990, *S. saprophyticus* ATCC 15305, *Enterococcus faecalis* CECT 795 (from Type Culture Spanish Collection), *Mycobacterium smegmatis* ATCC 19420, *Proteus mirabilis* CECT170, *Escherichia coli* ATCC 9637, *Pseudomonas aeruginosa* AK958 (from the University of British Columbia, Department of Microbiology collection), *Salmonella* spp. CECT 456, and *Candida albicans* CECT 1039.

Bacterial cultures were developed at 37 ◦C in nutrient broth (NB), except for *E. faecalis* and *M. smegmatis* that were grown in brain heart infusion broth (BHI), or *C. albicans* cultured in Sabouraud liquid medium. All media were purchased from Oxoid.

#### *2.2. Quinones and Others Antibacterial Compounds*

Celastrol and pristimerin were isolated, purified, and characterized from the roots of Celastraceae species as previously reported [37,38]. Pure compounds were dissolved in dimethyl sulfoxide (DMSO) before the evaluation. The reference antibacterial agents ciprofloxacin, rifampicin, tetracycline, penicillin, and clofoctol (Sigma-Aldrich, St. Louis, MO, USA) were used as controls according to the Clinical and Laboratory Standards Institute [39].

#### *2.3. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)*

The antimicrobial activity was determined for each compound in triplicate by broth microdilution method (range 0.08–40 μg/mL) in 96-well microtiter plates, according to the M07-A9 approved standard of the Clinical and Laboratory Standards Institute (CLSI) [40]. Wells with the same proportions of DMSO were used as controls and never exceeded 1% (*v*/*v*). The starting concentration of microorganism ranged between 1 and 5 × 105 colony-forming

units (CFU)/mL, and growth was monitored by measuring the increase in optical density at 550 nm (OD550) with a microplate reader (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland) and viable count in agar plates. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound that completely inhibits growth of the organisms compared to the untreated cells. All wells with no visible growth were subcultured in duplicate by transferring (100 μL) to nutrient, BHI, or Sabouraud agar plates. After overnight incubation, colony counts were performed and the minimum bactericidal concentration (MBC) was defined as the lowest compound concentration that produces ≥99.9% killing of the initial inoculum.

#### *2.4. Bacterial Killing Assays*

Overnight liquid cultures of *B. subtilis* were diluted in Monod's flask containing 10 mL of NB medium, to give a working concentration ranging between 1 and 5 × 105 CFU/mL. Celastrol (3 μg/mL) or pristimerin (10 μg/mL) was added at time 0 (lag phase) or after 3 h of incubation (log-phase, OD550 ≈ 0.4). These suspensions were incubated at 37 ◦C in a rotator shaker and growth was monitored by measuring the OD550 and CFU count on nutrient agar plates. Cultures with known antibiotics or without drugs were used as positive and negative controls, respectively. The assays were repeated at least three times.

#### *2.5. Inoculum Effect*

Overnight liquid cultures of *B. subtilis* were 10-fold serial diluted in NB medium to give different inoculum concentrations (10<sup>3</sup> to 10<sup>7</sup> CFU/mL). Celastrol (3 μg/mL) and pristimerin (10 μg/mL) were added and cultures were incubated at 37 ◦C in a rotatory shaker. Bacterial growth was monitored as described above. The assays were repeated at least three times.

### *2.6. Measurement of Radioactive Precursor Incorporation*

Cultures of *B. subtilis* were diluted to obtain 106 CFU/mL in Davis–Mingioli medium [41] supplemented with glucose (1%), asparagine (0.1 g/L), and casamino acids (2 g/L) (pH 7). The cultures were grown at 37 ◦C under shaking for at least 3 h to obtain an optical density (OD550) of 0.4. Volumes of 10 mL were transferred to prewarmed flasks containing celastrol (3 μg/mL) or pristimerin (10 μg/mL) and one of the precursors of DNA (1 μCi/mL [6-3H] + 2 μg/mL unlabeled thymidine), RNA (1 μCi/mL [5-3H] + 2 μg/mL unlabeled uridine), protein (5 μCi/mL [4,5-3H] + 2 μg/mL unlabeled leucine), or cell wall peptidoglycan (0.1 μCi/mL *N*-Acetyl-D-[1-14C] glucosamine). The samples were incubated at 37 ◦C under shaking. Volumes of 0.5 mL were collected and precipitated with 2 mL ice-cold 10% trichloroacetic acid (TCA) at different times. After 30 min of incubation in cold TCA, samples were filtered on glass microfiber filters grade GF/C (Whatman, Maidstone, UK)) and washed three times with 5 mL cold 10% TCA and once with 5 mL of 95% ethanol. The dried filters were placed in vials covered with a scintillation cocktail and counted in counter LKB Wallac Rackbeta (Perkin Elmer, Courtaboeuf, France). Macromolecular synthesis was measured by quantifying the incorporation of radiolabeled precursors (Amersham Biosciences Europe GmbH) into acid-insoluble material. Evaluations with DMSO added in the same proportion or a specific inhibitor of each biosynthetic pathway were included as negative and positive controls, respectively.
