Antibiofilm Properties Exhibited by the Prickly Pear ( Opuntia ficus-indica ) Seed Oil †

: Prickly pear [ Opuntia ficus-indica (L.) Mill.] is a succulent plant that is globally diffused. The oil obtained from its seeds has antimicrobial and antioxidant properties. We evaluated the antibiofilm of the oil and its capacity to block the metabolic changes taking place in the microbial cells included in the biofilm. The oil was capable to inhibit at 38.75% the biofilm of Escherichia coli , Pseudomonas aeruginosa , and Pectobacterium carotovorum (38.75%, 71.84%, and 63.06% inhibition, respectively). The metabolic activity of the microbial cells within the biofilm was also strongly inhibited. The action of the prickly pear seeds oil was effective also in blocking at 64.97% the metabolism of Listeria monocytogenes cells.


Introduction
Prickly or cactus pear [Opuntia ficus-indica (L.) Mill., 1768] is a succulent plant belonging to the Cactaceae family, native to Central America but now diffused both in the Mediterranean area (mainly Sicily, Calabria, Puglia, Sardinia, and Malta) and in the temperate areas of America, Africa, Asia, and Oceania. It is a drought-tolerant crop and needs low agronomic requirements and high water use efficiency. Generally, the fruits are used for human consumption, and cladodes (called pad) are consumed as animal feed. The fruit is an oval-shaped berry with an average weight of 100-200 g, constituted mainly by juicy pulp [1]. Seeds contribute 10% to 15% to the pulp weight. Oil obtained from the seed represents 7-15% of the whole seed weight [2]. Seeds also have a high content of oil (98.8 g/kg) [3], characterized by high levels of linoleic and oleic acids and other components such as phenols, all of which have a benefit on human health [4][5][6][7][8]. The health-promoting properties of prickly pear fruit is highly appealing, also for the development of nutraceutical and functional foods, as well as for the emphasis that consumers give toward the search of new products or components with high benefits.
Cactus pear contains several bioactive compounds exhibiting high antioxidant and antimicrobial activity [9].
The antimicrobial activity of the cactus pear seed oil has been demonstrated vs. different pathogens, such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa [10]. The oil is also useful as preserving agent to improve the shelf life of fresh food, such as sliced beef, meliorating concurrently the quality of the product and safeguarding it from a microbiological point of view [11]. The extract of cactus pear cladodes showed a noticeable activity in inhibiting the biofilm formation of St. aureus [12]. The aim of our work was to evaluate the capacity of the prickly pear seeds oil in inhibiting the formation of biofilm of different pathogens and in blocking the metabolic changes taking place in the microbial cells included in the biofilm.

Material and Methods
Organic prickly pear (Opuntia ficus-indica L.) seed oil, obtained through cold pressure, was purchased from the company Bionoble Cosmétiques BIO (Paris, France). The bacterial culture medium, PBS, DMSO, tetracycline, ciprofloxacin, and MTT were supplied by Sigma (Milano, Italy).

Microorganisms and Culture Conditions
Listeria monocytogenes ATCC 7644, EHEC, Escherichia coli DSM 8579, Pseudomonas aeruginosa DSM 50071, and the phytopathogen Pectobacterium carovotorum DSM 102074 were used as test bacterial strains. The strains were previously stored at −30 °C in sterile Luria Bertani (LB) broth (Sigma) supplemented with 20% sterile glycerol (Sigma). Bacteria were thawed and added (inoculum 2%) to LB broth. E. coli, L. monocytogenes, and P. aeruginosa were grown for 18 h at 37 °C and 80 rpm (Corning LSE, Pisa, Italy). P. carotovorum was grown at 28 °C and 80 rpm. Fresh cultures were used as inoculum (2% final concentration) and grown in the conditions described above.

Minimal Inhibitory Concentration (MIC)
The MIC values were calculated using the resazurin microtiter-plate assay [13]. Multiwell plates were prepared in triplicate and incubated at 37 °C for 24 h. The lowest concentration at which a color change occurred (from dark purple to colorless) revealed the MIC value.

Biofilm Inhibitory Activity
The effect of the prickly pear seeds oil on bacterial ability to form biofilm was assessed according to the method of O'Toole and Kolter [14] in flat-bottomed 96-well microtiter plates, using volumes of the oil (previously dissolved in sterile DMSO) ranging from 1 to 8 μL/mL. The overnight bacterial cultures were adjusted to 0.5 McFarland with fresh culture broth. Then, 10 μL of the diluted cultures was distributed in each well, and different volumes of the oil and sterile Luria-Bertani broth were added to reach a final volume of 250 μL/well. Microplates were completely covered with parafilm tape to avoid the evaporation of samples with relative loss of volume and incubated for 48 h at different temperatures (depending on the strain). Planktonic cells were removed and the attached cells were gently washed twice with sterile physiological saline. After that, 200 μL of methanol was added to each well, retaining it for 15 min to fix the sessile cells. Methanol was then discarded, and each plate was left until complete dryness of samples. The staining of the adhered cells was obtained by adding 200 μL of 2% w/v crystal violet solution to each well that was left for 20 min. Wells were gently washed with sterile physiological solution and left to dry. Two hundred microliters of glacial acetic acid 20% w/v were added to allow the release of the bound dye. The absorbance was measured at OD = 540 nm (Varian Cary Spectrophotometer model 50 MPR, Cernusco sul Naviglio, Italy). The percent value of biofilm inhibition was calculated with respect to control (cells grown without the presence of the EOs). The average results from triplicate tests were taken for reproducibility.

Metabolic Activity of Biofilm Cells
The effect of different volumes of prickly pear seeds oil, ranging from 1 to 8 μL/mL on the metabolic activity of biofilm cells, was evaluated through the MTT colorimetric method [15,16] using 96-well microtiter plates. The overnight bacterial cultures were adjusted to 0.5 McFarland and treated as described in Section "Biofilm Inhibitory Activity." After 48 h incubation, bacterial suspension was removed and 150 μL of sterile PBS and 30 μL of 0.3% MTT (Sigma, Milan, Italy) were added, keeping microplates at 37 °C. After 2 h, the MTT solution was removed, two washing steps were performed gently with 200 μL of sterile physiological solution, and 200 μL of DMSO was added to allow the dissolution of the formazan crystals, which were measured at OD = 570 nm (Varian). Triplicate tests were carried out and the average results were taken for reproducibility.

Results and Discussion
Based on the minimal inhibitory activity exhibited by the prickly pear seeds oil (Table 1), we provided to evaluate the potential effect that different volumes of this oil could have on the formation of biofilm of some pathogenic bacteria and assessed if the oil could exhibit some inhibitory effect also on the metabolism of the cells included in the biofilm.  The data acquired showed that the O. ficus-indica seeds oil has an interesting capability to inhibit the growth of different pathogens, confirming previous studies which demonstrated the effectiveness of this oil in inhibiting the growth of different Gram-positive and Gram-negative negative pathogens [10,17,18]. To our knowledge, this is the first time that the possible capability of the oil to inhibit the formation of biofilms by pathogenic microorganisms was evaluated. Therefore, it is also the first time that the effect of the oil on the metabolism of microbial cells trapped inside the biofilm has been studied. Results are shown in Tables 2 and 3, respectively. E. coli and P. carotovorum were sensitive to the action of the oil even when we tested one μL/mL, which caused an inhibition of the biofilm of 36.77% and 43.33%, respectively (Table 2). Table 2. Inhibitory action of Opuntia ficus-indica seeds oil on the formation of biofilm. Results are reported as percent of inhibition respect to the control (% = 0). They are the mean (± SD) of three experiments. Such action was much more incisive when we used 8 μL/mL: in this case, we detected percentages of biofilm inhibition of 63.06% (P. carotovorum) and 75.79% (E. coli). Ps. aeruginosa, which also proved to be resistant to the action of 1 μL/mL of the oil, showed percentages of biofilm inhibition that reached 73.84% using 8 μL/mL of the oil instead.
The metabolic activity of the microbial cells present within the biofilm was also strongly inhibited (Table 3) and, when the oil was tested against P. carotovorum, the microbial cell metabolism was completely inhibited. A similar behavior was exhibited by E. coli, which cell metabolism was almost completely inhibited (96.26%) using 8 μL/mL of the oil. The action of the prickly pear seeds oil was effective also in blocking at 64.97% the metabolism of L. monocytogenes cells, which therefore had conversely demonstrated to be more resistant compared to the other bacteria (31% of biofilm inhibition when we tested the highest volume of the O. ficus-indica seeds oil). This could suggest that, conversely to a general higher resistance exhibited by the Gram-negative bacteria to biocides [19], the Gram-positive strain used in these experiments, that is L. monocytogenes, could be slightly more resistant, for the same volume of oil used, during the biofilm formation phase, but not from a metabolic point of view. The activity exhibited by the oil against the phytopathogen P. carotovorum makes it a candidate to treat and prevent bacterial infections in crops for instance using new species-specific technologies, such as the encapsulation of the oil in mesoporous silica nanoparticles [20]. Data from the present study indicate an interesting applicative versatility of this oil, with potentialities for food, agriculture, and health purposes.