In Vitro Antimicrobial Activity and Effect on Biofilm Production of a White Grape Juice (Vitis vinifera) Extract

Background. The aim of the present study was to evaluate the antimicrobial effect of a white grape juice extract (WGJe) against a range of Gram-positive and Gram-negative bacteria, yeasts, and the fungus Aspergillus niger. WGJe was also tested on the production of bacterial biofilms in vitro. Results. WGJe inhibited in vitro most Gram-positive bacteria tested, Staphylococcus aureus ATCC 6538P being the most sensitive strain (MIC values of 3.9 μg/mL). The effect was bactericidal at the concentration of 500 μg/mL. Amongst the Gram-negative bacteria, Escherichia coli was the only susceptible strain (MIC and MBC of 2000 μg/mL). No effect on the growth of Candida sp. and the fungus Aspergillus niger was detected (MIC values > 2000 μg/mL). WGJe inhibited the biofilms formation of E. coli and Pseudomonas aeruginosa with a dose-dependent effect. Conclusions. WGJe exerted both bacteriostatic and bactericidal activity in vitro. The presented results could be used to develop novel strategies for the treatment of skin infections and against potential respiratory pathogens.


Introduction
A wide variety of compounds have been identified in grapes, most of which with health promoting properties. A number of studies have shown that beneficial effects of grape and grape products consumption are related to the presence of polyphenols, mainly flavonoids and phenolic acids, with antioxidant, anti-inflammatory, antimicrobial, antiviral, and cancer preventive properties [1][2][3][4]. Flavonoids, stilbene, and proanthocyanidins are considered the most abundant class of bioactive compounds in grapes. It has also been demonstrated that grape juices, both white and purple, represent an important source of minerals, which would help explain the antioxidant and antimutagenic properties of grapes [5]. We have recently shown that an extract from white grape juice could have a beneficial effect on radiocontrast medium toxicity in human renal proximal tubular cells [6] and exert neuroprotective effect in a mouse model of experimental autoimmune encephalomyelitis [7]. Due to the increased antibiotic resistance, the antimicrobial properties of natural compounds have been gaining attention: we have previously reported the antibacterial activity of polyphenols-rich natural products, including almonds [8,9], pistachios [10], Citrus plants [11], Vitis vinifera L. [12], Olea europaea L. [13], Citrus bergamia essential oil [14], and juice [15]. In particular, the identification of novel compounds with bactericidal rather than bacteriostatic effect has attracted interest in recent years. Inhibition and eradication of Gram-positive and Gram-negative biofilms have also been proven to be rather difficult with conventional antibiotics, with a need for novel antimicrobials able to treat biofilm-related infections. Yadav et al. [16] have recently demonstrated that eugenol was effective against methicillin-resistant and methicillinsensitive Staphylococcus aureus clinical strain biofilms. The effects of cranberry extracts have also been evaluated on the growth and biofilm production of Escherichia coli and Staphylococcus sp. [17].
In the present study we evaluated the antimicrobial effect of a polyphenols-rich white grape juice extract against Grampositive and Gram-negative bacteria, yeasts, and the fungus Aspergillus niger. Furthermore, the same extract was tested on the production of bacterial biofilms in vitro.

Susceptibility Studies.
For the susceptibility studies, WGJe was dissolved in sterile PBS at the concentration of 4 mg/mL; the pH of this solution was 7.10. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), and the minimum fungicidal concentration (MFC) of WGJe were determined by the broth microdilution method, according to CLSI [18]. Briefly, twofold serial dilutions of WGJe were added to MHB and inoculated into 96-microtiter plates with a final inoculum of approximately 5 × 10 5 CFU mL −1 . The tested concentrations ranged from 2000 to 3.9 g mL −1 , and no significant changes of the pH of the growth medium were detected after addition of WGJe. The MIC was considered as the lowest concentration, which completely inhibited bacterial growth after 20 h.
The MBCs were determined by seeding 20 L from all clear MIC wells onto Mueller-Hinton agar (MHA, Oxoid) plates. The MBC was defined as the lowest extract concentration that killed 99.9% of the final inocula after 24 h incubation.

Effect on Biofilm Formation.
The effect of different concentrations of WGJe on biofilm-forming ability was tested on polystyrene flat-bottomed microtiter plates as described by Nostro et al. [19].
Twofold serial dilutions of WGJe ranging from 500 to 62.5 g mL −1 were used for E. coli ATCC 25922 and P. aeruginosa ATCC 9027.
Cultures were grown overnight and suspensions adjusted to 10 6 CFUmL −1 . Aliquots of 100 mL were dispensed into each well of a sterile flat-bottomed 96-well polystyrene microtiter plates (Corning Inc., Corning, NY) in the presence of WGJe.
Planktonic growth was determined by spectrophotometric values (OD 492 nm) after 24 h. The medium was then aspirated and the wells, rinsed twice with phosphate-buffered saline (PBS), were fixed by drying for 2 h. Once the wells were fully dry, 200 mL of 0.1% safranin was added for 2 min. The content of the wells was then aspirated, and, after rinsing with water, 200 mL of 30% acetic acid (v/v) was added. OD 492 was measured by spectrophotometry using an ELISA reader.
Biofilm controls consisting of medium alone, medium plus strains (without WGJe), and medium plus WGJe (without strain) were included.
Each assay was performed in duplicate and repeated at least three times. Table 1. WGJe was active against all the Gram-positive strains included in the study (MIC values between 3.9 and 1000 g mL −1 ), whereas no activity was found against the Gram-negative bacteria, with the exception of E. coli (MIC of 2000 g mL −1 ), the yeasts, and A. niger at the concentrations tested.

Minimum Inhibitory Concentrations. The MIC and MBC values of WGJe against the strains tested are shown in
Amongst the Gram-positive bacteria, S. aureus ATCC 6538P showed the highest sensitivity, with MIC and MBC values equal to 3.9 and 500 g mL −1 , respectively.
Similarly, a good growth inhibitory activity was found against S. epidermidis and M. catarrhalis, with MIC values of 15.62 g mL −1 and MBC values of 2000 g mL −1 . WGJe (125 g mL −1 ) has also been able to inhibit the growth of L. monocytogenes, S. mutans, S. pyogenes, and S. pneumonia. MBC values of 1000 g mL −1 for S. pyogenes and S. pneumonia and 2000 g mL −1 for S. mutans were detected, whereas no bactericidal activity was found against L. monocytogenes at the concentrations tested. MIC values of 250, 500, and 1000 g mL −1 were also found against B. subtilis, E. durans, and E. hirae, respectively. A MBC value corresponding to the maximum concentration tested was detected for B. subtilis, whereas the same concentration showed bacteriostatic activity on E. durans and E. hirae.
Amongst the Gram-negative bacteria, a slight inhibitory activity was found against E. coli for which the value of MIC found, however, corresponds to the highest concentration used. No activity was observed against other Gram-negative strains. Table 2 shows the effect of WGJe on the biofilm formation of E. coli and P. aeruginosa. Since the MIC value was relatively high for E. coli and no inhibition was observed against P. aeruginosa (Table 1), the WGJe concentrations used ranged between 500 and 62.5 g mL −1 .

Effect on Biofilm Formation.
The results demonstrated that WGJe (500 and 250 g mL −1 ) produced a reduction on biofilm formation of 42.49% and 23.79% for E. coli and 30.47% and 23.88% for P. aeruginosa, respectively.

Discussion
The present study has demonstrated that WGJe was effective against a range of Gram-positive bacteria. Although WGJe did not affect the growth of any Gram-negative bacteria tested, with the exception of E. coli, a reduction on biofilm formation of both E. coli and P. aeruginosa was detected. A number of studies have previously investigated the antimicrobial effect of grape, wine, and their byproducts [20,21]. Red wine has been shown to prevent damage to the gastric mucosa induced by Helicobacter pylori, possibly through inhibition of the vacA gene [22]. Jayaprakasha et al. [23] have demonstrated that grape seed extracts have antimicrobial potential, Gram-positive bacteria being more sensitive than Gram-negative bacteria. Our study has also demonstrated that Gram-positive strains were more susceptible to WGJe compared with Gram-negative bacteria, with a bactericidal effect observed on all Gram-positive strains tested, with the exception of E. durans, E. hirae, Listeria spp., and S. epidermidis ATCC 49134. A strong inhibitory effect against Listeria monocytogenes has been found by grape juice and grape extracts derived from Vitis vinifera variety "Ribier" [24]. Sanhueza et al. [25] have recently reported an antibacterial effect of grape pomace extracts mainly against S. aureus and E. coli: the activity was directly related to the polar phenolic content. Grape seed extracts obtained from wine and table cultivars of Vitis vinifera L. were found to be active against Candida albicans sp. and their activity was related to the presence of polymeric flavan-3-ols [26].
The antibacterial activity of polyphenols has been recently reviewed [27]. In agreement with our study, several wine phenolic acids, mainly gallic acid and ethyl gallate, were able to inhibit the growth of respiratory pathogenic bacteria and potential respiratory pathogens including P. aeruginosa, S. aureus, M. catarrhalis, and E. faecalis [28]. However, unlike the report by Cueva et al. [28], we found Gram-positive bacteria were more sensitive to WGJe compared with the Gram-negative tested strains. Some phenolic acids, such as cinnamic acid, ferulic acid, p-coumaric acid, and caffeic acid, were also found to be active against Listeria spp. [29]. Chlorogenic acid extracted from blueberry fruit was able to inhibit 46% and 42% of S. epidermidis and P. aeruginosa biofilm formation, respectively [30]. We believe that the phenolic acids present in WGJe played an important role in reducing biofilm formation of E. coli and P. aeruginosa, in a dose-dependent way.

Conclusions
The results of the present study demonstrated that WGJe was effective against a range of Gram-positive bacteria, including