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

Authors: Angela  Filocamo; Carlo  Bisignano; Giuseppina  Mandalari; Michele  Navarra
Journal: Evidence-Based Complementary and Alternative Medicine
 (2015)

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## Abstract

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 fungusAspergillus niger. WGJe was also tested on the production of bacterial biofilmsin vitro.Results. WGJe inhibitedin 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 ofCandida sp. and the fungusAspergillus niger was detected (MIC values > 2000 μg/mL). WGJe inhibited the biofilms formation ofE. coli andPseudomonas aeruginosa with a dose-dependent effect.Conclusions. WGJe exerted both bacteriostatic and bactericidal activityin vitro. The presented results could be used to develop novel strategies for the treatment of skin infections and against potential respiratory pathogens.


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## Body

## 1. 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 [1–4]. 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 methicillin-sensitiveStaphylococcus aureus clinical strain biofilms. The effects of cranberry extracts have also been evaluated on the growth and biofilm production ofEscherichia coli andStaphylococcus sp. [17].In the present study we evaluated the antimicrobial effect of a polyphenols-rich white grape juice extract against Gram-positive and Gram-negative bacteria, yeasts, and the fungusAspergillus niger. Furthermore, the same extract was tested on the production of bacterial biofilmsin vitro.


## 2. Materials and Methods

### 2.1. Grape Juice Extract

A white grape (Vitis vinifera) juice extract (WGJe) was kindly provided by “Bono & Ditta” (Campobello di Mazzara, Trapani, Italy). The extract derived from a mixture of white grapes juice fromVitis viniferavar. Catarratto,Vitis vinifera var. Grillo, andVitis vinifera var. Insolia. WGJe was produced in its liquid form by passing the must-mute columns equipped with adsorbent resins, known to retain polyphenols. Molecules were then eluted with 4% NaOH and immediately passed through cationic resins, thus obtaining an acidic form. Products were collected, filtered, and then sprayed to obtain a dry powder kept at −20°C until further use [6, 7]. WGJe chemical composition, evaluated by UPLC/QqQ-MS/MS, has been previously reported [6, 7]. The major polyphenols identified in WGJe were quercetin-3-glucuronide, procyanidin B1, quercetin-3-glucoside, catechin, andt-coutaric acid. Minor identified compounds included a number of glucosides, such as kaempferol-3-glucuronide and kaempferol-3-glucoside, isorhamnetin-3-glucoside, and quercetin-3-glucoside-arabinoside. Phenolic acids, including vanillic acid, ellagic acid, ferulic acid, chlorogenic acid, caffeic acid, andp-coumaric acid, were also identified.


### 2.2. Microbial Strains and Culture Conditions

The following strains from the University of Messina’s in-house culture collection (Messina, Italy) were used for the antimicrobial testing:Staphylococcus aureus ATCC 6538P,Staphylococcus aureus MRSA ATCC 43300,Staphylococcus epidermidis ATCC 49134,Staphylococcus epidermidis ATCC 35984,Staphylococcus epidermidis ATCC 12228,Streptococcus pneumoniae ATCC 6003,Streptococcus pyogenes ATCC 19615,Streptococcus mutans ATCC 35668,Listeria monocytogenes ATCC 7644,Listeria monocytogenes ATCC 1392,Enterococcus hirae ATCC 10541,Moraxella catarrhalis ATCC 8176,Bacillus subtilis ATCC 8176,Enterococcus durans (wild-type strain),Escherichia coli ATCC 25922,Klebsiella pneumoniae (wild-type strain),Pseudomonas aeruginosa ATCC 27853,Pseudomonas aeruginosa ATCC 9027,Pseudomonas aeruginosa ATCC 15442,Proteus mirabilis (wild-type strain),Serratia marcescens (wild-type strain),Salmonella typhi ATCC 13311,Candida albicans ATCC 10231,Candida parapsilosis ATCC 29947, and the fungusAspergillus niger ATCC 16404.Bacteria were grown in Mueller-Hinton Broth (MHB, Oxoid, CM0405) at 37°C (24 h), whereas yeasts were cultured in Sabouraud Liquid Medium (SLM, Oxoid, CM0147) at 30°C (48 h). For solid media, 1.5% (w/v) agar (Difco) was added.Aspergillus niger was grown in Sabouraud Dextrose Agar at 30°C for 7 days as previously reported [10].


### 2.3. 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 × 105 CFU mL−1. The tested concentrations ranged from 2000 to 3.9 μg 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 20μL 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.


### 2.4. 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 62.5μg mL−1 were used forE. coli ATCC 25922 andP. aeruginosa ATCC 9027.Cultures were grown overnight and suspensions adjusted to 106 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 (OD492 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. OD492 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.


## 2.1. Grape Juice Extract

A white grape (Vitis vinifera) juice extract (WGJe) was kindly provided by “Bono & Ditta” (Campobello di Mazzara, Trapani, Italy). The extract derived from a mixture of white grapes juice fromVitis viniferavar. Catarratto,Vitis vinifera var. Grillo, andVitis vinifera var. Insolia. WGJe was produced in its liquid form by passing the must-mute columns equipped with adsorbent resins, known to retain polyphenols. Molecules were then eluted with 4% NaOH and immediately passed through cationic resins, thus obtaining an acidic form. Products were collected, filtered, and then sprayed to obtain a dry powder kept at −20°C until further use [6, 7]. WGJe chemical composition, evaluated by UPLC/QqQ-MS/MS, has been previously reported [6, 7]. The major polyphenols identified in WGJe were quercetin-3-glucuronide, procyanidin B1, quercetin-3-glucoside, catechin, andt-coutaric acid. Minor identified compounds included a number of glucosides, such as kaempferol-3-glucuronide and kaempferol-3-glucoside, isorhamnetin-3-glucoside, and quercetin-3-glucoside-arabinoside. Phenolic acids, including vanillic acid, ellagic acid, ferulic acid, chlorogenic acid, caffeic acid, andp-coumaric acid, were also identified.


## 2.2. Microbial Strains and Culture Conditions

The following strains from the University of Messina’s in-house culture collection (Messina, Italy) were used for the antimicrobial testing:Staphylococcus aureus ATCC 6538P,Staphylococcus aureus MRSA ATCC 43300,Staphylococcus epidermidis ATCC 49134,Staphylococcus epidermidis ATCC 35984,Staphylococcus epidermidis ATCC 12228,Streptococcus pneumoniae ATCC 6003,Streptococcus pyogenes ATCC 19615,Streptococcus mutans ATCC 35668,Listeria monocytogenes ATCC 7644,Listeria monocytogenes ATCC 1392,Enterococcus hirae ATCC 10541,Moraxella catarrhalis ATCC 8176,Bacillus subtilis ATCC 8176,Enterococcus durans (wild-type strain),Escherichia coli ATCC 25922,Klebsiella pneumoniae (wild-type strain),Pseudomonas aeruginosa ATCC 27853,Pseudomonas aeruginosa ATCC 9027,Pseudomonas aeruginosa ATCC 15442,Proteus mirabilis (wild-type strain),Serratia marcescens (wild-type strain),Salmonella typhi ATCC 13311,Candida albicans ATCC 10231,Candida parapsilosis ATCC 29947, and the fungusAspergillus niger ATCC 16404.Bacteria were grown in Mueller-Hinton Broth (MHB, Oxoid, CM0405) at 37°C (24 h), whereas yeasts were cultured in Sabouraud Liquid Medium (SLM, Oxoid, CM0147) at 30°C (48 h). For solid media, 1.5% (w/v) agar (Difco) was added.Aspergillus niger was grown in Sabouraud Dextrose Agar at 30°C for 7 days as previously reported [10].


## 2.3. 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 × 105 CFU mL−1. The tested concentrations ranged from 2000 to 3.9 μg 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 20μL 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.


## 2.4. 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 62.5μg mL−1 were used forE. coli ATCC 25922 andP. aeruginosa ATCC 9027.Cultures were grown overnight and suspensions adjusted to 106 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 (OD492 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. OD492 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.


## 3. Results

### 3.1. Minimum Inhibitory Concentrations

The MIC and MBC values of WGJe against the strains tested are shown in Table1. WGJe was active against all the Gram-positive strains included in the study (MIC values between 3.9 and 1000 μg mL−1), whereas no activity was found against the Gram-negative bacteria, with the exception ofE. coli (MIC of 2000 μg mL−1), the yeasts, andA. niger at the concentrations tested.Table 1
MICs and MBCs of WGJe (expressed asμg/mL) against Gram-positive bacteria, Gram-negative bacteria, yeasts, and the fungus A. niger.
MIC
MBC
Gram+
M. catarrhalis ATCC 8176
15.62
2000
B. subtilis ATCC 6633
250
2000
E. durans (wild-type strain)
500
>2000
E. hirae ATCC 10541
1000
>2000
L. monocytogenes ATCC 7466
125
>2000
L. monocytogenes ATCC 13932
250
>2000
S. aureus ATCC 6538P
3.9
500
S. aureus ATCCC 43300
62.5
500
S. epidermidis ATCC 49134
15.62
>2000
S. epidermidis ATCC 12228
31.25
2000
S. epidermidis ATCC 35984
15.62
2000
S. mutans ATCC 35668
125
2000
S. pyogenes ATCC 19615
125
1000
S. pneumoniae ATCC 6003
125
1000
Gram−
E. coli ATCC 25922
2000
2000
K. pneumoniae (wild-type strain)
>2000
>2000
P. aeruginosa ATCC 27853
>2000
>2000
P. aeruginosa ATCC 9027
>2000
>2000
P. aeruginosa ATCC15442
>2000
>2000
P. mirabilis (wild-type strain)
>2000
>2000
S. marcescens (wild-type strain)
>2000
>2000
S. typhi ATCC13311
>2000
>2000
Yeasts
C. albicans ATCC 10231
>2000
>2000
C. parapsilosis ATCC 29947
>2000
>2000
Fungi
A. niger ATCC 16404
>2000
>2000
MICs, minimal inhibitory concentrations; MBCs, minimal bactericidal concentrations; WGJe, white grape juice extract.Amongst the Gram-positive bacteria,S. aureus ATCC 6538P showed the highest sensitivity, with MIC and MBC values equal to 3.9 and 500 μg mL−1, respectively.Similarly, a good growth inhibitory activity was found againstS. epidermidis andM. catarrhalis, with MIC values of 15.62 μg mL−1 and MBC values of 2000 μg mL−1. WGJe (125 μg mL−1) has also been able to inhibit the growth ofL. monocytogenes,S. mutans,S. pyogenes, andS. pneumonia. MBC values of 1000 μg mL−1 forS. pyogenes andS. pneumonia and 2000 μg mL−1 forS. mutans were detected, whereas no bactericidal activity was found againstL. monocytogenes at the concentrations tested.MIC values of 250, 500, and 1000μg mL−1 were also found againstB. subtilis,E. durans, andE. hirae, respectively. A MBC value corresponding to the maximum concentration tested was detected forB. subtilis, whereas the same concentration showed bacteriostatic activity onE. durans andE. hirae.Amongst the Gram-negative bacteria, a slight inhibitory activity was found againstE. coli for which the value of MIC found, however, corresponds to the highest concentration used. No activity was observed against other Gram-negative strains.


### 3.2. Effect on Biofilm Formation

Table2 shows the effect of WGJe on the biofilm formation ofE. coli andP. aeruginosa. Since the MIC value was relatively high forE. coli and no inhibition was observed againstP. aeruginosa (Table 1), the WGJe concentrations used ranged between 500 and 62.5 μg mL−1.Table 2
Percentage biofilm reduction for twofold serial dilutions of WGJe ranging from 500 to 62.5µg mL−1  on E. coli ATCC 25922 and P. aeruginosa ATCC 9027.
500
250
125
62.5
E. coliATCC 10536
42.49  ±  0.75
23.79  ±  0.48
19.28  ±  0.33
10.55  ±  0.78
P. aeruginosaATCC 9027
30.47  ±  0.27
23.88  ±  0.14
21.20  ±  0.19
17.38  ±  0.37
WGJe, white grape juice extract.The results demonstrated that WGJe (500 and 250μg mL−1) produced a reduction on biofilm formation of 42.49% and 23.79% forE. coli and 30.47% and 23.88% forP. aeruginosa, respectively.


## 3.1. Minimum Inhibitory Concentrations

The MIC and MBC values of WGJe against the strains tested are shown in Table1. WGJe was active against all the Gram-positive strains included in the study (MIC values between 3.9 and 1000 μg mL−1), whereas no activity was found against the Gram-negative bacteria, with the exception ofE. coli (MIC of 2000 μg mL−1), the yeasts, andA. niger at the concentrations tested.Table 1
MICs and MBCs of WGJe (expressed asμg/mL) against Gram-positive bacteria, Gram-negative bacteria, yeasts, and the fungus A. niger.
MIC
MBC
Gram+
M. catarrhalis ATCC 8176
15.62
2000
B. subtilis ATCC 6633
250
2000
E. durans (wild-type strain)
500
>2000
E. hirae ATCC 10541
1000
>2000
L. monocytogenes ATCC 7466
125
>2000
L. monocytogenes ATCC 13932
250
>2000
S. aureus ATCC 6538P
3.9
500
S. aureus ATCCC 43300
62.5
500
S. epidermidis ATCC 49134
15.62
>2000
S. epidermidis ATCC 12228
31.25
2000
S. epidermidis ATCC 35984
15.62
2000
S. mutans ATCC 35668
125
2000
S. pyogenes ATCC 19615
125
1000
S. pneumoniae ATCC 6003
125
1000
Gram−
E. coli ATCC 25922
2000
2000
K. pneumoniae (wild-type strain)
>2000
>2000
P. aeruginosa ATCC 27853
>2000
>2000
P. aeruginosa ATCC 9027
>2000
>2000
P. aeruginosa ATCC15442
>2000
>2000
P. mirabilis (wild-type strain)
>2000
>2000
S. marcescens (wild-type strain)
>2000
>2000
S. typhi ATCC13311
>2000
>2000
Yeasts
C. albicans ATCC 10231
>2000
>2000
C. parapsilosis ATCC 29947
>2000
>2000
Fungi
A. niger ATCC 16404
>2000
>2000
MICs, minimal inhibitory concentrations; MBCs, minimal bactericidal concentrations; WGJe, white grape juice extract.Amongst the Gram-positive bacteria,S. aureus ATCC 6538P showed the highest sensitivity, with MIC and MBC values equal to 3.9 and 500 μg mL−1, respectively.Similarly, a good growth inhibitory activity was found againstS. epidermidis andM. catarrhalis, with MIC values of 15.62 μg mL−1 and MBC values of 2000 μg mL−1. WGJe (125 μg mL−1) has also been able to inhibit the growth ofL. monocytogenes,S. mutans,S. pyogenes, andS. pneumonia. MBC values of 1000 μg mL−1 forS. pyogenes andS. pneumonia and 2000 μg mL−1 forS. mutans were detected, whereas no bactericidal activity was found againstL. monocytogenes at the concentrations tested.MIC values of 250, 500, and 1000μg mL−1 were also found againstB. subtilis,E. durans, andE. hirae, respectively. A MBC value corresponding to the maximum concentration tested was detected forB. subtilis, whereas the same concentration showed bacteriostatic activity onE. durans andE. hirae.Amongst the Gram-negative bacteria, a slight inhibitory activity was found againstE. coli for which the value of MIC found, however, corresponds to the highest concentration used. No activity was observed against other Gram-negative strains.


## 3.2. Effect on Biofilm Formation

Table2 shows the effect of WGJe on the biofilm formation ofE. coli andP. aeruginosa. Since the MIC value was relatively high forE. coli and no inhibition was observed againstP. aeruginosa (Table 1), the WGJe concentrations used ranged between 500 and 62.5 μg mL−1.Table 2
Percentage biofilm reduction for twofold serial dilutions of WGJe ranging from 500 to 62.5µg mL−1  on E. coli ATCC 25922 and P. aeruginosa ATCC 9027.
500
250
125
62.5
E. coliATCC 10536
42.49  ±  0.75
23.79  ±  0.48
19.28  ±  0.33
10.55  ±  0.78
P. aeruginosaATCC 9027
30.47  ±  0.27
23.88  ±  0.14
21.20  ±  0.19
17.38  ±  0.37
WGJe, white grape juice extract.The results demonstrated that WGJe (500 and 250μg mL−1) produced a reduction on biofilm formation of 42.49% and 23.79% forE. coli and 30.47% and 23.88% forP. aeruginosa, respectively.


## 4. 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 ofE. coli, a reduction on biofilm formation of bothE. coli andP. 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 byHelicobacter 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 ofE. durans,E. hirae,Listeria spp., andS. epidermidis ATCC 49134. A strong inhibitory effect againstListeria monocytogenes has been found by grape juice and grape extracts derived fromVitis vinifera variety “Ribier” [24]. Sanhueza et al. [25] have recently reported an antibacterial effect of grape pomace extracts mainly againstS. aureus andE. coli: the activity was directly related to the polar phenolic content. Grape seed extracts obtained from wine and table cultivars ofVitis vinifera L. were found to be active againstCandida 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 includingP. aeruginosa,S. aureus,M. catarrhalis, andE. 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 againstListeria spp. [29]. Chlorogenic acid extracted from blueberry fruit was able to inhibit 46% and 42% ofS. epidermidis andP. aeruginosa biofilm formation, respectively [30]. We believe that the phenolic acids present in WGJe played an important role in reducing biofilm formation ofE. coli andP. aeruginosa, in a dose-dependent way.


## 5. Conclusions

The results of the present study demonstrated that WGJe was effective against a range of Gram-positive bacteria, including potential respiratory pathogens, andE. coli amongst Gram-negative strains. The exerted activity was both bacteriostatic and bactericidal. Furthermore, WGJe was able to inhibit the biofilm formation ofE. coli andP. aeruginosa in vitro.



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