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Research article

J. Microbiol. Biotechnol. 2019; 29(8): 1177-1183

Published online August 28, 2019 https://doi.org/10.4014/jmb.1905.05022

Copyright © The Korean Society for Microbiology and Biotechnology.

Anti-Biofilm Activity of Grapefruit Seed Extract against Staphylococcus aureus and Escherichia coli

Ye Ji Song , Hwan Hee Yu , Yeon Jin Kim , Na-Kyoung Lee and Hyun-Dong Paik *

Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Republic of Korea

Correspondence to:Hyun-Dong  Paik
hdpaik@konkuk.ac.kr

Received: May 10, 2019; Accepted: July 29, 2019

Abstract

Grapefruit seed extract (GSE) is a safe and effective preservative that is used widely in the food industry. However, there are few studies addressing the anti-biofilm effect of GSE. In this study, the anti-biofilm effect of GSE was investigated against biofilm-forming strains of Staphylococcus aureus and Escherichia coli. The GSE minimum inhibitory concentration (MIC) for S. aureus and E. coli were 25 μg/ml and 250 μg/ml, respectively. To investigate biofilm inhibition and degradation effect, crystal violet assay and stainless steel were used. Biofilm formation rates of four strains (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) were 55.8%, 70.2%, 55.4%, and 20.6% at 1/2 × MIC of GSE, respectively. The degradation effect of GSE on biofilms attached to stainless steel coupons was observed (≥ 1 log CFU/coupon) after exposure to concentrations above the MIC for all strains and 1/2 × MIC for S. aureus 7. In addition, the specific mechanisms of this anti-biofilm effect were investigated by evaluating hydrophobicity, auto-aggregation, exopolysaccharide (EPS) production rate, and motility. Significant changes in EPS production rate and motility were observed in both S. aureus and E. coli in the presence of GSE, while changes in hydrophobicity were observed only in E. coli. No relationship was seen between auto-aggregation and biofilm formation. Therefore, our results suggest that GSE might be used as an anti-biofilm agent that is effective against S. aureus and E. coli.

Keywords: Anti-biofilm, grapefruit seed extract, biofilm, Staphylococcus aureus, Escherichia coli

Introduction

The World Health Organization (WHO) estimated that the consumption of contaminated food causes illness in 1 in 10 people every year, and as a result, leads to 420,000 deaths per year [1]. According to the Centers for Disease Control and Prevention (CDC), Escherichia coli, Listeria spp., Salmonella spp., Campylobacter spp., and Staphylococcus aureus are some of the most common causes of food poisoning [2]. The most common symptoms of food poisoning are diarrhea, fever, vomiting, nausea, stomach cramps, and stomachache. Food poisoning pathogens can contaminate food products at any time during processing, distribution, or storage. It is very important to limit the growth and development of food poisoning pathogens such as S. aureus and E. coli; however, elimination of these organisms is difficult because of their ability to form biofilms on a variety of surfaces [3]. In addition, many outbreaks of food poisoning have been found to be associated with biofilm-forming pathogens in the dairy, poultry, meat, and ready-to-eat (RTE) food industries [4].

Biofilms are a survival and protection strategy for pathogens, and the cycle of biofilm formation, maturation, and dispersal is the main cause of surface-to-food cross-contamination [5, 6]. After bacterial cells attach to a surface, they produce a polymer matrix, composed of exopoly-saccharides (EPS), extracellular proteins, and extracellular DNA, to defend themselves against antibiotics and other chemical compounds [7]. It is well recognized that bacteria within biofilms show increased antimicrobial resistance compared to planktonic cells grown in suspension [8]. It is important to use appropriate sanitization procedures in the processing environment to both ensure the removal of pathogens and to prevent contamination of the final products [9, 10].

In order to prevent biofilm formation, several studies were conducted to discover natural antimicrobial agents that affect the viability of bacteria in biofilms [11-13]. Furthermore, a shift in consumer preferences towards food containing safe preservatives has led to the emergence of a demand for natural antimicrobial agents [14]. Plant extracts from leaves, roots, and seeds have been reported to demonstrate anti-biofilm activity against S. aureus, E. coli, Listeria spp., and Campylobacter spp. [3, 15-18].

In this study, we chose to evaluate the anti-biofilm activity of grapefruit seed extract (GSE), as it is already known to be a safe and effective preservative. Previous studies have demonstrated that GSE has an antimicrobial effect on gram-positive bacteria, gram-negative bacteria, and yeasts [19-21]. However, there have been no specific reports on the effect of GSE on biofilms. Therefore, the purpose of this study was to investigate the anti-biofilm effect of GSE on two food poisoning pathogens (S. aureus and E. coli) to determine if it may be effective for biofilm removal in the food industry.

Materials and Methods

Bacterial Strains and Growth Conditions

S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125, were used in this study through biofilm-forming screening. S. aureus strains isolated from bovine were supplied by Seoul National University (Korea). E. coli ATCC 25922 and E. coli O157:H4 FRIK 125 were supplied from Korean Culture Center of Microorganisms (KCCM; Korea) and IOWA State University, respectively. These strains were cultured in the Tryptic Soy Broth (TSB; Difco Laboratories, USA) for 24 h at 37°C and stocked at -80°C with 20% glycerol. In biofilm assay, the strains were cultured in TSB containing 0.25% glucose (D-(+)-Glucose; Sigma, Germany) to promote biofilm formation.

Minimum Inhibitory Concentration (MIC) of GSE

Grapefruit seed extract (GSE; ES Food, Korea) was solubilized in TSB with 0.05% Tween 80. A double broth dilution method was used to determine the GSE MIC in 96-well plates [11]. GSE was serially diluted two-fold and 50 µl of each concentration was transferred into 96-well plates. Each well was then inoculated with the same volume of bacterial suspension at a concentration of 1.0 × 105 colony-forming units (CFU)/ml and the microplate was incubated for 24 h at 37°C. The MIC was measured as the lowest concentration of GSE that led to complete inhibition of visible growth.

Biofilm Assay

Biofilm inhibition and degradation by GSE were assessed using a crystal violet assay, as previously described [22]. To investigate the inhibitory effect of GSE on initial stages of biofilm formation, bacterial cultures were diluted to a final concentration of 1.0 × 105 CFU/ml and 50 µl was transferred to a 96-well polystyrene microtiter plate (SPL, Korea) with 50 µl GSE at a range of concentrations from 3.125 to 50 µg/ml for S. aureus and 31.25 to 500 µg/ml for E. coli (1/8 × MIC – 2 × MIC) and incubated for 24 h at 37°C. Control was prepared in TSB without GSE for each strain. The culture medium was removed, and the wells were washed twice with distilled water to remove planktonic cells. Plates were incubated at 37°C for 15 min to completely dry, and then 100 µl 1% crystal violet solution was added to each well to stain the attached biofilm cells. After 30 min, the wells were washed gently with tap water followed by distilled water. Then, 100 µl dissolving solution (30% methanol and 10% acetic acid) was added to each well to dissolve the crystal violet, and the optical density (OD) was measured at 570 nm using a microplate reader (Emax, Molecular Devices, USA).

To investigate the degradation effect of GSE on mature biofilms, 100 µl cultured cell suspension was transferred to 96-well plates, without GSE. After biofilm maturation (24 h at 37°C), the cell suspension was replaced with 100 µl of each concentration of GSE solution (1/8 × MIC – 2 × MIC) and plates were incubated for a further 24 h at 37°C. The biofilm was quantified as described above. The biofilm formation rate (%) was calculated using the following equation:

Biofilm formation rate (%)=ODtreatment/ODcontrol×100

where ODtreatment and ODcontrol refer to the absorbance at 570 nm in each well with and without GSE, respectively, after the addition of dissolving solution.

Hydrophobicity and Auto-Aggregation

The cell surface hydrophobicity of biofilm-forming strains was determined in the presence of GSE as previously described, with some modifications [23]. Cells were treated with a sub-inhibitory concentration of GSE (12.5 µg/ml for S. aureus and 125 µg/ml for E. coli) for 4 h at 37°C; nontreated cells were used as a control. Cells were centrifuged (14,240 ×g for 5 min at 4°C) and the resulting pellets were washed twice and resuspended in phosphate buffered saline (PBS, pH 7.4; Hyclone, USA) to an OD of 0.5 ± 0.05 at 600 nm (ODinitial). Toluene (0.5 ml) was added to each suspension (2 ml), followed by vortexing for 2 min. After 15 min, the OD of the lower aqueous layer was measured at 600 nm (ODtreatment) and hydrophobicity (%) was calculated using the following equation:

Hydrophobicity (%)=(1-ODtreatment/ODinitial)×100

After washing the incubated cells, the cells were resuspended in 4 ml PBS to an OD of 0.5 ± 0.05 at 600 nm (ODinitial). The suspensions were incubated for 6 h at 37°C and then the OD at 600 nm was measured for each suspension (ODtreatment). Auto-aggregation (%) was calculated using the following equation:

Auto-aggregation (%)=(1-ODtreatment/ODinitial)×100

Total EPS Production Rate

Total EPS production rate of biofilm-forming strains was determined as previously described, with some modifications [24]. Bacterial cultures were treated with 1/2 × MIC of GSE (S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml) in TSB with 2% sucrose for 12 h at 37°C; nontreated cells were used as a control. Cultures were then centrifuged at 8,000 ×g for 10 min at 4°C and 1 ml supernatant was combined with 2 ml 99% ethyl alcohol and incubated overnight at 4°C. After incubation, the suspension was centrifuged (14,240 ×g at 4°C for 5 min) and resuspended in 500 µl distilled water. Then, 1 ml 5% phenol and 5 ml 95% sulfuric acid were added to 100 µl cell suspension, which was mixed by vortexing and left to stand for 10 min at 30°C. Absorbance was measured at 490 nm and total EPS production rate (%) was calculated using the following equation:

Total EPS production rate (%)=(1-ODtreatment/ODcontrol)×100

where ODtreatment and ODcontrol refer to the absorbance of the phenol-sulfuric acid solution at 490 nm with and without GSE, respectively.

Motility of GSE-Treated Pathogens

Ten microliters S. aureus cultures were stab-inoculated to the center of tryptic soy agar (TSA) containing 0.3% agar (colony spreading) and GSE at 1/2 × MIC (treatment) or no GSE (control) and incubated without lids for up to 20 h at 37°C [25]. For E. coli, 5 µl cultures were stab-inoculated to the center of TSA containing 0.3% and 0.6% agar for swimming and swarming motility, respectively, and GSE at 1/2 × MIC (treatment) or no GSE (control) and incubated without lids for up to 12 h at 37°C [25]. After incubation, the diameter of the zone traveled by the bacteria from the point of inoculation was measured.

Biofilm Degradation Effect of GSE on Stainless Steel

S. aureus and E. coli were incubated for 24 h at 37°C in TSB and then diluted to a final concentration of 105 CFU/ml, and 2.4 ml of these suspensions were loaded into each well of 24-well culture plates (SPL) containing stainless steel coupons (type 304, 10 mm × 15 mm × 2 mm; i-Nexus Inc., Korea). Plates were then incubated for 24 h at 37°C. After biofilm formation, the coupons were rinsed with 0.1% peptone water to remove planktonic cells and transferred to another 24-well plate. GSE solution was added (from 1/4 × MIC to 4 × MIC), and plates were incubated for 24 h at 37°C. After treatment, the coupons were washed again with 0.1% peptone water and vortexed for 1 min with 4-5 autoclaved glass beads in 10 ml 0.1% peptone water. The suspensions were diluted 10-fold with 0.1% peptone water and spread on TSA. TSA plates were incubated for 24 h at 37°C, and the number of CFU were determined and expressed as log CFU/coupon.

Scanning Electron Microscopy (SEM) Analysis

SEM was carried out to evaluate bacterial adhesion to stainless steel in the presence of GSE [11]. Biofilms formed on the stainless steel coupons were fixed in 2.5% glutaraldehyde for 1 h at 4°C. The fixed samples were washed three times with PBS and dehydrated in 50%, 70%, 80%, 90%, and 100% ethanol (15 min each), then the ethanol was replaced with isoamyl acetate and the samples were freeze-dried. Stainless steel samples were sputter coated with gold (15 mV for 1.5 min) and observed using a field-emission scanning electron microscope (FESEM; SU8010; Hitachi High-Technologies Co., Japan).

Statistical Analysis

Statistical analysis was performed using SPSS version 18.0 (SPSS Inc., USA). All experiments were repeated three times. The results are stated as the mean ± standard deviation. Significant differences among the means were evaluated using one-way analysis of variance (ANOVA).

Results and Discussion

Determination of Minimum Inhibitory Concentration (MIC)

The antimicrobial effect of GSE was assessed by determining the MIC against selected food poisoning pathogens. GSE inhibited the growth of S. aureus and E. coli at 25 µg/ml and 250 µg/ml, respectively. Several studies have shown that GSE has an antimicrobial effect on various pathogens including gram-positive bacteria, gram-negative bacteria, and yeasts [19-21]. For example, the mean MIC of GSE against Salmonella sp. and L. monocytogenes was 15 ± 0.62 µg/ml and 64 ± 0.24 µg/ml, respectively [19]. In another study, the MIC of GSE against S. aureus and E. coli (O:157 and O:128) was 8.25% (w/v) and 4.13% (w/v), respectively [20]. The same study also confirmed that GSE contains high levels of polyphenol compounds, which are produced by plants in self-defense against plant pathogens [20]. Furthermore, Heggers et al. [21] demonstrated that the antimicrobial mechanism of GSE involves destruction of the cell membrane, leading to bacterial cell death.

Biofilm Inhibition and Degradation Effects of GSE

The inhibitory effect of GSE on biofilms formed by food poisoning pathogens is shown in Fig. 1A. Biofilm inhibition effect represented inhibitory effects in the initial stage of biofilm formation [22]. In the crystal violet staining assay, S. aureus and E. coli biofilms were significantly inhibited at concentrations above 1/4 × MIC (6.25 µg/ml) and 1/8 × MIC (31.25 µg/ml), respectively (p < 0.05). Biofilm formation levels for S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125 at 1/2 × MIC were 55.8%, 70.2%, 55.4%, and 20.6%, respectively. Interestingly, GSE inhibited the formation of biofilms at a concentration below the MIC. These results suggested that GSE can be used as an antibiofilm candidate at lower concentration than MIC, especially against the formation of biofilms by pathogens.

Figure 1. Effect of grapefruit seed extract (GSE) on the biofilm of food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) in 96-well plates. (A) Inhibitory effect of GSE on biofilms when co-incubated for 24 h with different concentrations of GSE. (B) Degradation effect of 24 h GSE treatment on mature biofilms. GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each organism. The MIC values of S. aureus and E. coli strains are 25 µg/ml and 250 µg/ml, respectively. a-eValues with different letters in the same bar indicate significant differences (p < 0.05).

Biofilm degradation effects refer to the effects on mature biofilms of pathogens [22]. GSE had a significant degradation effect on mature S. aureus and E. coli biofilms at 1/2 × MIC (12.5 µg/ml) and 1/8 × MIC (31.25 µg/ml), respectively (p < 0.05; Fig. 1B). In biofilm degradation effect, biofilm formation rates at 1/2 × MIC were 64.8%, 63.1%, 35.4%, and 17.6% for S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125, respectively.

Compared to inhibition of biofilm formation (Fig. 1A) and degradation of matured biofilm (Fig. 1B), biofilm formation rate at the initial stage was higher than matured biofilm. This is because once formed, biofilm becomes difficult to remove, and inhibition of biofilm formation has been reported at the initial stage.

A number of studies have demonstrated the anti-biofilm effects of plant extracts and phytochemicals; for example, one group found that grapefruit juice has an anti-biofilm effect against E. coli O157:H7 and S. Typhimurium by interfering with quorum sensing [26]. However, the anti-biofilm effect of GSE had not been reported yet.

Mode of GSE Anti-Biofilm Effect

The changes in biofilm forming properties of each pathogen, in response to GSE, are shown in Table 1. Hydrophobicity and auto-aggregation are important for colonization, adhesion, and biofilm growth and these differ depending on strain type [27]. According to Choi et al. [27], cell surface hydrophobicity varied according to the hydrocarbons used but generally S. aureus rather than E. coli appeared high. We investigated hydrophobicity by assessing pathogen adhesion to toluene in the presence of GSE at 1/2 × MIC. GSE reduced cell surface hydrophobicity significantly in E. coli (p < 0.05) but did not have a significant reduction in S. aureus (Table 1). Thus, GSE concentration of 1/2 × MIC did not affect the cell surface hydrophobicity of S. aureus, the gram-positive bacteria. Auto-aggregation of E. coli and S. aureus was not significantly affected by GSE.

Table 1 . Changes in cell surface hydrophobicity, auto-aggregation, EPS production, and motility in food poisoning pathogens treated with 1/2 × MIC of GSE..

Pathogens

S. aureus 7S. aureus 8E. coli ATCC 25922E. coli O157:H4 FRIK 125
Hydrophobicity (%)
Control97.72 ± 1.9798.53 ± 0.3459.47 ± 0.3466.85 ± 2.34
Treated91.98 ± 3.6498.53 ± 0.8730.55 ± 9.96*62.12 ± 1.44*
Auto-aggregation (%)
Control34.33 ± 2.2741.53 ± 1.2124.39 ± 2.6023.63 ± 2.30
Treated34.23 ± 2.0941.04 ± 0.6027.96 ± 1.5520.13 ± 1.63
EPS (%)
Control100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00
Treated45.13 ± 0.21***39.18 ± 0.12***71.15 ± 0.43***97.36 ± 0.63**
Motility
Colony spreading (mm)
Control3.25 ± 0.754.75 ± 0.25--
Treated2.5.0 ± 0.76**3.50 ± 1.00**--
Swimming (mm)
Control--34.50 ± 0.505.00±0.50
Treated--26.67 ± 0.83***2.33±0.17**
Swarming (mm)
Control--5.17 ± 0.175.50 ± 0.29
Treated--3.33 ± 0.33**2.67 ± 0.33**

GSE, grapefruit seed extract; MIC, minimum inhibitory concentration; Control, nontreated GSE; Treated, 1/2 × MIC of GSE (S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml); EPS, exopolysaccharide..

All values are mean ± standard error (* p < 0.05, ** p < 0.01, *** p < 0.001)..



Biofilm forming bacteria can overcome external stress following colonization and biofilm maturation. Cells within biofilms secrete EPS, which helps to trap nutrients [6]. In this study, EPS production was significantly reduced in all pathogens treated with GSE at 1/2 × MIC (p < 0.05). Compared to a control, EPS production in S. aureus 7 and S. aureus 8 decreased by 54.87% and 60.82%, respectively; whereas, in E. coli ATCC 25922 and E. coli O157:H4 FRIK 125, it decreased by 28.85% and 2.64%, respectively.

Bacterial motility has been shown to play a role in biofilm formation and attachment of cells to a surface [28]. Specifically, flagella-mediated motility of L. monocytogenes was shown to be essential in the initial stage of biofilm formation and the motility of E. coli is thought to be essential for enabling cells to reach to the surface and to facilitate biofilm growth and spread [29, 30]. In this study, the GSE-treated group had significantly reduced motility compared to the control (p < 0.01).

These results provide insight into how GSE inhibits and degrades biofilms (Fig. 1). Our results suggest that GSE influences EPS production and motility in both S. aureus and E. coli and hydrophobicity only in E. coli. The main composition of the cell wall of gram-positive strains is known as peptidoglycan, which is highly influenced by EPS production. Meanwhile gram-negative strains have cell walls composed of lipopolysaccharides, and are therefore influenced by hydrophobicity as shown in these results.

SEM Analysis of Biofilm Degradation on Stainless Steel

GSE has a significant degradation effect on biofilms on stainless steel at concentrations above the MIC (p < 0.05; Fig. 2). The SEM image of biofilm cells attached to stainless steel (Fig. 3) demonstrate that as the concentration of GSE increases, the attached cells gradually decrease, and the cells are destroyed.

Figure 2. Anti-biofilm effect of 24 h treatment with grapefruit seed extract (GSE) on the biofilm of food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) on stainless steel coupons. The MIC values of S. aureus and E. coli strains are 25 µg/ml and 250 µg/ml, respectively. a-fValues with different letters in the same bar showed significant differences (p < 0.05).
Figure 3. Scanning electron micrographs of S. aureus 7 (A) and E. coli O157:H4 FRIK 125 (B) biofilms on stainless steel-type 304 after treatment with grapefruit seed extract (GSE) (× 5,000 magnification). GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each strain.

In the absence of GSE (control), S. aureus cells were observed as cocci, where several cells were assembled in a layer; whereas in the presence of GSE at 4 × MIC (S. aureus, 100 µg/ml; E. coli, 1,000 µg/ml), individual cells were contracted and damaged (Fig. 3A). Similarly, E. coli cells were observed as bacilli in a biofilm layer in the control experiments, and as the GSE concentration increased, the number of cells attached to the stainless steel surface decreased (Figs. 2 and 3B).

This study is important because, to date, there has been no investigation of the anti-biofilm effects of the natural preservative GSE against S. aureus and E. coli. These results demonstrate that GSE has an anti-biofilm effect on both gram-positive and gram-negative bacteria, by reducing EPS production and motility in S. aureus and E. coli. Therefore, GSE has the potential to be used to safely prevent problems caused by biofilms in the food industry, while avoiding the use of the chemical compounds that are causing concern to consumers.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Effect of grapefruit seed extract (GSE) on the biofilm of food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) in 96-well plates. (A) Inhibitory effect of GSE on biofilms when co-incubated for 24 h with different concentrations of GSE. (B) Degradation effect of 24 h GSE treatment on mature biofilms. GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each organism. The MIC values of S. aureus and E. coli strains are 25 µg/ml and 250 µg/ml, respectively. a-eValues with different letters in the same bar indicate significant differences (p < 0.05).
Journal of Microbiology and Biotechnology 2019; 29: 1177-1183https://doi.org/10.4014/jmb.1905.05022

Fig 2.

Figure 2.Anti-biofilm effect of 24 h treatment with grapefruit seed extract (GSE) on the biofilm of food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 FRIK 125) on stainless steel coupons. The MIC values of S. aureus and E. coli strains are 25 µg/ml and 250 µg/ml, respectively. a-fValues with different letters in the same bar showed significant differences (p < 0.05).
Journal of Microbiology and Biotechnology 2019; 29: 1177-1183https://doi.org/10.4014/jmb.1905.05022

Fig 3.

Figure 3.Scanning electron micrographs of S. aureus 7 (A) and E. coli O157:H4 FRIK 125 (B) biofilms on stainless steel-type 304 after treatment with grapefruit seed extract (GSE) (× 5,000 magnification). GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each strain.
Journal of Microbiology and Biotechnology 2019; 29: 1177-1183https://doi.org/10.4014/jmb.1905.05022

Table 1 . Changes in cell surface hydrophobicity, auto-aggregation, EPS production, and motility in food poisoning pathogens treated with 1/2 × MIC of GSE..

Pathogens

S. aureus 7S. aureus 8E. coli ATCC 25922E. coli O157:H4 FRIK 125
Hydrophobicity (%)
Control97.72 ± 1.9798.53 ± 0.3459.47 ± 0.3466.85 ± 2.34
Treated91.98 ± 3.6498.53 ± 0.8730.55 ± 9.96*62.12 ± 1.44*
Auto-aggregation (%)
Control34.33 ± 2.2741.53 ± 1.2124.39 ± 2.6023.63 ± 2.30
Treated34.23 ± 2.0941.04 ± 0.6027.96 ± 1.5520.13 ± 1.63
EPS (%)
Control100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00100.00 ± 0.00
Treated45.13 ± 0.21***39.18 ± 0.12***71.15 ± 0.43***97.36 ± 0.63**
Motility
Colony spreading (mm)
Control3.25 ± 0.754.75 ± 0.25--
Treated2.5.0 ± 0.76**3.50 ± 1.00**--
Swimming (mm)
Control--34.50 ± 0.505.00±0.50
Treated--26.67 ± 0.83***2.33±0.17**
Swarming (mm)
Control--5.17 ± 0.175.50 ± 0.29
Treated--3.33 ± 0.33**2.67 ± 0.33**

GSE, grapefruit seed extract; MIC, minimum inhibitory concentration; Control, nontreated GSE; Treated, 1/2 × MIC of GSE (S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml); EPS, exopolysaccharide..

All values are mean ± standard error (* p < 0.05, ** p < 0.01, *** p < 0.001)..


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