Anti-Biofilm Activity of Grapefruit Seed Extract Against Staphylococcus

J. Microbiol. Biotechnol. (2019), 29(8), 1177–1183 https://doi.org/10.4014/jmb.1905.05022 Research Article Special Topic - Biofilm jmb

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

Received: May 10, 2019 Revised: July 17, 2019 Grapefruit seed extract (GSE) is a safe and effective preservative that is used widely in the Accepted: July 29, 2019 food industry. However, there are few studies addressing the anti-biofilm effect of GSE. In this First published online study, the anti-biofilm effect of GSE was investigated against biofilm-forming strains of August 1, 2019 Staphylococcus aureus and Escherichia coli. The GSE minimum inhibitory concentration (MIC)

*Corresponding author for S. aureus and E. coli were 25 µg/ml and 250 µg/ml, respectively. To investigate biofilm Phone: +82-2-2049-6011; inhibition and degradation effect, crystal violet assay and stainless steel were used. Biofilm Fax: +82-2-455-3082; E-mail: [email protected] 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 pISSN 1017-7825, eISSN 1738-8872 is effective against S. aureus and E. coli.

Introduction biofilms on a variety of surfaces [3]. In addition, many outbreaks of food poisoning have been found to be The World Health Organization (WHO) estimated that associated with biofilm-forming pathogens in the dairy, the consumption of contaminated food causes illness in 1 in poultry, meat, and ready-to-eat (RTE) food industries [4]. 10 people every year, and as a result, leads to 420,000 Biofilms are a survival and protection strategy for deaths per year [1]. According to the Centers for Disease pathogens, and the cycle of biofilm formation, maturation, Control and Prevention (CDC), Escherichia coli, Listeria spp., and dispersal is the main cause of surface-to-food cross- Salmonella spp., Campylobacter spp., and Staphylococcus contamination [5, 6]. After bacterial cells attach to a surface, aureus are some of the most common causes of food they produce a polymer matrix, composed of exopoly- poisoning [2]. The most common symptoms of food saccharides (EPS), extracellular proteins, and extracellular poisoning are diarrhea, fever, vomiting, nausea, stomach DNA, to defend themselves against antibiotics and other cramps, and stomachache. Food poisoning pathogens can chemical compounds [7]. It is well recognized that bacteria contaminate food products at any time during processing, within biofilms show increased antimicrobial resistance distribution, or storage. It is very important to limit the compared to planktonic cells grown in suspension [8]. It is growth and development of food poisoning pathogens important to use appropriate sanitization procedures in the such as S. aureus and E. coli; however, elimination of these processing environment to both ensure the removal of organisms is difficult because of their ability to form pathogens and to prevent contamination of the final

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products [9, 10]. a crystal violet assay, as previously described [22]. To investigate In order to prevent biofilm formation, several studies the inhibitory effect of GSE on initial stages of biofilm formation, were conducted to discover natural antimicrobial agents bacterial cultures were diluted to a final concentration of 1.0 × 5 that affect the viability of bacteria in biofilms [11-13]. 10 CFU/ml and 50 µl was transferred to a 96-well polystyrene Furthermore, a shift in consumer preferences towards food 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 containing safe preservatives has led to the emergence of a 500 µg/ml for E. coli (1/8 × MIC – 2 × MIC) and incubated for 24 h demand for natural antimicrobial agents [14]. Plant extracts at 37°C. Control was prepared in TSB without GSE for each strain. from leaves, roots, and seeds have been reported to The culture medium was removed, and the wells were washed demonstrate anti-biofilm activity against S. aureus, E. coli, twice with distilled water to remove planktonic cells. Plates were Listeria spp., and Campylobacter spp. [3, 15-18]. incubated at 37°C for 15 min to completely dry, and then 100 µl In this study, we chose to evaluate the anti-biofilm 1% crystal violet solution was added to each well to stain the activity of grapefruit seed extract (GSE), as it is already attached biofilm cells. After 30 min, the wells were washed gently known to be a safe and effective preservative. Previous with tap water followed by distilled water. Then, 100 µl studies have demonstrated that GSE has an antimicrobial dissolving solution (30% methanol and 10% acetic acid) was effect on gram-positive bacteria, gram-negative bacteria, added to each well to dissolve the crystal violet, and the optical and yeasts [19-21]. However, there have been no specific density (OD) was measured at 570 nm using a microplate reader reports on the effect of GSE on biofilms. Therefore, the (Emax, Molecular Devices, USA). To investigate the degradation effect of GSE on mature purpose of this study was to investigate the anti-biofilm biofilms, 100 µl cultured cell suspension was transferred to 96- effect of GSE on two food poisoning pathogens (S. aureus well plates, without GSE. After biofilm maturation (24 h at 37°C), and E. coli) to determine if it may be effective for biofilm the cell suspension was replaced with 100 µl of each concentration removal in the food industry. 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 Materials and Methods above. The biofilm formation rate (%) was calculated using the following equation: Bacterial Strains and Growth Conditions

S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4 Biofilm formation rate (%) = ODtreatment/ODcontrol × 100 FRIK 125, were used in this study through biofilm-forming screening. S. aureus strains isolated from bovine were supplied by where ODtreatment and ODcontrol refer to the absorbance at 570 nm in Seoul National University (Korea). E. coli ATCC 25922 and E. coli each well with and without GSE, respectively, after the addition O157:H4 FRIK 125 were supplied from Korean Culture Center of of dissolving solution. Microorganisms (KCCM; Korea) and IOWA State University, respectively. These strains were cultured in the Tryptic Soy Broth Hydrophobicity and Auto-Aggregation (TSB; Difco Laboratories, USA) for 24 h at 37°C and stocked at The cell surface hydrophobicity of biofilm-forming strains was -80°C with 20% glycerol. In biofilm assay, the strains were determined in the presence of GSE as previously described, with cultured in TSB containing 0.25% glucose (D-(+)-Glucose; Sigma, some modifications [23]. Cells were treated with a sub-inhibitory Germany) to promote biofilm formation. 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. Minimum Inhibitory Concentration (MIC) of GSE Cells were centrifuged (14,240 ×g for 5 min at 4°C) and the Grapefruit seed extract (GSE; ES Food, Korea) was solubilized resulting pellets were washed twice and resuspended in in TSB with 0.05% Tween 80. A double broth dilution method was phosphate buffered saline (PBS, pH 7.4; Hyclone, USA) to an OD used to determine the GSE MIC in 96-well plates [11]. GSE was of 0.5 ± 0.05 at 600 nm (ODinitial). Toluene (0.5 ml) was added to serially diluted two-fold and 50 µl of each concentration was each suspension (2 ml), followed by vortexing for 2 min. After transferred into 96-well plates. Each well was then inoculated 15min, the OD of the lower aqueous layer was measured at with the same volume of bacterial suspension at a concentration 600 nm (ODtreatment) and hydrophobicity (%) was calculated using of 1.0 × 105 colony-forming units (CFU)/ml and the microplate the following equation: was incubated for 24 h at 37°C. The MIC was measured as the lowest concentration of GSE that led to complete inhibition of Hydrophobicity (%) = (1 − ODtreatment / ODinitial) × 100 visible growth. After washing the incubated cells, the cells were resuspended in

Biofilm Assay 4 ml PBS to an OD of 0.5 ± 0.05 at 600 nm (ODinitial). The suspensions Biofilm inhibition and degradation by GSE were assessed using were incubated for 6 h at 37°C and then the OD at 600 nm was

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measured for each suspension (ODtreatment). Auto-aggregation (%) plates were incubated for 24 h at 37°C, and the number of CFU was calculated using the following equation: were determined and expressed as log CFU/coupon.

Auto-aggregation (%) = (1 − ODtreatment/ODinitial) × 100 Scanning Electron Microscopy (SEM) Analysis SEM was carried out to evaluate bacterial adhesion to stainless Total EPS Production Rate steel in the presence of GSE [11]. Biofilms formed on the stainless Total EPS production rate of biofilm-forming strains was steel coupons were fixed in 2.5% glutaraldehyde for 1 h at 4°C. determined as previously described, with some modifications The fixed samples were washed three times with PBS and [24]. Bacterial cultures were treated with 1/2 × MIC of GSE dehydrated in 50%, 70%, 80%, 90%, and 100% ethanol (15 min (S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml) in TSB with 2% sucrose each), then the ethanol was replaced with isoamyl acetate and the for 12 h at 37°C; nontreated cells were used as a control. Cultures samples were freeze-dried. Stainless steel samples were sputter were then centrifuged at 8,000 ×g for 10 min at 4°C and 1ml coated with gold (15 mV for 1.5 min) and observed using a field- supernatant was combined with 2 ml 99% ethyl alcohol and emission scanning electron microscope (FESEM; SU8010; Hitachi incubated overnight at 4°C. After incubation, the suspension was High-Technologies Co., Japan). 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 Statistical Analysis were added to 100 µl cell suspension, which was mixed by Statistical analysis was performed using SPSS version 18.0 vortexing and left to stand for 10 min at 30°C. Absorbance was (SPSS Inc., USA). All experiments were repeated three times. The measured at 490 nm and total EPS production rate (%) was results are stated as the mean ± standard deviation. Significant calculated using the following equation: differences among the means were evaluated using one-way analysis of variance (ANOVA).

Total EPS production rate (%) = ODtreatment / ODcontrol × 100 Results and Discussion where ODtreatment and ODcontrol refer to the absorbance of the phenol-sulfuric acid solution at 490 nm with and without GSE, Determination of Minimum Inhibitory Concentration respectively. (MIC) The antimicrobial effect of GSE was assessed by Motility of GSE-Treated Pathogens determining the MIC against selected food poisoning Ten microliters S. aureus cultures were stab-inoculated to the pathogens. GSE inhibited the growth of S. aureus and E. coli center of tryptic soy agar (TSA) containing 0.3% agar (colony spreading) and GSE at 1/2 × MIC (treatment) or no GSE (control) at 25 µg/ml and 250 µg/ml, respectively. Several studies and incubated without lids for up to 20 h at 37°C [25]. For E. coli, have shown that GSE has an antimicrobial effect on various 5 µl cultures were stab-inoculated to the center of TSA containing pathogens including gram-positive bacteria, gram-negative 0.3% and 0.6% agar for swimming and swarming motility, bacteria, and yeasts [19-21]. For example, the mean MIC of respectively, and GSE at 1/2 × MIC (treatment) or no GSE GSE against Salmonella sp. and L. monocytogenes was 15 ± (control) and incubated without lids for up to 12 h at 37°C [25]. 0.62 µg/ml and 64 ± 0.24 µg/ml, respectively [19]. In After incubation, the diameter of the zone traveled by the bacteria another study, the MIC of GSE against S. aureus and E. coli from the point of inoculation was measured. (O:157 and O:128) was 8.25% (w/v) and 4.13% (w/v), respectively [20]. The same study also confirmed that GSE Biofilm Degradation Effect of GSE on Stainless Steel contains high levels of polyphenol compounds, which are S. aureus and E. coli were incubated for 24 h at 37°C in TSB and produced by plants in self-defense against plant pathogens then diluted to a final concentration of 105 CFU/ml, and 2.4 ml of [20]. Furthermore, Heggers et al. [21] demonstrated that the these suspensions were loaded into each well of 24-well culture plates (SPL) containing stainless steel coupons (type 304, 10 mm × antimicrobial mechanism of GSE involves destruction of 15 mm × 2 mm; i-Nexus Inc., Korea). Plates were then incubated the cell membrane, leading to bacterial cell death. for 24 h at 37°C. After biofilm formation, the coupons were rinsed with 0.1% peptone water to remove planktonic cells and Biofilm Inhibition and Degradation Effects of GSE transferred to another 24-well plate. GSE solution was added The inhibitory effect of GSE on biofilms formed by food (from 1/4 × MIC to 4 × MIC), and plates were incubated for 24 h poisoning pathogens is shown in Fig. 1A. Biofilm inhibition at 37°C. After treatment, the coupons were washed again with effect represented inhibitory effects in the initial stage of 0.1% peptone water and vortexed for 1 min with 4-5 autoclaved biofilm formation [22]. In the crystal violet staining assay, glass beads in 10 ml 0.1% peptone water. The suspensions were S. aureus and E. coli biofilms were significantly inhibited at diluted 10-fold with 0.1% peptone water and spread on TSA. TSA

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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 Fig. 1. Effect of grapefruit seed extract (GSE) on the biofilm of E. coli appeared high. We investigated hydrophobicity by food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli assessing pathogen adhesion to toluene in the presence of ATCC 25922, and E. coli O157:H4 FRIK 125) in 96-well plates. GSE at 1/2 × MIC. GSE reduced cell surface hydrophobicity (A) Inhibitory effect of GSE on biofilms when co-incubated for 24 h significantly in E. coli (p < 0.05) but did not have a with different concentrations of GSE. (B) Degradation effect of 24 h significant reduction in S. aureus (Table 1). Thus, GSE GSE treatment on mature biofilms. GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each concentration of 1/2 × MIC did not affect the cell surface organism. The MIC values of S. aureus and E. coli strains are 25 µg/ml hydrophobicity of S. aureus, the gram-positive bacteria. and 250 µg/ml, respectively. a-eValues with different letters in the Auto-aggregation of E. coli and S. aureus was not same bar indicate significant differences (p < 0.05). significantly affected by GSE. Biofilm forming bacteria can overcome external stress following colonization and biofilm maturation. Cells concentrations above 1/4 × MIC (6.25 µg/ml) and 1/8 × within biofilms secrete EPS, which helps to trap nutrients MIC (31.25 µg/ml), respectively (p < 0.05). Biofilm formation [6]. In this study, EPS production was significantly reduced levels for S. aureus 7, S. aureus 8, E. coli ATCC 25922, and in all pathogens treated with GSE at 1/2 × MIC (p < 0.05). E. coli O157:H4 FRIK 125 at 1/2 × MIC were 55.8%, 70.2%, Compared to a control, EPS production in S. aureus 7 and 55.4%, and 20.6%, respectively. Interestingly, GSE inhibited S. aureus 8 decreased by 54.87% and 60.82%, respectively; the formation of biofilms at a concentration below the MIC. whereas, in E. coli ATCC 25922 and E. coli O157:H4 FRIK These results suggested that GSE can be used as an 125, it decreased by 28.85% and 2.64%, respectively. antibiofilm candidate at lower concentration than MIC, Bacterial motility has been shown to play a role in especially against the formation of biofilms by pathogens. biofilm formation and attachment of cells to a surface [28]. Biofilm degradation effects refer to the effects on mature Specifically, flagella-mediated motility of L. monocytogenes biofilms of pathogens [22]. GSE had a significant degradation was shown to be essential in the initial stage of biofilm effect on mature S. aureus and E. coli biofilms at 1/2 × MIC formation and the motility of E. coli is thought to be (12.5 µg/ml) and 1/8 × MIC (31.25 µg/ml), respectively (p < essential for enabling cells to reach to the surface and to 0.05; Fig. 1B). In biofilm degradation effect, biofilm facilitate biofilm growth and spread [29, 30]. In this study,

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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 7 S. aureus 8 E. coli ATCC 25922 E. coli O157:H4 FRIK 125 Hydrophobicity (%) Control 97.72 ± 1.97 98.53 ± 0.34 59.47 ± 0.34 66.85 ± 2.34 Treated 91.98 ± 3.64 98.53 ± 0.87 30.55 ± 9.96* 62.12 ± 1.44* Auto-aggregation (%) Control 34.33 ± 2.27 41.53 ± 1.21 24.39 ± 2.60 23.63 ± 2.30 Treated 34.23 ± 2.09 41.04 ± 0.60 27.96 ± 1.55 20.13 ± 1.63 EPS (%) Control 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 Treated 45.13 ± 0.21*** 39.18 ± 0.12*** 71.15 ± 0.43*** 97.36 ± 0.63** Motility Colony spreading (mm) Control 3.25 ± 0.75 4.75 ± 0.25 - - Treated 2.5.0 ± 0.76** 3.50 ± 1.00** - - Swimming (mm) Control - - 34.50 ± 0.50 5.00±0.50 Treated - - 26.67 ± 0.83*** 2.33±0.17** Swarming (mm) Control - - 5.17 ± 0.17 5.50 ± 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). 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 Fig. 2. Anti-biofilm effect of 24 h treatment with grapefruit stainless steel at concentrations above the MIC (p < 0.05; seed extract (GSE) on the biofilm of food poisoning pathogens Fig. 2). The SEM image of biofilm cells attached to stainless (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli steel (Fig. 3) demonstrate that as the concentration of GSE O157:H4 FRIK 125) on stainless steel coupons. increases, the attached cells gradually decrease, and the The MIC values of S. aureus and E. coli strains are 25 µg/ml and cells are destroyed. 250 µg/ml, respectively. a-fValues with different letters in the same In the absence of GSE (control), S. aureus cells were bar showed significant differences (p < 0.05).

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Fig. 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. observed as cocci, where several cells were assembled in a while avoiding the use of the chemical compounds that are layer; whereas in the presence of GSE at 4 × MIC (S. aureus, causing concern to consumers. 100 µg/ml; E. coli, 1,000 µg/ml), individual cells were contracted and damaged (Fig. 3A). Similarly, E. coli cells Conflict of Interest were observed as bacilli in a biofilm layer in the control experiments, and as the GSE concentration increased, the The authors have no financial conflicts of interest to number of cells attached to the stainless steel surface declare. decreased (Figs. 2 and 3B). This study is important because, to date, there has been References no investigation of the anti-biofilm effects of the natural preservative GSE against S. aureus and E. coli. These results 1. World Health Organization (WHO). 2018. Fact sheets. demonstrate that GSE has an anti-biofilm effect on both Available from https://www.who.int/news-room/fact-sheets/ gram-positive and gram-negative bacteria, by reducing detail/food-safety. Accessed Apr. 10, 2019. EPS production and motility in S. aureus and E. coli. 2. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson Therefore, GSE has the potential to be used to safely MA, Roy SL, et al. 2011. Foodborne illness acquired in the United States-major pathogens. Emerg. Infect. Dis. 17: 7-15. prevent problems caused by biofilms in the food industry,

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