The Antibiofilm Effect of Ginkgo biloba Extract Against Salmonella and Listeria Isolates from Poultry

Abstract

Salmonella spp. and Listeria spp. are common foodborne pathogens in poultry and have caused a large number of outbreaks worldwide. Biofilm formation is common in the food industry and is also a mechanism of antimicrobial resistance. The aim of this work was to investigate the antimicrobial effect and mechanism of Ginkgo biloba extract against the biofilm formation of Salmonella and Listeria isolates from poultry at retail markets. Bacteria detection, isolation, and enumeration were carried out on 27 chicken and 29 ducks at retail markets. The effects of temperature and G. biloba extract against biofilm formation of Salmonella and Listeria isolates were measured using the crystal violet assay and swimming and swarming motilities. The monitoring results of Salmonella and Listeria in 56 poultry carcasses at retail markets in Korea showed that the prevalence of Salmonella spp. in poultry was low (5.4%), but the prevalence of Listeria spp (78.6%) was high. L. innocua was the predominant serotype (80%) in the isolated Listeria species. Temperature, strain, and surface affected the biofilm formation of Salmonella spp. and Listeria spp. L. innocua showed the best biofilm formation ability on a 96-well plate, while Salmonella Enteritidis formed the most biofilm on a glass slide. Biofilm formation abilities of Salmonella spp. and Listeria spp. were increased with the increase of temperature. G. biloba extract at 75 μg/mL significantly inhibited biofilm formation of Salmonella spp. and Listeria spp (p < 0.05). The mechanism of the antibiofilm effect of the G. biloba extract showed that the motility reduction may be one of the mechanisms of G. biloba extract against some serotypes of Salmonella and Listeria, but not L. monocytogenes. The findings of this study provided the basis for the application of G. biloba extract as a food additive to promote the quality and safety of poultry products.

Introduction

S almonella spp. and Listeria monocytogenes are common foodborne pathogens and have caused a large number of outbreaks worldwide. Center for Disease Control and Prevention (CDC) estimated that Salmonella caused 1 million illnesses in the United States, with 19,000 hospitalizations and 380 deaths (Scallan et al., 2011). During the period of 2011–2014, 183 persons were infected with the outbreak-associated strain of L. monocytogenes, resulting in 39 deaths (CDC, 2014). European Centre for Disease Prevention and Control and European Food Safety Authority (ECDC and EFSA, 2013; ECDC and EFSA, 2014) also reported that eight strong-evidence foodborne outbreaks caused by L. monocytogenes in Europe resulted in 71 cases and 9 deaths in 2011–2012. Recently, 1214 broilers were collected from an integrated broiler supply chain in Korea (Choi et al., 2014). Salmonella spp. was detected in 16% of the broilers from the supply chain. The highest prevalence of Salmonella was observed in the broiler transporting truck (71.43%), followed by the slaughterhouse (63.89%) and broiler farm (16.05%). Salmonella Hadar was the most frequently isolated serotype (83.08%). Moreover, the high prevalence of Listeria species was also detected in poultry carcasses (Alonso-Hernando et al., 2012) and other raw meat from retail markets (Yücel et al., 2005).

Biofilm is an architecturally complex community of cells that are attached to either a biotic or abiotic substrate and enclosed in a self-produced extracellular matrix. The extracellular matrix, which holds the bacterial biofilms together, is a complex mixture, including exopolysaccharides, proteins, and extracellular DNA. Some of the Salmonella and Listeria species are able to form a biofilm on various surfaces such as glass, stainless steel, and plastic (Stepanović et al., 2004; Bonsaglia et al., 2014; Casarin et al., 2014; Corcoran et al., 2014). Outbreaks of pathogens may not only depend on the characteristics of the virulence phenotypes but they may also be involved with the biofilm formation ability (Roberts et al., 2009; Valderrama and Cutter, 2013). Thus, biofilm formation ability of these pathogens is a great problem for food quality and human health.

Bacteria have developed the biofilm strategy to protect themselves against adverse conditions. Commonly used disinfectants can inhibit the growth of planktonic cells, but are not so effective against biofilms (Kostaki et al., 2012; Corcoran et al., 2014). The bacterial cells inside the biofilm are well protected from access by antimicrobial compounds, and thus, biofilm poses a greater threat to the human health than planktonic cells. Therefore, novel strategies are needed to control biofilm formation.

Plants have developed advanced defense mechanisms against bacteria, and many plants have secondary metabolites that are a major source of antimicrobial agents and pharmaceuticals (Wallace, 2004; Schmidt et al., 2012). Recently, several phytochemicals that were able to inhibit Salmonella spp. and Listeria spp. biofilm formation have been identified. For example, essential oils of peppermint and lemongrass have been found to inhibit Salmonella Enteritidis biofilm formation (Valeriano et al., 2012). Thyme oil, oregano oil, and phenolic carvacrol have been reported to inhibit and inactivate the biofilm of Salmonella Typhimurium (Soni et al., 2013). Yarrow essential oil, Cymbopogon sp. essential oils, eugenol, and carvacrol have all been identified as Listeria biofilm inhibitor (Oliveira et al., 2010; Pérez-Conesa et al., 2011; Jadhav et al., 2013). However, few biofilm inhibitors can instantly be applied to the food industry due to the safety of these components.

Ginkgo biloba is one of the oldest tree species with no living relatives. It has long been used as a traditional medicine and its extract is one of the top-selling herbal supplements in the United States (Lee et al., 2014). In addition, the G. biloba extract has various biological activities, such as neurons protective (Oyama et al., 1996), radical scavenging (Maitra et al., 1995), memory improving (Rigney et al., 1999), and antibacterial activities (Sati and Joshi, 2011). Recently, it is reported that G. biloba extract and ginkgolic acids have the inhibition effect against enterohemorrhagic Escherichia coli O157:H7 (EHEC) and Staphylococcus aureus biofilm formation (Lee et al., 2014). However, the antibiofilm effect of G. biloba extract against Salmonella and Listeria species has not been studied. Therefore, the purposes of this work were to investigate (i) the biofilm formation abilities of Salmonella and Listeria isolates from poultry at various temperatures, (ii) the effect of Ginkgo biloba extract against Salmonella spp. and Listeria spp. biofilm formation, and (iii) the mechanism of antibiofilm effect of G. biloba extract against Salmonella spp. and Listeria spp.

Materials and Methods

Isolation, identification, and enumeration of Salmonella spp. and Listeria spp. from poultry

From April 2014 to August 2014, 56 packaged chicken and duck samples were purchased from 34 butcher shops and 22 supermarkets in Seoul, Korea. The district location of butcher shops and supermarkets for the sample was selected according to the population of each district among 25 districts in Seoul. Two major supermarkets in each district were selected for sample collection. Samples were placed in an icebox (6°C) and transported to the food safety laboratory. For the microbiological test, each sample was weighed, marked, and placed in a Ziploc plastic bag (33.3 × 39.7 cm, 3500 mL, THAI GRIPTECH CO., LTD.) with 400 mL of 0.15% peptone water (BD). The average weight of chicken and duck was 954 g and 1782 g, respectively. The average pH and Aw of these samples were 0.977 and 5.29, respectively. The whole carcass was manually shaken for 1 min to ensure the internal and external parts of the poultry carcass had full contact with the peptone water. The poultry rinse was used for monitoring and enumeration of Salmonella and Listeria species (MFDS, 2014a).

Bacteria detection, isolation, and enumeration were carried out in 56 poultry samples according to the USDA-FSIS (2014) methods with some modification. To detect Salmonella spp., 30 mL of 1% buffered peptone water was added to a 30 mL poultry rinse in a 250-mL conical flask and then incubated under aerobic conditions at 36°C for 24 h. Following the pre-enrichment, the selective enrichment was implemented by inoculating 0.1 mL primary enrichment suspension to 10 mL Rappaport Vassiliadis broth (Oxoid) and incubated at 42°C for 24 h. Then, 0.1 mL of the resulting culture was streaked onto a xylose lysine dextrose (XLD) agar plate (Oxoid) and incubated at 36°C overnight. Presumptively positive colonies were later confirmed with an API 20E kit (bioMérieux). The Salmonella serotypes were identified by the Korea Animal and Plant Quarantine Agency.

To detect Listeria spp., 25 mL of the poultry rinse was added to a 225 mL University of Vermont-modified (UVM) Listeria enrichment broth (Oxoid) in a stomacher bag and then incubated under aerobic conditions at 30°C for 24 h. Then, 0.1 mL of overnight UVM pre-enrichment culture was inoculated into 10 mL Fraser broth (Oxoid) for secondary enrichment. After incubation for 48 h at 36°C, 0.1 mL of a serial dilution of the resulting culture was streaked onto PALCAM agar (Oxoid) and incubated at 36°C for 24 h. Following incubation, suspect colonies were confirmed with an API Listeria kit (bioMérieux). Quantitative enumeration was also carried out by plating 0.1 mL of the poultry rinse on a PALCAM agar and XLD agar for Listeria and Salmonella, respectively (MFDS, 2014a).

Effect of temperature on biofilm formation of Salmonella and Listeria species

In total, six different strains were used for the screening of the biofilm formation ability. Strains of L. monocytogenes, L. innocua, L. welshimeri, Salmonella Typhimurium, and Salmonella Stanley were all isolated from the poultry carcasses. Salmonella Enteritidis (KCCM 12021) was purchased from Korea Culture Center of Microorganisms (KCCM). Isolated strains were all maintained at −80°C in Viabank™ bacterial storage beads (MWE), and Salmonella Enteritidis was maintained at −80°C in tryptone soya broth (TSB) (Oxoid) containing 20% of glycerol (Sigma-Aldrich).

For the biofilm formation assay, 10 μL of Salmonella Enteritidis stocks and one bead of each isolated strain from poultries, which were purchased at butcher shops and supermarkets, were incubated in a 250-mL flask containing 10 mL of TSB at 36°C with shaking at 140 rpm for 24 h. The bacterial cultures of Salmonella spp. and Listeria spp. were streaked on the tryptone soya agar (Oxoid). The colonies were inoculated into TSB by sterile loop and were homogenized to yield a 1.0 × 10⁶ colony-forming units per milliliter (CFU/mL) standard suspension. Biofilm was formed in the 96-well immune plate (SPL™) by adding 200 μL standard suspension culture in each well. The negative control wells only contained broth. After 24 h incubation at 4°C, 15°C, 30°C, and 36°C without shaking, biofilm production was measured using the modified crystal violet assay described by Stepanović et al. (2000) with some modifications. Briefly, the content of 96-well plate was aspirated, and each well was washed thrice with 250 μL of sterile distilled water. The plates were manually shaken to remove any unattached cells. The remaining attached cells were fixed with 200 μL of 99% methanol (Merck) per well for 15 min. Then, each well in plate was stained with 200 μL of 2% crystal violet (Junsei Chemical Co., Ltd.). After 5 min, the stain was rinsed off by placing the plate under running tap water. The dye bound to the attached cells was resolubilized with 200 μL of 33% (v/v) glacial acetic acid (Duksan) per well. The OD of each well was measured at 570 nm by an ELISA reader MQX200R (BioTek). To comparatively analyze the test results, the classifications of the biofilm formation abilities of each tested strain were divided into four categories as follows: nonbiofilm (−), weakly (+), moderately (++), and strongly (+++) biofilm, based upon the ODs of the bacterial biofilm. Optical density of the negative (ODc) was defined as three standard deviations above the mean OD of the negative control.

The biofilm formation abilities of each strain were classified as follows:

OD ≤ ODc, nonbiofilm;

ODc< OD ≤2 × ODc, weakly;

2 × ODc< OD ≤4 × ODc, moderately;

4 × ODc< OD, strongly.

Antibiofilm effect of Ginkgo biloba extract against Salmonella and Listeria species

To test the antibiofilm activity of the G. biloba extract against Salmonella spp. and Listeria spp., a static biofilm formation assay was performed in a 96-well immune plate (Sandasi et al., 2011). The G. biloba leave extract (99% methanol extract) was purchased from the Korea Plant Extract Bank. The G. biloba extract was dissolved in sterile distilled water. The supernatant was collected by centrifuging the water solution of G. biloba extract at 2000 rpm for 15 min. A total of 100 μL of extract (25, 50, 75, or 100 μg/mL) was transferred into wells of a 96-well plate. Then, 100 μL of standardized cultures of Salmonella and Listeria was added into the wells and incubated for 8 h at 36°C without shaking. Total biofilm formation was quantified by the modified crystal violet assay mentioned in the biofilm formation assay (Stepanović et al., 2000).

Fluorescence microscope assay for antibiofilm effect of Ginkgo biloba extract

To investigate the effect of Ginkgo biloba extract on the viability of Salmonella spp. and Listeria spp. and to allow for the visualization of bacteria within the biofilm, all biofilms were formed in a SPL eight-well cell culture slide (Percival et al., 2008). Each well of the plate was inoculated with 0.4 mL of each standard bacterial culture (10⁶ CFU/mL) and was incubated at 36°C for 24 h with the G. biloba extract (100 μg/mL); sterile distilled water was used as a negative control. All biofilms were stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen). The LIVE/DEAD BacLight bacterial viability kits utilize mixtures of SYTO 9 green fluorescent nucleic acid stain, red fluorescent nucleic acid stain, and propidium iodide. SYTO 9 stain generally labels all live and dead bacteria. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain fluorescence when both dyes are present. Thus, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. Stain of biofilm was prepared following the manufacturer's instruction (Invitrogen) with some modification. The mixture of equal volumes of SYTO 9 and propidium iodide was diluted by sterile distilled water to a concentration of 0.3 μL/mL. Cells attached to the slide were stained with the standard SYTO 9/propidium iodide solution for 15 min in the dark and observed using a fluorescence microscope (Nikon Eclipse800) equipped with FITC and TRITC filters (Nikon). The pictures of cells on the slide stained with green were taken by a digital camera (Nikon) to detect live and dead cells.

The effect of the G. biloba extract on swimming and swarming motilities

Both Salmonella spp. and Listeria spp. biofilm formation are influenced by cell motility. Thus, the effects of the G. biloba extract on flagella that mediated swimming and swarming motilities were investigated. The motility assay was performed on a semisolid agar. To assess the swimming motility, TSB containing 0.3% agar was used. For the swarming motility, TSB supplemented with 0.5% glucose and 0.5% agar was used. The 100 μg/mL of G. biloba extracts was mixed with motility agar before solidifying (115°C). Sterile distilled water was used as a control. Strains of Listeria and Salmonella were grown overnight in TSB at 36°C, and an inoculating needle dipped in each culture was used to spot into the motility agar in triplicate. The plates were incubated at room temperature.

Statistical analysis

Each experiment was replicated at least twice at different times with duplicate treatments in each replication. The significance of differences was determined by one-way ANOVA, followed by Duncan's test for multiple comparisons using SAS (version 9.3, SAS Institute, Inc.). A probability level of p < 0.05 was considered statistically significant.

Results and Discussion

Prevalence and contamination level of Salmonella spp. and Listeria spp. in poultry carcass

A total of 56 poultry carcasses (chicken: 27, duck: 29) from super markets and butcher shops from Seoul, Korea, were analyzed for the presence of Listeria spp. and Salmonella spp. Table 1 presents the types, numbers, and sources of the poultry samples analyzed in this study. Overall, the prevalence of the Listeria spp. in the poultry carcasses from the butcher shops (91%) was higher than the poultry carcasses from the supermarkets (61%), demonstrating that the microbiological quality of poultry products from supermarkets is better than that from butcher shops.

Table 1.

Prevalence and Contamination Level of Salmonella and Listeria Species in Poultry Carcass from Retail Markets

			Quantitative analysis
Location	Sample	Identified strain	No. of positive sample	No. of sample ^b	Contamination level (CFU/carcass)
Supermarket	Chicken (10)^a	L. innocua	2	1	1200
	Duck (13)	Salmonella Stanley	1	1	—
		L. innocua	5	4	20000
		L. welshimeri	1	-	125000
		Listeria spp.	6	5	2000
Total	23		15	11
Butcher shop	Chicken (17)	L. innocua	7	7	—
		L. welshimeri	2	2	—
		L. monocytogenes	1	1	—
		Listeria spp.	6	6	—
	Duck (16)	Salmonella Typhimurium	2	1	28000
		L. innocua	9	2	245000
		L. welshimeri	2	2	—
		Listeria spp.	3	2	4000
Total	33		32	23

Number of sample.

Less than detection limit (34 samples out of 47).

CFU, colony-forming units.

According to the results of the qualitative test, Salmonella spp. was detected in only three ducks (5.4%), while Listeria spp. was isolated from 78.6% of the poultry samples (41% from chicken and 61% from duck). Three Listeria serotypes were isolated from the poultry carcasses. L. innocua was the predominant serotype (80%). Furthermore, 16.7% of the isolated samples were L. welshimeri (40% from chicken and 60% from duck), while L. monocytogenes was only detected in one chicken sample purchased from the butcher shop. In previous studies, the high prevalence of Listeria species was also detected in poultry carcasses (Alonso-Hernando et al., 2012) and other raw meat from retail markets (Yücel et al., 2005). Furthermore, all of these studies showed that L. innocua was the predominant Listeria serotype isolated from poultry and raw meat, indicating that L. innocua has the special ability to survive and contaminate in the poultry and raw meat industry. However, the prevalence of L. monocytogenes in the present study (2.3%) was much lower than previous studies (24.5–41%). The results of the present study indicate the low incidence of foodborne outbreak in Korea due to L. monocytogenes.

As for the Salmonella, the prevalence of Salmonella spp. in the poultry carcasses was quite high in various countries (Antunes et al., 2003; Bohaychuk et al., 2006; Álvarez-Fernández et al., 2012). However, the prevalence of Salmonella spp. in this current study was only 5.4%. Our result was more similar to the other study, in that only 3 of the 205 fresh chickens in the retail markets yielded Salmonella spp (Soultos et al., 2003). Among the three Salmonella isolates from duck samples in the present study, two of them were Salmonella Typhimurium from the butcher shop and one of them was Salmonella Stanley from the supermarket, which are pathogenic strains related to outbreaks due to Salmonella spp. Salmonella Stanley was the primary serotype prevalent in Asia; however, in recent years, this serotype also caused a lot of outbreaks in Europe (Kinross et al., 2014; Springer et al., 2014). Considering the high demand for poultry, special attention has to be made to the raw and processed poultry at the retail markets.

According to the result of quantitative analysis, a very low contamination level (<1 log CFU/g) was measured for the Salmonella and Listeria species in the poultry carcasses. Only one of three of the Salmonella isolates was more than 1 log CFU/g (Salmonella Typhimurium: 28000 CFU/carcass, 1.24 log CFU/g), and contamination range of 12 Listeria isolates from poultry was 1200–245000 CFU/carcass (Table 1).

Effect of temperature on biofilm formation of Salmonella and Listeria species

Biofilm formation abilities of Salmonella and Listeria isolates from poultry and Salmonella Enteritidis (KCCM 12021) at various temperatures are shown in Figure 1. Overall, all of the tested bacteria have the ability to form biofilm, and biofilm formation abilities were affected by strain and temperature. Temperature significantly affected the quantity of the biofilm formation for all of the tested strains (p < 0.05). The quantity of biofilm formed by all of the tested bacteria except for L. innocua increased with increased temperature. Among the strains, L. monocytogenes (Fig. 1A), L. welshimeri (Fig. 1C), and Salmonella Stanley (Fig. 1F) formed weak biofilm at 4°C, 15°C, 30°C, and 36°C. The L. innocua had the best biofilm formation ability, and the most biofilm was formed at 15°C, while it formed a weak biofilm at 4°C (Fig. 1B). The Salmonella Enteritidis (KCCM 12021) formed a moderate biofilm at 36°C and the smallest amount of biofilm at 4°C, 15°C, and 30°C with no significant differences between the quantity of biofilm formed at these temperatures (p > 0.05) (Fig. 1D). In contrast, the Salmonella Typhimurium did not form any biofilm at 4°C. However, it formed a weak biofilm at 15°C and moderate biofilms at 30°C and 36°C (Fig. 1E). The biofilm formations of Salmonella Stanley at 30°C and 36°C were more than that at 4°C and 15°C, and there was no significant difference between 30°C and 36°C (p > 0.05) (Fig. 1F).

FIG. 1.

Biofilm formation of Salmonella and Listeria strains at various temperatures. OD, optical density. Means (n = 6) followed by the different letters are significantly different (p < 0.05). (A) L. monocytogenes, (B) L. innocua, (C) L. welshimeri, (D) Salmonella Enteritidis, (E) Salmonella Typhimurium, (F) Salmonella Stanley.

Few studies have been done for biofilm formation of other Listeria species, and this current study has revealed that L. innocua can form strong biofilms at various temperatures. This is one of the reasons or may be the only reason of the high contamination rate of L. innocua in poultry carcasses in this current work. The biofilm formation abilities of 30 strains of Salmonella spp. were also tested in a previous study (Stepanović et al., 2003). The largest quantity of biofilm formed at 30°C after 24 h incubation. In this current study, the Salmonella Typhimurium isolate from poultry also formed the largest quantity of biofilm at 30°C, but Salmonella Enteritidis (KCCM 12021) formed a significantly higher quantity of biofilm at 36°C compared to the other temperatures. The same phenomenon was also observed in the Listeria species. L. monocytogenes formed the largest quantity of biofilm at 36°C, while L. innocua formed the largest quantity of biofilm at 15°C. It seems that the difference of biofilm formation ability existed between the different serotypes of Salmonella spp. and Listeria spp. It was reported that the virulence regulator PrfA positively regulates virulence gene expression and biofilm formation of L. monocytogenes, but not L. innocua (Lemon et al., 2010; Zhou et al., 2011), and the virulence genes are required for biofilm formation of Salmonella (Barak et al., 2005). The expression of the virulence genes of Listeria and Salmonella increased with increased temperature (Leimeister-Wächter et al., 1992; Clements et al., 2001; Johansson et al., 2002). Therefore, pathogenic Salmonella and Listeria usually formed the largest quantity of biofilm at relatively high temperatures, like 36°C, and the virulence genes were also highly expressed at this temperature. In contrast, nonpathogenic Salmonella and Listeria did not always form the largest quantity of biofilm at the higher temperature.

Antibiofilm effect of Ginkgo biloba extract against Salmonella and Listeria species

The results of the G. biloba extract against Salmonella spp. and Listeria spp. biofilm formation are shown in Figure 2. Overall, the G. biloba extract had the antibiofilm effect against all of the tested Listeria and Salmonella species. The G. biloba extract at 25 μg/mL had no antibiofilm effects against L. monocytogenes, L. innocua, Salmonella Enteritidis, and Salmonella Typhimurium (Fig. 2A, B, D, E), while this concentration did have an effect on inhibiting biofilm formation of L. welshimeri and Salmonella Stanley (Fig. 2C, F). The difference of the L. monocytogenes biofilm formation between the G. biloba extract at 50 μg/mL level and the negative control was significant (p < 0.05). However, the G. biloba extract at 75 and 100 μg/mL significantly decreased the L. monocytogenes biofilm formation (p < 0.05), and there was no significant difference between the 75 μg/mL and 100 μg/mL levels (p > 0.05). The G. biloba extract at 50 μg/mL significantly reduced the biofilm formation of L. innocua, and the biofilm formation of L. innocua was almost completely inhibited at 75 μg/mL (Fig. 2B). The antibiofilm effects of the G. biloba extract against L. monocytogenes, L. welshimeri, Salmonella Typhimurium, and Salmonella Stanley were concentration dependent; the antibiofilm effects increased with the increased concentration of the G. biloba extract (Fig. 2A, C, E, F). As for the Salmonella Enteritidis, the G. biloba extract at 25, 50, and 75 μg/mL failed to inhibit the biofilm formation compared to control (0 μg/mL) and the 100 μg/mL of the G. biloba extract showed the antibiofilm effect for Salmonella Enteritidis.

FIG. 2.

The effect of G. biloba extract against Listeria and Salmonella biofilm formation on 96-well plate. Means (n = 4) followed by the different letters are significantly different (p < 0.05). (A) L. monocytogenes, (B) L. innocua, (C) L. welshimeri, (D) Salmonella Enteritidis, (E) Salmonella Typhimurium, (F) Salmonella Stanley.

The fluorescence microscope pictures of the biofilm formed in glass are shown in Figure 3. In the present study, Salmonella Enteritidis and L. innocua cells show strong biofilm formation ability. Thus, the antibiofilm effect of G biloba extract against Salmonella Enteritidis and L. innocua cells was also observed qualitatively and shown in Fig. 3A and Fig. 3B, respectively. The results of LIVE/DEAD BacLight Bacterial staining also confirmed the live (green) biofilm cells. In addition, the 100 μg/mL of the G. biloba extract reduced the number of live cells attached to the glass. It was recently reported that the G. biloba extract at 100 μg/mL can inhibit biofilm formation of E. coli O157:H7 and Staphylococcus aureus without affecting bacterial growth, and the active component was ginkgolic acid (Lee et al., 2014). In another study, ginkgolic acid was reported to inhibit the growth and biofilm formation of Streptococcus mutans (He et al., 2013). The standardized preparation of the Ginkgo leaf extract (EGb 761) contained two main bioactive constituents, flavonoid glycosides and terpene lactones, along with less than 5 ppm of the allergenic component, ginkgolic acid (Mahadevan and Park, 2008). The toxicological impact of the G. biloba extract remains controversial. The German and Korean government limit the concentration of ginkgolic acid in commercial phytopreparation to 5 μg/g (Ndjoko et al., 2000; MFDS, 2014b), and no toxic effects are expected in man at this concentration (Ahlemeyer and Krieglstein, 2003). The presence of ginkgolic acid in the G. biloba extract was confirmed using HPLC by Lee et al. (2014) and reported that the concentration of ginkgolic acid in the G. biloba extract was 50 μg/mg. The maximum concentration of the G. biloba extract used in this current study was 100 μg/mL and was equivalent to a total amount of ginkgolic acid of 5 μg/mL. The ginkgolic acid at a concentration of less than 5 μg/mL efficiently inhibited the biofilm formation of Salmonella and Listeria species that were isolated from poultry carcasses in this current study.

FIG. 3.

Fluorescence microscope analysis for the effects of G. biloba extract on biofilm formation on glass. The scale bar represents 50 μm. (A) Salmonella Enteritidis, (B) L. innocua.

In considering the effect of the surface materials on biofilm formation, the amount of biofilm formed from Salmonella Enteritidis (Fig 1D) on the 96-well plate was less than that of L. innocua (Fig 1B). However, according to the result of the fluorescence microscope, Salmonella Enteritidis cells (Fig 3A) attached to the glass surface were more than those of L. innocua (Fig 3B). These results indicated that the biofilm formation ability of Salmonella Enteritidis on glass was better than on plastic. The results of this current study also agreed with the previous study that showed the biofilm formation of Salmonella Enteritidis observed on glass and stainless steel was significantly more than on a polyethylene surface (Manijeh et al., 2008). There are many factors that affect biofilm formation of Listeria and Salmonella. Di Bonaventura et al. (2008) reported that the hydrophobicity and biofilm level of L. monocytogenes increased with increased temperature. Hydrophobicity is one of the many factors involved in the initial attachment of L. monocytogenes to polystyrene, but not to glass (Chae et al., 2006), and the reduction of hydrophobicity decreased the adherence of cell to the hydrophobicity surface, such as polystyrene (Paul and Jeffrey, 1985).

Effect of Ginkgo biloba extract on swimming and swarming motilities

Flagella-mediated motility is critical for Listeria and Salmonella species (Solano et al., 2002; Lemon et al., 2007). Flagella are necessary for the initial attachment and bacterial interaction. To investigate the effect of G. biloba extract on the motility of Salmonella and Listeria species isolated from poultry carcasses, flagella-mediated swarming motility (cells moving side-by-side on a solid surface) and swimming motility (cells moving individually in a liquid medium) were conducted on a semisolid motility agar (Kim et al., 2003; Gueriri et al., 2008), and the results are shown in Figure 4. The G. biloba extract induced swarming motility of L. monocytogenes and had no significant impact on the swimming motility (Fig 4A), suggesting that there is no correlation between the antibiofilm effect of the G. biloba extract against L. monocytogenes and the effect of the G. biloba extract on the motility of L. monocytogenes. L. innocua and L. welshimeri have no swarming motility, and thus, the addition of the G. biloba extract did not affect the swarming motility, but reduced the swimming motility of L. innocua and L. welshimeri. In contrast, Salmonella Enteritidis showed both swarming and swimming motility (Fig. 4D). The G. biloba extract reduced the swarming motility, but induced the swimming motility, suggesting that the swarming motility rather than the swimming motility influenced the Salmonella Enteritidis biofilm formation, unlike the L. innocua and L. welshimeri. The Salmonella Typhimurium isolated from the poultry carcasses at the retail markets showed no swarming motility, and the G. biloba extract had no influence on the swimming motility of Salmonella Typhimurium (Fig. 4E). The mechanism of the antibiofilm effect of the G. biloba extract against Salmonella Typhimurium also had no relation with the motility. However, other isolates from the poultry carcasses, the Salmonella Stanley showed both the swarming and swimming motility abilities like Salmonella Enteritidis and the G. biloba extract also reduced the swarming motility of Salmonella Stanley (Fig. 4F). However, the swimming motility was not affected by the G. biloba extract, indicating that the biofilm formation of Salmonella Stanley may depend on the swarming motility but not swimming motility. It was recently reported that the G. biloba extract and ginkgolic acids reduced EHEC swarming motility, but increased its swimming motility, and they inhibited biofilm formation (Lee et al., 2014). Furthermore, carvacrol and citrus flavonoid were found to reduce the motility of Salmonella Typhimurium (Vikram et al., 2011; Inamuco et al., 2012). Plant-derived trans-cinnamaldehyde, carvacrol, thymol, and eugenol were reported to repress motility associated with the gene and to inhibit the biofilm formation of L. monocytogenes (Upadhyay et al., 2013). These results indicated that motility control by biofilm inhibitors is not rare in the plant kingdom, and further study of the safety and commercial settings for these materials is necessary.

FIG. 4.

Effect of G. biloba extract on swarming and swimming motility of Salmonella and Listeria. (A) L. monocytogenes, (B) L. innocua, (C) L. welshimeri, (D) Salmonella Enteritidis, (E) Salmonella Typhimurium, (F) Salmonella Stanley.

Furthermore, the bioactive compounds in the G. biloba extract are stable with high temperature (Jianxin, 2002). Our results also show that the antibiofilm and antimicrobial activities remained even when G. biloba extract was sterilized at 121°C for 15 min. Flavonoid glycosides (myricetin and quercetin) and terpenoids (ginkgolides and bilobalides) are the major functional compounds in the G. biloba extract. Quercetin in onion was reported to be stable with cooking methods, like boiling and frying (Price et al., 1997). Myricetin in potato was also reported to be stable with various cooking methods (Blessington et al., 2010). Since the bioactivities of G. biloba extract added in food are stable during the heat processing, the G. biloba extract can be recommended to the food industry as antibiofilm and antimicrobial agents. Thus, the application of G. biloba extract as a food additive or coating film for packaging materials may contribute to the quality and safety of poultry products.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Ahlemeyer

, Krieglstein

. Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci, 2003; 60:1779–1792.

Alonso-Hernando

, Prieto

, García-Fernández

, Alonso-Calleja

, Capita

. Increase over time in the prevalence of multiple antibiotic resistance among isolates of Listeria monocytogenes from poultry in Spain. Food Control, 2012; 23:37–41.

Álvarez-Fernández

, Alonso-Calleja

, García-Fernández

, Capita

. Prevalence and antimicrobial resistance of Salmonella serotypes isolated from poultry in Spain: Comparison between 1993 and 2006. Int J Food Microbiol, 2012; 153:281–287.

Antunes

, Réu

, Sousa

, Peixe

, Pestana

. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Int J Food Microbiol, 2003; 82:97–103.

Barak

, Gorski

, Naraghi-Arani

, Charkowski

. Salmonella enterica virulence genes are required for bacterial attachment to plant tissue. Appl Environ Microbiol, 2005; 71:5685–5691.

Blessington

, Nzaramba

, Scheuring

, Hale

, Reddivari

, Miller

Jr . Cooking methods and storage treatments of potato: Fffects on carotenoids, antioxidant activity, and phenolics. Am J Potato Res, 2010; 87:479–491.

Bohaychuk

, Gensler

, King

, Manninen

, Sorensen

, Wu

, Stiles

, McMullen

. Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. J Food Prot, 2006; 69:2176–2182.

Bonsaglia

ECR

, Silva

NCC

, Fernades Júnior

, Araújo Júnior

, Tsunemi

, Rall

VLM

. Production of biofilm by Listeria monocytogenes in different materials and temperatures. Food Control, 2014; 35:386–391.

Casarin

, Brandelli

, de Oliveira Casarin

, Soave

, Wanke

, Tondo

. Adhesion of Salmonella Enteritidis and Listeria monocytogenes on stainless steel welds. Int J Food Microbiol, 2014; 191:103–108.

10.

Center for Disease Control and Prevention (CDC).. National Center for Emerging and Zoonotic Infectious Diseases (NCEZID), Division of Foodborne, Waterborne, and Environmental Diseases (DFWED). 2014. Available at: http//www.cdc.gov/Listeria/outbreaks/index.html, Accessed March 10, 2014 .

11.

Chae

, Schraft

, Truelstrup Hansen

, Mackereth

. Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass. Food Microbiol, 2006; 23:250–259.

12.

Choi

S-W

, Ha

J-S

, Kim

B-Y

, Lee

D-H

, Park

J-K

, Youn

H-N

, Hong

Y-H

, Lee

S-B

, Lee

J-B

, Park

S-Y

. Prevalence and characterization of Salmonella species in entire steps of a single integrated broiler supply chain in Korea. Poult Sci, 2014; 93:1251–1257.

13.

Clements

, Eriksson

, Tezcan-Merdol

, Hinton

, Rhen

. Virulence gene regulation in Salmonella enterica . Ann Med, 2001; 33:178–185.

14.

Corcoran

, Morris

, De Lappe

, O'Connor

, Lalor

, Dockery

, Cormican

. Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials. Appl Environ Microbiol, 2014; 80:1507–1514.

15.

Di Bonaventura

, Piccolomini

, Paludi

, D'orio

, Vergara

, Conter

, Ianieri

. Influence of temperature on biofilm formation by Listeria monocytogenes on various food contact surfaces: Relationship with motility and cell surface hydrophobicity. J Appl Microbiol, 2008; 104:1552–1561.

16.

European Centre for Disease Prevention and Control (ECDC) and European Food Safety Authority (EFSA). The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011. EFSA Journal, 2013; 11(4):3129.

17.

18.

Gueriri

, Cyncynatus

, Dubrac

, Arana

, Dussurget

, Msadek

. The DegU orphan response regulator of Listeria monocytogenes autorepresses its own synthesis and is required for bacterial motility, virulence and biofilm formation. Microbiology, 2008; 154:2251–2264.

19.

, Wang

, Wu

, Cao

, Xu

, Zhou

. Effects of ginkgoneolic acid on the growth, acidogenicity, adherence, and biofilm of Streptococcus mutans in vitro. Folia Microbiol, 2013; 58:147–153.

20.

Inamuco

, Veenendaal

AKJ

, Burt

, Post

, Tjeerdsma-van Bokhoven

JLM

, Haagsman

, Veldhuizen

EJA

. Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells. Veterinary Microbiol, 2012; 157:200–207.

21.

Jadhav

, Shah

, Bhave

, Palombo

. Inhibitory activity of yarrow essential oil on Listeria planktonic cells and biofilms. Food Control, 2013; 29:125–130.

22.

Jianxin

. Studies on the antimicrobial characteristics of Ginkgo biloba (EGb) extract. Food Sci, 2002; 9:033.

23.

Johansson

, Mandin

, Renzoni

, Chiaruttini

, Springer

, Cossart

. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes . Cell, 2002; 110:551–561.

24.

Kim

, Killam

, Sood

, Surette

. Swarm-cell differentiation in Salmonella enterica serovar Typhimurium results in elevated resistance to multiple antibiotics. J Bacteriol, 2003; 185:3111–3117.

25.

Kinross

, Alphen

, Urtaza

, Struelens

, Takkinen

, Coulombier

, Mäkelä

, Bertrand

, Mattheus

, Schmid

, Kanitz

, Rücker

, Krisztalovics

, Pászti

, Szögyényi

, Lancz

, Rabsch

, Pfefferkorn

, Hiller

, Mooijman

, Gossner

. Multidisciplinary investigation of a multicountry outbreak of Salmonella Stanley infections associated with turkey meat in the European Union, August 2011 to January 2013. Euro Surveill, 2014; 19: pii: 20801.

26.

Kostaki

, Chorianopoulos

, Braxou

, Nychas

G-J

, Giaouris

. Differential biofilm formation and chemical disinfection resistance of sessile cells of Listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions. Appl Environ Microbiol, 2012; 78:2586–2595.

27.

Lee

J-H

, Kim

Y-G

, Ryu

, Cho

, Lee

. Ginkgolic acids and Ginkgo biloba extract inhibit Escherichia coli O157:H7 and Staphylococcus aureus biofilm formation. Int J Food Microbiol, 2014; 174:47–55.

28.

Leimeister-Wächter

, Domann

, Chakraborty

. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J Bacteriol, 1992; 174:947–952.

29.

Lemon

, Freitag

, Kolter

. The virulence regulator PrfA promotes biofilm formation by Listeria monocytogenes . J Bacteriol, 2010; 192:3969–3976.

30.

Lemon

, Higgins

, Kolter

. Flagellar motility is critical for Listeria monocytogenes biofilm formation. J Bacteriol, 2007; 189:4418–4424.

31.

Mahadevan

, Park

. Multifaceted therapeutic benefits of Ginkgo biloba L.: Chemistry, efficacy, safety, and uses. J Food Sci, 2008; 73:R14–R19.

32.

Maitra

, Marcocci

, Droy-Lefaix

, Packer

. Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761. Biochem Pharmacol, 1995; 49:1649–1655.

33.

Manijeh

, Mohammad

, Roha

. Biofilm formation by Salmonella enteritidis on food contact surfaces. J Biol Sci, 2008; 8:502–505.

34.

Ministry of Food and Drug Safety (MFDS). The processing criteria and component specification of animal-origin product. 2014a. Available at: http://mfds.go.kr/index.do?mid=687&pageNo=26&seq=7691&cmd=v, accessed March 11, 2014

35.

Ministry of Food and Drug Safety (MFDS). Food Additives Code. Korea Food Drug Admin 2014b. Available at: http://fse.foodnara.go.kr/residue/RS/jsp/menu_02_01_01.jsp, accessed November 19, 2014

36.

Ndjoko

, Wolfender

J-L

, Hostettmann

. Determination of trace amounts of ginkgolic acids in Ginkgo biloba L. leaf extracts and phytopharmaceuticals by liquid chromatography–electrospray mass spectrometry. J Chromatogr B Biomed Sci Appl, 2000; 744:249–255.

37.

Oliveira

MMMd

, Brugnera

, Cardoso

MdG

, Alves

, Piccoli

. Disinfectant action of Cymbopogon sp. essential oils in different phases of biofilm formation by Listeria monocytogenes on stainless steel surface. Food Control, 2010; 21:549–553.

38.

Oyama

, Chikahisa

, Ueha

, Kanemaru

, Noda

. Ginkgo biloba extract protects brain neurons against oxidative stress induced by hydrogen peroxide. Brain Res, 1996; 712:349–352.

39.

Paul

, Jeffrey

. Evidence for separate adhesion mechanisms for hydrophilic and hydrophobic surfaces in Vibrio proteolytica . Appl Environ Microbiol, 1985; 50:431–437.

40.

Percival

, Bowler

, Woods

. Assessing the effect of an antimicrobial wound dressing on biofilms. Wound Repair Regen, 2008; 16:52–57.

41.

Pérez-Conesa

, Cao

, Chen

, McLandsborough

, Weiss

. Inactivation of Listeria monocytogenes and Escherichia coli O157: H7 biofilms by micelle-encapsulated eugenol and carvacrol. J Food Prot, 2011; 74:55–62.

42.

Price

, Bacon

, Rhodes

. Effect of storage and domestic processing on the content and composition of flavonol glucosides in onion (Allium cepa). J Agric Food Chem, 1997; 45:938–942.

43.

Rigney

, Kimber

, Hindmarch

. The effects of acute doses of standardized Ginkgo biloba extract on memory and psychomotor performance in volunteers. Phytother Res, 1999; 13:408–415.

44.

Roberts

, Williams

, Wiedmann

, Nightingale

. Some Listeria monocytogenes outbreak strains demonstrate significantly reduced invasion, inlA transcript levels, and swarming motility in vitro. Appl Environ Microbiol, 2009; 75:5647–5658.

45.

Sandasi

, Leonard

, Van Vuuren

, Viljoen

. Peppermint (Mentha piperita) inhibits microbial biofilms in vitro. S Afr J Bot, 2011; 77:80–85.

46.

Sati

, Joshi

. Antibacterial activities of Ginkgo biloba L. leaf extracts. Sci World J, 2011; 11:2237–2242.

47.

Scallan

, Hoekstra

, Angulo

, Tauxe

, Widdowson

M-A

, Roy

, Jones

, Griffin

. Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis, 2011; 17:7–15.

48.

Schmidt

, Khalid

, Romanha

, Alves

, Biavatti

, Brun

, Da Costa

, de Castro

, Ferreira

, de Lacerda

. The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases—part I. Curr Med Chem, 2012; 19:2128–2175.

49.

Solano

, García

, Valle

, Berasain

, Ghigo

, Gamazo

, Lasa

. Genetic analysis of Salmonella enteritidis biofilm formation: Critical role of cellulose. Mol Microbiol, 2002; 43:793–808.

50.

Soni

, Oladunjoye

, Nannapaneni

, Schilling

, Silva

, Mikel

, Bailey

. Inhibition and inactivation of Salmonella Typhimurium biofilms from polystyrene and stainless steel surfaces by essential oils and phenolic constituent carvacrol. J Food Prot, 2013; 76:205–212.

51.

Soultos

, Koidis

, Madden

. Presence of Listeria and Salmonella spp. in retail chicken in Northern Ireland. Lett Appl Microbiol, 2003; 37:421–423.

52.

Springer

, Allerberger

, Kornschober

. Letter to the editor: Salmonella Stanley outbreaks–a prompt to reevaluate existing food regulations. Euro surveillance: Bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin, 2014; 19.

53.

Stepanović

, Ćirković

, Mijač

, Švabić-Vlahović

. Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiol, 2003; 20:339–343.

54.

Stepanović

, Ćirković

, Ranin

. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett Appl Microbiol, 2004; 38:428–432.

55.

Stepanović

, Vuković

, Dakić

, Savić

, Švabić-Vlahović

. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods, 2000; 40:175–179.

56.

United States Department of Agriculture-Food Safety and Inspection Service(USDA-FSIS).. Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish Products and Carcass and Environmental Sponges. 2014. Available at: http://www.fsis.usda.gov/wps/portal/fsis/topics/science/laboratories-and-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/microbiology-laboratory-guidebook, Accessed November 19, 2015

57.

Upadhyay

, Upadhyaya

, Kollanoor-Johny

, Venkitanarayanan

. Antibiofilm effect of plant derived antimicrobials on Listeria monocytogenes . Food Microbiol, 2013; 36:79–89.

58.

Valderrama

, Cutter

. An ecological perspective of Listeria monocytogenes biofilms in food processing facilities. Crit Rev Food Sci Nutr, 2013; 53:801–817.

59.

Valeriano

, De Oliveira

TLC

, De Carvalho

, Cardoso

MdG

, Alves

, Piccoli

. The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel. Food Control, 2012; 25:673–677.

60.

Vikram

, Jesudhasan

, Jayaprakasha

, Pillai

, Jayaraman

, Patil

. Citrus flavonoid represses Salmonella pathogenicity island 1 and motility in Salmonella Typhimurium LT2. Int J Food Microbiol, 2011; 145:28–36.

61.

Wallace

. Antimicrobial properties of plant secondary metabolites. Proc Nutr Soc, 2004; 63:621–629.

62.

Yücel

, Çıtak

, Önder

. Prevalence and antibiotic resistance of Listeria species in meat products in Ankara, Turkey. Food Microbiol, 2005; 22:241–245.

63.

Zhou

, Feng

, Wang

, Feng

, Yin

, Luo

. Virulence regulator PrfA is essential for biofilm formation in Listeria monocytogenes but not in Listeria innocua . Current Microbiol, 2011; 63:186–192.