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Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus
Journal Pre-proof Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus
Rui Li, Jieyuan Lu, Hebo Duan, Jun Yang, Changbo Tang PII:
S0956-7135(20)30064-5
DOI:
https://doi.org/10.1016/j.foodcont.2020.107148
Reference:
JFCO 107148
To appear in:
Food Control
Received Date:
04 December 2019
Accepted Date:
28 January 2020
Please cite this article as: Rui Li, Jieyuan Lu, Hebo Duan, Jun Yang, Changbo Tang, Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus, Food Control (2020), https://doi.org/10.1016/j.foodcont.2020.107148
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Journal Pre-proof Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus Rui Lia, Jieyuan Lu a, Hebo Duana,Jun Yanga, Changbo Tangb,⁎ a College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, 430023, China； b College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China. Correspondence
Changbo Tang, E-mail: [email protected]. Tel: 08602584399702. Mailing address: College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China.
This manuscript has been thoroughly edited by a native English speaker.
1
Journal Pre-proof ABSTRACT Vibrio mimicus is a relatively rare food-borne pathogen in seafood and water. Rare reports have been published to investigate the biofilm of Vibrio mimicus.
(-)-
epigallocatechin-3-gallate (EGCG), the major polyphenolic component of tea, can interfere with bacterial biofilm formation. This study showed that Vibrio mimicus was capable of forming high amounts of biofilms in various culture media. Sub-MICs of EGCG significantly reduced the biofilm production of Vibrio mimicus at 15 ℃, 28 ℃ and 37 ℃. Confocal laser-scanning microscope (CLSM) observation proved that the architecture of Vibrio mimicus biofilm was affected by EGCG at the concentration of 64 µg/mL (1/4 MIC). EGCG reduced Vibrio mimicus autoaggregation and swimming motility. EGCG also increased membrane permeability and ROS production, caused cell membrane damage and led to potassium leakage. These may contribute to the antibiofilm efficacy of EGCG against Vibrio mimicus. Our work showed the inhibitory effects of sub-MICs of EGCG on Vibrio mimicus biofilm for the first time, supporting the potential application of EGCG as a natural antibiofilm agent in the food industry.
Keywords: Vibrio mimicus; biofilm; EGCG; inhibition; sub-MICs
2
Journal Pre-proof 1. Introduction Vibrio mimicus is closely related to Vibrio cholerae and carries a number of potential virulence factors, including cholera-like enterotoxin, heat-stable enterotoxin, heat-labile enterotoxin, hemolysin, etc (Ali, Nelson, Lopez, & Sack, 2015). Vibrio mimicus is found in freshwater and seawater and can be potentially pathogenic for some animals, like mussel, fish, shrimp, and humans (Abd, Valeru, Sami, Saeed, & Sandström, 2010). Human infections with this organism are normally associated with ingestion of contaminated seafood and water. Reported cases are usually sporadic, but outbreaks have also occurred. For example, large food-borne outbreak caused by Vibrio mimicus involving 306 people occurred in Thailand in 2004. The probable cause of the outbreak was seafood soup (Chitov, Kirikaew, Yungyune, Ruengprapan, & Sontikun, 2009). In Shandong province Yiyuan county, China, 27 people were infected with Vibrio mimicus due to consumption of contaminated razor clam on 2011, 5th July. A cluster of severe diarrheal disease caused by Vibrio mimicus infection among four persons who had consumed crayfish occurred in Washington state, USA (Kay et al., 2012). Although Vibrio mimicus are not targets in food risk monitoring, isolation of the organism from clinical samples has been reported in many countries (Shinoda et al., 2004).
In natural environments, Vibrio species can attach to abiotic or biotic surfaces and form biofilms (Teschler et al., 2015). Biofilms are matrix-enclosed communities of microorganisms and the extracellular substances they produce (Flemming & Wingender, 2010). Bacteria in a biofilm are more resistant to sanitizers and antibiotics compared with planktonic bacteria. Vibrio cholerae has become a model organism in biofilm research, and the formation of its biofilm has been studied intensively (Yan, Sharo, Stone, Wingreen, & Bassler, 2016). However, unlike Vibrio cholerae, little 3
Journal Pre-proof information about Vibrio mimicus biofilm has been reported. Earlier work by TerceroAlburo et al. showed the biofilm formation of Vibrio mimicus at 37 °C in TSB broth by using scanning electron microscopy (Tercero-Alburo, Gonzßlez-Mßrquez, BonillaGonzßlez, Qui, & Vßzquez-Salinas, 2014). However, the inhibition of biofilm formation by Vibrio mimicus has not been studied yet. As pathogenic bacteria may thrive in biofilms, biofilm-associated infections pose a major health threat and there is a pressing need for antibiofilm agents (Li & Lee, 2017). Tea polyphenols are present in abundance in teas and have remarkable inhibitory effects against bacteria (Slobodníková, Fialová, Rendeková, Kováč, & Mučaji, 2016). Among tea polyphenols, (-)-epigallocatechin-3-gallate (EGCG), which is the major polyphenolic component of tea, exhibits antibiofilm activities (Bansal et al., 2013). But some reports indicated that EGCG seems inefficient against or may even sometimes promote biofilms which rely on types of biofilm matrix (Hengge, 2019). Special interests have been assigned to EGCG as an antibiofilm agent (Asahi et al., 2014; Serra, Mika, Richter, & Hengge, 2016), but so far the inhibitory effects of EGCG on Vibrio mimicus biofilm remain unknown. In the present study, we present data that reveal the following: (i) antibiofilm efficacy of EGCG against Vibrio mimicus; and (ii) the possible underlying mechanisms of action. 2. Materials and methods 2.1. Chemicals and bacteria strain EGCG monomer (99% purity) was purchased from Guang Run Biotechnology Co. Ltd (Nan Jing, China). An environmental Vibrio mimicus strain SNJ was isolated from fish and used in the study. 2.2. Biofilm development 4
Journal Pre-proof Vibrio mimicus was cultured overnight at 37 ℃ under shaking in LB broth. The bacteria cells were collected and adjusted to get an OD600 value of 1, then diluted 1:25 in saline solution. Aliquots (50 µL) of this dilution were inoculated into six wells of a flat-bottomed, 96-well polystyrene tissue culture plate (Corning Costar, USA). The outermost wells were not used for the assay, because these wells suffer edge effects. Various media (TSB, LB, NB, BHI, MH and 1/2LB) were prepared to evaluate nutrient availability. To prepare 1/2LB medium, LB broth was diluted 2-fold with distilled water. Aliquots (150 µL) of each medium were dispensed in the microtiter plate wells and mixed with portions of diluted bacterial suspension to obtain final concentration of 105 CFU/mL. Each medium without bacterial cells was placed in six wells of each plate as negative controls. The plates were incubated at 28 ℃ under static conditions. At each time point, the medium was removed from each well and the plates were rinsed with sterile phosphate-buffered saline (PBS, pH 7.4). The plates were air dried and stained with 0.1 % crystal violet for 15min. Then the wells were rinsed again and destained with 95% ethanol. The amount of biofilm was measured as the absorbance at 595 nm using a microplate reader (Tecan, Austria). Two extreme values (the highest and the lowest OD values) were discarded to decrease error, and the four OD595 values were calculated to obtain the average OD of each sample. Final OD for the biofilm measurement was calculated by subtracting the average OD of the negative control wells from the average OD of test sample wells. Three independent experiments were performed. 2.3. Determination of minimum inhibitory concentration The minimum inhibitory concentration (MIC) of EGCG was determined by the standard broth microdilution method as described previously (Du, Zhou, Liu, Chen, & Li, 2018). EGCG was diluted by 2-fold serial dilution with MH broth. Aliquots (50 µL) 5
Journal Pre-proof of diluted EGCG were mixed with an inoculum (50 µL of 106 CFU/mL) of Vibrio mimicus, and the control group was not inoculated with bacteria. After incubation at 37 ℃ for 24 h, the lowest concentration inhibiting bacterial growth was defined as the MIC. The MIC of EGCG against the Vibrio mimicus was determined in triplicate. 2.4. biofilm inhibition The effects of EGCG on biofilm formation by Vibrio mimicus were assessed by using crystal violet staining method. Briefly, Vibrio mimicus was cultured and diluted with LB broth as described above. This suspension (105 CFU/mL) was used as inoculum and placed into a 96-well polystyrene microtitre plate. Then EGCG solution was added to the wells to obtain final concentrations of 64 µg/mL, 128 µg/mL and 256 µg/mL. Bacterial cultures without addition of EGCG were used as positive controls. Broth only (microtitre wells containing uninoculated LB medium) was used as negative control. The plates were incubated at 15 ℃ for up to 11 days, 28 ℃ for 6 days and 37 ℃ for 5 days under static conditions. After incubation, the biofilm amount was measured as described above. Three independent experiments were performed with at least three replicates for each sample. 2.5. Swimming and swarming motility assays Swimming and swarming assays were performed as previously reported with some modifications (Inoue, Shingaki, & Fukui, 2008; Ondrusch & Kreft, 2011). For swimming assay, aliquots (3 mL) of LB agar supplemented with EGCG (final concentrations were 64 µg/mL, 128 µg/mL and 256 µg/mL) were added into 6-well culture plates. Vibrio mimicus was cultured overnight at 37 ℃ under shaking in LB broth. Then cells were point inoculated onto the the center of the agar with a sterile inoculating needle. The plates were then incubated at 28 ℃ for 24 h or 37 ℃ for 20 h. To observe swimming motility at 15 ℃, one-microliter aliquots of cultures were 6
Journal Pre-proof inoculated onto 6-well plates containing 3 mL LB (0.3% agar) and supplemented with increasing concentrations of EGCG. Then the plates were incubated at 15 ℃ for 72 h. The diameters of the swimming zones were measured after incubation. EGCG-free LB agar was used as positive control. Swarming experiments were performed on LB agar plates (0.5% agar) supplemented with different concentrations of EGCG (final concentrations were 64 µg/mL and 128 µg/mL). The bacteria cells were gently inoculated using a sterile inoculating needle at the center of the agar surface, and the plates were incubated at 28 ℃ for 24 h. Each experiment was carried out three times with at least three replicates for each sample. 2.6.Confocal laser scanning microscope ( CLSM) observation Biofilms were developed on an 8-well glass bottom chamber slide (LabTek II, Thermo Fisher Scientific, USA). Vibrio mimicus was grown overnight. Then cultures were determined at an OD600 of 1 and diluted 50 times with LB broth prior to inoculations (bacteria concentration was about 105 CFU/mL). Each chamber was inoculated with 200 µL of the culture and 200 µL of EGCG solution at final concentration of 64 µg/mL. The chamber slide was incubated statically at 35 °C for 72 h. After incubation, medium was removed from the well. The resident biofilm was washed gently with sterile PBS, and stained with 20 µg/mL FITC-conA in darkness for 30 min. Then biofilm was rinsed again and stained with 10 µg/mL PI in darkness for 30 min. Image was viewed with confocal laser scanning microscope (FV1000 IX81, Olympus, Japan). The excitation wavelengths were set to 488 nm and 543 nm for FTIC and PI, respectively. Three-dimensional projections of the biofilms were reconstructed from the CLSM acquisitions using Image J software. Image stack sections were composed of a series of images with 1-µm intervals in z-section from the substrate (disk) 7
Journal Pre-proof to the top of the biofilm. Quantitative structural parameters were analyzed by the computer program COMSTAT. Assays were repeated with at least three biological replicates. 2.7. Autoaggregation assay As EGCG interfered with the bacterial suspension color, aggregation ability of Vibrio mimicus was examined as previously reported with some modifications (Sorroche, Spesia, Zorreguieta, & Giordano, 2012). Vibrio mimicus was grown overnight at 37 ℃ and co-cultivated with different concentrations of EGCG at 37 ℃ for 2 h on a shaking incubator (150 rpm). Then the cells were then transferred into a centrifuge tube, harvested by centrifugation at 10000rpm for 10 min, washed twice and resuspended in PBS to the turbidity of the 0.5 McFarland standard. The bacterial suspensions were allowed to settle for 6 h at room temperature. A 4-mL aliquot of the upper portion of the suspension was transferred onto a turbidimetric cup to determine autoaggregation
with
a
turbidimeter
(MHY-2847,
BeiJing
MeiHuaYi
Technology Co., LTD., China). The autoaggregation coefficient (AC) was calculated as : A C (%) =（Ti-Tt）/ Ti × 100. where Ti is the initial turbidity of the microbial suspension and Tt is the final turbidity measured after 6 h of incubation. 2.8. Fluorescent probe assays N-phenyl-1-napthylamine (NPN) was used to measure the outer membrane permeability and Nile red as lipid stain for membrane. All fluorescence measurements were done on a spectrofluorometer (F96pro, ShangHai lengguang technology co. LTD.) as previously reported with some modifications (Sorroche, Spesia, Zorreguieta, & Giordano, 2012; Tang et al., 2010). The Vibrio mimicus cells in exponential phase 8
Journal Pre-proof （OD600= 0.4-0.6）were obtained and adjusted to get an OD600 of 1 for fluorescence probe assays. Bacterial suspension was first mixed with either NPN (100μM in acetone) or Nile red (1 mg/ml in methanol) to a final probe concentration of 20 μM for NPN and 10 μg/ml for Nile red. Next, 100 μL of the bacterial suspension was added to a Corning 96 well black plate containing 100 μL of EGCG solution. For negative controls, 100μL of PBS buffer was added to the 96 well plate instead of EGCG solution. Citric acid was proved to be a permeabilizer for E. coli as assayed by NPN uptake (Helande & Mattila-Sandholm, 2000). Therefore, E. coli ATCC 25922 was treated with citric acid and used as positive control. Fluorescence measurements were then taken, and the fluorescence intensity was measured at intervals. For NPN, excitation and emission wavelengths were set at 350 and 420nm, respectively. For Nile red, fluorescence was recorded at an excitation wavelength of 542nm and an emission wavelength of 618nm. 2.9. Potassium leakage The method of previous report was used for this assay (Thirumurugan, Rao, & Dhanaraju, 2016). The Vibrio mimicus cells in exponential phase were obtained and adjusted to get an OD600 of 1. Then PBFI probe (ShangHai MaoKang Bio technology Co., LTD., China) was added and incubated at 37 ℃ for 90 min. The cells were washed, collected and resuspended with PBS buffer. Aliquots (100 μL) of bacterial suspension were transferred to a Corning 96 well black plate, and 100 μL of various concentrations of EGCG were dispensed in the microtiter plate wells. The negative and positive controls were maintained with PBS-treated and 5 mmoL/L KCltreated cells, respectively. The assessment of the potassium ions present in medium was 9
Journal Pre-proof carried out using a fluorospectro photometer (F96pro, ShangHai lengguang technology co. LTD., China) at an excitation wavelength of 346 nm and an emission wavelength of 505 nm. Fluorescence intensity of both excitation and emission peaks was monitored over time. 2.10. Membrane damage The Vibrio mimicus cells in exponential phase were obtained and adjusted to get an OD600 of 1. Cells were then treated with EGCG, isopropanol or PBS at 37℃ for 2h under shaking. Cells were centrifuged once at 8000 rpm for 5 min, washed twice and resuspended in PBS. Then, 500 μL of each sample were mixed with 10 μL propidium iodide (PI; Sigma-Aldrich). The final concentration of PI was 10 μg/mL. Samples were incubated for 45 min in the dark at 37℃. PI uptake was measured by flow cytometry (CytoFLEX LX，Beckman Coulter, USA). PBS-treated cells were used as negative control and isopropanol-treated cells as positive control. For each sample, 10000 events were counted. 2.11. measurement of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) Vibrio mimicus was incubated to an OD600 of 1 in LB broth, harvested, centrifuged and then treated with PBS or EGCG at 37℃ for 2h under shaking. The cells were then used for the following assays. The PBS-treated bacterial cells were used as control. Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the intracellular levels of ROS in cells (Beyotime Institute of Biotechnology, China). Cells were collected, washed with PBS buffer, and incubated for 40 min in the same buffer containing DCFH-DA (final concentration was 1μM). Next, aliquots (100 μL) of the mixtures were transferred to a 96 well black plate and the relative fluorescent intensity (RFI) was measured (Excitation:488 nm; Emission:525 nm). Aliquots taken from each 10
Journal Pre-proof mixture were diluted and placed onto LB agar plates simultaneously. The intracellular ROS level was defined as the formula (Li et al., 2016): ROS level=Intracellular RFI/viable bacterial number. Hydrogen peroxide (H2O2) production was measured using the commercial kit (Beyotime Institute of Biotechnology, China). The Vibrio mimicus cells were cultured and treated with EGCG as described above. A standard curve was used to quantify the concentrations of hydrogen peroxide in the treated cells. The intracellular H2O2 level was defined as the formula: H2O2 level per cell = H2O2 concentration/viable bacterial number. For SOD activity assays, The Vibrio mimicus cells were cultured and treated with with PBS or EGCG at 37℃ for 2h. Bacteria were harvested, washed and resuspended in PBS buffer. Bacteria were then ultrasonically lysed , and cellular debris was removed by centrifugation. SOD activity and protein in the supernatant was determined with the total SOD assay kit with WST-1 (Nanjing Jiancheng Bioengineering Institute, China ) and BCA protein assay kit (TaKaRa Dalian Biotechnology, Dalian, China), respectively. One unit of SOD activity was defined as the amount of enzyme to inhibit 50 % of the reduction of WST-1 formazan. The relative SOD activity/μg protein was calculated according to the manufacturer’s instructions. Three independent experiments were performed. 2.12. Statistical analysis The results of triplicate experiments conducted for each of the above assays were analyzed with SPSS software (SPSS-Statistical Package for the Social Sciences, Inc., USA). Differences between the experimental group and the untreated control group were compared by t-test and one-way ANOVA. T-test was used to compare the difference between two groups. For a comparison of more than two groups, ANOVA was used. P values below 0.05 were considered to be statistically significant. P < 0.01 was considered highly significant. 11
Journal Pre-proof 3. Results and Discussion 3.1. Biofilm formation by Vibrio mimicus In the present study, biofilm formation was tested in various media to identify the effects of nutrients on biofilm formation. Biofilm formation was evaluated at 28 °C, as environmental temperature ranges from 20-28 °C in most cases. Biofilm formation can be divided into the following steps: attachment, growth, maturation and dispersal. In the present study, biofilm biomass reached the highest after incubation for several hours. Then biofilm formation decreased when incubation time prolonged (Fig.1). The results indicated that Vibrio mimicus is capable of producing biofilm in both nutrient-poor medium (eg. MH) and nutrient-rich medium (eg. BHI). Nutrient availability may not be the key factor that affects the biofilm formation of Vibrio mimicus. 3.2. Biofilm control In the present study, the minimum inhibitory concentration (MIC) of EGCG for Vibrio mimicus was determined to be 256 µg/mL. The biofilm forming ability of Vibrio mimicus was examined in the absence and presence of EGCG at three temperatures (15 °C, 28 °C and 37 °C), using the 96-well microtiter plate assay. As shown in figure 2A and 2C, at 15 °C and 37 °C, the biofilm levels were significantly reduced in the presence of EGCG, even at the concentration as low as 64 µg/mL (P < 0.01). At 28 °C, addition of EGCG caused the reduction of biofilm formation by Vibrio mimicus, and the degree of biofilm inhibition exhibited an obvious dose effect correlation (Fig. 2B). The effect of biofilm inhibition gradually enhanced with the increased concentration of EGCG. Food preservatives and disinfectants are widely used to control microorganisms and biofilms in the food processing plants. However, the effectiveness of these compounds is influenced by many factors including the characteristics of the tissue surface. Bacterial cells may find shelter where access by such chemicals is restricted and may 12
Journal Pre-proof expose to sub-MICs of antimicrobials. The stimulation of biofilm formation by subMICs of antimicrobials has been reported for multiple Gram positive and Gram negative species (Ranieri, Whitchurch, & Burrows, 2018). For example, benzalkonium chloride at sub-MICs can enhance the biofilm-forming ability of listeria monocytogenes strains (Rodriguez-Melcon, Capita, Rodríguez-Jerez, Martinez-Suarez, & Alonso-Calleja, 2019). Sub-MICs of colistin and polymyxin B can promote Acinetobacter baumannii biofilm formation (Sato, Unno, Ubagai, & Ono, 2018). These findings suggest the importance of avoiding sub-MICs of disinfectants or antimicrobials in food processing environments. The results of this study showed that sub-MICs of EGCG reduced the biofilm production of Vibrio mimicus, suggesting that EGCG may be applied as a good alternative antibiofilm and antibacterial agent. In addition, it was notable that in this study the absorbance values for biofilm formation in controls at 15 °C ranged from 0.10 to 0.99, in contrast to those at 28 °C (1.10 to 1.59) and at 37 °C (0.94 to 1.55). The results indicated that higher amounts of biofilms were formed at 28 °C and 37 °C compared with 15 °C (Fig. 2). Incubation temperature is a critical determinant for biofilm formation by Vibrio mimicus. 3.3. CLSM observation The results of this study indicated that sub-MICs of EGCG effectively inhibited the biofilm formation of Vibrio mimicus. These findings were also evidenced by visualization of biofilms by CLSM (Fig. 3). To allow the development of a stable biofilm on the glass surface, slides were incubated at 28 °C for 3 days. then data from controls and EGCG-treated samples are presented to measure the differences in the biofilm architecture. As shown in figure 3, a thick coating of biofilm was observed in control, whereas a visible reduction was observed in the biofilm of EGCG-treated sample (Fig. 3). The CLSM-captured images were then analyzed quantitatively using 13
Journal Pre-proof the COMSTAT software. Five features from the COMSTAT were calculated to identify the biofilm structures: bio-volume, mean thickness, surface to volume ratio, average diffusion distance and roughness coefficient. EGCG at the concentration of 64 µg/mL was found to be effective in reducing the biomass, average diffusion distance and mean thickness (Table 1) (P <0.01). However, addition of EGCG increased the surface to volume ratio and roughness coefficient of biofilm in control. Biofilm roughness is an indicator of biofilm heterogeneity. The results proved that sub-MIC EGCG influenced the total biomass of the biofilm, the morphology of the biofilm, and reduced the biofilm production. 3.4. Motility inhibition Swimming motility is reported to be flagellum-dependent (Brown & Häse, 2001). For swarming motility, some Vibrio species have been reported to form specialized elongated swarm cells (Brown & Häse, 2001). It is generally accepted that flagellummediated motility is critical for biofilm formation on abiotic surfaces, as it is essential to propel cells towards the surface before attachment (Lemon, Higgins, & Kolter, 2007). In the present study, the swarming motility of Vibrio mimicus was not altered regardless of EGCG addition (Fig. 4). However, the Swimming motility of Vibrio mimicus was decreased in the presence of sub-MICs (64 µg/mL and128 µg/mL) of EGCG (Fig. 4). At the selected three temperatures (15 °C, 28 °C and 37 °C), significant differences were observed between EGCG-treated sample and EGCG-free control (P < 0.01) (Fig. 5). As sub-MICs of EGCG was used in our experiments and the biofilm inhibition was not due to growth inhibition, the motility inhibition may play a part role in inhibiting biofilm formation. 3.5. Autoaggregation inhibition In this study, the effect of EGCG on autoaggregation by Vibrio mimicus was 14
Journal Pre-proof investigated (Fig. 6). EGCG decreased autoaggregation abilities of Vibrio mimicus cells and more cells remained in suspension, whereas untreated-control cells settled to the bottom of the tube and showed higher AC values (Fig. 6). Autoaggregation plays an important role in maintaining the stability of bacterial biofilm colonies. A positive correlation has been found between autoaggregation and biofilm formation abilities in Sinorhizobium meliloti (Sorroche, Spesia, Zorreguieta, & Giordano, 2012) and Myroides odoratus (Jacobs & Chenia, 2009). Kaplan et al. also found that methicillin induced biofilm formation and autoaggregation in Staphylococcus aureus (Kaplan et al., 2012). For Vibrio species, a connection between aggregation and biofilm formation ability is not clear and needs to be explored in the future. 3.6. Membrane permeability ,potassium leakage and membrane damage N-phenyl-1-napthylamine (NPN) is used as the fluorescent probe in outer membrane permeability studies because its fluorescence increases when it moves from a hydrophilic to hydrophobic location in the cells. Nile red is a hydrophobic probe, which localizes and stains lipids existing in the cells. Nile red is almost non-fluorescent in water, whereas its fluorescence intensity is greatly enhanced in lipid droplets or bilayers of cells. In this study, both NPN and Nile red's fluorescence intensities in E. coli rapidly increased after the addition of citric acid (Fig. 7 and Fig. 8). These results are consistent with the findings that citric acid acted as permeabilizer in E. coli cells (Helande & Mattila-Sandholm, 2000). Compared with PBS-treated cells, EGCG caused higher NPN fluorescence intensities in the cells (Fig. 7), indicating that EGCG increased outer membrane permeability of Vibrio mimicus. For Nile red staining, no significant differences were observed in the EGCG and PBS-treated cells (Fig. 8). Both of them showed high intensities at the starting time. Then stained cells began to lose their fluorescence when staining time prolonged. It may due to the limitation of Nile 15
Journal Pre-proof red
that
it
quickly
photo-bleaches
when
exposed
to
light
(Govender, Ramanna, Rawat, & Bux, 2012). Potassium leakage is an indication of increased membrane permeability and membrane damage in microorganisms. It was observed that cells treated with EGCG presented an increase in the potassium leakage in a dose-dependent manner (Fig. 9). With the concentration of EGCG increased, the efflux of potassium increased. The DNA-binding probe PI penetrates cells only when membranes are damaged. In this study, the changes in the integrity of the cytoplasmic membrane were assessed with PI by using flow cytometry. The result showed that Isopropanol killed-control cells showed the highest fluorescence intensity (Fig. 10). EGCG induced PI influx compared to the PBS-treated bacterial cells (Fig. 10). Taken together, the results obtained in the study showed that EGCG caused cell membrane damage and increased membrane permeability. In accordance with this, EGCG leads to the efflux of small molecules including potassium. Some antimicrobials exerted antibiofilm activity by increasing membrane permeability and causing membrane damage (Choi & Lee, 2012; Rosseti, Rocha, & Costa, 2015). We suspect that EGCG may impact the Vibrio mimicus biofilm by its membrane disrupting-action. 3.7. Endogenous oxidative stress In the present study, EGCG increased the intracellular ROS level and H2O2 concentration, and decreased SOD activity (Fig. 11). EGCG acted as prooxidant to inhibit bacteria as previously reported (Xiong et al., 2017). The function of oxidative stress in biofilm formation is controversial. Some authors found that some antimicrobials inhibited biofilm formation by promoting oxidative stress. For example, Diphenyl diselenide stimulated ROS production, increased cell membrane permeability and inhibited biofilm formation in Candida albicans (Rosseti, Rocha, & Costa, 2015). Some authors observed the opposite results. Geier et al. reported that autoinducer-2 16
Journal Pre-proof triggers the oxidative stress response in Mycobacterium avium, thus leading to biofilm formation (Geier, Mostowy, Cangelosi, Behr, & Ford, 2008). Bacterial cells in Biofilm in response to different oxidative stress levels may induce several different signaling pathways, including the switch between planktonic and sessile forms (Gambino & Cappitelli, 2016). Therefore, it is hard to conclude that EGCG generates ROS to reduce biofilm formation by Vibrio mimicus. 4. Conclusion Biofilms may enhance the ability of Vibrio species to survive in aquatic ecosystems, supporting infectivity in the host (Yildiz & Visick, 2009). Thus, the eradication and reduction of Vibrio species biofilm in the food processing environment is needed. the inhibitory effects of EGCG on Vibrio mimicus biofilm remained unknown until this work. As EGCG is a natural product, it may take part in the fight against Vibrio mimicus infections. Biofilm is a multifactorial phenomenon, understanding the mechanisms regulating biofilm development in response to antimicrobial exposure is challenging. This study may provide an understanding of the mechanism of action of EGCG against Vibrio biofilm.
Acknowledgments This work was funded by the National Key R&D Program of China (Grant No.2017YFC1600100) ； Partially supported by Chongqing performance guidance fund (17427). Conflict of interest: No conflict of interest declared References 17
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Journal Pre-proof Du, W., Zhou, M., Liu, Z., Chen, Y., & Li, R. (2018). Inhibition effects of low conce ntrations of epigallocatechin gallate on the biofilm formation and hemolytic activity
of
Listeria
monocytogenes.
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Control,
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Figure Captions Figure 1. The effects of different culture media on biofilm production of Vibrio mimicus at 28 ℃, as assessed by crystal violet staining and measuring absorbance at 595 nm. Mean values of three independent experiments and standard deviation are shown. Figure 2. The inhibitory effects of different concentrations of EGCG against biofilm formation by Vibrio mimicus as quantified by crystal violet staining. (A): Vibrio 23
Journal Pre-proof mimicus biofilm developed at 15 ℃. (B): Vibrio mimicus biofilm developed at 28 ℃. (C): Vibrio mimicus biofilm developed at 37 ℃. Mean values of triplicate independent experiments and standard deviation are shown. Figure 3. Laser confocal micrographs of Vibrio mimicus biofilm after incubation with 64 µg/mL EGCG at 28 ℃ for 72h. Representative reconstructed biofilm images from untreated-control (A) and EGCG-treated sample (B) are shown. Figure 4. Swimming motility of Vibrio mimicus grown at 28 ℃. Representative swim rings are shown in the upper panels (A-C). EGCG-free agar (A) and agars mixed with EGCG at final concentrations of 64 mg/mL (B) and 128 mg/mL (C) are demonstrated. Swarming motility of Vibrio mimicus grown at 28 ℃. Representative swarm zones are shown in the lower panels (D-F). EGCG-free agar (D) and agars mixed with EGCG at final concentrations of 64 mg/mL (E) and 128 mg/mL (F) are demonstrated. Figure 5. Efficacy of different concentrations of EGCG in reducing swimming motility of Vibrio mimicus. Mean values of triplicate independent experiments and standard deviation are shown.
** P < 0.01.
Figure 6. Efficacy of different concentrations of EGCG in reducing autoaggregation ability of Vibrio mimicus. Mean values of triplicate independent experiments and standard deviation are shown.
** P < 0.01. * P < 0.05.
Figure 7. Outer membrane permeability measured by NPN uptake. Vibrio mimicus cells were incubated NPN in the presence of various concentrations of EGCG or PBS. E. coli cells incubated with NPN and citric acid were used as positive control. Mean values of triplicate independent experiments and standard deviation are shown. Figure 8. The uptake of Nile red probe by bacteria over time. The fluorescence spectra of Nile red after the addition of various concentrations of EGCG or PBS to Vibrio 24
Journal Pre-proof mimicus cells was measured. E. coli cells incubated with Nile red and citric acid were used as positive control. Mean values of triplicate independent experiments and standard deviation are shown. Figure 9. Release of intracellular K+ ions in Vibrio mimicus as measured by PBFI uptake. The negative and positive controls were maintained with PBS-treated and 5 mmoL/L KCl-treated cells, respectively. Mean values of triplicate independent experiments and standard deviation are shown. Figure 10. Membrane damage of Vibrio mimicus, using PI through flow cytometry. Experiments were conducted in triplicates and representative results are shown. A total of 10,000 cells were acquired for each flow cytometry analysis. Histograms of Vibrio mimicus cells labeled with PI and treated with different concentrations of EGCG (B, C and D), PBS (A) and isopropanol (E) for 2h. Percentage of PI uptake by Vibrio mimicus as quantified from flow cytometry data is shown(F).These data represent mean (±SD) of three independent experiments (** P < 0.01). Figure 11. Intracellular ROS level (A), H2O2 production (B) and SOD activity (C) in Vibrio mimicus. Bacterial cells were treated with various concentrations of EGCG or PBS (control) for 2h. Mean values of triplicate independent experiments and standard deviation are shown.
25
** P < 0.01. * P < 0.05.
Journal Pre-proof Author statement Rui Li: Conseptualization, Methodology, Writing-Original draft preparation. Jieyuan Lu : Investigation, Formal analysis. Hebo Duan: Visualization. Jun Yang:Investigation. Changbo Tang: Funding acquisition, Writing-Reviewing and Editing.
Journal Pre-proof Conflict of interest: No conflict of interest declared
Journal Pre-proof Highlights: 1. Sub-MICs of EGCG inhibited Vibiro mimicus biofilm. 2. EGCG reduced autoaggregation and swimming motility. 3. EGCG increased ROS and membrane permeability. EGCG induced membrane damage and potassium leakage.
1
Journal Pre-proof Table1 Comparison of different variables for biofilm formation by Vibrio mimicus by using CLSM/COMSTAT analysis. Samples Biomass Thickness Maximum Roughness Surface to Average thickness coefficient biovolume diffusion ratio distance Control EGCGTreated Differential analysis
(µm)
(µm)
(µm)
(Ra*)
(µm-1)
(µm)
3.59±0.41 2.33±0.05
7.27±0.47 5.31±0.65
9.32±1.76 6.52±0.44
0.07±0.00 0.10±0.02
1.64±0.06 2.47±0.32
0.73±0.00 0.54±0.04
P <0.01
P <0.01
P <0.05
P >0.05
P <0.01
P <0.01
Rui Li, Jieyuan Lu, Hebo Duan, Jun Yang, Changbo Tang PII:
S0956-7135(20)30064-5
DOI:
https://doi.org/10.1016/j.foodcont.2020.107148
Reference:
JFCO 107148
To appear in:
Food Control
Received Date:
04 December 2019
Accepted Date:
28 January 2020
Please cite this article as: Rui Li, Jieyuan Lu, Hebo Duan, Jun Yang, Changbo Tang, Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus, Food Control (2020), https://doi.org/10.1016/j.foodcont.2020.107148
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Biofilm inhibition and mode of action of Epigallocatechin Gallate against Vibrio mimicus Rui Lia, Jieyuan Lu a, Hebo Duana,Jun Yanga, Changbo Tangb,⁎ a College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, 430023, China； b College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China. Correspondence
Changbo Tang, E-mail: [email protected]. Tel: 08602584399702. Mailing address: College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China.
This manuscript has been thoroughly edited by a native English speaker.
1
Journal Pre-proof ABSTRACT Vibrio mimicus is a relatively rare food-borne pathogen in seafood and water. Rare reports have been published to investigate the biofilm of Vibrio mimicus.
(-)-
epigallocatechin-3-gallate (EGCG), the major polyphenolic component of tea, can interfere with bacterial biofilm formation. This study showed that Vibrio mimicus was capable of forming high amounts of biofilms in various culture media. Sub-MICs of EGCG significantly reduced the biofilm production of Vibrio mimicus at 15 ℃, 28 ℃ and 37 ℃. Confocal laser-scanning microscope (CLSM) observation proved that the architecture of Vibrio mimicus biofilm was affected by EGCG at the concentration of 64 µg/mL (1/4 MIC). EGCG reduced Vibrio mimicus autoaggregation and swimming motility. EGCG also increased membrane permeability and ROS production, caused cell membrane damage and led to potassium leakage. These may contribute to the antibiofilm efficacy of EGCG against Vibrio mimicus. Our work showed the inhibitory effects of sub-MICs of EGCG on Vibrio mimicus biofilm for the first time, supporting the potential application of EGCG as a natural antibiofilm agent in the food industry.
Keywords: Vibrio mimicus; biofilm; EGCG; inhibition; sub-MICs
2
Journal Pre-proof 1. Introduction Vibrio mimicus is closely related to Vibrio cholerae and carries a number of potential virulence factors, including cholera-like enterotoxin, heat-stable enterotoxin, heat-labile enterotoxin, hemolysin, etc (Ali, Nelson, Lopez, & Sack, 2015). Vibrio mimicus is found in freshwater and seawater and can be potentially pathogenic for some animals, like mussel, fish, shrimp, and humans (Abd, Valeru, Sami, Saeed, & Sandström, 2010). Human infections with this organism are normally associated with ingestion of contaminated seafood and water. Reported cases are usually sporadic, but outbreaks have also occurred. For example, large food-borne outbreak caused by Vibrio mimicus involving 306 people occurred in Thailand in 2004. The probable cause of the outbreak was seafood soup (Chitov, Kirikaew, Yungyune, Ruengprapan, & Sontikun, 2009). In Shandong province Yiyuan county, China, 27 people were infected with Vibrio mimicus due to consumption of contaminated razor clam on 2011, 5th July. A cluster of severe diarrheal disease caused by Vibrio mimicus infection among four persons who had consumed crayfish occurred in Washington state, USA (Kay et al., 2012). Although Vibrio mimicus are not targets in food risk monitoring, isolation of the organism from clinical samples has been reported in many countries (Shinoda et al., 2004).
In natural environments, Vibrio species can attach to abiotic or biotic surfaces and form biofilms (Teschler et al., 2015). Biofilms are matrix-enclosed communities of microorganisms and the extracellular substances they produce (Flemming & Wingender, 2010). Bacteria in a biofilm are more resistant to sanitizers and antibiotics compared with planktonic bacteria. Vibrio cholerae has become a model organism in biofilm research, and the formation of its biofilm has been studied intensively (Yan, Sharo, Stone, Wingreen, & Bassler, 2016). However, unlike Vibrio cholerae, little 3
Journal Pre-proof information about Vibrio mimicus biofilm has been reported. Earlier work by TerceroAlburo et al. showed the biofilm formation of Vibrio mimicus at 37 °C in TSB broth by using scanning electron microscopy (Tercero-Alburo, Gonzßlez-Mßrquez, BonillaGonzßlez, Qui, & Vßzquez-Salinas, 2014). However, the inhibition of biofilm formation by Vibrio mimicus has not been studied yet. As pathogenic bacteria may thrive in biofilms, biofilm-associated infections pose a major health threat and there is a pressing need for antibiofilm agents (Li & Lee, 2017). Tea polyphenols are present in abundance in teas and have remarkable inhibitory effects against bacteria (Slobodníková, Fialová, Rendeková, Kováč, & Mučaji, 2016). Among tea polyphenols, (-)-epigallocatechin-3-gallate (EGCG), which is the major polyphenolic component of tea, exhibits antibiofilm activities (Bansal et al., 2013). But some reports indicated that EGCG seems inefficient against or may even sometimes promote biofilms which rely on types of biofilm matrix (Hengge, 2019). Special interests have been assigned to EGCG as an antibiofilm agent (Asahi et al., 2014; Serra, Mika, Richter, & Hengge, 2016), but so far the inhibitory effects of EGCG on Vibrio mimicus biofilm remain unknown. In the present study, we present data that reveal the following: (i) antibiofilm efficacy of EGCG against Vibrio mimicus; and (ii) the possible underlying mechanisms of action. 2. Materials and methods 2.1. Chemicals and bacteria strain EGCG monomer (99% purity) was purchased from Guang Run Biotechnology Co. Ltd (Nan Jing, China). An environmental Vibrio mimicus strain SNJ was isolated from fish and used in the study. 2.2. Biofilm development 4
Journal Pre-proof Vibrio mimicus was cultured overnight at 37 ℃ under shaking in LB broth. The bacteria cells were collected and adjusted to get an OD600 value of 1, then diluted 1:25 in saline solution. Aliquots (50 µL) of this dilution were inoculated into six wells of a flat-bottomed, 96-well polystyrene tissue culture plate (Corning Costar, USA). The outermost wells were not used for the assay, because these wells suffer edge effects. Various media (TSB, LB, NB, BHI, MH and 1/2LB) were prepared to evaluate nutrient availability. To prepare 1/2LB medium, LB broth was diluted 2-fold with distilled water. Aliquots (150 µL) of each medium were dispensed in the microtiter plate wells and mixed with portions of diluted bacterial suspension to obtain final concentration of 105 CFU/mL. Each medium without bacterial cells was placed in six wells of each plate as negative controls. The plates were incubated at 28 ℃ under static conditions. At each time point, the medium was removed from each well and the plates were rinsed with sterile phosphate-buffered saline (PBS, pH 7.4). The plates were air dried and stained with 0.1 % crystal violet for 15min. Then the wells were rinsed again and destained with 95% ethanol. The amount of biofilm was measured as the absorbance at 595 nm using a microplate reader (Tecan, Austria). Two extreme values (the highest and the lowest OD values) were discarded to decrease error, and the four OD595 values were calculated to obtain the average OD of each sample. Final OD for the biofilm measurement was calculated by subtracting the average OD of the negative control wells from the average OD of test sample wells. Three independent experiments were performed. 2.3. Determination of minimum inhibitory concentration The minimum inhibitory concentration (MIC) of EGCG was determined by the standard broth microdilution method as described previously (Du, Zhou, Liu, Chen, & Li, 2018). EGCG was diluted by 2-fold serial dilution with MH broth. Aliquots (50 µL) 5
Journal Pre-proof of diluted EGCG were mixed with an inoculum (50 µL of 106 CFU/mL) of Vibrio mimicus, and the control group was not inoculated with bacteria. After incubation at 37 ℃ for 24 h, the lowest concentration inhibiting bacterial growth was defined as the MIC. The MIC of EGCG against the Vibrio mimicus was determined in triplicate. 2.4. biofilm inhibition The effects of EGCG on biofilm formation by Vibrio mimicus were assessed by using crystal violet staining method. Briefly, Vibrio mimicus was cultured and diluted with LB broth as described above. This suspension (105 CFU/mL) was used as inoculum and placed into a 96-well polystyrene microtitre plate. Then EGCG solution was added to the wells to obtain final concentrations of 64 µg/mL, 128 µg/mL and 256 µg/mL. Bacterial cultures without addition of EGCG were used as positive controls. Broth only (microtitre wells containing uninoculated LB medium) was used as negative control. The plates were incubated at 15 ℃ for up to 11 days, 28 ℃ for 6 days and 37 ℃ for 5 days under static conditions. After incubation, the biofilm amount was measured as described above. Three independent experiments were performed with at least three replicates for each sample. 2.5. Swimming and swarming motility assays Swimming and swarming assays were performed as previously reported with some modifications (Inoue, Shingaki, & Fukui, 2008; Ondrusch & Kreft, 2011). For swimming assay, aliquots (3 mL) of LB agar supplemented with EGCG (final concentrations were 64 µg/mL, 128 µg/mL and 256 µg/mL) were added into 6-well culture plates. Vibrio mimicus was cultured overnight at 37 ℃ under shaking in LB broth. Then cells were point inoculated onto the the center of the agar with a sterile inoculating needle. The plates were then incubated at 28 ℃ for 24 h or 37 ℃ for 20 h. To observe swimming motility at 15 ℃, one-microliter aliquots of cultures were 6
Journal Pre-proof inoculated onto 6-well plates containing 3 mL LB (0.3% agar) and supplemented with increasing concentrations of EGCG. Then the plates were incubated at 15 ℃ for 72 h. The diameters of the swimming zones were measured after incubation. EGCG-free LB agar was used as positive control. Swarming experiments were performed on LB agar plates (0.5% agar) supplemented with different concentrations of EGCG (final concentrations were 64 µg/mL and 128 µg/mL). The bacteria cells were gently inoculated using a sterile inoculating needle at the center of the agar surface, and the plates were incubated at 28 ℃ for 24 h. Each experiment was carried out three times with at least three replicates for each sample. 2.6.Confocal laser scanning microscope ( CLSM) observation Biofilms were developed on an 8-well glass bottom chamber slide (LabTek II, Thermo Fisher Scientific, USA). Vibrio mimicus was grown overnight. Then cultures were determined at an OD600 of 1 and diluted 50 times with LB broth prior to inoculations (bacteria concentration was about 105 CFU/mL). Each chamber was inoculated with 200 µL of the culture and 200 µL of EGCG solution at final concentration of 64 µg/mL. The chamber slide was incubated statically at 35 °C for 72 h. After incubation, medium was removed from the well. The resident biofilm was washed gently with sterile PBS, and stained with 20 µg/mL FITC-conA in darkness for 30 min. Then biofilm was rinsed again and stained with 10 µg/mL PI in darkness for 30 min. Image was viewed with confocal laser scanning microscope (FV1000 IX81, Olympus, Japan). The excitation wavelengths were set to 488 nm and 543 nm for FTIC and PI, respectively. Three-dimensional projections of the biofilms were reconstructed from the CLSM acquisitions using Image J software. Image stack sections were composed of a series of images with 1-µm intervals in z-section from the substrate (disk) 7
Journal Pre-proof to the top of the biofilm. Quantitative structural parameters were analyzed by the computer program COMSTAT. Assays were repeated with at least three biological replicates. 2.7. Autoaggregation assay As EGCG interfered with the bacterial suspension color, aggregation ability of Vibrio mimicus was examined as previously reported with some modifications (Sorroche, Spesia, Zorreguieta, & Giordano, 2012). Vibrio mimicus was grown overnight at 37 ℃ and co-cultivated with different concentrations of EGCG at 37 ℃ for 2 h on a shaking incubator (150 rpm). Then the cells were then transferred into a centrifuge tube, harvested by centrifugation at 10000rpm for 10 min, washed twice and resuspended in PBS to the turbidity of the 0.5 McFarland standard. The bacterial suspensions were allowed to settle for 6 h at room temperature. A 4-mL aliquot of the upper portion of the suspension was transferred onto a turbidimetric cup to determine autoaggregation
with
a
turbidimeter
(MHY-2847,
BeiJing
MeiHuaYi
Technology Co., LTD., China). The autoaggregation coefficient (AC) was calculated as : A C (%) =（Ti-Tt）/ Ti × 100. where Ti is the initial turbidity of the microbial suspension and Tt is the final turbidity measured after 6 h of incubation. 2.8. Fluorescent probe assays N-phenyl-1-napthylamine (NPN) was used to measure the outer membrane permeability and Nile red as lipid stain for membrane. All fluorescence measurements were done on a spectrofluorometer (F96pro, ShangHai lengguang technology co. LTD.) as previously reported with some modifications (Sorroche, Spesia, Zorreguieta, & Giordano, 2012; Tang et al., 2010). The Vibrio mimicus cells in exponential phase 8
Journal Pre-proof （OD600= 0.4-0.6）were obtained and adjusted to get an OD600 of 1 for fluorescence probe assays. Bacterial suspension was first mixed with either NPN (100μM in acetone) or Nile red (1 mg/ml in methanol) to a final probe concentration of 20 μM for NPN and 10 μg/ml for Nile red. Next, 100 μL of the bacterial suspension was added to a Corning 96 well black plate containing 100 μL of EGCG solution. For negative controls, 100μL of PBS buffer was added to the 96 well plate instead of EGCG solution. Citric acid was proved to be a permeabilizer for E. coli as assayed by NPN uptake (Helande & Mattila-Sandholm, 2000). Therefore, E. coli ATCC 25922 was treated with citric acid and used as positive control. Fluorescence measurements were then taken, and the fluorescence intensity was measured at intervals. For NPN, excitation and emission wavelengths were set at 350 and 420nm, respectively. For Nile red, fluorescence was recorded at an excitation wavelength of 542nm and an emission wavelength of 618nm. 2.9. Potassium leakage The method of previous report was used for this assay (Thirumurugan, Rao, & Dhanaraju, 2016). The Vibrio mimicus cells in exponential phase were obtained and adjusted to get an OD600 of 1. Then PBFI probe (ShangHai MaoKang Bio technology Co., LTD., China) was added and incubated at 37 ℃ for 90 min. The cells were washed, collected and resuspended with PBS buffer. Aliquots (100 μL) of bacterial suspension were transferred to a Corning 96 well black plate, and 100 μL of various concentrations of EGCG were dispensed in the microtiter plate wells. The negative and positive controls were maintained with PBS-treated and 5 mmoL/L KCltreated cells, respectively. The assessment of the potassium ions present in medium was 9
Journal Pre-proof carried out using a fluorospectro photometer (F96pro, ShangHai lengguang technology co. LTD., China) at an excitation wavelength of 346 nm and an emission wavelength of 505 nm. Fluorescence intensity of both excitation and emission peaks was monitored over time. 2.10. Membrane damage The Vibrio mimicus cells in exponential phase were obtained and adjusted to get an OD600 of 1. Cells were then treated with EGCG, isopropanol or PBS at 37℃ for 2h under shaking. Cells were centrifuged once at 8000 rpm for 5 min, washed twice and resuspended in PBS. Then, 500 μL of each sample were mixed with 10 μL propidium iodide (PI; Sigma-Aldrich). The final concentration of PI was 10 μg/mL. Samples were incubated for 45 min in the dark at 37℃. PI uptake was measured by flow cytometry (CytoFLEX LX，Beckman Coulter, USA). PBS-treated cells were used as negative control and isopropanol-treated cells as positive control. For each sample, 10000 events were counted. 2.11. measurement of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) Vibrio mimicus was incubated to an OD600 of 1 in LB broth, harvested, centrifuged and then treated with PBS or EGCG at 37℃ for 2h under shaking. The cells were then used for the following assays. The PBS-treated bacterial cells were used as control. Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the intracellular levels of ROS in cells (Beyotime Institute of Biotechnology, China). Cells were collected, washed with PBS buffer, and incubated for 40 min in the same buffer containing DCFH-DA (final concentration was 1μM). Next, aliquots (100 μL) of the mixtures were transferred to a 96 well black plate and the relative fluorescent intensity (RFI) was measured (Excitation:488 nm; Emission:525 nm). Aliquots taken from each 10
Journal Pre-proof mixture were diluted and placed onto LB agar plates simultaneously. The intracellular ROS level was defined as the formula (Li et al., 2016): ROS level=Intracellular RFI/viable bacterial number. Hydrogen peroxide (H2O2) production was measured using the commercial kit (Beyotime Institute of Biotechnology, China). The Vibrio mimicus cells were cultured and treated with EGCG as described above. A standard curve was used to quantify the concentrations of hydrogen peroxide in the treated cells. The intracellular H2O2 level was defined as the formula: H2O2 level per cell = H2O2 concentration/viable bacterial number. For SOD activity assays, The Vibrio mimicus cells were cultured and treated with with PBS or EGCG at 37℃ for 2h. Bacteria were harvested, washed and resuspended in PBS buffer. Bacteria were then ultrasonically lysed , and cellular debris was removed by centrifugation. SOD activity and protein in the supernatant was determined with the total SOD assay kit with WST-1 (Nanjing Jiancheng Bioengineering Institute, China ) and BCA protein assay kit (TaKaRa Dalian Biotechnology, Dalian, China), respectively. One unit of SOD activity was defined as the amount of enzyme to inhibit 50 % of the reduction of WST-1 formazan. The relative SOD activity/μg protein was calculated according to the manufacturer’s instructions. Three independent experiments were performed. 2.12. Statistical analysis The results of triplicate experiments conducted for each of the above assays were analyzed with SPSS software (SPSS-Statistical Package for the Social Sciences, Inc., USA). Differences between the experimental group and the untreated control group were compared by t-test and one-way ANOVA. T-test was used to compare the difference between two groups. For a comparison of more than two groups, ANOVA was used. P values below 0.05 were considered to be statistically significant. P < 0.01 was considered highly significant. 11
Journal Pre-proof 3. Results and Discussion 3.1. Biofilm formation by Vibrio mimicus In the present study, biofilm formation was tested in various media to identify the effects of nutrients on biofilm formation. Biofilm formation was evaluated at 28 °C, as environmental temperature ranges from 20-28 °C in most cases. Biofilm formation can be divided into the following steps: attachment, growth, maturation and dispersal. In the present study, biofilm biomass reached the highest after incubation for several hours. Then biofilm formation decreased when incubation time prolonged (Fig.1). The results indicated that Vibrio mimicus is capable of producing biofilm in both nutrient-poor medium (eg. MH) and nutrient-rich medium (eg. BHI). Nutrient availability may not be the key factor that affects the biofilm formation of Vibrio mimicus. 3.2. Biofilm control In the present study, the minimum inhibitory concentration (MIC) of EGCG for Vibrio mimicus was determined to be 256 µg/mL. The biofilm forming ability of Vibrio mimicus was examined in the absence and presence of EGCG at three temperatures (15 °C, 28 °C and 37 °C), using the 96-well microtiter plate assay. As shown in figure 2A and 2C, at 15 °C and 37 °C, the biofilm levels were significantly reduced in the presence of EGCG, even at the concentration as low as 64 µg/mL (P < 0.01). At 28 °C, addition of EGCG caused the reduction of biofilm formation by Vibrio mimicus, and the degree of biofilm inhibition exhibited an obvious dose effect correlation (Fig. 2B). The effect of biofilm inhibition gradually enhanced with the increased concentration of EGCG. Food preservatives and disinfectants are widely used to control microorganisms and biofilms in the food processing plants. However, the effectiveness of these compounds is influenced by many factors including the characteristics of the tissue surface. Bacterial cells may find shelter where access by such chemicals is restricted and may 12
Journal Pre-proof expose to sub-MICs of antimicrobials. The stimulation of biofilm formation by subMICs of antimicrobials has been reported for multiple Gram positive and Gram negative species (Ranieri, Whitchurch, & Burrows, 2018). For example, benzalkonium chloride at sub-MICs can enhance the biofilm-forming ability of listeria monocytogenes strains (Rodriguez-Melcon, Capita, Rodríguez-Jerez, Martinez-Suarez, & Alonso-Calleja, 2019). Sub-MICs of colistin and polymyxin B can promote Acinetobacter baumannii biofilm formation (Sato, Unno, Ubagai, & Ono, 2018). These findings suggest the importance of avoiding sub-MICs of disinfectants or antimicrobials in food processing environments. The results of this study showed that sub-MICs of EGCG reduced the biofilm production of Vibrio mimicus, suggesting that EGCG may be applied as a good alternative antibiofilm and antibacterial agent. In addition, it was notable that in this study the absorbance values for biofilm formation in controls at 15 °C ranged from 0.10 to 0.99, in contrast to those at 28 °C (1.10 to 1.59) and at 37 °C (0.94 to 1.55). The results indicated that higher amounts of biofilms were formed at 28 °C and 37 °C compared with 15 °C (Fig. 2). Incubation temperature is a critical determinant for biofilm formation by Vibrio mimicus. 3.3. CLSM observation The results of this study indicated that sub-MICs of EGCG effectively inhibited the biofilm formation of Vibrio mimicus. These findings were also evidenced by visualization of biofilms by CLSM (Fig. 3). To allow the development of a stable biofilm on the glass surface, slides were incubated at 28 °C for 3 days. then data from controls and EGCG-treated samples are presented to measure the differences in the biofilm architecture. As shown in figure 3, a thick coating of biofilm was observed in control, whereas a visible reduction was observed in the biofilm of EGCG-treated sample (Fig. 3). The CLSM-captured images were then analyzed quantitatively using 13
Journal Pre-proof the COMSTAT software. Five features from the COMSTAT were calculated to identify the biofilm structures: bio-volume, mean thickness, surface to volume ratio, average diffusion distance and roughness coefficient. EGCG at the concentration of 64 µg/mL was found to be effective in reducing the biomass, average diffusion distance and mean thickness (Table 1) (P <0.01). However, addition of EGCG increased the surface to volume ratio and roughness coefficient of biofilm in control. Biofilm roughness is an indicator of biofilm heterogeneity. The results proved that sub-MIC EGCG influenced the total biomass of the biofilm, the morphology of the biofilm, and reduced the biofilm production. 3.4. Motility inhibition Swimming motility is reported to be flagellum-dependent (Brown & Häse, 2001). For swarming motility, some Vibrio species have been reported to form specialized elongated swarm cells (Brown & Häse, 2001). It is generally accepted that flagellummediated motility is critical for biofilm formation on abiotic surfaces, as it is essential to propel cells towards the surface before attachment (Lemon, Higgins, & Kolter, 2007). In the present study, the swarming motility of Vibrio mimicus was not altered regardless of EGCG addition (Fig. 4). However, the Swimming motility of Vibrio mimicus was decreased in the presence of sub-MICs (64 µg/mL and128 µg/mL) of EGCG (Fig. 4). At the selected three temperatures (15 °C, 28 °C and 37 °C), significant differences were observed between EGCG-treated sample and EGCG-free control (P < 0.01) (Fig. 5). As sub-MICs of EGCG was used in our experiments and the biofilm inhibition was not due to growth inhibition, the motility inhibition may play a part role in inhibiting biofilm formation. 3.5. Autoaggregation inhibition In this study, the effect of EGCG on autoaggregation by Vibrio mimicus was 14
Journal Pre-proof investigated (Fig. 6). EGCG decreased autoaggregation abilities of Vibrio mimicus cells and more cells remained in suspension, whereas untreated-control cells settled to the bottom of the tube and showed higher AC values (Fig. 6). Autoaggregation plays an important role in maintaining the stability of bacterial biofilm colonies. A positive correlation has been found between autoaggregation and biofilm formation abilities in Sinorhizobium meliloti (Sorroche, Spesia, Zorreguieta, & Giordano, 2012) and Myroides odoratus (Jacobs & Chenia, 2009). Kaplan et al. also found that methicillin induced biofilm formation and autoaggregation in Staphylococcus aureus (Kaplan et al., 2012). For Vibrio species, a connection between aggregation and biofilm formation ability is not clear and needs to be explored in the future. 3.6. Membrane permeability ,potassium leakage and membrane damage N-phenyl-1-napthylamine (NPN) is used as the fluorescent probe in outer membrane permeability studies because its fluorescence increases when it moves from a hydrophilic to hydrophobic location in the cells. Nile red is a hydrophobic probe, which localizes and stains lipids existing in the cells. Nile red is almost non-fluorescent in water, whereas its fluorescence intensity is greatly enhanced in lipid droplets or bilayers of cells. In this study, both NPN and Nile red's fluorescence intensities in E. coli rapidly increased after the addition of citric acid (Fig. 7 and Fig. 8). These results are consistent with the findings that citric acid acted as permeabilizer in E. coli cells (Helande & Mattila-Sandholm, 2000). Compared with PBS-treated cells, EGCG caused higher NPN fluorescence intensities in the cells (Fig. 7), indicating that EGCG increased outer membrane permeability of Vibrio mimicus. For Nile red staining, no significant differences were observed in the EGCG and PBS-treated cells (Fig. 8). Both of them showed high intensities at the starting time. Then stained cells began to lose their fluorescence when staining time prolonged. It may due to the limitation of Nile 15
Journal Pre-proof red
that
it
quickly
photo-bleaches
when
exposed
to
light
(Govender, Ramanna, Rawat, & Bux, 2012). Potassium leakage is an indication of increased membrane permeability and membrane damage in microorganisms. It was observed that cells treated with EGCG presented an increase in the potassium leakage in a dose-dependent manner (Fig. 9). With the concentration of EGCG increased, the efflux of potassium increased. The DNA-binding probe PI penetrates cells only when membranes are damaged. In this study, the changes in the integrity of the cytoplasmic membrane were assessed with PI by using flow cytometry. The result showed that Isopropanol killed-control cells showed the highest fluorescence intensity (Fig. 10). EGCG induced PI influx compared to the PBS-treated bacterial cells (Fig. 10). Taken together, the results obtained in the study showed that EGCG caused cell membrane damage and increased membrane permeability. In accordance with this, EGCG leads to the efflux of small molecules including potassium. Some antimicrobials exerted antibiofilm activity by increasing membrane permeability and causing membrane damage (Choi & Lee, 2012; Rosseti, Rocha, & Costa, 2015). We suspect that EGCG may impact the Vibrio mimicus biofilm by its membrane disrupting-action. 3.7. Endogenous oxidative stress In the present study, EGCG increased the intracellular ROS level and H2O2 concentration, and decreased SOD activity (Fig. 11). EGCG acted as prooxidant to inhibit bacteria as previously reported (Xiong et al., 2017). The function of oxidative stress in biofilm formation is controversial. Some authors found that some antimicrobials inhibited biofilm formation by promoting oxidative stress. For example, Diphenyl diselenide stimulated ROS production, increased cell membrane permeability and inhibited biofilm formation in Candida albicans (Rosseti, Rocha, & Costa, 2015). Some authors observed the opposite results. Geier et al. reported that autoinducer-2 16
Journal Pre-proof triggers the oxidative stress response in Mycobacterium avium, thus leading to biofilm formation (Geier, Mostowy, Cangelosi, Behr, & Ford, 2008). Bacterial cells in Biofilm in response to different oxidative stress levels may induce several different signaling pathways, including the switch between planktonic and sessile forms (Gambino & Cappitelli, 2016). Therefore, it is hard to conclude that EGCG generates ROS to reduce biofilm formation by Vibrio mimicus. 4. Conclusion Biofilms may enhance the ability of Vibrio species to survive in aquatic ecosystems, supporting infectivity in the host (Yildiz & Visick, 2009). Thus, the eradication and reduction of Vibrio species biofilm in the food processing environment is needed. the inhibitory effects of EGCG on Vibrio mimicus biofilm remained unknown until this work. As EGCG is a natural product, it may take part in the fight against Vibrio mimicus infections. Biofilm is a multifactorial phenomenon, understanding the mechanisms regulating biofilm development in response to antimicrobial exposure is challenging. This study may provide an understanding of the mechanism of action of EGCG against Vibrio biofilm.
Acknowledgments This work was funded by the National Key R&D Program of China (Grant No.2017YFC1600100) ； Partially supported by Chongqing performance guidance fund (17427). Conflict of interest: No conflict of interest declared References 17
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Figure Captions Figure 1. The effects of different culture media on biofilm production of Vibrio mimicus at 28 ℃, as assessed by crystal violet staining and measuring absorbance at 595 nm. Mean values of three independent experiments and standard deviation are shown. Figure 2. The inhibitory effects of different concentrations of EGCG against biofilm formation by Vibrio mimicus as quantified by crystal violet staining. (A): Vibrio 23
Journal Pre-proof mimicus biofilm developed at 15 ℃. (B): Vibrio mimicus biofilm developed at 28 ℃. (C): Vibrio mimicus biofilm developed at 37 ℃. Mean values of triplicate independent experiments and standard deviation are shown. Figure 3. Laser confocal micrographs of Vibrio mimicus biofilm after incubation with 64 µg/mL EGCG at 28 ℃ for 72h. Representative reconstructed biofilm images from untreated-control (A) and EGCG-treated sample (B) are shown. Figure 4. Swimming motility of Vibrio mimicus grown at 28 ℃. Representative swim rings are shown in the upper panels (A-C). EGCG-free agar (A) and agars mixed with EGCG at final concentrations of 64 mg/mL (B) and 128 mg/mL (C) are demonstrated. Swarming motility of Vibrio mimicus grown at 28 ℃. Representative swarm zones are shown in the lower panels (D-F). EGCG-free agar (D) and agars mixed with EGCG at final concentrations of 64 mg/mL (E) and 128 mg/mL (F) are demonstrated. Figure 5. Efficacy of different concentrations of EGCG in reducing swimming motility of Vibrio mimicus. Mean values of triplicate independent experiments and standard deviation are shown.
** P < 0.01.
Figure 6. Efficacy of different concentrations of EGCG in reducing autoaggregation ability of Vibrio mimicus. Mean values of triplicate independent experiments and standard deviation are shown.
** P < 0.01. * P < 0.05.
Figure 7. Outer membrane permeability measured by NPN uptake. Vibrio mimicus cells were incubated NPN in the presence of various concentrations of EGCG or PBS. E. coli cells incubated with NPN and citric acid were used as positive control. Mean values of triplicate independent experiments and standard deviation are shown. Figure 8. The uptake of Nile red probe by bacteria over time. The fluorescence spectra of Nile red after the addition of various concentrations of EGCG or PBS to Vibrio 24
Journal Pre-proof mimicus cells was measured. E. coli cells incubated with Nile red and citric acid were used as positive control. Mean values of triplicate independent experiments and standard deviation are shown. Figure 9. Release of intracellular K+ ions in Vibrio mimicus as measured by PBFI uptake. The negative and positive controls were maintained with PBS-treated and 5 mmoL/L KCl-treated cells, respectively. Mean values of triplicate independent experiments and standard deviation are shown. Figure 10. Membrane damage of Vibrio mimicus, using PI through flow cytometry. Experiments were conducted in triplicates and representative results are shown. A total of 10,000 cells were acquired for each flow cytometry analysis. Histograms of Vibrio mimicus cells labeled with PI and treated with different concentrations of EGCG (B, C and D), PBS (A) and isopropanol (E) for 2h. Percentage of PI uptake by Vibrio mimicus as quantified from flow cytometry data is shown(F).These data represent mean (±SD) of three independent experiments (** P < 0.01). Figure 11. Intracellular ROS level (A), H2O2 production (B) and SOD activity (C) in Vibrio mimicus. Bacterial cells were treated with various concentrations of EGCG or PBS (control) for 2h. Mean values of triplicate independent experiments and standard deviation are shown.
25
** P < 0.01. * P < 0.05.
Journal Pre-proof Author statement Rui Li: Conseptualization, Methodology, Writing-Original draft preparation. Jieyuan Lu : Investigation, Formal analysis. Hebo Duan: Visualization. Jun Yang:Investigation. Changbo Tang: Funding acquisition, Writing-Reviewing and Editing.
Journal Pre-proof Conflict of interest: No conflict of interest declared
Journal Pre-proof Highlights: 1. Sub-MICs of EGCG inhibited Vibiro mimicus biofilm. 2. EGCG reduced autoaggregation and swimming motility. 3. EGCG increased ROS and membrane permeability. EGCG induced membrane damage and potassium leakage.
1
Journal Pre-proof Table1 Comparison of different variables for biofilm formation by Vibrio mimicus by using CLSM/COMSTAT analysis. Samples Biomass Thickness Maximum Roughness Surface to Average thickness coefficient biovolume diffusion ratio distance Control EGCGTreated Differential analysis
(µm)
(µm)
(µm)
(Ra*)
(µm-1)
(µm)
3.59±0.41 2.33±0.05
7.27±0.47 5.31±0.65
9.32±1.76 6.52±0.44
0.07±0.00 0.10±0.02
1.64±0.06 2.47±0.32
0.73±0.00 0.54±0.04
P <0.01
P <0.01
P <0.05
P >0.05
P <0.01
P <0.01