Tea polyphenols can be developed into new types of disinfectants for drinking water. The antibacterial effect of epigallocatechin gallate (EGCG) on Escherichia coli (E. coli) in the presence of Ca2+ is affected by the Ca2+ concentration. The oxidative damage mechanism and oxidative damage process of EGCG in E. coli under the presence of Ca2+ were deeply analyzed under three aspects: reactive oxygen species (ROS), antioxidant system, and oxidative stress response in E. coli to provide a theoretical basis for the use of EGCG as a disinfectant in drinking water disinfection. EGCG leads to excessive production of superoxide anion in E. coli and the presence of Ca2+ promotes further imbalance of superoxide anion in E. coli; Ca2+ has little effect on EGCG hindering the scavenging of hydroxyl radicals in bacteria; EGCG can hinder the effect of antioxidant enzymes in E. coli, and Ca2+ has a particular regulatory effect on antioxidant enzymes, thus hindering the oxidative damage of EGCG to E. coli; Ca2+ can cause the expression of the oxyR and DPS genes, protect bacterial DNA, and prevent EGCG from damaging bacterial DNA. In the presence of a high concentration of Ca2+, it may activate the cell efflux pump through the soxS gene, resulting in E. coli resistance to EGCG.
EGCG has potential for disinfection of drinking water.
The antibacterial effect of EGCG on E. coli in the presence of Ca2+ is affected by the Ca2+ concentration.
EGCG leads to excessive production of superoxide anion in E. coli.
Ca2+ has an effect on EGCG hindering the scavenging of hydroxyl radicals in bacteria.
Ca2+ may activate the cell efflux pump through the soxS gene, resulting in E. coli resistance to EGCG.
Polyphenols are a class of natural biologically active polyphenols present in tea (Xiong et al. 2019). Catechins are the main polyphenolic compounds and include eight different monomers, namely catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (CG), epicatechin gallate (ECG), gallocatechin gallate (GCG), and epigallocatechin gallate (EGCG) (Li et al. 2020). Tea polyphenols have antioxidant, antibacterial, anti-cancer, anti-viral, anti-radiation, and other biological activities (Xiaolin et al. 2019) and they have been proven to have great disinfectant effects in water disinfection processes (Xuanqi et al. 2019). Among all tea polyphenols, EGCG is the main component with an antibacterial effect.
Drinking water contains numerous beneficial metal elements. Specifically, calcium concentrations vary between 100 and 200 mg/L (Licai et al. 2012; Wei 2014). Calcium is an essential second messenger in regulating cell function, as well as affecting cell activity and metabolism (Zhang & Zhang 2019). Notably, changes in the concentration of extracellular Ca2+ affect cell growth and proliferation of bacteria and can even interfere with the antibacterial effects of bacteriostatic agents.
Previous studies have shown that the Ca2+ concentration can impact the bacteriostatic effect of EGCG. Macroscopically, in the presence of a relatively low concentration of Ca2+ (1–5 mM), the bacteriostatic effect of EGCG is weakened, while in the relatively high concentrations of Ca2+ (5–10 mM), EGCG inhibits bacterial growth by accessing the bacterial body and exerting a bacteriostatic effect (Xu et al. 2021).
In this manuscript, we focus on the interrelationship between Ca2+ and the damage generated by reactive oxygen species (ROS), antioxidant system, and the oxidative stress response within Escherichia coli (E. coli) to then rationalize the destructive effect enhanced by EGCG in the bacterial cell milieu.
MATERIALS AND METHODS
E. coli cells were obtained from the China Industrial Microorganism Culture Collection and Management Center, in the form of a freeze-dried powder with low activity. Resuscitation was performed by culturing in the fresh bacterial suspension. The bacterial count was approximately 108 CFU/mL. The contents of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in E. coli were determined using, respectively, the SOD, the CAT, and the microbially reduced GSH ELISA kits (96 T) purchased from Shanghai Enzyme Link Biotechnology Co., Ltd.
Activation, culture, and preservation of E. coli
After dissolving the lyophilized E. coli vial in a small amount of sterile water, the cells were inoculated in 10 mL of sterilized nutrient broth medium, sealed, and placed in a constant temperature shaker to be cultured overnight at 37 °C and 220 rpm, obtaining the first-generation bacterial culture. Second- and third-generation bacterial cultures were prepared by successive re-inoculation in 10 mL of fresh medium and overnight culture in the same experimental conditions. Eight 10-mL test tubes tilt-filled with fresh sterile agar medium were inoculated with the third-generation bacterial culture with a sterile loop. The tubes were sealed with a rubber stopper and cultured at 37 °C constant temperature in an incubator until the colony was visible, and stored at 4 °C. Throughout the study, the solid cultures were used to prepare E. coli suspension cultures by inoculating scraps of the colony into fresh medium and culturing in the same experimental conditions mentioned above, to an estimated bacterial count of 108 CFU/mL.
Detection of antibacterial activity
The bacteriostatic activity of EGCG was characterized by measuring the diameter of the inhibition zone, the minimum inhibitory concentration (MIC), and the growth curve of E. coli.
Bacterial inhibition circle assays were performed as follows: after diluting a bacterial suspension to 106 CFU/mL with sterile distilled water, 100 μL of the solution was applied to nutrient agar medium plates under aseptic conditions. Three sterile Oxford cups were gently placed onto the plates with forceps in a triangle arrangement and filled with 100 μL of 2 mM EGCG (blank), 100 μL of 1–10 mM CaCl2 (control), or 100 μL of 2 mM EGCG and 1–10 mM CaCl2. The diameter of the inhibition circle was then measured after the incubation of plates for 24 h at 37 °C in a constant temperature incubator.
The MIC of EGCG on E. coli, before and after the presence of CaCl2, was determined by the test tube doubling-dilution method. Each experiment was repeated three times.
Determination of calcium ion concentration
Compared with the blank group, E. coli growth in the medium with Ca2+ only did not exhibit significant differences. E. coli growth in 1 and 10 mM Ca2+ conditions entered the logarithmic phase after 2 h (largest bacterial growth rate in the medium enriched with nutrients), while the plateau (stable phase of bacterial growth) was reached after 12 h. In contrast, in the medium added with EGCG only, E. coli entered the logarithmic phase only after 4 h, and the bacterial growth rate was significantly lower than that of the blank group. Moreover, the cell density of the bacteria decreased significantly, E. coli entered the stable phase after 8 h, and the bacterial mortality rate exceeded the proliferation rate.
As can be seen from figure 4, the decay of EGCG under different conditions in the 0–4 h time window was in the following order: EGCG + 1 mM Ca2+ > EGCG + 10 mM Ca2+ > EGCG. The reason for the slower decay of EGCG without Ca2+ is twofold: first, the initial bacterial content (102 CFU/mL) was low and EGCG had not yet entered the cells; secondly, the concentration of EGCG was relatively high, and the pH of the bacterial solution decreased due to H+ ionization, leading to a lack of EGCG oxidation, hence slow EGCG decay.
Detection of antibacterial functional groups
As the superoxide anion ( ) reacts with hydroxylamine hydrochloride, it generates . Under the action of p-aminobenzenesulfonic acid and α-naphthylamine, forms a red azo compound, with a characteristic absorption peak at 530 nm. By detecting the absorbance at a wavelength of 530 nm with a visible light spectrophotometer, we calculated the content in each experimental group. Through the ‘Fenton reaction’, H2O2/Fe2+ generates hydroxyl radicals that can oxidize Fe2+ in phenanthroline-Fe2+ aqueous solution to Fe3+, resulting in a decrease in the absorbance of the solution at 536 nm. The detection of changes in absorbance at 536 nm allowed us to back-calculate the scavenging rate of hydroxyl radicals.
In this experiment, E. coli cells were cultured for 8 h up to 108 CFU/mL, to then split into three culturing subgroups: (i) 2 mM EGCG; (ii) 2 mM EGCG and 1 mM Ca2+; (iii) 2 mM EGCG and 10 mM Ca2. Cells were cultured at 37 °C for an additional 8 h, to be then diluted with Phosphate-Buffered Saline (PBS) (pH 7.2–7.4) to 106 CFU/mL and frozen-and-thawed repeatedly to lyse the cells and release the intracellular components. The lysates were centrifuged at 2,500 rpm for 20 min and the supernatants were collected. The bacterial lysate before the addition of ECGC was used as a blank. The enzyme kits mentioned above were used to detect the changes in SOD enzyme, CAT enzyme, and GSH content in E. coli (and compared to a blank group of E. coli cultured for 6 h in a medium without either EGCG or Ca2+).
Three 1-mL E. coli cultures were collected after 8 h culturing up to 108 CFU/mL. Samples were mixed with 2 mM EGCG, or 2 mM EGCG and 1 mM Ca2+, or 2 mM EGCG and 10 mM Ca2+, to be then cultured further in a constant temperature incubator at 37 °C for 6 h. RNA was then extracted with the RNA extraction kit, reverse-transcribed into cDNA, then quantified by q-Polymerase Chain Reaction (PCR) with SYBR Green fluorescent dye and specifically designed primers obtained from Sangon Biotech®.
RESULTS AND DISCUSSION
ROS levels in E. coli cell milieu
ROS are one-electron reduction products obtained from oxygen participating in life processes. ROS include superoxide anion (), hydrogen peroxide, hydroxyl radical (·OH), etc. and reflect the oxidation state of E. coli cells. In response to excessive ROS production by the cell, antioxidant systems are activated to help protect from redox damage. is initially generated in the early stages of oxygen bio-reactivity. ·OH is the most active and most reactive ROS, it is easily combined with other molecules and has great oxidative potency. In this experiment, the changes of and ·OH after E. coli exposure to EGCG in combination with different Ca2+ concentrations were detected, to explore E. coli levels of internal oxidation and antioxidant consequent responses.
Changes in superoxide anion () content
Superoxide anion () is related to several physiological activities in cells. Under normal physiological conditions, in cells is maintained at relatively balanced levels, but in the presence of external stimuli, its concentration can reach excessive amounts. can damage DNA, proteins, and other biomacromolecules, leading to apoptosis (Min et al. 2020).
Figure 5 shows that the content of in the blank group was low, between 0.110 and 0.115 μmol/mL, indicating that the cells were in a state of dynamic redox equilibrium.
When E. coli cells were treated with EGCG in combination with 1–10 mM Ca2+, the content increased within 2 h. This phenomenon indicated that the oxidation of EGCG would stimulate E. coli to produce , but 10 mM Ca2+ inhibited EGCG oxidation.
Interestingly, when E. coli cells were treated with EGCG only, the levels increased at first, then decreased and increased again significantly after 2 h, reaching a maximum value at 6 h, to then finally decrease again, indicating that EGCG affected the balance in E. coli. Notably, even though the amounts decreased in this process, they never recovered to the initial content. The putative reasons behind this are twofold: (1) the oxidation of EGCG within E. coli caused oxidative stress, thus antioxidant enzymes helped to decompose some of the excess; and (2) the is further converted into a hydroxyl radical by the Fenton reaction or the Haber–Weiss reaction (Lan 2017). Generally, concentration was increased in the presence of Ca2+ and the increasing trend was stronger after 4 h of exposure, which indicates that the presence of Ca2+ could inhibit the antioxidant systems in E. coli. The oxidative stress response was weakened, so that the produced by EGCG oxidation was not counter-attacked by antioxidant enzymes, resulting in further oxidative damage in E. coli.
Changes in hydroxyl radicals (·OH) scavenging rate
Compared to , hydroxyl radicals are more hydrophobic and more reactive to phospholipid biomolecules in cell membranes and generally, can react with almost all biological macromolecules when they enter the cell. Excessive ·OH in the organism results in DNA bases and deoxyribose decomposition and leads to cell apoptosis (Shuyan et al. 2021). EGCG contains multiple phenolic hydroxyl groups with strong antioxidant properties and can scavenge ·OH to prevent cells from being oxidized to detrimental extents. When the concentration of EGCG is relatively high, it promotes pro-oxidative effects and accelerates cell oxidation (Xianqiang 2013). Therefore, the effect of EGCG on ·OH in the presence of Ca2+ is affected by the scavenging rate of EGCG on ·OH.
Figure 6 shows that the hydroxyl radical scavenging rate of E. coli in the blank group remained between 40 and 50% after culturing for 2 h and the overall change was not significant. This is due to the fact that the bacterial physiological metabolism is relatively strong under sufficient nutrient conditions and no external interference, and the excess hydroxyl free radicals generated during the growth and reproduction of bacteria can be rapidly decomposed. It is possible to maintain a relatively balanced concentration of free radicals in cells.
We observed an overall increasing trend of the hydroxyl radical scavenging rate in each group, although small differences were detected. During the 2–4 h incubation period, each experimental group's hydroxyl radical scavenging rate showed a similar downward trend (20% for cells treated with EGCG or EGCG + 1 mM Ca2+; 16% for cells treated with EGCG + 10 mM Ca2+). This indicates that the addition of EGCG did not enhance the free radical scavenging. However, to a certain extent, EGCG addition hinders the scavenging effect of E. coli antioxidant system on ·OH. Finally, our data also show that EGCG still has this hindering effect in the presence of Ca2+.
After 4 h of culture, the scavenging rate of ·OH increased significantly, specifically of 59, 51, and 57% in E. coli cells treated with EGCG, EGCG + 1 mM Ca2+, and EGCG + 10 mM Ca2+, respectively, after 8 h of growth. The clearance rate was significantly higher than that of the blank group.
In conclusion, the addition of 2 mM EGCG can inhibit ·OH scavenging in bacteria. However, with prolonged exposure times, EGCG concentration is attenuated, and the scavenging effect of ·OH is enhanced. Ca2+ had little effect on ·OH in our experimental conditions. If EGCG is not added in an effective concentration, it will be difficult to inhibit the antioxidant reaction in bacteria.
Internal antioxidant system of E. coli
The E. coli internal antioxidant system includes antioxidant enzymes and antioxidant substances, which constitute two barriers of the antioxidant system in living cells.
Changes in the content of antioxidant enzymes
SOD catalyzes the decomposition of in cells to generate oxygen and hydrogen peroxide. From the data in Figure 7, it can be seen that the SOD content in each experimental condition was greater than that in the blank group at the beginning of the experiment. Combined with the data reported in Figure 5, this implies that the excess of produced by E. coli in the presence of EGCG increased the amount of SOD. After 2 h, the SOD content in each experimental group was lower than that of the blank group and the data in Figure 5 showed that the content was still high at this time point. The results showed that the presence of EGCG inhibited the activity of the SOD enzyme, resulting in SOD being unable to remove the excess of intracellular . In addition, compared to the cells treated with EGCG, the SOD content of the cells treated with EGCG + 1 mM Ca2+ after 4 h was always lower. This indicates that 1 mM Ca2+ could enhance the EGCG inhibitory effect on the SOD enzyme, thereby enhancing the oxidative effect of EGCG on bacterial cells. However, the content of SOD enzyme in the cells treated with EGCG + 10 mM Ca2+ was significantly higher at 4 h and then lower after that. This indicates that Ca2+ has a robust regulatory effect on SOD at high concentrations, which is related to the inhibitory effect of EGCG on SOD, an antagonistic correlation consistent with previously reported results (Chunyan et al. 2022). EGCG exerts a better oxidative and bacteriostatic effect by inhibiting SOD, but the concentration of Ca2+ significantly affects this process. EGCG's inhibitory effect on SOD can be strengthened by 1 mM Ca2+, but it is weakened by 10 mM Ca2+.
CAT catalyzes the decomposition of hydrogen peroxide to generate oxygen and water in cells. As shown in Figure 8, the content of CAT is higher in E. coli cells treated with EGCG compared to the blanks, indicating that EGCG promotes the production of hydrogen peroxide in bacteria via increased CAT content. Compared to the cells treated with EGCG, the CAT content of the cells treated with EGCG + 1 mM Ca2+ was significantly lower, and the changing trend was similar to the blank group, indicating that CAT and hydrogen peroxide in the bacteria maintained a relatively balanced state at this time. 1 mM Ca2+ could stimulate CAT to decompose excess hydrogen peroxide, leading to a significant reduction in CAT activity, so EGCG is unlikely to cause oxidative damage to E. coli when it produces excess hydrogen peroxide in the presence of 1 mM Ca2+. Conversely, the CAT content in the cells treated with EGCG + 10 mM Ca2+ was similar to that of those treated with EGCG only, within 4 h of culture, but it was 19% higher after 6 h of culture. This indicates that in the presence of 10 mM Ca2+, EGCG could promote E. coli to produce more hydrogen peroxide and CAT with time. EGCG consumption would also increase correspondingly, resulting in a decrease in the oxidative and bacteriostatic effects of EGCG. In conclusion, EGCG can stimulate E. coli to produce excessive hydrogen peroxide to achieve the bacteriostatic effect, whereas Ca2+ will weaken the persistence of EGCG oxidation and bacteriostasis.
Changes in the content of antioxidant substances
The data reported in Figure 9 show that, compared with the blank group, the overall content of GSH in E. coli cells treated with EGCG is lower. However, the GSH content was higher after 8 h of culture. The reason for this is presumably that the sulfhydryl groups of GSH can combine with the hydroxyl group of the EGCG molecule and a small amount of EGCG is consumed when the GSH content declines, preventing EGCG from reacting with the sulfhydryl groups of the cell membrane proteins to maintain membrane integrity (Shunni & Li 2020). Therefore, EGCG reduces E. coli GSH content. The increase of GSH amounts in the later stages of the reaction due to the excess of hydrogen peroxide produced by E. coli stimulates the antioxidant system to produce a large amount of GSH to ensure the balance of free radicals, while the EGCG content is low in the later stages of the reaction and cannot react with GSH, resulting in an increase of free GSH. Compared with the cells treated with ECGC only, the GSH content in the cells treated with EGCG + 1 mM Ca2+ decreased over time, although the GSH content gradually approached that of the blank group after 4 h of culture, indicating that 1 mM Ca2+ could promote the production of GSH and accelerate the consumption of EGCG, hence the oxidation and antioxidant response in E. coli to restore the balance gradually. Although the GSH content change trend in the cells treated with EGCG + 10 mM Ca2+ is similar to that of cells treated with EGCG + 1 mM Ca2+, the actual GSH content after 2 h is significantly higher in the former group, indicating that the presence of 10 mM Ca2+ stimulates the bacteria to produce excessive H2O2. In addition, the antioxidant system in E. coli produces a large amount of GSH, indicating that Ca2+ could increase the GSH content in bacteria and weaken the oxidative effect of EGCG to a certain extent.
Oxidative stress response of E. coli and its changes
EGCG can stimulate E. coli to produce a large amount of ROS, such as s and hydrogen peroxide. When the antioxidant system of E. coli cannot completely remove ROS excess, it causes cellular oxidative stress, hence inhibition of cell growth and even apoptosis. SoxRS and oxyR regulators are important defense systems against oxidative stress in E. coli cells. Once E. coli is exposed to oxidative stress, these two regulators are activated and change gene expression and regulate ROS. In this way, they restore the oxidative balance and exert their antioxidant effects in bacteria (Baez & Shiloach 2013). SoxRS regulon is a defense system against excessive stimulation (Kaur & Benov 2020) and its most relevant genes are sodA, soxR, and soxS. The oxyR regulon is a defense system against excess hydrogen peroxide stimulation and its related genes include oxyR, oxyS, ahpC, adpCF, DPS, gor, and katG.
Figure 10 illustrates that, in contrast to the cells cultured with EGCG only, in the presence of Ca2+ the expression levels of sodA, soxR, and soxS defense genes are significantly increased. The expression level of soxS gene in the cells cultured with EGCG + 10 mM Ca2+ was nine times higher than that observed in the presence of EGCG only. The protein transcribed from soxS gene activates E. coli efflux pumps and promotes the active excretion of drugs from the cytosol, thereby making the bacteria resistant to it (Zhang et al. 2017). This indicates that E. coli is less sensitive to EGCG and resistant to EGCG in the presence of 10 mM Ca2+, and this experimental condition could cause more bacteria to be injured to a certain extent (Xu et al. 2021). The presence of EGCG at 10 mM Ca2+ could inhibit bacterial damage.
The oxyR gene is mainly responsible for the recognition and regulation of hydrogen peroxide and plays a vital role in the gene expression of CAT in bacteria. DPS protein binds to DNA forming a tight DPS-DNA complex that protects DNA from oxidative damage (Wen et al. 2015). As can be seen from Figure 11, compared with the cells cultured with EGCG only, the addition of 1 mM Ca2+ induced a twofold higher expression of most genes related to the oxyR regulator, which indicated that the content of hydrogen peroxide in this group was lower than the control group, and 1 mM Ca2+ might attenuate the oxidative damage of EGCG to bacterial DNA.
The expression of oxyR gene in the cells cultured with EGCG + 10 mM Ca2+ was slightly higher, indicating that in these experimental conditions, more hydrogen peroxide is produced in E. coli, triggering the defense system related to hydrogen peroxide. However, the expression level of other genes was significantly lower. For example, the DPS expression level was only one-third of that in the control group, suggesting that the hydrogen peroxide content observed with EGCG + 10 mM Ca2+ exceeds the hydrogen peroxide defense ability of E. coli, and the DNA oxidative damage was more evident, which was beneficial to the EGCG-dependent oxidative damage.
Ca2+ plays a significant role in the oxidative stress defense system of E. coli cells. 1 mM Ca2+ improves the hydrogen peroxide defense ability in E. coli cells and weakens oxidative stress damage in bacteria. 10 mM Ca2+ promotes the production of hydrogen peroxide and decreases the expression of defense system-related genes, enhancing the EGCG-dependent oxidative damage to bacterial DNA.
In the presence of Ca2+, the permeability of E. coli cell membrane is enhanced, leading to an increased entry of EGCG into the cell. EGCG uses its own oxidation to promote the increase of and hydrogen peroxide content in E. coli, augmenting the level of ROS in bacteria. Due to the inhibitory effect of EGCG on the antioxidant enzymes and antioxidant substances in the antioxidant system, the level of ROS in E. coli exceeds its own antioxidant capacity, thereby causing oxidative stress in E. coli and resulting in oxidative damage. Nevertheless, Ca2+ stimulates the expression of oxyR, DPS, and soxS genes, reducing the damage of EGCG to bacterial DNA, and Ca2+ may activate efflux pumps, leading to the resistance of E. coli to EGCG and leaving the bacteria susceptible to disinfection damage.
EGCG can be complexed with metal ions and the metal complexes formed to have different inhibitory activities and can improve the bacterial inhibitory effect of EGCG. The mechanism of bacterial inhibition is shown in its effect on bacterial cell membranes, proteins, genetic material, and other aspects. EGCG is used for disinfection of drinking water and shows good disinfection characteristics.
Currently, the highest content of metal ions in drinking water is Ca2+. Experimental results show that EGCG has a strong inhibitory effect on the growth of E. coli under high concentrations of Ca2+, which can improve the killing ability of EGCG on E. coli. In the future, EGCG can also be used in conjunction with new drinking water treatment technologies such as ultrafiltration membranes to ensure the safety of water quality in water supply networks.
This research is funded by National Natural Science Foundation of China (51678026), Open Research Fund Program of Key Laboratory of Urban Stormwater System and Water Environment (Beijing University of Civil Engineering and Architecture), Ministry of Education (2020) and Beijing University of Civil Engineering and Architecture Postgraduate Innovation Project (PG2022061). The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn) for the expert linguistic services provided.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.