Abstract
Background
In previous studies, authors have completed the total synthesis of several phloroglucinol natural products and synthesized a series of their derivatives, which were tested with good biological activities.
Objectives
To discover anti-MRSA lead compound and study their mechanism of action.
Methods
Phloroglucinol derivatives were tested to investigate their activities against several gram-positive strains including Methicillin-resistant Staphylococcus aureus (MRSA). The mechanism study was conducted by determining extracellular potassium ion concentration, intracellular NADPH oxidase content, SOD activity, ROS amount in MRSA and MRSA survival rate under A5 treatment. The in vitro cytotoxicity test of A5 was conducted.
Results
The activity of monocyclic compounds was stronger than that of bicyclic compounds, and compound A5 showed the best MIC value of 0.98 μg/mL and MBC value of 1.95 μg/mL, which were 4–8 times lower than that of vancomycin. The mechanism study of A5 showed that it achieved anti-MRSA effect through membrane damage, which is proved by increased concentration of extracellular potassium ion after A5 treatment. Another possible mechanism is the over ROS production induced cell death, which is suggested by observed alternation of several reactive oxygen species (ROS) related indicators including NADPH concentration, superoxide dismutase (SOD) activity, ROS content and bacterial survival rate after A5 treatment. The cytotoxicity results in vitro showed that A5 was basically non-toxic to cells.
Conclusion
Acylphloroglucinol derivative A5 showed good anti-MRSA activity, possibly via membrane damage and ROS-mediated oxidative stress mechanism. It deserves further exploration to be a potential lead for the development of new anti-MRSA agent.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1007/s40199-024-00503-4.
Keywords: Acylphloroglucinol derivatives, Drug resistance, Antimicrobial activities, Anti-MRSA, Mechanism of anti-MRSA
Introduction
Bacterial infection has become one of the most serious threats to human health all over the world. Although many antibiotics are being used in clinic, the emergence of drug-resistant bacteria has brought a great threat to clinical treatment [1–3]. MRSA was considered as the most intractable pathogenic bacterium due to high morbidity and mortality rates it caused [4–7]. Most of the clinically available antibacterial drugs were encountering drug resistance on account of MRSA with numerous resistance mechanisms [8, 9]. Therefore, the discovery of novel antimicrobial agents to overcome multidrug-resistant is essential for maintaining the public health in the future.
Phloroglucinol compounds are found in different types of ferns in nature, and were proved have good bactericidal, antiviral, tumor suppressing and insect repellent effects in Modern pharmacological studies [10–12]. For example, some phloroglucinol compounds isolated from Dryopteris fragrans (L.) Schott had good biological activity [13–15]. According to the report, there has been six new phloroglucinol compounds isolated from Dryopteris championii, including monocyclic,and bicyclic compounds [16]. Monocyclic and bicyclic compounds were mostly studied, such as monocyclic aspidinol and pseudoaspidinol, bicycles flavaspidic acid (AB) and albaspidin (AA) [17–19]. In previous studies, authors have completed the total synthesis, derivatization and activity evaluation of some monocyclic phloroglucinols and noticed that the monocyclic derivatives have a wide range of biological activities [17]. Recently, a great deal of researches reported that phloroglucinol compounds showed potent anti-MRSA activities [20–22].
In this study, authors selected some monocyclic and bicyclic phloroglucinol derivatives (Fig. 1) to investigate their activities against several gram-positive strains, and most derivatives exhibited good antibacterial activity. Compound A5 indicated excellent antibacterial activity against MRSA, similar to Vancomycin's antibacterial activity. In order to study the mechanism of compound A5 with good anti-MRSA activity, the effect of A5 on several components in MRSA was tested. In combination with the cytotoxicity results, A5 was recognized as a good candidate for anti-MRSA treatment, deserving further development.
Fig. 1.
Monocyclic and bicyclic phloroglucinol derivatives
Methods
Materials
The solvents and reagents were purchased from commercial vendors. The compounds were prepared according to the references [17] and the NMR chart of representative compounds can be found in SI. The bacterial were provided by Guangdong Pharmaceutical University.
Bacteria culture
Antibacterial activity of compounds was determined against MRSA (ATCC43300), Staphylococcus aureus, Bacillus subtilis and Staphylococcus albus. The bacterial were provided by Guangdong Pharmaceutical University. Subsequent experiments were conducted on a super clean workbench. Firstly, the bacterial strains were taken from the refrigerator and thawed. A small account of the bacterial solution was dipped with an inoculum ring and inoculated on agar plates. The plates were inverted and put in a 37 ℃ constant temperature incubator for cultivation until a single colony grew, and the colony with the best growth status was selected and removed to the liquid medium by inoculum ring for cultivation at 37 ℃ until the medium became turbid. 100 μL bacterial solution and 100 μL 50% sterilized glycerol were mixed in a sterile tube and stored in a low-temperature refrigerator. The bacterial suspension was prepared by adding an appropriate amount of culture medium. Subsequent experiments could be conducted after the operations mentioned above were completed.
The preparation of stock solutions
Stock solutions of all compounds were prepared in DMSO, filtered with 0.22 μm membrane, and diluted with liquid culture before experiments. DMSO content was ensured in a safe range (0.05%-0.1%), which could reduce the impact on bacterial growth. Stock solution of Vancomycin was prepared in sterile water, filtered with 0.22 μm membrane, and diluted with liquid culture before experiments. The stock solution of DMSO was diluted with liquid culture and filtered with 0.22 μm membrane before experiments.
Kirby-Bauer disk diffusion susceptibility test protocol
Under sterile conditions, the stock solutions of all compounds and Vancomycin were diluted with culture medium to 500 μg/mL, filtered with 0.22 μm membrane, and 20 μL drug solutions were placed on 6 mm drug-sensitive tablets. After the solvent was evaporated, the tablets were stored in brown sealed bottles. The concentration of four gram-positive bacterial suspensions was adjusted to 1.5 × 108 CFU/mL, and 100 μL bacterial suspension of each bacteria was absorbed into an agar plate medium, coated with sticks evenly, covered with petri dishes, and placed at room temperature for 5 min. Then, the drug-sensitive tablets were pasted on the surface of the solid medium (medium was used as blank control group and 0.1% dimethyl sulfoxide (DMSO) as negative control group, Vancomycin as positive control group). The tested bacteria were incubated at 37 °C for 18 ~ 24 h. The bacteriostatic circle size was measured and recorded by the vernier caliper crossing method. All assays were performed in triplicate.
Broth microdilution experiment
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined according to the Clinical Laboratory and Standards Institute (CLSI) guidelines. The bacteria suspensions were transferred to 96-well plates at a concentration of 1.5 × 106 CFU/mL with 100 μL culture medium. The stock solutions of compounds and Vancomycin were prepared and added the culture medium for two-fold serial dilutions (500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μg/mL). The tested bacterial were incubated at 37 °C for 18 ~ 24 h. Wells without compounds in the medium were used as the negative control and wells with Vancomycin were used as the positive controls. MIC was calculated as the lowest concentration that inhibited visible growth of the bacteria. 10 μL bacterial solution was absorbed from the wells without invisible growth of the bacteria, evenly applied to each plate, and incubated at 37 °C for 18 ~ 24 h. MBC was calculated as the lowest concentration that inhibited visible growth of the bacterial colony on the plate. All assays were performed in triplicate.
The bactericidal dynamic curve of MRSA
The concentration of the MRSA suspension in the solution was adjusted to 1 × 105~6 CFU/mL. The final concentrations of the drug in the solution were 0.5 × MIC, 1 × MIC and 2 × MIC, and bacterial suspension without drug was set as a negative control. After 0, 1, 2, 4, 8, 12, 16, and 24 h respectively, 50 μL suspension of each drug concentration was absorbed and gradient diluted with multiple of 104, 105 and 106 times respectively by MH broth medium. And 5 μL diluted suspension was applied to the agar medium, cultured overnight at 37 °C and calculated the number of bacteria colony [23]. All assays were performed in triplicate.
Assay of effect of A5 on potassium ion in MRSA
A5 was added to the pre-prepared bacterial liquid (1 × 105~6 CFU/mL), bringing the drug concentrations to 1 × MIC and 3 × MIC, and cultured at 37 ℃ respectively. 3 mL suspension was taken every 30 min, centrifuged at 8900 g for 10 min, and supernatant fluid was taken and determined its potassium ion concentration with K+ kit. The experiment was parallel for 3 times.
Assay of effect of A5 on the NADPH oxidase of MRSA
MRSA suspension was cultured at 37 °C for 24 h, centrifuged for 10 min. The precipitation was suspended in PBS solution and the bacterial concentration was adjusted to 1 × 105~6 CFU/mL. Bacteria were cultured in a nutrient broth containing different concentrations of A5 (0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC) at 37 °C for 4 h, then centrifuged at 4500 g for 5 min and suspended with phosphate buffer saline (PBS 50 mM, pH 7.0). Then bacteria dissolved in 0.5 mg/mL lysonase for 1 h under 4 °C. Then, 100 μL suspension was added to buffer (2.8 mL) containing NADPH (0.15 mM) and the absorbent value at 340 nm was measured with a UV–VIS spectrophotometer [24]. With the A5 concentration as the transverse coordinate and the NADPH oxidase activity (U/mg protein) as the longitudinal coordinate. The experiment was parallel for 3 times.
Assay of effect of A5 on the SOD in MRSA
MRSA concentration was adjusted to 1 × 105~6 CFU/mL. Then, bacteria were cultured in a nutrient broth containing different drug concentrations of A5 (0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC) at 37 °C for 4 h. The medium was centrifuged at 4500 g and the precipitation was cleaned with PBS. Then bacteria dissolved in 0.5 mg/mL lysonase for 1 h under 4 °C, centrifuged at 4 °C and for 5 min at 13400 g. The supernatant fluid was collected for detecting the activity of the SOD. 20 μL untested sample was mixed with 200 μL substrate working fluid and 20 μL enzyme working fluid, incubated at 37 °C for 20 min, and the absorbent value at 450 nm was detected with a UV–VIS spectrophotometer. The inhibition percentage was calculated as follows: the inhibition percentage = {(Absorbance of control group – Absorbance of control blank group) – (Absorbance of administration group – Absorbance of administration blank group)}/(Absorbance of control group – Absorbance of control blank group) × 100%. The SOD inhibition rate under each drug concentration was calculated, and 50% inhibition rate was defined as an enzyme activity unit [25]. The concentration of the protein was detected with the BCA kit. With the concentration of A5 as the transverse coordinate and the SOD activity (U/mg protein) as the longitudinal coordinate, the curve was repeated 6 times (P < 0.01, n = 6).
Assay of effect of A5 and antioxidant catalase on ROS in MRSA
The concentration of MRSA suspension was adjusted to 1 × 105~6 CFU/mL, centrifuged at 4500 g for 10 min, and suspended in PBS solution. Bacteria were cultured in a nutrient broth containing different drug concentrations of A5 (0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC) at 37 °C for 4 h. Then, 1 mg/mL NBT was added to incubate at 37 °C for 30 min, and nutrient broth was used as negative control. 100 μL of 0.1 M HCl was added to terminate the experiment. The suspension was centrifuged at 1500 g for 10 min, then 600 μL DMSO and 500 μL PBS were added to each tube, and the absorbent value at 575 nm was determined by a UV–VIS spectrophotometer. The drugs were treated with different concentrations of antioxidant catalase (100 and 200 U/mL), and the changes of ROS were detected according to the same method [26].
Assay of effect of antioxidant catalase on the survival rate of MRSA after A5 treatment
The concentration of MRSA suspension was adjusted to 1 × 105~6 CFU/mL, centrifuged at 4500 g for 10 min. Antioxidant peroxidase (100 and 200 U/mL) were added and acted at 37 °C for 15 min. Different drug concentrations of A5 (0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC) were added and reacted with them at 37 °C for 4 h. Then, 10 μL suspension was taken from each sample, and applied to each plate, and counted after 24 h. The curve was drawn with the concentration of A5 as the abscissa and the survival rate as the ordinate. Each experiment was repeated 3 times [27].
Cytotoxicity of A5
Methyl thiazolyl tetrazolium (MTT) assays were used to test the effect of A5 on the viability of LO2 cells [28]. 100 μL of LO2 cell solution were seeded in 96-well plates at a density of 5000 cells per well, and incubated with A5 at the indicated concentrations for at 37 °C for 24 h. After the culture medium was removed from each well, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well and cultured at 37 ℃ for 4 h. After that, the MTT solution was replaced with 100 μL of DMSO, followed by shaking for 15 min. The absorbance was measured at 490 nm on a UV–VIS spectrophotometer with Skanlt Software 3.2 (Multiskan GO, Thermo Scientific).
Statistical analysis
The data were analyzed using SPSS 10.0 software, and expressed as mean ± SD from three separate experiments. The values of MIC and MBC were determined by broth microdilution method. The differences between groups were analyzed using Student's t test (two-sided). Differences were statistically significant at P < 0.05.
Results
Inhibition zone of acylphloroglucinol compounds against four bacteria
The antibacterial activities of twenty-five novel acylphloroglucinol derivatives against gram-positive Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Bacillus subtilis and Staphylococcus albus were screened with Kirby-Bauer Disk Diffusion Susceptibility Test Protocol, using Vancomycin as positive control. The inhibition zone diameter of those compounds were used to determine the antibacterial activity in vitro, the results were shown in Table 1. The inhibition zone diameter of five series compounds revealed that compounds with one rings exhibited better antibacterial activity than compounds with two rings. The antibacterial activity of acylphloroglucinol derivatives decreased when one acyl group was removed. Compound A5 showed the best inhibitory effect against all tested strains among these derivatives, it has lower inhibitory activity against three strains than that of Vancomycin, but higher activity against MRSA than that of Vancomycin, with an inhibition zone diameter of 18.98 mm to 15.35 mm. The results suggested that the acyl side chain was a prerequisite for antibacterial activities of these derivatives.
Table 1.
Inhibitory zone diameter of acylphloroglucinol compounds
| Compound | Inhibitory zone diameter(mm) | |||
|---|---|---|---|---|
| MRSA | S.albus | S.aurus | B.Sub | |
| A1 | 11.47++ | 9.08+ | 11.50++ | 8.47+ |
| A2 | 13.89++ | 10.94++ | 13.12++ | 8.72+ |
| A3 | 15.89++ | 9.93+ | 18.74+++ | 14.17++ |
| A4 | 8.45+ | 12.40++ | 10.98++ | - |
| A5 | 18.98+++ | 16.21+++ | 27.60++++ | 18.00+++ |
| B1 | 6.45+ | - | 7.45+ | - |
| B2 | 6.73+ | - | 6.95+ | 6.34+ |
| B3 | 6.89+ | 6.89+ | 7.25+ | 6.56+ |
| B4 | - | 6.73+ | 7.30+ | - |
| B5 | 7.24+ | 7.75+ | 8.83+ | 7.31+ |
| C1 | 6.25+ | 7.36+ | 7.33+ | - |
| C2 | 6.25+ | 6.83+ | 7.65+ | - |
| C3 | 8.46+ | - | 11.45++ | - |
| C4 | 7.50+ | - | 7.86+ | - |
| C5 | 9.89+ | - | 16.46+++ | 6.39+ |
| D1 | 6.41+ | - | 6.20+ | - |
| D2 | 6.75+ | - | 6.51+ | - |
| D3 | 6.23+ | 7.34+ | 6.63+ | - |
| D4 | 6.38+ | - | 6.26+ | - |
| D5 | 6.96+ | - | 6.44+ | - |
| E1 | - | 6.75+ | 6.81+ | 6.86+ |
| E2 | - | 6.78+ | 6.30+ | 6.93+ |
| E3 | - | 6.53+ | 6.88+ | 6.87+ |
| E4 | 6.54+ | 6.27+ | 6.82+ | 6.76+ |
| E5 | - | 6.54+ | 6.63+ | - |
| Vancomycin | 15.35+++ | 27.01++++ | 27.94++++ | 28.95++++ |
Notes: The diameter of bacteriostatic ring is used as the criterion for determining the bacteriostatic activity of Kirby-Bauer Disk Diffusion Susceptibility Test Protocol (test was performed as described by the CLSI) [29, 30]: if the diameter ≤ 6 mm, the drug sensitivity is determined as -; If 6 mm ≤ diameter < 10 mm, it is determined as + ; If 10 mm ≤ diameter < 16 mm, it is determined as + + ; If 16 mm ≤ diameter < 26 mm, it is determined as + + ; If the diameter ≥ 26 mm, it is determined as + + + +
MIC and MBC values of acylphloroglucinol compounds against three gram-positive bacteria
The antibacterial activities of those acylphloroglucinol derivatives against four strains mentioned above were screened with broth microdilution method. Vancomycin was used as positive control. The test results were reported by recording minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) listed in Table 2. The MIC and MBC results of these compounds indicated that the A series has the best antibacterial activity. And compound A5 had excellent antibacterial activity against MRSA with the value of MIC was 0.98 μg/mL and the value of MBC was 1.95 μg/mL. The MIC effect of A5 on three Gram-positive bacteria was excellent, as results showed in Fig. 2.
Table 2.
Antibacterial activity (MIC, MBC) of acylphloroglucinol compounds (μg/mL)
| Compound | MRSA | S.albus | S.aurus | B.Sub | ||||
|---|---|---|---|---|---|---|---|---|
| MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | |
| A1 | 3.91 | 62.50 | 15.63 | 31.25 | 7.81 | 15.63 | 31.25 | 125 |
| A2 | 0.98 | 31.25 | 7.81 | 15.63 | 3.91 | 7.81 | 1.95 | 31.25 |
| A3 | 0.98 | 15.63 | 0.98 | 7.81 | 1.95 | 1.95 | 3.91 | 15.63 |
| A4 | 0.98 | 15.63 | 0.98 | 7.81 | 1.95 | 1.95 | 3.91 | 15.63 |
| A5 | 0.98 | 1.95 | 0.98 | 3.91 | 0.98 | 1.95 | 1.95 | 7.81 |
| B1 | 500 | > 500 | 62.5 | > 500 | 250 | > 500 | 500 | > 500 |
| B2 | 250 | > 500 | 250 | > 500 | 31.25 | 62.50 | 125 | > 500 |
| B3 | 15.63 | 31.25 | 15.63 | 62.50 | 15.63 | 500 | 125 | > 500 |
| B4 | 250 | 500 | 125 | 500 | 62.50 | > 500 | 31.25 | > 500 |
| B5 | 3.91 | 7.81 | 7.81 | 7.81 | 15.63 | 62.50 | 62.50 | 125 |
| C1 | 125 | 62.50 | 500 | > 500 | 62.50 | 500 | 250 | > 500 |
| C2 | 15.63 | 125 | 125 | 500 | 125 | 125 | 15.63 | > 500 |
| C3 | 62.50 | > 500 | 7.81 | 62.50 | 15.63 | 15.63 | 31.25 | > 500 |
| C4 | 1.95 | 250 | 31.25 | 125 | 250 | 250 | 7.81 | 250 |
| C5 | 1.95 | 31.25 | 3.91 | 3.91 | 15.63 | 15.63 | 250 | 125 |
| D1 | > 500 | > 500 | 125 | > 500 | 500 | > 500 | > 500 | > 500 |
| D2 | 250 | > 500 | 250 | > 500 | 500 | > 500 | > 500 | > 500 |
| D3 | 62.50 | > 500 | 31.25 | > 500 | 125 | > 500 | 250 | > 500 |
| D4 | 31.25 | > 500 | 500 | > 500 | 250 | 500 | > 500 | > 500 |
| D5 | 250 | > 500 | 125 | > 500 | 250 | > 500 | 250 | > 500 |
| E1 | 500 | > 500 | 250 | > 500 | > 500 | > 500 | > 500 | > 500 |
| E2 | 500 | > 500 | 500 | > 500 | > 500 | > 500 | > 500 | > 500 |
| E3 | 250 | > 500 | 500 | > 500 | 500 | > 500 | 500 | > 500 |
| E4 | > 500 | > 500 | 250 | > 500 | > 500 | > 500 | 125 | 500 |
| E5 | > 500 | > 500 | 500 | > 500 | > 500 | > 500 | > 500 | > 500 |
| Vancomycin | 3.91 | 15.63 | 1.95 | 3.91 | 0.98 | 0.98 | 0.98 | 7.81 |
Fig. 2.
Effects of compound A5 on three gram-positive bacteria (a) MRSA (b) Staphylococcus aureus (c) Staphylococcus alba
Effects of A5 on the survival of MRSA
The bactericidal dynamic curve of A5 on MRSA was shown in Fig. 3. The results showed that the 1 × MIC administration group kept the number of bacteria almost unchanged within the tested time, and the bacteria in the 2 × MIC administration group were killed quickly within 4 h and became stable after 4 h. The number of bacteria in the control group (Broth) increased rapidly within 4 h and slowly after that time. The number of bacteria in the 0.5 × MIC administration group increased rapidly within 4 h, slowly after 4 h, and became stable after 8 h. The above results illustrated that A5 had good bactericidal effect on MRSA. Combined with MIC and MBC results, the anti-MRSA effect of A5 was proved excellent.
Fig. 3.
Time-killing kinetics of A5 against MRSA (ATCC43300)
Effects of A5 on the concentration of extracellular potassium ion of MRSA
The membrane damage of bacteria can be reflected by the changes in cell electrolytes, such as the concentration of extracellular potassium ion. The effect of A5 on the extracellular K+ content of MRSA was shown in Fig. 4. in the 1 × MIC and 3 × MIC administration group, the concentration of extracellular potassium ion increased significantly compared with the control group within 3 h. A5 achieved the anti-MRSA effect by increasing the concentration of extracellular potassium ion, which may be caused by bacterial membrane damage.
Fig. 4.
Effect of A5 on K+ in MRSA
Effects of A5 on the NADPH oxidase in MRSA
In recent years, the antibacterial pathway of ROS and the damage of ROS to cellular macromolecules have attracted much attention. The activation of NADPH oxidase is one of the common ways to induce ROS production. Therefore, the effect of A5 on NADPH oxidase in MRSA was investigated and the results was shown in Fig. 5. The concentration of NADPH oxidase increased with the increase of administration concentration, in which the concentration of NADPH oxidase in untreated group was 1.80 ± 0.15 U/mg, while in the 0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC administration group they were 2.73 ± 0.21, 4.50 ± 0.62, 6.57 ± 0.39 and 8.45 ± 0.25 U/mg, respectively. Therefore, it can be concluded that the activation of NADPH oxidase is one of the reasons for the anti-MRSA activity of A5.
Fig. 5.
Effect of A5 on the NADPH oxidase in MRSA. **indicate that the concentration of NADPH oxidase increased significantly, **p ≤ 0.01
Effects of A5 on the SOD in MRSA
The production of excess ROS in bacterial will activate its defense systems of free radical clearing enzymes (such as superoxide dismutase (SOD), catalase, glutathione reductase, and glutathione peroxidase) and non-enzymes (such as glutathione, arginine, guionine and zinc), which protect bacterial from oxidative damage. Among them, SOD and GSH are representative enzymes and non-enzyme antioxidants that can jointly maintain the redox balance in cells. SOD catalyze the disambiguation of O2 to H2O2, which acts as the first line of defense against damage causes by excess ROS in the bacteria. The effect of A5 on the SOD in MRSA was investigated. Sod kit was used to detect SOD activity. The darker the color of reactants, the lower the superoxide-forming enzyme activity and the higher the SOD activity. As shown in Fig. 6, the activity of SOD decreased significantly with the increase of administration concentration, in which the concentration of SOD in untreated MRSA was 26.34 ± 2.31 U/mL, the concentrations in the 0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC administration group were 24.22 ± 2.25, 14.67 ± 0.23, 8.23 ± 1.21 and 3.37 ± 1.59 U/mL, respectively.
Fig. 6.
Effect of A5 on the SOD of MRSA. **indicate SOD activity protein decreased significantly, **p ≤ 0.01
Effects of antioxidant catalase on treatment of MRSA with A5
To further demonstrate that A5 induced MRSA death by increasing ROS content, the effects of different concentrations of A5 on ROS in MRSA were investigated by enzyme marker assay. Then, antioxidant catalase (100 U/mL and 200 U/mL) were added to the bacterial suspension, and treated with different concentrations of A5 to investigate the effect of antioxidant catalase on ROS in MRSA. Results were shown in Fig. 7, the concentration of ROS increased significantly with the increase of A5 concentration. The values of OD at 575 nm in untreated group were 0.35 ± 0.03, while in the 0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC administration group, they were 0.42 ± 0.02, 0.60 ± 0.03, 1.08 ± 0.04 and 1.28 ± 0.15, respectively. The effect of A5 on ROS was significantly weakened by addition of antioxidant catalase, with slight difference between different concentrations of antioxidant catalase. The values of OD at 575 nm were varied from 0.33 ± 0.02, 0.36 ± 0.03, 0.38 ± 0.04 and 0.45 ± 0.15 in 100 U/mL antioxidant catalase group to 0.32 ± 0.04, 0.16 ± 0.04, 0.27 ± 0.06, and 0.41 ± 0.04 in 200 U/mL antioxidant catalase group. Above results proved that A5 increased the amount of ROS in MRSA, and catalase could reduce the amount of ROS at high drug concentrations.
Fig. 7.
Effect of antioxidant catalase on the ROS level of MRSA after A5 treatment. *indicate the absorbance decreased significantly with the addition of catalyst when the concentration is greater than or equal to MIC, *p ≤ 0.05
Effects of antioxidant catalase on bacterial survival under A5 treatment
To demonstrate whether A5 is an anti-MRSA agent by upregulating ROS, the effect of antioxidant catalase on bacterial survival under A5 treatment was determined by microscopic counting, and the survival rate was expressed as percentage of the control group. As shown in Fig. 8, the survival rate decreased significantly with the increase of A5 concentration, with the survival rate of 55.34 ± 1.45%, 35.12 ± 0.77%, 8.96 ± 3.34% and 4.45 ± 2.12% in the 0.5 × MIC, 1 × MIC, 2 × MIC and 4 × MIC administration group, respectively. However, when antioxidant catalase was added, the bacterial survival rate increased significantly in two different concentrations (100 U/mL and 200 U/mL) in the 4 × MIC administration group, with the survival rate of 66.34 ± 1.89% and 73.67 ± 0.80%. The results showed that antioxidant catalase significantly improved bacterial survival, indicating that the mechanism of A5 is mainly through the upregulation of ROS in cells.
Fig. 8.
Effect of antioxidant catalase on the survival rate of MRSA. *indicate the survival rate increased with the addition of catalyst, *p ≤ 0.05
Cell viability of LO2 cells under different concentrations of A5
Above results shows that A5 has good antibacterial activity. However, A5 should not affect the proliferation of normal cells as an antibacterial material. Thus, the cytotoxicity of different concentrations of A5 was detected using MTT method and the cell viability of LO2 cells was measured after 24 h of administration. The results was shown in Fig. 9, the cell survival was nearly 90% at 62.5 μg/mL, which indicate that the toxic effect of A5 on cells is small. Thus, the growth and function of LO2 cells are not significantly affected by A5.
Fig. 9.
Cytotoxicity of A5 under different concentrations by MTT assay
Discussion
Phloroglucinol and its derivatives have been proved a variety of antimicrobial properties. Previous articles have described their antibacterial activity and mechanism of actions. Callistemonols A and B showed excellent inhibitory activity against MRSA better than Vancomycin [31]. The antibacterial potency of them was results of depolarization of the bacterial membrane and bacterial cell lysis effect [32]. Aspidin BB has a better bactericidal activity against several drug-sensitive strains [33]. One of its possible action mechanisms against S. aureus was the elevated production of ROS [34]. In the current research, the activity test results showed that the acyl side chains were a prerequisite of antibacterial activity, and the activity of monocyclic compounds were stronger than that of bicyclic compounds. Phloroglucinol derivatives A5 with mono ring and di-acyl side chains showed the best anti-MRSA activity with lower MIC and MBC values, which are better than that of Vancomycin.
Interpreting bacterial cell membrane biosynthesis to alter cell homeostasis is an important strategy for antibacterial therapies. In this study, the anti-MRSA mechanism study of A5 showed that the concentration of extracellular potassium ion increased with the increased concentration of administrated A5, suggesting A5 functionalized in a similar way to Daptomycin. An ion channel was formed after A5 treatment to efflux intracellular potassium ion, causing damage to the bacterial membrane and contribute to bacterial death. This mechanism of altering cell membrane to achieve bactericidal activity means that it is difficult to develop cross resistance with most existing antibiotics, showing its talent to be a promising antibacterial drug again.
In recent years, intensive studies have proposed that bactericidal antibiotics induced reactive oxygen species formation contribute to drug-mediated bacterial death [35]. Because of their low redox potential, phenolic compounds were regarded as promising pro-oxidants. They can generate a significant quantity of ROS to induce damage to bacteria’s macromolecules and subsequent lethality. Above results suggested us that A5 could played the antibacterial role in correlation to ROS-mediated oxidative stress. The activation of NADPH oxidase is one of the common ways to induce ROS production, which has been confirmed by other groups [36]. In this study, the NADPH oxidase concentration increased with the increase of A5 concentration, suggesting A5 activated the ROS generation pathway mediated by NADPH oxidase. The ROS content determination results showed that with its concentration increased, A5 upregulated ROS content in MRSA and consequently lead to lower MRSA survival rate. Whereas in the same condition, SOD activity was decreased, which can catalyze the disambiguation of O2 to H2O2 and acts as the first line of defense against excess ROS amounts within the bacteria. In contrast, the addition of antioxidant catalase weakened the antibacterial activity of A5 under high drug concentration, with a dramatic decrease in the ROS content in cells and an increase in the MRSA survival rate. The alternation of those key ROS-related indicators suggesting that A5 functionalized as antibacterial agent through ROS-mediated pathway. The cytotoxicity of A5 to LO cell was particularly small. The cell survival rate was greater than 90% at 62.5 μg/mL, which is much greater than the MIC value of A5 against MRSA, indicating that A5 has good safety.
Conclusion
In conclusion, this study discovered phloroglucinol derivative with highly active anti-MRSA activity and investigated their preliminary mechanisms. Phloroglucinol derivative A5 deserve further exploration as a potential lead for the discovering new anti-MRSA drug for fighting against antimicrobial resistance.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The work was supported by Innovation and strengthening project of Guangdong Pharmaceutical University- Special Projects in Key Fields of General Colleges and Universities in Guangdong Province(2022ZDZX2030) and Guangdong Province Graduate Education Innovation Program in 2021 (2021JGXM071), Medical Scientific Research Foundation of Guangdong Province (B20234214), Scientific Research Project of Guangdong Provincial Bureau of traditional Chinese Medicine (20231205).
Funding
No funding is applicable.
Declarations
Competing interest
The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.
Footnotes
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