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Article

Research article

J. Microbiol. Biotechnol. 2021; 31(9): 1288-1294

Published online September 28, 2021 https://doi.org/10.4014/jmb.2105.05015

Copyright © The Korean Society for Microbiology and Biotechnology.

Synergistic Antibacterial Activity of an Active Compound Derived from Sedum takesimense against Methicillin-Resistant Staphylococcus aureus and Its Clinical Isolates

Eun-Tak Jeong1,2, Seul-Ki Park3, Du-Min Jo2, Fazlurrahman Khan4, Tae Ho Choi1, Tae-Mi Yoon1, and Young-Mog Kim2*

1DYNE SOZE Co., Ltd., U-TOWER, Yongin 16827, Republic of Korea
2Department of Food Science and Technology, Pukyong National University, Busan 48513, Republic of Korea
3Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea
4Research Center for Marine Integrated Bionics Technology, Pukyong National University, Busan 48513, Republic of Korea

Correspondence to:Young-Mog Kim,   ymkim@pknu.ac.kr

Received: May 12, 2021; Revised: July 17, 2021; Accepted: July 19, 2021

Abstract

There are a growing number of reports of hospital-acquired infections caused by pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). Many plant products are now being used as a natural means of exploring antimicrobial agents against different types of human pathogenic bacteria. In this research, we sought to isolate and identify an active molecule from Sedum takesimense that has possible antibacterial activity against various clinical isolates of MRSA. NMR analysis revealed that the structure of the HPLC-purified compound was 1,2,4,6-tetra-O-galloyl-glucose. The minimum inhibitory concentration (MIC) of different extract fractions against numerous pathogenic bacteria was determined, and the actively purified compound has potent antibacterial activity against multidrug-resistant pathogenic bacteria, i.e., MRSA and its clinical isolates. In addition, the combination of the active compound and β-lactam antibiotics (e.g., oxacillin) demonstrated synergistic action against MRSA, with a fractional inhibitory concentration (FIC) index of 0.281. The current research revealed an alternative approach to combating pathogenesis caused by multi-drug resistant bacteria using plant materials. Furthermore, using a combination approach in which the active plant-derived compound is combined with antibiotics has proved to be a successful way of destroying pathogens synergistically.

Keywords: Antibiotic resistance, clinical isolates, methicillin-resistant, Sedum takesimense, Staphylococcus aureus, synergy

Introduction

Various antibiotics have been used to treat microbiological infections or diseases; however, several organizations, including the CDC (Centers for Disease Control and Prevention) and the U.S. Food and Drug Administration (U.S. FDA) have issued repeated warnings regarding their overuse [1]. Furthermore, antibiotic overuse is a growing concern among global public health authorities, and carries with it the significant risk of propagating antibiotic-resistant bacteria. According to CDC reports, at least 2 million people become infected each year, and 23,000 people die in the United States as a result of infection with multi-drug resistant pathogenic bacteria [2]. Every year, the number of people infected by antibiotic-resistant bacteria rises, particularly methicillin-resistant Staphylococcus aureus (MRSA), which poses a severe threat to human health [3]. MRSA is resistant to antibiotics with methicillin groups, with the exception of glycopeptides such as vancomycin and teicoplanin [4]. Thus, overuse of vancomycin to treat MRSA infection resulted in the development of vancomycin-resistant S. aureus (VRSA) [5]. Furthermore, numerous studies have shown that not only the type strain of MRSA, but also several clinically isolated MRSA, exhibit antibiotic resistance [6]. As a result, various alternative strategies for controlling infections caused by antibiotic-resistant bacteria have been developed, including the use of natural or synthetic medicines, as well as the combination of these treatments with conventional antibiotics to produce synergistic antimicrobial effects [7, 8].

Many studies on the antibacterial effects of phytochemicals derived from plants as natural therapies have been published [9]. These plant-derived antibacterial compounds have also been widely used in combination therapies in conjunction with conventional antibiotics [10-13]. Choi et al. reported that eckol, a compound derived from Ecklonia cava, has a bactericidal impact on MRSA and has synergistic bactericidal action when combined with β-lactam group antibiotics [10]. Similarly, plant extracts from Camellia sinensis, Delonix regia, and Holarhena antidysenterica have been shown in studies to have a bactericidal effect on MRSA, methicillin-susceptible S. aureus (MSSA), and clinical isolates. [11, 12]. Furthermore, when combined with antibiotics such as tetracycline, chloramphenicol, and ampicillin, these plant extracts have synergistic bactericidal properties. [11, 12]. Recently, it was revealed that a cryptotanshinone compound isolated and purified from Salvia miltiorrhiza had antibacterial activity against MRSA, VRSA, and MSSA [13]. When combined with β-lactam antibiotics such as oxacillin, ampicillin, and vancomycin, this pure compound also exhibited a synergistic effect [13].

Here, we achieved our goal of isolating and purifying a bioactive component from S. takesimense capable of efficiently killing MRSA and its clinical isolates. Furthermore, combination therapies using isolated molecules and β-lactam antibiotics were used to kill MRSA and its clinical isolates in a synergistic manner. The present study provides new insight on the use of a plant-derived natural product as an alternative strategy for killing multi-drug resistant bacterial infections..

Material and Methods

Extraction of Plant Material

The aerial part of S. takesimense was purchased from Arim SG Co., Ltd. botanical garden in Gyeongbuk Province, the Republic of Korea, in July 2019, and dried at 80°C for 4 days using a dry oven. The dried S. takesimense was rigorously extracted with a 10-time volume of 94% ethanol at room temperature for 30 h. The yield of the extracted and concentrated compound was found to be 15% (w/w). The extract was stored at -20°C before purification and future experiment in the anti-MRSA test.

Purification and Characterization of Active Compounds

The crude extract of powdered S. takesimense (20 g) was subjected to a silica gel column (3.5 by 60.0 cm; Kiesel gel 60, 150 g, 230 to 400 mesh; E. Merck, Darmstadt, Germany) with an eluting solvent of chloroform/methanol/distilled water/acetic acid (55:36:8:1 v/v) to get first fractions. After the first fraction, 4 fractions were successfully separated using a Sephadex LH20 column (3.5 by 60 cm; 130 g, 70 to 100 μm; Sigma-Aldrich, USA) in the presence of methanol as described earlier [14, 15]. These 4 fractions were further purified using a preparative high-performance liquid chromatographic (prep-HPLC) system (Capcell Pak C18, 20 by 250 mm; Shiseido, Japan). The substances were eluted with an isocratic solvent system such as methanol/water (20:80 v/v) at a flow rate of 15.0 ml/min and detected at a wavelength of 270 nm using a Waters 996 PDA detector (Waters, USA). With the help of prep-HPLC systems, a purified compound can be obtained.

The chemical structure of the purified compound was determined using proton nuclear magnetic resonance (1H-NMR) spectroscopy. The pure compound was also identified using a mass spectroscopy MSD1100 single quadrupole equipped with an ESI (Electrospray ionization, Hewlett-Packard Co., USA). 1H-NMR spectra were recorded by dissolving the pure compound in the deuterated methanol (CD3OD; Merck) and analyzed using a Bruker AMX-500 (Bruker Analytische Messtechnik GmbH) spectrometer, which was operated at 500 MHz for 1 h. The chemical shifts (parts per million) are pertinent to tetramethylsilane, which was used as an internal standard [15]. Coupling constants are reported in hertz, and the chemical structure of the purified single compound was analyzed based on the ESI-MS and 1H-NMR spectral data and comparisons of previously reported values [16-24].

Bacterial Strains and Antibiotics

The reference pathogenic bacteria used in the present study for the antimicrobial screening were purchased from the Korean Collection for Type Culture (KCTC; Korea) and included Escherichia coli (EC, KCTC 1682), Pseudomonas aeruginosa (PA, KCTC 4562), Salmonella Typhimurium (ST, KCTC 1925), Vibrio parahaemolyticus (VP, NCTC 11344), Bacillus cereus (BC, KCTC 3624), Listeria monocytogenes (LM, KCTC 3569), Staphylococcus aureus (SA, KCTC 1927), Staphylococcus epidermidis (SE, KCTC 3958), and Cutibacterium acnes (CA, KCTC 3314). The standard bacterial strain of methicillin-resistant Staphylococcus aureus (MRSA, KCCM 40511) used in this study was purchased from the Korean Culture Center of Microorganisms (KCCM; Korea). The seven clinical MRSA isolates were generously provided by Dong-A University Hospital (Korea). All bacterial strains were aerobically cultivated at 37°C in Mueller-Hinton broth (MHB; Difco, USA) or tryptic soy broth (TSB; Difco) growth media. All antibiotics and standard compounds used in this study were of analytical grade and purchased from Sigma (Sigma-Aldrich, USA).

Determination of Antibacterial Activity and MICs of the Extracts and Purified Compound

The antimicrobial activity of the crude extracts from S. takesimense was firstly screened based on the appearance of the inhibitory zone using the agar diffusion method as described by Dickson et al. [25] with a slight modification. The zone of inhibition that appeared on the tryptic soy agar (TSA) plate after 24 h of incubation was recorded. In one well, DMSO was also loaded as a negative control, and its value was subtracted from the test sample. The MICs were determined using a two-fold serial dilution method as recommended by the National Committee for Clinical Laboratory Standards (USA) [26].

Synergistic Effects of Active Compounds in Combination with Antibiotics Against MRSA

The interaction between active components present in the extract or purified compounds, with conventional antibiotics such as ampicillin, oxacillin, and clavulanic acid against MRSA, was tested as recommended by the checkerboard method [27]. The synergistic effect was evaluated by summing up the FICs to calculate the FIC index, which indicated synergy when the index values were: ≤ 0.5, synergic; > 0.5 to ≤ 1, additive; > 1 to ≤ 2, independent; > 2, antagonistic [28].

Results and Discussion

Purification and Structural Determination of the Compound

The ethanolic extract of S. takesimense was purified using chromatography strategies with silica gel columns, Sephadex LH-20 columns, and a prep-HPLC analytical system. As described in Materials and Methods, several strategies were tried to isolate and purify a single compound from S. takesimense. In the crude extract using ethanol, the extraction yield was 15% (w/w), and 30 g of extract was obtained from 200 g of raw material. Afterwards, 1.4 g of extract was obtained using Sephadex LH 20 column chromatography. Then, using the 1.4 g of extract, 3 fractions were obtained using the prep HPLC (fractions 4, 5, and 6), and among them, TOGG was isolated and purified from fraction 5 (63.2 mg). The HPLC chromatogram of the compound TOGG with retention time 15.2 min from fraction 5 is provided in the supplementary material (Fig. S1). The chemical structure of the purified compound was determined with the help of proton nuclear magnetic resonance (1H-NMR) spectroscopy. The NMR spectrum of the compound is given in the supporting information (Fig. S2). The chemical shifts of hydroxyl proton in galloyl and glucose in the TOGG are also summarized in Table S1.

Furthermore, the obtained spectra have also been compared with the previously reported spectra [29]. Hence, based on the spectroscopic studies, the isolated compound was identified as 1,2,4,6-tetra-O-galloyl-β-glucose (TOGG). Fig. 1 shows the chemical structure of pure compounds isolated from aerial parts of S. takesimense. Two standard compounds, penta-galloyl-glucose (PGG) and pyrogallol (PG), were also selected to evaluate the anti-MRSA activity, and the obtained results were compared with the identified compound (TOGG). Furthermore, PGG and PG were also used to evaluate the synergistic effect in combination with β-lactam antibiotics towards MRSA and its clinical isolates.

Figure 1. Chemical structure of purified compound (1,2,4,6-tetra-O-galloyl-β-glucose) identified from aerial parts of Sedum takesimense.

Screening of Antibacterial Activity of Crude Extracts and Purified Compound towards Different Pathogenic Bacteria

The antibacterial activity of the crude extract was first tested using the agar diffusion method, and the obtained MIC results are shown in Table 1. These results indicate that the crude extract of S. takesimense at two different concentrations (1 mg and 5 mg) has bactericidal activity against nine tested pathogenic bacteria. The zones of inhibition in diameter (mm) towards different bacterial strains were found in the order of SE > VP > ST > PA = BC > EC > CA > SA > LM, respectively. Meanwhile, towards gram-positive bacteria such as SA and LM, a lower inhibition zone was recorded as compared to the other pathogenic bacteria. The representative agar plate showing the zone of inhibition for each bacterium is also provided in the supporting information (Fig. S3). Also, among the gram-negative bacteria, CA had the lowest inhibition zone. These MIC results are in close agreement with the previous result of the antibacterial activity of S. hybridum extract [30]. According to Odontuya et al. [30], the prepared crude extract from S. hybridum has been reported to exhibit the lowest inhibition zone against the CA.

Table 1 . Antibacterial activity and MIC values of crude extract, its fraction, and pure compound from S. takesimense..

StrainAntimicrobial activity of 40% ethanolic extract

Zone of inhibition (mm)MIC (μg/mL)


CECEFrc. 4Frc. 5Frc. 6TOGG

1mg5mg
Escherichia coli KCTC 1682-9.0±0.51024102425625632
Pseudomonas aeruginosa KCTC 4562-9.7±0.51024512128512128
Salmonella typhimurium KCTC 1925-11.3±0.510241024128512128
Vibrio parahaemolyticus NCTC 11344-11.7±0.5-1024---
Bacillus cereus KCTC 3624-9.7±0.5512512326432
Listeria monocytogenes KCTC 3569-2.7±0.8512643264128
Staphylococcus aureus KCTC 1927-7.3±0.55122561625616
Staphylococcus epidermidis KCTC 39588.3±0.515±0.825664166416
Cutibacterium acnes KCTC 3314-7.7±0.5-----

*CE, Crude extract of S.takesimense with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; TOGG, and single compound (1,2,4,6-tetra-O-galloyl-β-glucose)..



MIC screening for antibacterial activity was carried out using crude extracts (CE), fractions 4, 5, 6 (F4, F5, F6), and purified compound (TOGG) towards different pathogenic bacterial species as shown in Table 1. Among them, F5 showed the highest effect on the inhibition of pathogenic bacteria growth. The MIC value of the F5 fraction required to inhibit pathogenic bacterial growth was found to be 256 μg/ml for EC, 128 μg/ml for PA and ST, 32 μg/ml for BC and LM, and 16 μg/ml for SA and SE. The pure compound (TOGG) showed the highest inhibitory activity at lower concentrations as compared to other fractions. It required a concentration of 32 μg/ml to inhibit EC and BC growth, 128 μg/ml against PA, ST, and LM, and 16 μg/ml against SA and SE. The purified compound (TOGG) showed higher activity in inhibiting the growth of gram-positive bacteria such as Staphylococcus species. The obtained results are in close agreement with the results reported by several previous studies [30-33]. These studies reported that a purified compound extracted from plants effectively inhibits gram-positive bacteria. Therefore, the inhibitory activity against MRSA and its clinical isolates, as well as the synergistic effect with existing antibiotics, were examined using crude extracts, different fractions (F4, F5, and F6), and purified compound (TOGG).

Bactericidal Effect of Active Compound Against MRSA and its Clinical Isolates

The results of the MIC values against MRSA and its clinical isolates were shown in Table 2. The results showed that fractions 1, 2 and 3 had no bactericidal effect on these pathogens (data not shown). The different extracts derived from S. takesimense, such as crude extract, F4, F5, F6, and purified TOGG, and standard compounds (PGG, and PG), inhibited cell growth of all bacterial strains. The inhibitory activity of these extracts and pure compounds was found to differ according to the type of sample or fractions. Among all of the tested compounds, TOGG showed the most potent in vitro antibacterial activity against MRSA and its clinical isolates with MIC values ranging from 16 to 256 μg/ml. In particular, the MIC value of TOGG towards standard strain and clinically isolated strains 6, 8, 10, and 12 appeared to be 16 μg/ml, whereas the MIC values of TOGG against clinically isolated strains 13 and 18 were found to be 32 μg/ml. This singly purified compound derived from S. takesimense exhibits a higher inhibitory effect on bacterial cell growth as compared to the standard compounds such as PGG and PG. Several other studies have reported that extracts prepared from natural sources such as plants or seaweeds have high efficacy of antibacterial activity [31, 32]. According to Bensouici et al. [31], compounds isolated from Sedum caeruleum have antibacterial activities against clinically isolated pathogenic bacteria such as E. coli, P. aeruginosa, Klebsiella pneumoniae, S. aureus, respectively. Also, Lee et al. [32] reported a synergistic antibacterial effect of dieckol isolated from Ecklonia stolonifera algae in combination with β-lactam-type antibiotics against MRSA. Hence, based on previous and present studies, it was concluded that compounds isolated from plant materials exhibit potent antibacterial activities and show a synergy when applied in combination with β-lactam antibiotics. Therefore, the present study was carried out to determine the anti-MRSA activity of compounds derived from S. takesimense towards both clinical and non-clinical isolates and also to investigate the synergistic effect in combination with β-lactam antibiotics.

Table 2 . Minimum inhibitory concentration (MIC) values of crude extracts, fractions, standard compounds, and singly purified compound against methicillin-resistant Staphylococcus aureus..

StrainSource or ReferenceMinimum inhibitory concentration (μg/mL) *

CEF4F5F6SCPGGPG
KCCM 40511Standard strain25664163216128128
Isolated 6Clinical isolate**102410246464166464
Isolated 81024102464641664128
Isolated101024102432641664128
Isolated125123216641612864
Isolated13102451216, 32643264256
Isolated1410245125126425664128
Isolated181024102432643264512

*CE, Crude extract of S. takesimense with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; SC, single compound; PGG, penta-galloyl-glucose(standard compound 1); PG, pyrogallol (standard compound 2)..

**MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital..



MIC of β-Lactam Antibiotics Against Clinically Isolated MRSA

MRSA and all its clinical isolates were found to be highly resistant to the β-lactam antibiotics, including ampicillin and oxacillin, as evidenced from the MIC values where they were found to be either equal to or greater than 64 μg/ml (Table 3). However, in clavulanic acid, the MIC values were either equal to or less than 64 μg/mL towards the MRSA standard strain and its 7 clinical isolates. Several studies have reported that the clinical isolates of MRSA exhibit a varied range of MIC values [33-35]. Other previous studies also reported that clavulanic acid has a bactericidal effect towards MRSA, and especially, that it is more effective when applied in combination with other antibiotics such as amoxicillin [34, 35]. The present study showed a similar bactericidal effect of β-lactam antibiotics such as ampicillin, oxacillin, and clavulanic acid towards MRSA and its clinical isolates.

Table 3 . Minimum inhibitory concentration (MIC) values of antibiotics (ampicillin, oxacillin, and clavulanic acid) against methicillin-resistant Staphylococcus aureus and its clinical isolates..

Strain*Minimum inhibitory concentration (μg/mL)

AmpicillinOxacillinClavulanic acid
KCCM 4051125612832
Isolated 612812832
Isolated 82566464
Isolated101286464
Isolated121282568
Isolated13646416
Isolated14646416
Isolated181286432

*MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital..



The MIC values of TOGG compound that were isolated from S. takesimense were found to be in the range of 16-32 μg/ml towards MRSA and all clinical isolates, except for isolate No. 14 (Table 2). The MIC values of pure compound TOGG were less than those of the β-lactam- class antibiotics against MRSA and its clinical isolates. The MIC determination result for the antibiotics showed that the MRSA isolate No. 12 was highly sensitive to clavulanic acid as compared to other antibiotics such as ampicillin and oxacillin (Table 3). Similar results have also been found for MRSA KCCM 40511, and MRSA isolate No. 6. Among β-lactam antibiotics, ampicillin was the least effective against MRSA and its isolates, followed by oxacillin, whereas clavulanic acid inhibits the growth of MRSA and its isolates most effectively.

Synergistic Effects of Active Compound in Combination with β-Lactam Antibiotics Against MRSA

The present study determined the synergistic effects of an active compound derived from S. takesimense towards MRSA and several of its clinical isolates in combination with commercial β-lactam antibiotics such as ampicillin, oxacillin, and clavulanic acid. As shown in Table 4, the MIC value of crude extract combined with oxacillin against MRSA strain KCCM 40511 was reduced dramatically from 256 to 0.25 μg/ml. Similarly, the MIC values of F5 and TOGG in combination with oxacillin reduced from 16 to 4 μg/ml and 6 to 2 μg/ml, respectively. The FIC indices of crude extract, F5, and TOGG, when combined with ampicillin, were the same value, i.e., > 1.0. However, the FIC values of the TOGG compound, when combined with oxacillin, were found to be in the range of 0.281 to 0.562 (Table 4). The FIC value of TOGG in combination with oxacillin was found to be 0.281, which exhibited a high effect on MRSA. Fig. S4 shows a representative 96-well microtiter plate used for the checkerboard assays of crude extract (CE), fraction No. 5 (F5), and single compound (TOGG) with ampicillin antibiotic against methicillin-resistant S. aureus KCCM 4051. These findings suggest that the combination of TOGG and oxacillin synergistically inhibits MRSA cell growth. Previous reports showed varied synergistic inhibition of MRSA cell growth by β-lactam in combination with epigallocatechin gallate (EGCg) and dieckol [32, 33].

Table 4 . Fractional inhibitory concentration (FIC) indexes (FICIs) of combined effects among major active compounds (crude extract, fraction 5, and pure compound), standard compounds (Penta-galloyl-glucose and pyrogallol), and antibiotics against methicillin-resistant Staphylococcus aureus KCCM 40511..

StrainAntibioticsCompound**MIC (μg/mL)∑FICmaxa)∑FICminb)FICIc)

AloneCombination
MRSA KCCM 40511*AmpicillinCE256322.2500.6251.031
AMP256128
F5160.0161.2500.7501.031
AMP256256
SC1621.2500.6251.031
AMP256256
PGG128641.0630.6250.844
AMP256128
PG12821.0000.1250.262
AMP25664
OxacillinCE2560.251.5000.5010.531
OXA12864
F51640.6250.5000.562
OXA12832
SC1620.3130.1880.281
OXA1288
PGG12810.1250.0640.070
OXA1288
PG12842.0000.2500.773
OXA12864
Clavulanic acidCE2562562.0002.0002.000
CLA3232
F51682.0001.5001.750
CLA3232
SC1682.0001.5001.750
CLA3232
PGG128321.1250.2810.434
CLA328
PG128----
CLA32-

a)ΣFICmax, maximum FIC value; b)ΣFICmin, minimum FIC value; c)FICI, The FIC index indicated synergistic effect: <0.5, marked synergy; 0.5 to <1.0, weak synergy; 1.0, additive; >1.0 to <2.0, subadditive; 2.0, indifferent; >2.0, antagonistic..

*MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital. **CE, Crude extract with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; SC, single compound; PGG, pentagalloyl-glucose (standard compound 1); PG, pyrogallol (standard compound 2)..


Conclusion

In this study, we isolated an active compound from the plant species S. takesimense utilizing solvent extraction procedures and chromatographic techniques. NMR analysis was also used to analyze the isolated component. The bioactive molecule identified from S. takesimense has been given the name 1,2,4,6-tetra-O-galloyl-glucose (TOGG). The isolated compound TOGG was found to show effective bactericidal activity towards MRSA and several of its clinical isolates. In addition, this compound also potentiates the efficacy of conventional antibiotics when applied in combination, and the results were found to be a synergistic way of killing MRSA and its clinical isolates. With the increase in vancomycin prescription, the sensitivity of MRSA to vancomycin has decreased in recent years [32]. Therefore, there is also a need to discover new drugs and alternative therapies to combat vancomycin-resistant microbial pathogens. The present study provides new insight for applying plant-derived bioactive compounds to effectively combat the pathogenesis caused by bacteria, especially MRSA. Future study is required to explore the molecular mechanism involved in killing these pathogenic bacteria by the newly isolated TOGG. Further investigation is also needed to address the mechanism of synergistic killing of this compound when combined with conventional antibiotics.

Supplemental Materials

Acknowledgments

This research was financially supported by the Ministry of Small and Medium-sized Enterprises (SMEs) and Startups (MSS), Korea, under the “Regional Specialized Industry Development Plus Program (R&D, S2874391)” supervised by the Korea Institute for Advancement of Technology (KIAT). This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2019R1A2C1087156 and 2021R1A6A1A03039211).

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Chemical structure of purified compound (1,2,4,6-tetra-O-galloyl-β-glucose) identified from aerial parts of Sedum takesimense.
Journal of Microbiology and Biotechnology 2021; 31: 1288-1294https://doi.org/10.4014/jmb.2105.05015

Table 1 . Antibacterial activity and MIC values of crude extract, its fraction, and pure compound from S. takesimense..

StrainAntimicrobial activity of 40% ethanolic extract

Zone of inhibition (mm)MIC (μg/mL)


CECEFrc. 4Frc. 5Frc. 6TOGG

1mg5mg
Escherichia coli KCTC 1682-9.0±0.51024102425625632
Pseudomonas aeruginosa KCTC 4562-9.7±0.51024512128512128
Salmonella typhimurium KCTC 1925-11.3±0.510241024128512128
Vibrio parahaemolyticus NCTC 11344-11.7±0.5-1024---
Bacillus cereus KCTC 3624-9.7±0.5512512326432
Listeria monocytogenes KCTC 3569-2.7±0.8512643264128
Staphylococcus aureus KCTC 1927-7.3±0.55122561625616
Staphylococcus epidermidis KCTC 39588.3±0.515±0.825664166416
Cutibacterium acnes KCTC 3314-7.7±0.5-----

*CE, Crude extract of S.takesimense with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; TOGG, and single compound (1,2,4,6-tetra-O-galloyl-β-glucose)..


Table 2 . Minimum inhibitory concentration (MIC) values of crude extracts, fractions, standard compounds, and singly purified compound against methicillin-resistant Staphylococcus aureus..

StrainSource or ReferenceMinimum inhibitory concentration (μg/mL) *

CEF4F5F6SCPGGPG
KCCM 40511Standard strain25664163216128128
Isolated 6Clinical isolate**102410246464166464
Isolated 81024102464641664128
Isolated101024102432641664128
Isolated125123216641612864
Isolated13102451216, 32643264256
Isolated1410245125126425664128
Isolated181024102432643264512

*CE, Crude extract of S. takesimense with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; SC, single compound; PGG, penta-galloyl-glucose(standard compound 1); PG, pyrogallol (standard compound 2)..

**MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital..


Table 3 . Minimum inhibitory concentration (MIC) values of antibiotics (ampicillin, oxacillin, and clavulanic acid) against methicillin-resistant Staphylococcus aureus and its clinical isolates..

Strain*Minimum inhibitory concentration (μg/mL)

AmpicillinOxacillinClavulanic acid
KCCM 4051125612832
Isolated 612812832
Isolated 82566464
Isolated101286464
Isolated121282568
Isolated13646416
Isolated14646416
Isolated181286432

*MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital..


Table 4 . Fractional inhibitory concentration (FIC) indexes (FICIs) of combined effects among major active compounds (crude extract, fraction 5, and pure compound), standard compounds (Penta-galloyl-glucose and pyrogallol), and antibiotics against methicillin-resistant Staphylococcus aureus KCCM 40511..

StrainAntibioticsCompound**MIC (μg/mL)∑FICmaxa)∑FICminb)FICIc)

AloneCombination
MRSA KCCM 40511*AmpicillinCE256322.2500.6251.031
AMP256128
F5160.0161.2500.7501.031
AMP256256
SC1621.2500.6251.031
AMP256256
PGG128641.0630.6250.844
AMP256128
PG12821.0000.1250.262
AMP25664
OxacillinCE2560.251.5000.5010.531
OXA12864
F51640.6250.5000.562
OXA12832
SC1620.3130.1880.281
OXA1288
PGG12810.1250.0640.070
OXA1288
PG12842.0000.2500.773
OXA12864
Clavulanic acidCE2562562.0002.0002.000
CLA3232
F51682.0001.5001.750
CLA3232
SC1682.0001.5001.750
CLA3232
PGG128321.1250.2810.434
CLA328
PG128----
CLA32-

a)ΣFICmax, maximum FIC value; b)ΣFICmin, minimum FIC value; c)FICI, The FIC index indicated synergistic effect: <0.5, marked synergy; 0.5 to <1.0, weak synergy; 1.0, additive; >1.0 to <2.0, subadditive; 2.0, indifferent; >2.0, antagonistic..

*MRSA KCCM 40511 is a standard strain purchased from KCCM; isolated strains were provided by Dong-A University Hospital. **CE, Crude extract with 40% Ethanol; F4, fraction 4; F5, fraction 5; F6, fraction 6; SC, single compound; PGG, pentagalloyl-glucose (standard compound 1); PG, pyrogallol (standard compound 2)..


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