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Introduction

Over the last few decades, the misuse and overuse of

antibiotics has contributed to the development and rapid

spread of multidrug-resistant (MDR) bacteria, leading to

global threat due to the increased therapeutic failures (Lamers

et al., 2013). MDR bacteria exposed to antibiotics have

developed their own survival strategies underlying several

mechanisms such as enzyme production and efflux pump

activity (Rumbo et al., 2013). The development of resistance

to β-lactam antibiotics in Staphylococcus aureus is mainly

attributed to the production of β-lactamases (Sibanda and

Okoh, 2007). S. aureus produces β-lactamases that hydrolyze

β-lactam antibiotics, responsible for the variants of methicillin-

resistant S. aureus (MRSA) (Fuda et al., 2005). In addition, the

mutational alterations in and overproduction of penicillin-

binding proteins (PBPs) result in the increased resistance to β-

lactam antibiotics (Sun et al., 2014). The discovery of new

classes of antibiotic has become a global priority. However,

the development of novel antibiotics has been faced with

difficulty because it lags far behind the rapid evolution of

antibiotic resistance in bacteria. Therefore, novel strategy for

tackling antibiotic resistance problem is urgently needed to

protect global public health.

Recently, potentiators have gained growing attention

towards potential adjuvants used in combination with anti-

biotics (Zabawa et al., 2016). The potentiators act as inhibitors

of β-lactamases, known as β-lactamase inhibitors (BLIs)

such as clavulanate, sulbactam, and tazobactam (Bush and

Bradford, 2016). The combined treatments of β-lactams and

BLIs can extend the spectrum of antibiotics against not only

Gram-positive bacteria but also Gram-negative bacteria

(Sood, 2013). BLIs are commercially used for enhancing

broad-spectrum activity, including ampicillin/sulbactam,

Assessment of β-Lactamase Inhibitor Potential of Medicinal Plant

Extracts against Antibiotic-resistant Staphylococcus aureus

Jirapat Dawan1 and Juhee Ahn2*1Graduate Student, Department of Biomedical Science, Kangwon National University, Chuncheon 24341, Korea

2Professor, Department of Biomedical Science and Institute of Bioscience and Biotechnology,

Kangwon National University, Chuncheon 24341, Korea

Abstract - This study was designed to assess the possibility of using medicinal plant extracts as β-lactamase inhibitors to

control antibiotic-resistant Staphylococcus aureus. The susceptibilities of S. aureus ATCC 15564 (SAWT), ciprofloxacin-

induced S. aureus ATCC 15564 (SACIP), oxacillin-induced S. aureus ATCC 15564 (SAOXA), and clinically-isolated S.

aureus CCARM 3008 (SACLI) to ampicillin were determined in the absence and presence of medicinal plant extracts,

including Cleyera japonica (CJ), Carpinus laxiflora (CL), Euphorbia helioscopia (EH), Euscaphis japonica (EJ),

Oenothera erythrosepala (OE), and Rosa multiflora (RM). The phenotypic change in the clear inhibition zones around

ampicillin disc was observed for SAWT, SACIP, and SAOXA, indicating the production of ampicillinase. Compared to the

controls, the MICs of ampicillin against SAWT, SACIP, and SAOXA were decreased from 4 to 0.5 ㎍/mL in the presence of CL,

16 to 4 ㎍/mL in the presence of RM, and 32 to 2 ㎍/mL in the presence of CL, EH, and RM, respectively. The medicinal

plant extracts, OE, EJ, and CL, effectively inhibited the β-lactamase activities of SAWT (78%), SACIP (57%), and SAOXA

(76%) when compared to the control. This results suggest that the medicinal plant extracts can be used as BLIs to control the

antibiotic-resistant S. aureus.

Key words – Ampicillin, Antibiotic resistance, β-Lactamase inhibitor, β-Lactamase activity, Medicinal plant extract,

Staphylococcus aureus

*Corresponding author. E-mail : juheeahn@kangwon.ac.kr

Tel. +82-33-250-6564

ⓒ 2020 by The Plant Resources Society of Korea

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Korean J. Plant Res. 33(6):578-585(2020)

https://doi.org/10.7732/kjpr.2020.33.6.578

Print ISSN 1226-3591

Online ISSN 2287-8203

Original Research Article

Assessment of β-Lactamase Inhibitor Potential of Medicinal Plant Extracts against Antibiotic-resistant Staphylococcus aureus

- 579 -

cefoperazone/sulbactam, piperacillin/tazobactam, ticarcillin/

clavulanate, cefepime/clavulanate, and carbapenem/clavulanate

(Akova, 2008; Cheng et al., 2019). In addition to the enhanced

antibiotic activity, BLIs can extend the use of conventional

antibiotics by re-considering sub-potent and outdated

antibiotics (Zabawa, et al., 2016). Medicinal plants, which

show antimicrobial properties, are well recognized for

antibiotic development with less or no side effects (Lee et al.,

2019; Rubens et al., 2015). The screening of plant extracts

have gained more attention for the discovery of novel drugs

in the treatment of life-threatening bacterial infections.

Medicinal plants are desirable to consider possible sources of

BLIs due to the increasing demand for naturally occurring

antibiotics (Aparna et al., 2014). Therefore, the objective of

this study was to assess the possibility of using medicinal

plants as BLIs in combination with antibiotics that can fight

antibiotic-resistant S. aureus.

Materials and Methods

Bacterial strains and culture conditions

Strains of Staphylococcus aureus ATCC 15564 (SAWT) and

clinically-isolated S. aureus CCARM 3008 (SACLI) were

obtained from American Type Culture Collection (ATCC,

Manassas, VA, USA) and Culture Collection of Antibiotic

Resistant Microbes (CCARM, Seoul, Korea), respectively.

SAWT was serially exposed to stepwise increasing concen-

trations of ciprofloxacin and oxacillin to induce antibiotic-

resistant strains (Michéa-Hamzehpour et al., 1994), which

were assigned as ciprofloxacin-induced S. aureus ATCC

15564 (SACIP) and oxacillin-induced S. aureus ATCC 15564

(SAOXA), respectively. All strains were cultured in typical soy

broth (TSB; BD, Becton, Dickinson and Co., Sparks, MD) at

37℃ for 20 h. The cultured cells were centrifuged with

phosphate-buffered saline (PBS, pH 7.2) at 6,000 × g for 10

min at 4℃ to remove cell debris and then suspended in PBS

to a concentration of 108 cfu/mL. No noticeable changes in

the antibiotic resistance profiles of SACIP and SAOXA were

observed through 10 serial passages in antibiotic-free TSB.

Plant materials

The extracts of medicinal plants, including Cleyera japonica

(CJ), Carpinus laxiflora (CL), Euphorbia helioscopia (EH),

Euscaphis japonica (EJ), Oenothera erythrosepala (OE), and

Rosa multiflora (RM) were kindly provided by Dr. Kil-Nam

Kim from the Korea Basic Science Institute (KBSI, Chun-

cheon, Korea). All medicinal plants were extracted with

water at 60℃ for 24 h. The extracts were lyophilized using a

freeze-dryer and stored at -20℃ prior to use. The extracts

were dissolved in water to obtain 30 ㎎/mL of stock solutions.

Hodge test

Modified Hodge test was used to evaluate the β-lactamase-

producing ability of SAWT, SACIP, SAOXA, and SACLI in the

presence of cefoxitin, ceftriaxone, imipenem, meropenem,

ampicillin, and piperacillin (Amjad et al., 2011; Anderson et

al., 2007). In brief, Escherichia coli as a control strain was

evenly spread on the surface of Mueller-Hinton (MH) agar.

The spread-plated MH agar was cut straightly from the center

to the edge using a blade smeared with SAWT, SACIP, SAOXA,

and SACLI. Antibiotic discs, including cefoxitin (30 ㎍),

ceftriaxone (30 ㎍), imipenem (10 ㎍), meropenem (10 ㎍),

ampicillin (10 ㎍), and piperacillin (100 ㎍), were placed on

the surface of MH agar and then incubated at 37℃ for 20 h.

Distortion in the clear zone of inhibition was observed to

determine the production of β-lactamases.

Antimicrobial susceptibility assay

The susceptibilities of SAWT, SACIP, SAOXA, and SACLI to

ampicillin in the absence and presence of medicinal plant

extracts, including CJ, CL, EH, EJ, OE, and RM, were

determined using a broth microdilution assay according to the

Clinical Laboratory Standards Institute (CLSI) procedure

(CLSI, 2015). The ampicillin (100 µL) was serially (1:2)

diluted with TSB in 96-well microtiter plates. Approximately

105 cfu/mL of each strain were inoculated, and the prede-

termined quarter MIC of each extract (MICs = 0.4-15 ㎎/mL)

was added in the 96-well microtiter plates. The prepared

microtiter plates were incubated at 37℃ for 20 h. MICs were

determined at the lowest concentration where no growths

were visually observed.

β-Lactamase activity assay

The β-lactamase activity of SAWT, SACIP, SAOXA, and SACLI

Korean J. Plant Res. 33(6) : 578~585 (2020)

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was evaluated by using a nitrocefin-hydrolyzing assay

(Matsumoto et al., 2011). Each strain was cultured in the

absence and the presence of a quarter MIC of medicinal plant

extracts (CJ, CL, EH, EJ, OE, and RM) at 37℃ for 20 h.

BLI-489 was used as a positive β-lactamase inhibitor. The

cultures were centrifuged at 6000 × g for 10 min at 4℃. The

cell-free supernatants were mixed with 10 μL of 1.5 mM

nitrocefin and incubated at 37℃ for 30 min. The absorbance

was measured every 5 min at 515 ㎚ using a microplate reader.

Statistical analysis

All experiments were conducted in three replicates. The

collected data were analyzed by the Statistical Analysis

System (SAS). The General Linear Model (GLM) and least

significant difference (LSD) procedures were used to

compare treatments at p < 0.05.

Results

Antibiotic susceptibilities of SAWT, SACIP, SAOXA, and

SACLI in the presence of medicinal plant extracts

The β-lactamase-producing strains were screened by the

modified Hodge assay (Fig. 1). Antibiotics are hydrolyzed by

β-lactamases produced by the test strains (SAWT, SACIP,

SAOXA, and SACLI), resulting in the growth of sensitive

control strain (E. coli). No noticeable phenotypic change in

the clear inhibition zones around cefoxitin, ceftriaxone,

imipenem, meropenem, and piperacillin discs was observed

at all test strains. In contrast, the distortion in the clear zone of

inhibition around ampicillin disc was observed at SAWT,

SACIP, and SAOXA, except SACLI. The ampicillin suscep-

tibilities of SAWT, SACIP, SAOXA, and SACLI were evaluated in

the absence and presence of medicinal plant extracts,

including Cleyera japonica (CJ), Carpinus laxiflora (CL),

Euscaphis japonica (EJ), Euphorbia helioscopia (EH),

Fig. 1. Modified Hodge test for β-lactamase-producing Staphylococcus aureus ATCC 15564 (SAWT), ciprofloxacin-induced S.

aureus ATCC 15564 (SACIP), oxacillin-induced S. aureus ATCC 15564 (SAOXA), and clinically-isolated S. aureus CCARM 3008

(SACLI) exposed to different antibiotics, including cefoxitin, ceftriaxone, imipenem, meropenem, ampicillin, and piperacillin.

Assessment of β-Lactamase Inhibitor Potential of Medicinal Plant Extracts against Antibiotic-resistant Staphylococcus aureus

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Oenothera erythrosepala (OE), and Rosa multiflora (RM)

(Table 1). SAOXA and SACLI (MIC=32 ㎍/mL) were highly

resistance to ampicillin in the absence of medicinal plant

extracts, followed by SACIP (MIC=16 ㎍/mL) and SAWT

(MIC=4 ㎍/mL). SAWT, SACIP, SAOXA, and SACLI were

susceptible to all medicinal plant extracts with the exception

of EH against SACLI, showing no change in the MIC (32 ㎍

/mL). The MICs of ampicillin against SAWT, SACIP, SAOXA,

and SACLI were decreased from 2 to 8 folds, 2 to 4 folds, 8

to16 folds, and 1 to 2 folds, respectively, in the presence of

CJ, CL, EH, EJ, OE, and RM.

Inhibitory effect of medicinal plant extracts on the produc-

tion of β-lactamases by SAWT, SACIP, SAOXA, and SACLI

The extracellular β-lactamase activities of SAWT, SACIP,

SAOXA, and SACLI were estimated in the presence of CJ, CL,

EH, EJ, OE, and RM, compared to the control (Fig. 2). The

highest β-lactamase activities were observed in SAWT, SACIP,

and SAOXA in the absence of medicinal plant extracts,

showing 39, 32, and 43 µmol/min/mL, respectively. SACLI

exhibited the lowest β-lactamase activity (3 µmol/min/mL),

which was not significantly changed in the presence of CJ,

CL, EH, EJ, OE, and RM (p > 0.05). The β-lactamase inhibitor,

Table 1. MICs of ampicillin against Staphylococcus aureus ATCC 15564 (SAWT), ciprofloxacin-induced S. aureus ATCC 15564

(SACIP), oxacillin-induced S. aureus ATCC 15564 (SAOXA), and clinically-isolated S. aureus CCARM 3008 (SACLI) treated without

and with medicinal plant extractsz

Strain AmpicillinAmpicillin with plant extractz

CJ CL EH EJ OE RM

SAWT 4 1 0.5 2 2 0.5 1

SACIP 16 8 8 8 8 8 4

SAOXA 32 4 2 2 4 4 2

SACLI 32 16 16 32 16 16 16

zMedicinal plant extracts include Cleyera japonica (CJ), Carpinus laxiflora (CL), Euscaphis japonica (EJ), Euphorbia helioscopia

(EH), Oenothera erythrosepala (OE), and Rosa multiflora (RM).

Fig. 2. Hydrolyzing activity of β-lactamases produced by Staphylococcus aureus ATCC 15564 (SAWT), ciprofloxacin-induced S.

aureus ATCC 15564 (SACIP), oxacillin-induced S. aureus ATCC 15564 (SAOXA), and clinically-isolated S. aureus CCARM 3008

(SACLI) treated with BLI-489 and medicinal plant extracts (Cleyera japonica, CJ; Carpinus laxiflora, CL; Euphorbia helioscopia,

EH; Euscaphis japonica, EJ; Oenothera erythrosepala, OE; Rosa multiflora, RM). Letters (a-c) on the bars are significantly different

among treatments within each strain at p < 0.05.

Korean J. Plant Res. 33(6) : 578~585 (2020)

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BLI-489, used as a positive control effectively reduced the β

-lactamase activities of SAWT, SACIP and SAOXA up to less

than 7 µmol/min/mL (> 80% reduction).

Discussion

Since some bacteria can evolve antibiotic resistance mediated

by the production of β-lactamases, the discovery of natural β

-lactamase inhibitors can provide a clue for the control of

antibiotic-resistant pathogens. Novel β-lactamase inhibitors

can help to make the best reuse of unnoticed and outdated

antibiotics.

The distortion in the clear zone of inhibition around

ampicillin disc (Fig. 1) suggests that the reduced susceptibility

of E. coli to ampicillin was mediated by ampicillin-hydrolyzing

enzymes produced by SAWT, SACIP, and SAOXA, whereas

SACLI did not produce β-lactamases. As a result, ampicillin

was selected for further β-lactamase inhibition study. The

specific types of β-lactamases, such as penicillinases and

cephalosporinases, have their own specificity for β-lactam

antibiotics based on the substrate specificity (Bauernfeind,

1986). The β-lactamases are divided into four classes, A

(Bla-A, SHV, and TEM), B (IMP, SPM, and VIM), C

(AmpC, Bla-B, and FOX), and D (OXA), and also classified

into class I through VI (Barry and Barlow, 2005; Bauern-

feind, 1986). The class A β-lactamases (class II, III, and V)

are resistant to ESBLs, associated with the overexpression of

AmpC and TEM (Féria et al., 2002). The class B β

-lactamases are resistant to most β-lactam antibiotics

(carbapenem) and β-lactamase inhibitors (clavulanic acid)

(Hanson and Sanders, 1999; Payne et al., 1994). The class C

β-lactamases (class I) include an inducible cephalosporinase

(Hanson and Sanders, 1999; Manchanda and Singh, 2003).

The class D β-lactamases (class IV) include OXA β

-lactamase (Bauernfeind, 1986).

Previous studies reported that the medicinal plant extracts,

CJ, CL, EH, EJ, OE, and RM, inhibited the growth of

Helicobacter pylori, Listeria monocytogenes, S. aureus, S.

epidermidis, Salmonella enterica, and Escherichia coli

(Cendrowski et al., 2020; Elizaquível et al., 2013; Hou et al.,

2003). The antimicrobial activity of CJ, CL, EH, EJ, OE, and

RM was not considered in this study because the ampicillin

treatments were combined with a quarter MIC of each extract

that might not affect the viability of SAWT, SACIP, SAOXA, and

SACLI. The decrease in the MICs of ampicillin combined with

CJ, CL, EH, EJ, OE, and RM might be due to the inhibition of

β-lactamase activity and/or PBP2a (Catteau et al., 2017). The

medicinal plant extracts containing flavonoids, tannins,

flavan-3-oleflavonoid, and epicatechin gallate can inhibit β

-lactamase activity, leading to the inhibition of hydrolysis of

β-lactam antibiotics (Eumkeb et al., 2012; Stapleton et al.,

2004; Zhao et al., 2002). Therefore, the increased suscepti-

bilities of SAWT, SACIP, and SAOXA to ampicillin in the

presence of medicinal plant extracts imply that the medicinal

plant extracts used in this study acted as β-lactamase inhibitor.

However, the ampicillin-resistant SACLI (32 ㎍/mL) might be

more associated with modified PBPs (PBP2a) that contributes

to the resistance to β-lactam antibiotics, rather than the

production of β-lactamases (Llarrull et al., 2009).

The β-lactamase inhibitor, BLI-489, effectively reduced

the β-lactamase activities of SAWT, SACIP and SAOXA (Fig. 2).

BLI-489 is a broad-spectrum β-lactamase inhibitor, which is

effective against class A, C, and D β-lactamases when

combined with piperacillin (Venkatesan et al., 2004). The

common β-lactamase inhibitors, sulbactam and clavulanic

acid, effectively inhibit the activity of extended-spectrum β

-lactamases (ESBLs), but less effective against AmpC (Liu et

al., 2016). The ability of CJ, CL, EH, EJ, OE, and RM to

inhibit β-lactamases varied with strains used in this study

(Fig. 2). The intrinsic β-lactamase activities of SAWT, SACIP,

and SAOXA were decreased by 55-78%, 27-57%, and 65-76%,

respectively, in the presence of medicinal plant extracts (Fig.

2). The highest reductions in β-lactamase activities of SAWT,

SACIP, and SAOXA were 78%, 57%, and 76%, respectively, for

OE, EJ, and CL. These results are in a good agreement with

previous report that medicinal plant extracts containing

quercetin, naringenin, and catechin could act as β-lactamase

inhibitors for enhancing antimicrobial activity (Boussoualim

and Abderrahmane, 2011; Eumkeb et al., 2010). These β

-lactamase inhibitors can directly interfere with β-lactamases

or compete for the binding sites of β-lactamases (Catteau, et

al., 2017).

In conclusion, the most significant finding was that

medicinal plant extracts, including CJ, CL, EH, EJ, OE, and

Assessment of β-Lactamase Inhibitor Potential of Medicinal Plant Extracts against Antibiotic-resistant Staphylococcus aureus

- 583 -

RM, potentiated the ampicillin activity against SAWT, SACIP,

and SAOXA. The β-lactamase activities of SAWT, SACIP, and

SAOXA were significantly reduced by 55-78%, 27-57%, and

65-76%, respectively, in the presence of CJ, CL, EH, EJ, OE,

and RM, which were corresponded to the increased

susceptibilities of SAWT, SACIP, and SAOXA treated with CJ,

CL, EH, EJ, OE, and RM. Therefore, the medicinal plant

extracts used in this study could be used as BLIs to enhance

the ampicillin activity against SAWT, SACIP, and SAOXA. The

antibiotic resistance mechanism-targeted approach can be a

promising alternative for the control of MRSA. However,

further study is needed to identify bioactive compounds in the

medicinal plant extracts and elucidate their mechanisms for

therapeutic use in combination with other antibiotics.

Abbreviations

Staphylococcus aureus ATCC 15564, SAWT; Clinically-

isolated Staphylococcus aureus CCARM 3008, SACLI; Cipro-

floxacin-induced Staphylococcus aureus ATCC 15564, SACIP;

Oxacillin-induced Staphylococcus aureus ATCC 15564,

SAOXA; Cleyera japonica, CJ; Carpinus laxiflora, CL;

Euphorbia helioscopia, EH; Euscaphis japonica, EJ; Oeno-

thera erythrosepala, OE; Rosa multiflora, RM.

Acknowledgements

This research was supported by Basic Science Research

Program through the National Research Foundation of Korea

(NRF) funded by the Ministry of Education (NRF-2016R1D1

A3B01008304).

Conflict of Interest

There are no conflicts of interest to declare.

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(Received 20 May 2020 ; Revised 14 September 2020 ; Accepted 14 September 2020)