Antimicrobial Activity of Native Thai Plant Extracts against Methicillin-Resistant
Staphylococcus aureus (MRSA)
Keerati Joyjamras1,*, Patamaporn Sukplang2, Acharawan Thongmee2,#
1
Biomedical Sciences Graduate Program, Rangsit University, Thailand
2
Microbiology Unit, Department of Medical Sciences, Faculty of Science,
Rangsit University, Thailand
*e-mail: j_keerati@hotmail.com, #e-mail: acharawan@rsu.ac.th
Abstract
Methicillin-resistant S.aureus (MRSA) is one of S.aureus strains that resists to a
variety of antibiotics, especially penicillin antibiotics. Its resistance to many available
antibiotics makes the treatment of MRSA infection more difficult because very few
antibiotics are effective against MRSA. In order to combat MRSA infections, enormous
efforts are being made to develop new compounds which can be used as potential
antimicrobial agents. An interesting alternatives include the use of natural products.
Therefore, the objectives of this research were to search for a potential antimicrobial agent
from native Thai plant extracts and to study the synergism between these extracts and
antibiotics against MRSA.
Eleven native Thai plants, i.e., Acanthus ebracteatus Vahl., Cissus quadrangularis
Lin, Kaempferia parviflora, Barleria lupulina Lindl, Orthosiphon aristatus (Blume) Miq,
Glycyrrhiza glabra L., Stevia rebaudiana Bertoni, Clitoria ternatea Linn., Curcuma longa
L.,Curcuma zedoaria (Berg.) Rosc. and Zingiber cassumunar Roxb were extracted with
water and 95% ethanol. These extracts were determined for antimicrobial activities against
S.aureus by agar diffusion method. Minimal inhibitory concentration (MIC) was also
determined using microdilution method.
The results of this study showed that Kaempferia parviflora ethanol extract possessed
antibacterial activity against only MRSA with inhibition zones of 15+3 mm. The MIC and
MBC of Kaempferia parviflora ethanol extract against MRSA was 46.87 g/mL and 96.75
g/mL, respectively. Study of synergism between plant extracts and antibiotics revealed that
when Kaempferia parviflora ethanol extract was combined with cefoxitin, the MIC and MBC
of cefoxitin against MRSA reduced from 23.44 g/mL to 1.46 g/mL and from 93.75 g/mL to
2.93 g/mL, respectively. Moreover, this combination reduced the MIC of Kaempferia
parviflora against MRSA from 46.87 g/mL to 23.44 g/mL. The results indicated that the
combination of Kaempferia parviflora and cefoxitin could be synergistic in antibacterial
effect against MRSA.
Keywords: MRSA, antibacterial activity, synergistic activity, native Thai plant extract
Introduction
The staphylococci are a group of gram positive bacteria that cause a variety of
diseases ranging from urinary tract infections to endocarditis. Staphylococcus aureus is the
most virulent staphylococcal species. S.aureus causes disease through both toxin-mediated
and non toxin-mediated mechanisms. S.aureus produces various human clinical diseases
including abscesses, bacteremia, central nervous system infections, endocarditis,
osteomyelitis, urinary tract infections, pneumonia, and a host of syndromes caused by
exotoxin, including bullous impetigo, food poisoning, scaled skin syndrome, and toxic shock
syndrome (1).
The treatment of choice for normal strains of S.aureus infection is penicillin. In many
countries, S. aureus strains have developed a resistance to penicillin due to production of an
enzyme by the bacteria called penicillinase. The introduction of the semisynthetic β-
lactamase-resistant penicillins such as methicillin and oxacillin in 1959 brought about a
general decline in the resistant S.aureus during early 1960. In 1961 Methicillin-resistant
Staphylococcus aureus (MRSA) were first detected in a hospital in the United Kingdom and
the first reported case of MRSA in the United States was in 1968. MRSA is a mutated form
of Staphylococcus aureus resistant to antibiotics, known as β-lactams, such as methicillin,
oxacillin, penicillin, and amoxicillin, and cephalosporins, such as cephalexin, and ceflactor
(2).
The emergence of MRSA has led to the development of novel antibiotics. Enormous
efforts are being made to synthesize and develop new compounds which can be used as
potential antimicrobial agents. Among the potential sources of new antimicrobial agents,
natural products could be an interesting alternative. An important source of natural products
is plants or herbs which are rich in a wide variety of active compounds such as tannin,
terpenoids, alkaloids and flavonoids. These compounds have been found in vitro to have
antimicrobial properties with lesser side effects and reduced toxicity when compared to
synthetic agents. Therefore, attention has been focused on natural products, in which they
contain complex compound and novel modes of action (3). The extracts of Senna alata,
Eupatorium odoratum, Garcinia mangostana and Barleria lupulina showed strong inhibit the
growth of Propionibacterium acnes (4). The extracts of Camellia sinensis, Delonix regia,
Holarrhena antidysenterica, Lawsonia inermis, Punica granatum, Terminalia chebula and
Terminalia belerica showed broad-spectrum antibacterial activity against Staphylococcus
aureus (5). Essential oils, distilled from plants such as lemongrass, lemon myrtle, mountain
savory, cinnamon and melissa, myrtle, exhibited zones of inhibition against MRSA. In
addition, these essential oils distilled from lemongrass combined with Eucalyptus globulus,
Eucalyptus australiana, Eucalyptus radiata, marjoram, pine, cypress, lavender, spruce,
peppermint and Eucalyptus citriodora oils exhibited inhibitory activity against MRSA (6).
An alternative approach to treat infectious diseases is the use of plant extracts in the
combination with antibiotics. Yang, et al. investigated the synergistic activity of 11
antibiotics with ethanol extract of Cinnamomum cassia against Acinetobacter baumannii,
Pseudomonas aeruginosa and Staphylococcus aureus. The extracts showed synergistic
effects with many tested antibiotics, including tetracycline, clindamycin, amikacin,
trimethoprim-sulfamethoxazole and carboxyethylpyrrole against S. aureus (3). The use of
drug combinations between natural products and antibiotics may improve the efficacy of
antibiotics against resistant bacteria (7). In addition, the synergistic interactions between
antibiotics and natural product may increase efficiency, reduce undesirable effects, increase
the stability or bioavailability of the reactive agents and obtain an adequate therapeutic effect
with relatively small doses (8).
Therefore, the efficiency of plant extracts to inhibit MRSA will be determined in this
study and the synergism between plant extracts and some antibiotic to inhibit MRSA will also
be evaluated in order to reduce the adverse effects and increase the antibiotic effects to inhibit
the resistant bacteria.
Methodology
Bacterial Strains
Methicillin resistant Staphylococcus aureus (MRSA) and Methicillin susceptible
Staphylococcus aureus (MRSA) were collected from clinical specimens sent to Clinical
Microbiology Laboratory at Thammasart University Hospital and National Cancer Institute.
These isolates were identified as S.aureus by standard microbiological method including
gram stain, catalase, coagulase and growth on mannitol salt agar. S.aureus ATCC25923 and
S.aureus ATCC43300 were also included in this study as reference strains for MSSA and
MRSA, respectively.
All isolates were screened for MRSA by using a 1 g oxacillin disk diffusion test. In
addition, Polymerase chain reaction (PCR) was performed to detect the mecA gene in MRSA
isolates (9).
Preparation of Plant Crude Extracts
Thai plants used in this study consisted of Acanthus ebracteatus Vahl, Cissus
quadrangularis L., Kaempferia parviflora Wall. ex Baker, Barleri alupulina Lindl.,
Orthosiphon aristatus (Blume) Miq., Glycyrrhiza glabra L., Stevia rebaudiana (Bertoni)
Bertoni., Clitoria bracteata Poir., Curcuma longa L., Curcuma zerumbet (Burg) Roxb.,
Zingiber cassumunar Roxb. These plants were extracted with water and 95% Ethanol. The
aqueous extracts were filtered through 0.45 m. membrane filter and stored at 4C. Ethanol
extracts were evaporated by rotary evaporator and stored at 4C.
Antimicrobial Activity of Plant Crude Extracts
Plant crude extracts were screened for their inhibitory activity against MRSA and
MSSA. The disc diffusion assay was performed by dipping a sterile cotton swab into the
diluted bacterial culture approximately108 CFU ml-1, and then the bacteria were swabbed
onto TSA plate. Sterile 6 mm filter paper discs impregnated with 20 L of sample solutions
were placed on the TSA plates. Ampicillin, Oxacillin, Cefoxitin and Tetracyclin disks were
used as control. The plates were then incubated at 35 °C for 24 h and the diameter of the
inhibition zone was measured. Zone of inhibition of plant crude extracts were compared with
controls containing corresponding solvent.
Minimum Inhibitory Concentration (MIC) was determined using a microdilution
method. MRSA and MSSA were cultured into 5ml of TSB and incubated in a shaker
incubator at 35˚C for 18-24h. The optical density (OD) of the bacteria was adjusted to the
standard of 0.5 McFarland with TSB solution to achieve a concentration of approximately 108
CFU ml-1. The plant crude extracts were dissolved in 20% dimethyl sulfoxide (DMSO).
Plant crude extracts or antibiotics were two-fold serial diluted in 100 l of TSB into 96 well
plates. 100 l of bacteria were added in each dilution and then the plate was incubated at
35˚C for 18-24 h. The MIC was taken as the minimum concentration of dilution that
inhibited the growth of bacteria. Minimum bactericidal concentration (MBC) was defined as
the lowest concentration of the compound to kill bacteria. All the wells of dilutions were used
in the MIC studies that did not show any turbidity of the bacteria were determined for MBC.
The suspension was streaked onto TSA and incubated at 35˚C for 18-24 h. The dilution not
showed single colony on TSA that was taken as MBC.
Determination of Synergism between Antibiotics and Plant Crude Extracts
Preliminary synergistic effect was assayed using disk diffusion technique. The
antibiotics used in this assay were ampicillin, cefoxitin and oxacillin. The filter paper disk
containing the plant crude extracts (20L/disc) and the different concentrations of each
antibiotic agent were placed on the TSA plates inoculated with bacteria, and the distances
were 30 mm of each disc. The plates were incubated at 37˚C for 18-24 h. The subsequent
zones of inhibition around each disk were measured in three different directions and mean
diameters were recorded.
The interaction between crude plant extracts and antimicrobial agents was estimated
by calculating the fractional inhibitory concentration (FIC index) of the combination. The
concentration of the individual compound in the combination of crude extracts and antibiotic
in which the growth of bacteria is completely inhibited is taken as the MIC of the individual
compound in the combination. The fractional inhibitory concentration was calculated as
follows:
FIC of compound 𝐚 (FICa ) =
MIC of compound 𝐚 in combination
MIC of compound 𝐚 alone
FIC of compound 𝐛 (FICb ) =
MIC of compound 𝐛 in combination
MIC of compound 𝐛 alone
The sum of fractional inhibitory concentration (FICs) indices of two compounds in the
combination was calculated as follows: FICa + FICb = FICs
Synergism was defined as an FIC index of 0.5 or less, additivity as a FIC index of
more than 0.5 and less than 4, and antagonism as FIC index of more than 4 (10).
Phytochemical Screening
Phytochemical screening of plant extracts was carried out qualitatively for the
presence of alkaloids, terpenoids, and flavonoids using standard method described by Goni,
et al. (11). Alkaloid detection was carried out by treating the extract with Meyer's and
Wagner's reagents. The samples were scored positive on the basis of turbidity. Flavonoids
were tested by treating the extract with ammonia solution. A yellow color demonstrated
positive test for flavonoids. In addition, Sulfuric acid was used for analysis of terpenoids.
Results
Antimicrobial Activity of Plant Crude Extracts
Eleven Thai plants, i.e., Acanthus ebracteatus Vahl., Cissus quadrangularis Lin,
Kaempferia parviflora, Barleria lupulina Lindl, Orthosiphon aristatus (Blume) Miq,
Glycyrrhiza glabra L. , Stevia rebaudiana Bertoni, Clitoria ternatea Linn., Curcuma longa
L.,Curcuma zedoaria (Berg.) Rosc. and Zingiber cassumunar Roxb were extracted with
water and 95% ethanol. These extracts were determined for antimicrobial activities against
S.aureus by agar diffusion method. Minimal inhibitory concentration (MIC) was also
determined using microdilution method. The results of this study showed that Kaempferia
parviflora ethanol extract possessed antibacterial activity against only MRSA with inhibition
zones of 15+3 mm (Table 1). From the screening result of the antimicrobial activity,
Kaempferia parviflora ethanol extract was evaluated for MIC and MBC determination. The
MICs and MBCs of K.parviflora ethanol extracts against MRSA and MSSA showed in Table
2.
Table 1. Inhibition zone of plant crude extracts against MRSA and MSSA
Zone of Inhibition (mm)
MSSA
Plant extracts
Acanthus ebracteatus Vahl
Cissus quadrangularis Lin
Kaempferia parviflora
Barleria lupulina Lindl
Orthosiphon aristatus (Blume) Miq
Glycyrrhiza glabra L
Stevia rebaudiana Bertoni
Clitoria ternatea Linn
Curcuma longa L
Curcuma zedoaria (Berg.) Rosc
Zingiber cassumunar Roxb
Aqueous
Extract
(5mg/ml)
-
MRSA
Ethanol extract
(5mg/ml)
-
Aqueous
Extract
(5mg/ml)
-
Ethanol extract
(5mg/ml)
15+3
-
Table 2. Minimum Inhibitory Concentration (MICs) and Minimum Bactericidal Concentration (MBCs) of the
K. parviflora ethanol extracts against MRSA and MSSA
Strains
MRSA
MSSA
MBCs (g/ml)
96.75
750
MICs (g/ml)
46.87
750
The Synergistic Activity
The synergistic effects of K.parviflora ethanol extract and antibiotics such as
oxacillin, ampicillin, and cefoxitin were tested. The results showed that inhibition zone of the
combination of K.parviflora and cefoxitin to MRSA was significantly greater than inhibition
zone of the extract or antibiotic alone (Table 3). In addition, the combination of K. parviflora
ethanol extract and Cefoxitin reduced the MIC and MBC of Cefoxitin against MRSA and
MSSA (Table 4).
Table 3 Synergistic activity between K. parviflora ethanol extract and antibiotics
compound
K. parviflora Ethanol extract
Oxacillin
Cefoxitin
Ampicillin
mixture of K. parviflora Ethanol extract and Oxacillin
mixture of K. parviflora Ethanol extract and Cefoxitin
mixture of K. parviflora Ethanol extract and Ampicillin
Zone of inhibition (mm.)
MSSA
MRSA
No inhibition zone
14
15
No inhibition zone
24
No inhibition zone
14
No inhibition zone
15
14
24
18
14
13
Table 4. Synergistic activity between K. parviflora ethanol extract and cefoxitin against MRSA and MSSA
Strain
MRSA
MSSA
compound
Cefoxitin
K. parviflora ethanol extract
Cefoxitin (in combination of K. parviflora extract)
Cefoxitin
K. parviflora ethanol extract
Cefoxitin (in combination of K. parviflora extract)
MIC (µg/ml)
23.44
46.87
1.46
1.17
750
1.17
MBC (µg/ml)
93.75
96.75
2.93
1.17
750
1.17
The interaction between K.parviflora ethanol extracts and cefoxitin against MRSA
was estimated by calculating the fractional inhibitory concentration (FIC index) of the
combination. K.parviflora ethanol extract at the concentration of MIC and MIC/2 and twofold serial dilution of Cefoxitin starting from the concentration of MIC were tested. Table 5
showed interaction effect of K.parviflora ethanol extracts and cefoxitin against MRSA. The
MICs between K.parviflora extracts and Cefoxitin against MRSA decreased from 46.87
g/ml to 23.44 g/ml and 23.44 g/ml to 1.46 g/ml respectively. FIC index calculated from
MICs of K.parviflora extracts and Cefoxitin was 0.56. The FIC index was interpreted as
additive. (Synergism  0.5, additive between 0.5 – 4, Antagonism  4).
Table 5. Interaction effect of K.parviflora ethanol extracts and Cefoxitin against MRSA
Strains
MRSA
K.parviflora
MICs
MICs
combination
(g/ml)
(g/ml)
46.87
23.44
Cefoxitin
MICs
(g/ml)
23.44
MICs
combination
(g/ml)
1.46
FIC index
Interpretation
0.56
additive
Phytochemical Screening of K.parviflora ethanol extracts
The phytochemical screening of the K.parviflora ethanol extract showed the presence
of flavonoids and terpenoids and absence of alkaloids (Table 6).
Table 6. Phytochemical screening of K.parviflora ethanol extract
Test
Flavonoids
Terpenoids
Alkaloids
K.parviflora extracts
+
+
-
Discussion and Conclusion
Resistance of MRSA strains to many antibiotics make it difficult to treat this pathogen
which is major cause of hospital associated and community acquired infections worldwide.
Several new strategies to treat MRSA have considered including the use of natural products
and synergistic effect between antibiotic and natural product extracts (12).
In this study eleven Thai plants, i.e., Acanthus ebracteatus Vahl, Cissus
quadrangularis L., Kaempferia parviflora Wall. ex Baker, Barleri alupulina Lindl.,
Orthosiphon aristatus (Blume) Miq., Glycyrrhiza glabra L., Stevia rebaudiana (Bertoni)
Bertoni., Clitoria bracteata Poir., Curcuma longa L., Curcuma zerumbet (Burg) Roxb.,
Zingiber cassumunar Roxb. were extracted with water and 95% Ethanol. The antimicrobial
activities of these crude extracts were screened by disk diffusion method. The Kaempferia
parviflora ethanol extract demonstrated highest antimicrobial zone against MRSA while the
aqueous extract did not show antimicrobial activity. This finding indicates that the active
antimicrobial compounds in these plants are most likely to be non-polar. This finding
corresponds to the phytochemical screening result that terpenoids and flavonoids are found
in the K. parviflora ethanol extract. Kaempferia parviflora are widely used in folk medicine
for different types of infectious diseases. Sopa et al. reported that the ethanol extract of K.
parviflora exhibited strong anti-fungal activity against Trichophyton rubrum, Trichophyton
mentagrophytes and Microsporum gypseum with MIC values of 62.5, 125 and 250 mg/ml,
respectively (13). In addition, Nuntaree demonstrated that Kaempferia parviflora could be
one of the effective herbs for potential prevention and treatment of H. pylori infection since
Kaempferia parviflora ethyl acetate extract inhibited the invasion of both H. pylori virulent
strains (cagA+) and non-virulent strains (cagA-) in HEp-2 cells (14).
The potential effect of combinations of different antibiotics or natural substances has
been exhaustively studied because the synergistic interaction between natural products and
antibiotic can be used to yield new products with broad spectrum biological activity (15).
Therefore, many plant extracts are expected that they will increase the synergism to several
antibiotics that bacteria resist. In this study the synergistic effects of Kaempferia parviflora
ethanol extract and antibiotics such as ampicillin, oxacillin and cefoxitin were tested. The
results showed that inhibition zone of the combination of Kaempferia parviflora ethanol
extract and cefoxitin to MRSA is greater than inhibition zone of the extract or antibiotic
alone. In addition, the combination of Kaempferia parviflora ethanol extract and cefoxitin
reduced the MIC of cefoxitin against MRSA. The results of the combination studies revealed
that Kaempferia parviflora ethanol extract showed the synergistic effect when combined with
cefoxitin against MRSA. However, further study should be performed to demonstrate the
mechanism of the synergistic effect.
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