Plants used for traditional medicine contain a wide range of substances which can be
used to treat various infectious diseases. Hence, antibacterial activities of ethanolic extracts
of 19 plant species were studied against multi-drug resistant clinical isolates using agar well
diffusion method. Extracts of Liquidambar orientalis, Vitis vinifera,?? ?????????????????????? ????????????????????,
Punica granatum, Cornus sanguinea, Euphorbia peplus, Ecballium elaterium, Inula viscosa
and Liquidambar orientalis showed broad-spectrum antibacterial activity with inhibition
zones ranging from 8 to 26 mm. The most resistant organisms were Escherichia coli (E. coli)
(Ampicillin-, amoxycillin- and sulfamethoxazole-resistant), Stenotrophomonas maltophilia
(S. maltophilia) (Amoxycillin- and nalidixic acid-resistant) and Klebsiella pneumoniae (K.
pneumoniae) (Ampicillin-, amoxycillin- and aztreonam-resistant), and the most susceptible
species were Staphylococcus aureus (S. aureus) (Penicillin G- and oxacillin-resistant),
Streptococcus pyogenes (S. pyogenes) (Penicillin G-, erythromycin- and clindamycin-resistant)
and Pseudomonas aeruginosa (P. aeruginosa) (Sulfamethoxazole- and novobiocin-resistant),
respectively. Minimum Inhibitory Concentrations (MIC) of crude extracts were determined for
the seven highly active plants showing activity against methicillin resistant S. aureus (MRSA),
E. coli, P. aeruginosa, S. pneumoniae and the reference bacteria (E. coli ATCC 11229 and
Kocuria rhizophila ATCC 9341 NA). MICs of active extracts ranged from 8 to 14.2 mg/mL
against one or other test bacteria.
Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research
(2009), 8 (4): 293-300
Received: May 2008
Accepted: January 2009
Copyright ? 2009 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Activity of Some Plant
Multi-Drug Resistant Human Pathogens
Dilek Oskay and Fatih Kalyoncu
Department of Biology, Faculty of Sciences
and Arts, Celal Bayar University, Campus of Muradiye, Manisa, Turkey.
Plants used for traditional medicine contain a
wide range of substances which can be used to treat various infectious
diseases. Hence, antibacterial activities of ethanolic extracts of 19 plant
species were studied against multi-drug resistant clinical isolates using
agar well diffusion method. Extracts of Liquidambar orientalis, Vitis
vinifera, Rosmarinus officinalis, Punica granatum, Cornus sanguinea,
Euphorbia peplus, Ecballium elaterium, Inula viscosa and
Liquidambar orientalis showed broad-spectrum antibacterial activity with inhibition
zones ranging from 8 to 26 mm. The most resistant organisms were Escherichia
coli (E. coli) (Ampicillin-, amoxycillin- and sulfamethoxazole-resistant),
Stenotrophomonas maltophilia (S. maltophilia) (Amoxycillin- and nalidixic
acid-resistant) and Klebsiella pneumoniae (K. pneumoniae) (Ampicillin-,
amoxycillin- and aztreonam-resistant), and the most susceptible species were
Staphylococcus aureus (S. aureus) (Penicillin G- and oxacillin-resistant),
Streptococcus pyogenes (S. pyogenes) (Penicillin G-, erythromycin- and
clindamycin-resistant) and Pseudomonas aeruginosa (P. aeruginosa) (Sulfamethoxazole-
and novobiocin-resistant), respectively. Minimum Inhibitory Concentrations
(MIC) of crude extracts were determined for the seven highly active plants
showing activity against methicillin resistant S. aureus (MRSA), E. coli,
P. aeruginosa, S. pneumoniae and the reference bacteria (E. coli ATCC 11229 and
Kocuria rhizophila ATCC 9341 NA). MICs of active extracts ranged from 8 to
14.2 mg/mL against one or other test bacteria.
One of the more alarming recent trends in
infectious diseases has been the increasing frequency of antimicrobial
resistance among microbial pathogens causing nosocomial and community-acquired
infections. Numerous classes of antimicrobial agents have become less effective
as a result of the emergence of antimicrobial resistance, often as a result of
the selective pressure of antimicrobial usage. Among the more important emerging
resistance problems are oxacillin resistance in staphylococci, penicillin
resistance in streptococci, vancomycin resistance in enterococci (and eventually
staphylococci), resistance to extended-spectrum cephalosporins and
fluoroquinolones in Enterobacteriaceae, and carbapenem resistance in P. aeruginosa (1). For example, in clinical isolates of
S. pneumoniae resistance to
antibiotics routinely used to treat infections is now at 40% in some European
countries. Similarly, a high level of ampicillin resistance is very significant
in E. coli, while it would be natural in most other enterobacteria. Escherichia
coli and Klebsiella spp. are the only ones generally susceptible to
narrow-spectrum cephalosporins (2). Also, MRSA, gained much attention in the
last decade, is a major cause of hospital-acquired infections (3). During the
last two decades a renewed interest in Corynebacterium species and other
non-spore-forming Gram-positive bacilli has emerged among clinicians and
microbiologists alike. Infections caused by these organisms are emerging, new
species are being recognized, and infections by toxigenic and nontoxigenic Corynebacteriumdiphtheriae strains are also being described with increasing
frequency, indeed, in countries where diphtheria had been totally or almost
Herbal medicines have been important sources of
products for the developing countries in treating common infectious diseases and
overcome the problems of resistance and side effects of the currently available
antimicrobial agents (5). The World Health Organisation (WHO) estimates that 80%
of the people living in developing countries almost exclusively use traditional
medicines. This means approximately 3300 million people use medicinal plants on
a regular basis. Medicinal plants used in traditional medicine should therefore
be studied for safety and efficacy (6).
Using plants for medicinal purposes is an important
part of the culture and the tradition in Turkey. Therefore, this in vitro study
was aimed at screening selected plants for their antibacterial activity and
evaluating their potential use in treating infections caused by multi-drug
resistant clinical bacteria.
Plant materials and preparation of the ethanolic
Plants were collected in different sites of Manisa
province and arounds of Turkey. Voucher specimens were deposited in the
Herbarium of Botany, Department of Biology, Celal Bayar University. The used
parts were leaves, stems, flowers, roots, young branches and, in some cases,
fruits (Table 1).
The plant parts were separated, washed with
distilled water, dried and then powdered finely using a blender. Thirty grams of
ground air-dried plant material were shaken in 150 mL 96% weight/volume (w/v)
ethanol (EtOH 96?) at room temperature for 60 h (180 cycles/min). The insoluble
material was filtered by filter paper (Whatman No. 4) and evaporated to dryness
in a water bath at 50?C. The extract was weighed and dissolved in EtOH 96? at a
concentration of 200 mg/mL and stored at +4?C for further experiments.
Clinical isolates of the following: bacteria MRSA
(Penicillin G- and oxacillin-resistant, and clindamycin-, vancomycin-,
erythromycin-, sulfamethoxazole- and teicoplanin-sensitive), E. coli (Ampicillin-,
amoxycillin- and sulfamethoxazole-resistant, and gentamicin-, cefuroxime-,
levofloxacin-, imipenem-, aztreonam- and netilmycin-sensitive), P. aeruginosa (Sulfamethoxazole-
and novobiocin-resistant, gentamicin- and netilmycin-intermediate, and
piperacillin-, aztreonam-, imipenem- and tobramycin-sensitive), S. maltophilia (Amoxycillin-
and nalidixic acid-resistant, and sulfamethoxazole- and levofloxacin-sensitive),
K. pneumoniae (Ampicillin-, amoxycillin- and aztreonam-resistant, and imipenem-,
netilmycin- and gentamicin-sensitive), S. pyogenes (Penicillin G-, erythromycin-
and clindamycin-resistant, and oxacillin-sensitive), S. pneumoniae (Sulfamethoxazole-
and penicillin G-resistant, and oxacillin- and lincosamine-sensitive) and
Corynebacterium sp. (Erythromycin-, vancomycin- and nalidixic acid-resistant,
and fusidic acid and clindamycin-sensitive) were kindly provided by the
Department of Medical Microbiology, Faculty of Medicine, Osmangazi University (Eskisehir/Turkey).
Also, Gram-negative Escherichia coli ATCC 11229 and Gram-positive Kocuria
rhizophila ATCC 9341 were used as reference strains for comparison of MIC and
Cultures of bacteria
All bacteria were cultured on Nutrient Agar plates, except for
S. pyogenes, K. pneumoniae and S. pneumoniae which were cultured on Blood Agar plates, and were
incubated for 24 h at 37?C. Few colonies from these cultures were inoculated
into Mueller-Hinton Broth and incubated at 37?C for 24 h before use. Nutrient
Agar (Merck) and Blood Agar were used to maintain the clinical isolates of the
Agar well diffusion assay
The assay was conducted as described by Perez et al. (7) with slight
modification according to the present experimental conditions. Bacterial strains
grown on nutrient agar at 37?C for 18 h were suspended in a saline solution
(0.85% NaCl) and adjusted to a turbidity of 0.5 MacFarland standards [106 Colony
Forming Units (CFU)/mL]. Briefly, 50 ?l inoculum was used to inoculate 90-mm
diameter petri plates containing 25 mL Mueller-Hinton Agar (MHA), with a sterile
non-toxic cotton swab on a wooden applicator. Wells with 6-mm diameter were
punched in the agar and filled with 100 ?l extract solution (4 mg/mL). The
dissolution of the organic extracts (ethanolic) was facilitated with the
addition of 5% (v/v) dimethyl sulfoxide (DMSO) which not affected the growth of
microorganisms (as shown by our control experiments). The dishes were
preincubated at 4?C for 2 h to allow uniform diffusion into the agar. After
preincubation, the plates were incubated at 37?C for 24 h. The antibacterial
activity was evaluated by measuring the inhibition zone diameter observed. In
addition, ampicillin (10 ?g) and gentamicin (10 ?g) were used as positive
control to determine the sensitivity of the strains by the disc diffusion method
(8). The experiments were performed in triplicate.
Determination of minimal inhibitory concentration
The Minimum Inhibitory Concentration (MIC) was determined for the seven highly
active plants which showed antibacterial activity against MRSA, E. coli,
P. aeruginosa, S. pneumoniae and the reference bacteria. Broth technique with
slight modification was used to determine MIC of extracts against selected test
bacteria as described by the Clinical and Laboratory Standards Institute (CLSI)
(9). In brief, the cultures were diluted in Mueller-Hinton broth at a density
adjusted to 0.5 McFarland turbidity and 0.5 mL of a bacterial suspension
containing 1.5?106 CFU/mL was added to 4.5 mL of susceptibility test broth
containing diluted extract solution which was already prepared by serial
two-fold dilution from the extract stock solution starting from 30 to 0.8 mg/mL,
in glass test tubes. Positive controls were made of broth and innoculum only.
The first row of tube served as the negative control (broth plus innoculum plus
solvent used to dilute the extracts). The contents of each tube were mixed on a
shaker at 250 rpm for 1 min and then incubated at 37?C for 24 h before being
read. MICs of ampicillin and gentamicin were used as standards determined in
parallel experiments in order to comparison. The MIC was considered the lowest
concentration of the sample that prevented visible growth. All samples were
examined in two separate experiments.
Statistical treatment of the results
The mean values were analysed with the MINITAB Release 13.20 program
statistically by the general one-way (unstacked) analysis of variance (ANOVA) to
find out the most effective plants and the most sensitive test organisms.
Results and Discussion
Antibacterial activity of nineteen plants belonging to seventeen botanical
families was evaluated in vitro against eight drug-resistant clinical isolates
and against two reference bacteria which are known to cause pneumonia, mucosal,
respiratory, skin, soft tissue and urinary tract infections in humans.
The antibacterial activity of the extracts and their potency was assessed by the
presence or absence of inhibition zone as given in Table 2. Results showed that
the most susceptible organisms were MRSA (clinical isolate) which was sensitive
to 17 extracts, P. aeruginosa and Corynebacterium sp. being sensitive to 15
plant extracts, S. pneumoniae being sensitive to 14 plant extracts, and
S. pyogenes being sensitive to 13 plant extracts. The most resistant species were
E. coli being resistant to 11 plants, S. maltophilia being resistant to 9
plants, and E. coli ATCC 11229 which was resistant to 7 plants. Maximum
inhibitions were observed with the extract of Cornus sanguinea against S. aureus
(26 mm) and that of R. officinalis against S. pyogenes (26 mm). The inhibition
zone against E. coli were produced by the extract of 8 plants, i.e. L. orientalis,
R. officinalisP. granatum, Conyza canadensis, E. peplus, Citrus
reticulata, V. vinifera and E. elaterium, in which the first and second ones
with a inhibition zone of 16 mm apperead to be highly active. However, negative
control (DMSO, 100 ?l) could not inhibit test bacteria (Table 2).
Similar report by Erdogrul on antibacterial activities of
R. officinalis leaves
showed various inhibitory effects against Gram-positive and Gram-negative
bacteria (7?16 mm inhibition zone), except the acetone extract against Yersinia
enterocolitica (10). In another study, ethanol extract of P. granatum against
P. aeruginosa, Bacillus cereus and S. pyogenes developed imhibition zones of 12, 24
and 26 mm, respectively, while Nerium oleander was found to be less active
against 14 pathogenic bacterial species (11). Our results confirm these studies.
Sensitivity of test strains, in decreasing order, was as follows:
S. aureus > P. aeruginosa > S. pyogenes > S. pneumoniae >
Corynebacterium sp.> K. pneumoniae >
K. rhizophila ATCC 9341> E. coli ATCC 11229 > S. maltophilia >
E. coli (Figure
1). Gram-negative bacteria were less sensitive than Gram-positive bacteria,
which may be due to their differences in the cell wall composition (3). It was
interesting to note that antibiotic-resistant bacteria showed more sensitivity
to the investigated plant extracts. This has clearly indicated that antibiotic
resistance does not interfere with the antibacterial action of plant extracts
and these extracts might have different modes of action on test organisms.
Most of the studied plants are potentially rich sources of antimicrobial agents.
However, the plants differ significantly in their activity against test
bacteria. The most active plants were V. vinifera, L. orientalis,
E. elaterium, P. granatum, C. sanguinea, I. viscosa,
E. peplus and Eucalyptus camaldulensis showed broad-spectrum antibacterial activity against resistant
bacteria. On the other hand, the least active plants were Pyracantha coccinea,
Lonicera japonica, Carpobrotus acinaciformis, Mirabilis jalapa, Citrus
reticulate, N. oleander and C. canadensis. However, Hypericum perforatum, Thuja
orientalis and Artemisia arborescens were moderately active plants (Figure 2).
The antibiotic susceptibility pattern of the clinical bacterial strains was
provided by Faculty of Medicine, Osmangazi University (Eskisehir/Turkey), and
only ampicillin and gentamicin were tested against test bacteria in our
laboratory. With the exception of K. rhizophila which had an inhibition zone of
22 mm, other bacteria were resistant to ampicillin (10 ?g/disk), indicating
their multi-drug resistance phenotype. We could not use these antibiotics as
therapeutic agents to treat diseases caused by the reference bacteria. A
comparision on the inhibition zones of the pathogenic bacteria showed that
gentamicin was effective against all ten bacterial species tested.
Significant antibacterial effects, expressed as MIC of crude extracts, were
observed against MRSA, E. coli, P. aeruginosa and S. pneumoniae (Table 3). The
maximal inhibition zones and MIC values for bacterial strains, which were
sensitive to the plant extracts were in the range of 14-26 mm and 13.4?10.2 mg/mL,
respectively. Extracts of selected plants were among the most active with the
MIC values ranging from 8.0-14.2 mg/mL. Among the plants tested, ethanolic
extract of R. officinalis and V. vinifera showed very strong activity against
MRSA with the best MIC (8.6 and 9.4 mg/mL, respectively). The lowest MIC
obtained with V. vinifera and I. viscosa extract, was 8.0 mg/mL for
P. aeruginosa, whereas the highest MIC was 14.2 mg/mL for V. vinifera and
L. orientalis extracts against E. coli ATCC 11229. MIC values of
R. officinalis, L. orientalis and P. granatum extracts for
E. coli were 8.6, 10.2 and 11.8 mg/mL,
Plant extracts have been studied against bacteria for years in the last three
decades. During this period, a lot of antimicrobial screening evaluations have
been published based on the traditional use of Turkish plant (15, 16, 19). Yet,
a comparative study of the MIC of plant extracts against drug-resistant
bacterial isolates have not been previously reported. Also, little information
is available about the activity of plants against drug-resistant hospital
isolates. Many previous researchers (13, 14, 17) reported the antibacterial
activity of medicinal plants but their findings were different from those of
present study. This discrepancy could be due to differences in the plants
physiological state, seasonal variation, environmental condition, studied part
of the plants, extraction procedure, concentration of crude extracts and strains
of test bacteria.
Several studies have shown that the occurrence of resistance is closely related
to the medical use of a drug, even though the association may be variable. This
association has also been demonstrated for antimicrobial agents used for growth
promotion. Also, in the hospital environment, antimicrobial use plays an
essential role in the emergence of resistant bacteria causing the spread of
resistant clones (2). Staphylococci are an important cause of both nosocomial
and community-acquired infections. In the last decade, staphylococcal infection
has reemerged as a cause for concern because of its numerical increase, the
spread of MRSA isolates in the community, and the emergence of isolates not
susceptible to vancomycin (18).
With the increase in resistance of microorganisms to the currently used
antibiotics and the high cost of production of synthetic compounds,
pharmaceutical companies are now looking for alternatives. Medicinal plants
could be those alternatives because most of them are safe with little side
effects if any, cost less, and affect a wide range of antibiotic resistant
microorganisms (11, 19).
The demand for new effective antimicrobials is urgent and of great importance in
the clinical health. Allied with this demand is the need for assays to detect
new and previously undiscovered antimicrobials from plant sources. From this
study, the plant extracts were found to have antibacterial activity against
drug-resistant clinical bacteria. However, to explain the mode of action, the
active phytocompounds of these plants used against multidrug-resistant bacteria
and their toxicity have to be determined by additional studies.
In conclusion, all of the plant extracts tested in this study had potential
antibacterial activities against the reference strains. Our results support the
use of these plants in traditional medicine and suggest that some of the plant
extracts possess compounds with good antibacterial properties that can be used
as antimicrobial agents in the search for new drugs.
We wish to express our profound gratitude to Prof. Dr. G?l Durmaz and Dr. Askin
Derya Aybey of the Department of Medical Microbiology, Faculty of Medicine,
Osmangazi University (Eskisehir/Turkey) for providing clinical bacteria and
Osman G?k for collecting some of the plants.
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