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Original Article
Activity of Some Plant Extractsof Some Plant ExtractsSome Plant ExtractsPlant ExtractsExtractsAgainst Multi-Drug Resistant Human Pathogens-Drug Resistant Human PathogensPathogens
Mustafa Oskay
*
, Dilek Oskay and Fatih Kalyoncu
 Department of Biology, Faculty of Sciences and Arts, Celal Bayar University, Campus of Muradiye, Manisa, Turkey.
Abstract
Plants used for traditional medicine contain a wide range of substances which can beused to treat various infectious diseases. Hence, antibacterial activities of ethanolic extractsof 19 plant species were studied against multi-drug resistant clinical isolates using agar welldiffusion 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 inhibitionzones 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 susceptiblespecies were
Staphylococcus aureus
(
S. aureus
) (Penicillin G- and oxacillin-resistant),
Streptococcus pyogenes
(
S. pyogenes
) (Penicillin G-,erythromycin- and clindamycin-resistant)and
 Pseudomonasaeruginosa
(
 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). MICs of active extracts ranged from 8 to 14.2 mg/mLagainst one or other test bacteria.
Keywords
: Antibacterial activity; Clinical isolate; Drug-resistant; Medicinal plants;Turkish.
Introduction
One of the more alarming recent trends ininfectious diseases has been the increasingfrequency of antimicrobial resistance amongmicrobial pathogens causing nosocomial andcommunity-acquired infections. Numerousclasses of antimicrobial agents have becomeless effective as a result of the emergence of antimicrobial resistance, often as a result of theselective pressure of antimicrobial usage. Amongthe more important emerging resistance problemsare oxacillin resistance in staphylococci, penicillin resistance in streptococci, vancomycinresistance in enterococci (and eventuallystaphylococci), resistance to extended-spectrum
Enterobacteriaceae, and carbapenem resistancein
 P. aeruginosa
(1). For example, in clinicalisolates of 
S. pneumoniae
resistance to antibioticsroutinely used to treat infections is now at 40%
* Corresponding author:E-mail:mustafa.oskay@bayar.edu.tr 
Copyright © 2009 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health ServicesIranian Journal of Pharmaceutical Research (2009), 8 (4): 293-300Received:May 2008Accepted:January 2009
 
Oskay M, Oskay D and Kalyoncu F / IJPR (2009), 8 (4): 293-300
in some European countries. Similarly, a high

in
 E. coli
, while it would be natural in most other enterobacteria.
 Escherichia coli
and
 Klebsiella
spp. are the only ones generally susceptibleto 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 renewedinterest in
Corynebacterium
species and other non-spore-forming Gram-positive bacilli hasemerged among clinicians and microbiologistsalike. Infections caused by these organisms areemerging, new species are being recognized,and infections by toxigenic and nontoxigenic
Corynebacterium diphtheriae
strains are also being described with increasing frequency,indeed, in countries where diphtheria had beentotally or almost eradicated (4).Herbal medicines have been importantsources of products for the developing countriesin treating common infectious diseases andovercome the problems of resistance and sideeffects of the currently available antimicrobialagents (5). The World Health Organisation(WHO) estimates that 80% of the people livingin developing countries almost exclusively usetraditional medicines. This means approximately3300 million people use medicinal plants on aregular basis. Medicinal plants used in traditionalmedicine should therefore be studied for safety

Using plants for medicinal purposes is animportant part of the culture and the tradition inTurkey. Therefore, this in vitro study was aimedat screening selected plants for their antibacterialactivity and evaluating their potential use intreating infections caused by multi-drug resistantclinical bacteria.
Experimental
 Plant materials and preparation of theethanolic extracts
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,

cases, fruits (Table 1).The plant parts were separated, washedwith distilled water, dried and then powdered

air-dried plant material were shaken in 150 mL96% weight/volume (w/v) ethanol (EtOH 96°) atroom temperature for 60 h (180 cycles/min). The

(Whatman No. 4) and evaporated to dryness ina water bath at 50°C. The extract was weighedand dissolved in EtOH 96° at a concentrationof 200 mg/mL and stored at +4°Cfor further experiments.
 Bacterial strains
Clinical isolates of the following: bacteriaMRSA(Penicillin G- and oxacillin-resistant,andclindamycin-, vancomycin-, erythromycin-,sulfamethoxazole- and teicoplanin-sensitive)
 , E. coli
(Ampicillin-, amoxycillin- andsulfamethoxazole-resistant, and gentamicin-,
cefuroxime-,

, imipenem-, aztreonam-
and netilmycin-sensitive),
 P. aeruginosa
(Sulfamethoxazole- and novobiocin-resistant,gentamicin- and netilmycin-intermediate,andpiperacillin-, aztreonam-, imipenem-and tobramycin-sensitive),
S. maltophilia
(Amoxycillin- and nalidixic acid-resistant, andsulfamethoxazole- and

-sensitive),
 K. pneumoniae
(Ampicillin-, amoxycillin- andaztreonam-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, andfusidic acid and clindamycin-sensitive) werekindly provided by the Department of MedicalMicrobiology,Faculty of Medicine, OsmangaziUniversity(Eskisehir/Turkey). Also, Gram-negative
 Escherichia coli
ATCC 11229 andGram-positive
 Kocuria rhizophila
ATCC 9341were used as reference strains for comparison of MIC and inhibition zones.
Cultures of bacteria
All bacteria were cultured on Nutrient Agar  plates, except for 
S. pyogenes
,
K. pneumoniae
 
 Antimicrobial Activity of Medicinal Plants
and
S. pneumoniae
which were cultured onBlood Agar plates, and were incubated for 24h at 37°C. Few colonies from these cultureswere inoculated into Mueller-Hinton Broth andincubated at 37°C for 24 h before use.NutrientAgar (Merck) and Blood Agar were used tomaintain the clinical isolates of the bacteria.
 Agar well diffusion assay
The assay was conducted as described by

to the present experimental conditions. Bacterialstrains grown on nutrient agar at 37°C for 18 hwere suspended in a saline solution (0.85% NaCl)and adjusted to a turbidity of 0.5 MacFarlandstandards [10
6
Colony Forming Units (CFU)/mL].

90-mm diameter petri plates containing 25 mLMueller-Hinton Agar (MHA), with a sterilenon-toxic cotton swab on a wooden applicator.Wells with 6-mm diameter were punched in the

mg/mL). The dissolution of the organic extracts(ethanolic) was facilitated with the addition of 5% (v/v) dimethyl sulfoxide (DMSO) whichnot affected the growth of microorganisms (asshown by our control experiments). The disheswere preincubated at 4°C for 2 h to allow uniformdiffusion into the agar. After preincubation, the plates were incubated at 37°C for 24 h. Theantibacterial activity was evaluated by measuringthe inhibition zone diameter observed. In

the sensitivity of the strains by the disc diffusionmethod (8). The experiments were performed intriplicate.
 Determination of minimal inhibitoryconcentration
The Minimum Inhibitory Concentration(MIC) was determined for the seven highlyactive plants which showed antibacterialactivity against MRSA,
 E. coli
,
 P. aeruginosa
,
S. pneumoniae
and the reference bacteria. Broth

to determine MIC of extracts against selectedtest bacteria as described by the Clinical and
Studied plants Family Plant part (s) Rate Collection time Origin
 Nerium oleander 
L.ApocynaceaeLF
a
, FL2:124/06/2005Campus
 Pyracantha coccinea
M. Roem.RosaceaeLF, RF2:124/06/2005Botanical Garden (BG)
Cornus sanguinea
L.CornaceaeLF, FL, S2:1:125/06/2005Campus
 Artemisia arborescens
L.CompositaeLF, FL, S2:1:125/06/2005BG
Thuja orientalis
L.CupressaceaeLF, RF2:124/06/2005BG
Carpobrotus acinaciformis
(L.) L. BolusAizoaceaeLF, S2:127/06/2005BG
 Punica granatum
L.PunicaceaeLF, FL2:128/06/2005BG
Conyza canadensis
(L.) Cronquist.CompositaeLF07/07/2005BG
Mirabilis jalapa
L.NyctaginaceaeLF, FL, S2:1:126/06/2005BG
 Euphorbia peplus
L.EuphorbiaceaeLF, FL, S, RT2:1:1:108/07/2005Campus, Yagcýlar 
Citrus reticulata
L.RutaceaeLF, RF2:106/07/2005BG
Vitis vinifera
L.VitaceaeLF, RF, S, YB2:1:1:124/06/2005BG
 Liquidambar orientalis
Mill.HamamelidaceaeLF24/06/2005BG
 Inula viscosa
(L.) Aiton.CompositaeLF10/07/2005Muradiye
 
L.LamiaceaeLF27/06/2005Campus
 Hypericum perforatum
L.GuttiferaeLF, FL, S2:1:127/06/2005Campus, Yagcýlar 
 Lonicera japonica
Thunb.CaprifoliaceaeLF27/06/2005BG
 Ecballium elaterium
A. RichardCucurbitaceaeLF, FR1:130/06/2005Yagcýlar 
 Eucalyptus camaldulensis
Dehnh.MyrtaceaeLF05/07/2005Campus
a

Table 1.
List of the studied plants.
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