Professional Documents
Culture Documents
ISSN 1517-8382
Mattana, C.M.1*; Satorres, S.E.1; Sosa, A.2; Fusco, M.2; Alcaráz L.E.1
1
Area Microbiologia, Facultad de Química, Bioquímica y Farmacia. Universidad Nacional de San Luis, Chacabuco y Pedernera,
5700 San Luis, Argentina; 2Area de Farmacognosia. Facultad de Química, Bioquímica y Farmacia. Universidad Nacional de San
Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina.
Submitted: June 23, 2009; Returned to authors for corrections: September 04, 2009; Approved: February 18, 2010.
ABSTRACT
Antibacterial activity of organic and aqueous extracts of Acacia aroma was evaluated against methicillin-
resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA) and
methicillin-resistant Staphylococcus epidermidis. Inhibition of bacterial growth was determined using agar
diffusion and bioautographic methods. Among all assayed organic extracts only ethanolic and ethyl acetate
extracts presented highest activities against all tested Staphylococcus strains with minimal inhibitory
concentration (MIC) values ranging from 2.5 to 10 mg/ml and from 2.5 to 5 mg/ml respectively. The
aqueous extracts show little antibacterial activity against Staphylococcus strains. The bioautography assay
demonstrated well-defined growth inhibition zones against S. aureus in correspondence with flavonoids
and saponins. A. aroma would be an interesting topic for further study and possibly for an alternative
treatment for skin infections.
*Corresponding Author. Mailing address: Area Microbiología, Facultad de Química, Bioquímica y Farmacia. Universidad Nacional de San Luis, Chacabuco y
Pedernera, 5700 San Luis, Argentina.; Tel: +54-02652-423789/424689 Int.127 Fax: +54-02652-431301.; E-mail: cmmattan@unsl.edu.ar
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
practice in the popular medicine of most cultures, although the methicillin-resistant S. aureus (MRSA), methicillin-sensitive S.
precise cause of disease and mechanism of cure is not always aureus (MSSA) and methicillin-resistant S. epidermidis
understood. Staphylococcus aureus and Staphylococcus isolates in infected skin wounds. The extracts with the highest
epidermidis are ubiquitous colonizer of the skin and mucous antibacterial effectiveness could be of therapeutic usefulness
membranes of humans and animals. Many strains of S. aureus for subsequent application in pharmaceutical formulations
carry resistance genes for penicillin, tetracyclines, methicillin against Staphylococcus spp.
and now, vancomycin. Methicillin-resistant Staphylococcus
aureus (MRSA) is a major pathogen causing nosocomial MATERIALS AND METHODS
MRSA) cause infections in healthy individuals without January 2007, in the Northwestern region of the Province of
predisponent risk factor and outside the hospital setting. MRSA San Luis, Argentina. Acacia aroma (Fabaceae) was
and CA-MRSA presents a significant threat to public health authenticated by Dr. Del Vitto, Botany Department, San Luis
and are difficult to manage (14). Vancomycin and other National University (UNSL). A voucher specimen was
glycopeptides antibiotics are the current mainstay of therapy deposited in the herbarium of UNSL under the number 487.
for infections caused by MRSA. However, therapy with The parts used were leaves.
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
followed: I-Local clinical isolates: methicillin resistant activity was expressed as the mean of inhibition diameters
Staphylococcus aureus (n=20), methicillin sensitive S. aureus (mm) produced.
(n=18), methicillin resistant Staphylococcus epidermidis
(n=16) obtained from skin diseases. The written informed Minimal inhibitory concentration (MIC)
consent was obtained from all subjects prior to the study. II- Serial agar macro-dilution method: The tests were
Reference strains: Staphylococcus aureus ATCC 29213, performed in MHA medium. After cooling and drying, the
Staphylococcus aureus ATCC 43300 and Staphylococcus plates were inoculated with swabs containing each bacterial
epidermidis ATCC 12228. cell suspension (1 × 108 CFU/ml). Then, the surface of the agar
The strains were identified by the use of Biochemical was punched with 6-mm-diameter wells. Serial two-fold
profiles according to the recommendations of the Manual of dilution of each extract were added in each well in a
Clinical Microbiology (15). All organisms were maintained in concentration ranging from 20 mg/ml to 2.5 mg/ml. The plates
brain-heart infusion (BHI medium) containing 30% (v/v) were incubated aerobically for 24 hours at 37°C. A growth
glycerol at −20°C. The inocula were prepared by adjusting the control of each tested strain was included.
turbidity of the suspension to match the 0.5 Mc Farland. MIC was defined as the lowest concentration of A. aroma
extract in which no colony was observed after incubation
Antibacterial testing
Antibacterial activity of the crude organic extracts and Phytochemical screening
aqueous extract were determined by the modified agar well Characterization of chemical metabolites:
diffusion method of Perez et al. (19). The different organic • Detection of glucides: Regular methods were
extracts were dissolved in dimethylsulfoxide (DMSO) and the carried out using developers: Molisch reactive ( -
aqueous extracts were dissolved in water. The extracts were Naftol and H2SO4) for the detection and
sterilized by filtration through a 0.2 membrane filter. differentiation of glucides. Positive result was
indicated by purple coloring.
Agar diffusion assay • Detection of tannin: 5ml of plant extract was
The agar well diffusion method was employed. Mueller- evaporated and the remains were dissolved in 10
Hinton agar (MHA, Laboratorios Britania, Argentina) 25 ml ml of distilled water. The aqueous extract was
was poured into each petri plate. Once the agar solidified, the divided into three parts: 1-2 drops of 10% ferric
microorganisms were inoculated on the surface of the plates (1 chloride solution were added to one of the parts, a
8
× 10 CFU/ml). Subsequently, the surface of the agar was blue coloring indicated the possible presence of
punched with a 6-mm-diameter wells. Each well was filled hydrolysable tannin. The remaining parts were
with 50 l of each plant extract. The concentration of the poured with gelatin, and lead acetate solution at
extracts employed was 20 mg/ml. Simultaneously, gentamicin 10%. The occurrence of white precipitate
sulfate was used as positive control (1µg per well). Control indicated a positive result for the tannin test.
wells containing the same volume of DMSO, diethyl acetate, • Detection of flavonoids: For the visualization of
ethanol, petroleum ether, dichloromethane, and distilled water flavonoids UV light was used, previously
were made. After a 24-hour incubation at 35ºC, all plates were exposing the specimen to ammonia vapors
observed for zones of growth inhibition, and the diameter of (ionization in basic medium) giving an intense
these zones was measured in millimeters. yellow coloring for positive results.
All tests were performed in duplicate and the antibacterial • Detection of saponins: The reaction was
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
• performed in a test tube evaporating 5 ml of the heat. Three micro liters of rutine, quercetin, oleanolic acid and
extract, then it was shaken vigorously, and left to ß-amirin were used as standards. TLC plates were dried
settle for a period of 15 to 20 min. The presence overnight in a sterile room for complete removal of solvent.
of saponins was classified by the height and
permanence of the foam formation, being Bioautography
observed for 5 min. Developed TLC plates were covered with 1-2 mm layer of
• Detection of alkaloids: 30 ml of the plant extract soft medium (BHI agar 0.6%) containing 0.1 % (w/v) 2,3,5
was dried, and then, adding 5 ml of hydrochloric triphenyltetrazolium chloride (tetrazolium red) and a
acid at 10% to it. After filtering, some drops of suspension of S. aureus at a final concentration of 107
alkaloid precipitation reactive were added. A CFU/ml. The plates were placed in a sterile tray, sealed to
slight turbidity or precipitate colored proves the prevent the thin agar layer from drying, and incubated at 37°C
possible presence of alkaloids. for 24 h. Where bacterial growth has been inhibited, an
The determinations were done according to the uncolored area can be seen on the deep pink-red background.
recommendations of the Manual of Farmacognosis (12). The plates were run in duplicate.
Table 1. Antibacterial activity of organic and aqueous extracts of Acacia aroma by agar diffusion method.
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
Table 2. The MIC values of ethyl acetate and ethanolic extracts of A. aroma against strains of Staphylococcus aureus and
Staphylococcus epidermidis. Results are the mean of MIC values followed by the standard deviation.
Microorganisms n MIC (mg/ml) MIC (mg/ml)
ethyl acetate extract ethanolic extract
Methicillin-sensitive S. aureus 18 2.5±1.1 2.5±1.0
Methicillin-resistant S. aureus 20 2.5±1.7 2.5±1.4
S. aureus ATCC 43300 1 10.0±0.1 2.5±0.0
S. aureus ATCC 25923 1 10.0±0.0 2.5±0.1
Methicillin-resistant S. epidermidis 16 10.0±0.6 5.0±0.4
S. epidermidis (ATCC 12228) 1 10.0±0.0 5.0±0.2
n: strain number; results are the mean of inhibition zone diameter followed by the standard deviation.
Methicillin resistant S. aureus and methicillin resistant S. ethyl acetate and ethanolic extracts were analyzed by TLC on
epidermidis strains which are already known to be silica gel. TLC plates were run in duplicate and one set was
multirresistant to drugs were highly susceptible to ethyl acetate used as the reference chromatogram, and the other set was
and ethanolic extracts. Likewise, methicillin susceptible S. assayed for bioautography. TLC analysis revealed the presence
aureus strains were also inhibited by both extracts; that is to of flavonoids: rutine (Rf of 0.71) and quercetin (Rf of 0.94) and
say, the Staphylococcus spp. susceptibility to the most active sapogenines: oleanolic acid (Rf of 0.87) and amirine (Rf of
extracts of A. aroma did not correlate with the susceptibility or 0.50). This is in agreement with observations by other authors
resistance to oxacillin, in agreement with Arias et al. (3). The bioautography assay for qualitative antibacterial
To obtain some information on the active components, activity detection demonstrated well-defined inhibition zones
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
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Mattana, C.M. et al. Antibacterial activity of extracts of A. aroma
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