Bacterial resistance to antibiotics is a serious global problem and includes strains of beta-lactam-resistant Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Novel antimicrobials and/or new approaches to combat the problem are urgently needed. The aim of this study was to investigate the antimicrobial activity of alcoholic and aqueous extract of 23 medical plants species of Golestan province on clinical and standard stains of MRSA and MSSA. Twenty three medicinal plants were collected from their natural habitat in Golestan province in north of Iran. Their ethanolic and aqueous extract obtained by percolation methods. Antibacterial effects were assessed by disk diffusion method and the Minimum Inhibitory Concentration (MIC) of the extracts was determined by the micro broth dilution against 14 clinical and standard strains of methicillin resistant and sensitive of Staphylococcus aureus. The ethanolic and aqueous extract of 8, plants showed best anti staphylococcal effect, respectively. The ethanolic extract of Artemisia, herbaalba, Nigella sativa, Punica granatum, possed the most outstanding in vitro antibacterial activity which the maximum inhibition zone was 22.4-18 mm, respectively and the lowest MIC values was measured in Punica granatum, as 0.01 mg mL-1 against MRSA The results showed that ethanolic extract had better antibacterial effect than aqueous extract and anti staphylococcal activity of Ethanolic extract of plants against MRSA was better than MSSA strains. Ethanolic and aqueous extract of Punica granatum had the best antibacterial activity against the tested microorganisms. The result obtained from these plants might be considered sufficient for further studies.
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Staphylococcus aureus is a major Human pathogen responsible for a wide spectrum disease. It is an important cause of community and hospital acquired infections (Edine et al., 2004).
It is an organism commonly found in the nose and on the skin of healthy people. It can cause infection with clinical manifestations ranging from pustules to sepsis and death. Approximately 25 to 30% of the population is colonized in the nose with staph bacteria at a given time (North Dakota Department of Health, 2005).
According to WHO reports increase of antibacterial resistance is a growing problem in many countries (North Dakota Department of Health, 2005). And S. aureus strains acquired infections in the hospital are often resistant to antibiotics and soon after methicillin was introduced in to clinical use, methicillin-resistant S. aureus (MRSA) strains were reported over the last three decades and it was problem in hospital through the world (Akinyemi et al., 2005).
S. aureus first acquired resistance to methicillin and other penicillin-based antibiotics in the 1960 in England and emerged in the United States in the mild 1980 s (North Dakota Department of Health, 2005). MRSA strains are frequently resistant to many different classes of antimicrobial drugs and Many investigators have reported an increase in the incidence of MRSA during recent years (Edine et al., 2004). Most of which originated from wounds (North Dakota Department of Health, 2005). MRSA is responsible for approximately 25% of nosocomial infections (Shopsin and Kreswirth, 2001). Also recent study of isolates obtained in the United States reports that 41-43% of S. aureus isolates are methicillin-resistant (Drew et al., 2000).
The data were collected from January 1999 through December 2002, in Europe show that MRSA prevalence varied from <10% in northern Europe to >10% in southern and western Europe (Edine et al., 2004).
Study in Iran show that 9.9% of all S. aureus isolated from patients and health care workers was MRSA and the rate of MRSA and antibacterial resistance was higher in the S. aureus isolated from clinical infection (25%), compared with those from carries (7.9%) and in another study in Iran prevalence of MRSA strains in health care workers was 11.8%. The control of MRSA has become a significant problem in hospital (Mansouri and Khaleghi, 1997).
Medical plants have been used for centuries as remedies for human disease because they contain components of therapeutic value and source of both traditional and modern medicine and by increasing resistance of pathogenic bacteria to antibiotics in last few decades, many new compounds as a substitute for non- effective antibiotic. Compounds and extracts of the plants could be part of these substitutes. In this study we assessed efficacy of 23 medical plants that obtained in Golestan province in north of Iran against 14 standard and local isolated MRSA and MSSA strains.
MATERIALS AND METHODS
Plants: Fresh plant materials of 23 plants species commonly used in folk medicine in Iran were collected from natural habitat in Golestan Province in north of IRAN. Mature plants and their parts were collected from different places during the months of April-May 2005. Their botanical identities were determined and authenticated Samples were deposited in the Department Herbarium. The plants used in study were as follows (Table 1).
Ethanol extract: Plants parts were dried, ground to find texture and after which it, 70% ethanol were added to 50 g of dried plants powder in decanter for extended periods and the resultant extracts were obtained in period of 24 h. The resultant extracts were concentrated, under reduced pressure finally each samples were diluted with propylene glycol for provide 4 concentrations: 200, 100, 50 and 25 mg mL-1 (Dulgar and Gonuz, 2004; Mashhadian and Rakhshandeh, 2004; Murphy, 1999).
Aqueous extract: Hundred millilitre of hot sterile distilled water, 70-80°C, was added to the 30 g powder samples which were allowed to soak for 24 h in water bath at 45-50°C. The extracts were filtered by using filter- paper and the resultant extracts were transformed to sterile glass dishes. Finally obtained 4 dilutions: 1, 1/2, 1/4 and 1/8 (Mashhadian and Rakhshandeh, 2004).
Bacterial strains: The S. aureus strains used in this study were clinical isolates from patients presenting of S. aureus associated disease. The isolates were identified by standard method. The MRSA isolated were identified by screening tests were done on Mueller Hinton Agar (MH agar) supplemented with 4% NaCl and the oxacillin disc contain 1 mg mL-1 placed on it for isolating MRSA (Roberts et al., 2002).
|Ethonbotanic data of studied plants
Finally we obtained 8 MRSA strains and 4 MSSA strains from patients. Two standard strains, ATCC 25923 (MRSA) and PTCC 1341 (MSSA), that obtained from Scientific and Technological Research center in Iran, also were used in this study.
The organisms were maintained on agar slope at 4°C and sub-cultured for 24 h before use.
Bacterial susceptibility testing
Disk diffusion method: sterile paper Blank disk previously soaked in different concentration of ethanolic extract (200, 100, 50, 25 mg mL-1) with the final amount of extract in disks: 4, 2, 1 and 0.5 mg per disk and disks previously soaked in different dilution of aqueous extract with specific amount of it were prepared too. Mueller Hinton Agar plates were cultured with a standardized inoculums(1-2x108 cfu mL-1 equal to 0.5 macfarland) of each bacterial strain then the blank disks contain specific amount of extracts were carefully placed at the labeled seeded plate (Nostro, 2000).
The plates were incubated aerobically at 37°C and examined for zones of inhibition after 24 h. The inhibition zones were measured with a ruler and compared with the control disks (disk containing only propylene glycol that used as diluents of ethanolic extract and disk containing vancomycin 30 mg as positive control) (El Astal et al., 2005). Each test was repeated 3 times and means inhibition zone were recorded. Inhibitory zone ≥12 mm used as good inhibitory effect of extract (Nostro et al., 2000).
Micro dilution broth method: The ethanolic extracts of plants that showed an inhibition zone≥12 mm in disk diffusion method were chosen to assay the Minimum Inhibitory Concentration (MIC) with the Broth micro dilution method. Two fold serial dilutions of extracts were obtained with propylene glycol at a final concentration ranging from 200 to 0.01 mg mL-1. Hundred microliter of each diluted extract and 100 mL of each bacterial suspension on Mueller Hinton broth (final inoculums of 105 bacteria mL-1), were added in to each Elisa wells. The bacterial suspensions were used as negative control and vancomicin 30 mg were used as positive control. The OD of each well, were determine in 630 nm, by Elisa reader instrument, after 24 h of incubation at 37°C. the lowest concentration of the extracts in the wells that dont showed any addition on OD after this time, signed as the MIC value (National Committee for Clinical Laboratory Standards, 1993; Thornsberry and Dougal, 1983). Each test was assayed in triplicate.
The results of antibacterial activity of the ethanolic extracts of these plants revealed that, the high concentration only 8 ethanol extracts (4 mg mL-1) of the 23 plants had good inhibitory effect against Staphylococcal strains with inhibition zones between 12.2 to 22.5 mm and the largest zone belong to punica granatum (22.5 mm).
|Inhibition zone (mm) of ethanolic and aqueous extract of 23 plants at various concentration on MRSA
|Inhibition zone (mm) of ethanolic and aqueous extract of 23 plants at various concentration on MSSA
|MIC value of effective ethanolic plants extracts on MRSA and MSSA
|Percent activity of effective ethanolic plants extract on MRSA and MSSA
|Percent activity of effective aqueous plants extract on MRSA and MSSA
Seven out of 8 plants with good anti Staphylococcal effect, means Hypericum perforatum, Nigella sativa, Peganum harmalla, Punica granatum, Eucalyptys global, Berberis vulgaris and Tamarix aphylla showed good antibacterial activity against all MRSA and MSSA strains but Artemisia herbaalba had very strong antibacterial activity against MRSA but low activity against MSSA strain (Table 2 and 3).
Although the best inhibition action of these plants were seen in 4 mg mL-1 but in Hypericum Perforatum the best effect were seen in 0.5 mg mL-1 (Table 2 and 3) among 23 medical studied plants only The aqueous extract of 3 plants: Punica granatum, Eucalyptys global and Artemisia herbaalba have good inhibition zone against both MRSA and MSSA and Punica granatum had the best antibacterial activity and having inhibition zone 16.7 mm (Table 2 and 3).
Data in Table 2 and 3 clearly illustrates that ethanolic extracts of 8 plants and aqueous extract of 3 plants had a broad action against most of the tested microorganisms. All of the other extracts at one or more tested concentrations showed some activity against one or more microorganisms.
The results of MIC tests in Table 4 were shown that: the highest MIC values were measured as 0.78 mg mL-1 in Berberis vulgaris against MRSA with 12.5 mm DIZ (Diameter Inhibition Zone) and the lowest MIC values were measured in Punica granatum, as 0.01 mg mL-1 against MRSA (Table 4).
Percent activities of each effective plant extract were calculated. Ethanolic extract of E. global, P. granatum, P. hermala and aqueous extract of P. granatum had a 100% activity on MRSA and MSSA (Table 5 and 6).
The potencies of traditional antibiotics are decreasing steadily since drug-resistant bacteria are globally increasing.
MRSA poses and increasingly serious health care problem in many parts of the world. Several studies have reported increased morbidity and mortality associated with MRSA compared to Methicillin-suceptible S. aureus (MSSA) infections.
Medical plants have been used for a wide variety of purposes for many thousand of years in countries. The increasing use of plant extracts in the pharmaceutical industries suggests that, in order to find active compounds of medical plants is very important.
Recently, the antimicrobial activity of various plant extracts has been studied on the growth of many microorganisms.
Duglar and Gonuz (2004) observed antimicrobial activity of ethanolic extracts of 16 Turkish plants against nine bacterial species using the disk diffusion method. Of the 16 plant tested, ten showed antimicrobial activity and S. aureus is more susceptible to the extracts. Although, the results of the study on 6 Nigerian medical plants indicated that both ethanol and aqueous extracts of four out of six medical plants showed good activity against MRSA strains (Akinyemi et al., 2005) and the antibacterial activity of aqueous, ethanolic and phenolic extracts from three Palestinian medical plants in addition to their commercial oils against ten pathogen microorganisms, among the 10 tested microorganisms S. aureus was, the most susceptible microbe to most extracts of the three plants studied (El Astal et al., 2005). We here report a comparative study on the antimicrobial properties of two different extracts of 23 plants in order to choose that which gives the most efficient anti microbial compounds.
The results obtained in this study indicate a considerable difference in antimicrobial activity between ethanolic and aqueous extracts.
Both aqueous and ethanol extract of P. granatum, A. herbaalba and E. global were effect on MRSA strains. Ethanolic and aqueous extract of P. granatum had the best antibacterial activity against microorganisms. This activity was more pronounced against MRSA strains than MSSA.
The results obtained from ethanolic and aqueous extracts were compared, it was determined that the ethanolic extracts have higher antibacterial activity and MRSA strains were more susceptible to the extracts (p<0.05).
The plants differ significantly in their activity against tested microorganisms (MRSA and MSSA). This differences may attributed to fact that photochemical properties and differences a many species and for the evaluation of plants which are naturally grown in Golestan province are potential useful resources, additional studies will be beneficial from medicinal and economic stand point.
In conclusion, ethanolic, especially the extracts of P. granatum, can be used for protection against MRSA and MSSA strains.
It seems important to recommend that, further studies using isolated constituents instead of whole extract must be done in this field.
I would like to express my deep thanks to laboratories in Gorgan for providing us with S. aureus isolates also Medical Plants Co. Niak Cytopharma Laboratory and Golestan University of Medical Sciences for help in this study.
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