Antibacterial activity of Quercus infectoria extracts against bacterial isolated from wound infection

This study determines the nature of microbial wound colonization in191 patients with wound infection attending Internal Lab of Teaching Hospital and Emergency Hospital in Erbil city during the period 1-January-2007 to31-July-2007.A total of 241 bacterial isolates were identified after culturing the swabs on different culture media. The results indicated that the most frequent isolates were Staphylococcus aureus (32.78%), Pseudomonas aeruginosa (24.90%) , Escherichia coli(14.94%), Enterobacter spp. (9.96%), Proteus mirabilis (8.71%), Klebsiella pneumoniae (6.64%) ,Klebsiella oxytoca (1.24%) and Citrobacter freundii (0.83%). Most of the isolates showed high level resistance to commonly available antibiotic. The present study also undertaken to assess the antimicrobial effect of aqueous,methanol and ethanol extracts of Quercus infectoria on the isolated bacterial species.The minimum inhibitory concentration (MIC) of ethanolic extract on Staphylococcus aureus was 3.125 mg/ml and that effect on Escherichia coli,Klebsiella pneumoniae and Citrobacte freundii were 6.25 mg/ml while the aqueous, methanolic and ethanolic extracts had effect at 25.0,12.5 and 6.25 mg/ml respectively on Pseudomonas aeruginosa Proteus mirabilis and Klebsiella pneumonia respectively.


Introduction
A wound is a breach in the skin or mucosa membrane, which may allow the entry of microorganisms, possibly leading to infection (Bowler et al., 2001) . Wound tissue provides the rich environment necessary for the proliferation of microbes. It is characterized by hypoxia, necrosis and often an accompanying impaired immune response owing to suboptimal delivery of immune effectors molecules through damaged blood vessels (Bowler et al., 1999) . This compromised, necrotic, slough tissue provides a warm, moist and nutritive environment, perfect for replication of colonizing bacteria. Bacteria species which were previously harmless commensally of the human body, most commonly on the skin, may become pathogenic in a wound environment (Robson, 1997). Wound healing is the process of repair that follows injury to the skin and other soft tissues. Initial stages of wound healing involve an acute inflammatory phase followed by synthesis of collagen and other extracellular matrix which are later remodeled to form scar (Shittu et al., 2002). Topical antimicrobial therapy is one of the most important methods of wound care. The goal of topical antimicrobial therapy in wound care is to control microbial colonization and subsequent proliferation thus promoting the healing of the wounds (Odimegwu et al., 2008) . Some medicinal plants have been employed in folk medicine since time immemorial for wound care (Muhammad & Muhammad, 2005;Samy et al., 2006 andKudi &Ngbede, 2006). Some of these plants either promote direct wound repair or exhibit antimicrobial and other related properties which are beneficial in overall wound care. Antimicrobial principles have been isolated from some of the medicinal plants used in folk medicine for wound care. Quercus infectoria is one of such plants employed by herbalists in the treatment of sores and boils.In this research, Quercus infectoria was studied in order to investigate its antibacterial properties. Quercus infectoria is a small tree or a shrub belonging to the Fagaceae (Quercaceae) family. They are found in the Mediterranean area, mainly in Greece, Asia Minor, Syria and Iran. The galls arise on young branches of this tree as a result of attack by the gall-wasp Adleria gallaetinctoria (Dor, 1976). The galls of Q. infectoria have been shown to have many medicinal properties such as astringent, antidiabetic, antifungal, antiviral, antibacterial, larvicidal and anti-inflammatory activities (Digrak et al., 1999;Hwang et al., 2000;Kaur et al., 2004& Rahman et al., 2006. The chemical constituents of the galls have been reported to comprise a large amount of tannins and small amounts of free gallic acids, ellagic acid and synergic acid (Dor, 1976, Ikram & Nowshad 1977. The purpose of this study was to elucidate the pattern of bacteriological isolates, which are responsible for wound infections and to evaluate the in vitro antibacterial activity of aqueous, ethanol and methanol extracts against bacterial species isolated from surface wounds.

Collection of wound swabs and identification:
Surface wound swabs were collected from 190 patients attending Hawler Ferkari Lab and Emergency Hospitals in Erbil city. The wounds were first cleaned using sterile cotton swabs soaked in sterile normal saline. The specimens were collected by gently rotating sterile swab in the wound and then transported to the laboratory immediately. The swab samples were inoculated on Blood agar, MacConky agar and Chocolate agar plates and incubated overnight at 37 Cº for 24 hours aerobically. Bacterial pathogens were identified by conventional biochemical methods according to standard microbiological techniques (Norrell & Messley, 1997).

Disc diffusion method:
Antimicrobial susceptibility was performed on Mueller Hinton agar by the standard disc diffusion method recommended by the National committee for clinical laboratory standards (NCCLS) using the following antibiotics: Gentamicin (10µg) ,-Ceftazidime (30µg),-Amoxillin(25µg), - The surface of the Mueller-Hinton agar was inoculated with the isolated species. High potency discs were placed on the agar. After 18 hours of incubation, the plates were examined and the sensitivity result was interpreted according to NCCLS (NCCLS, 2006).

Preparation of extracts:
The galls of Q. infectoria used in this study were collected from different area of Erbil city. The galls were washed with distilled water, and dried in air. The galls were crushed in mechanical mortar. Aqueous, methanol and ethanol extractions were performed by the following method. 50 gm of gall powders were used with 300 ml of solvents with an extraction period 24-72 hours. The extracts were filtered using filter paper and the solvents were evaporated using rotary distillation apparatus. In order to obtain a completely dry extract, the resultant extracts were transferred to glass dishes, and were left in 50 Cº ovens for 24 hours. Then, they were left at 4 Cº until assessment of their antimicrobial activities (Mashhadian & Rakhshandeh, 2004).

Minimum inhibitory concentration (MIC):
A quantity of 0.2 g of each extract was dissolved in 4 ml sterile nutrient broth which yields an initial concentration of 50 mg/ml. Subsequently, two folds serial dilution were made from the stock of 4 ml containing 50 mg/ml. Nutrient broth was used to obtain the following concentrations 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.390 and 0.195mg/ml. One milliliter of standardized inoculums (1×10 8 cells/ml) of each isolated species was introduced into each extract nutrient broth mixture and then incubated at 37Cº for 24 h. The MIC was considered the lowest concentration of the extract that prevents visible growth in the liquid media (NCCLS, 2006).

Discussion
Wounds are known to be easy portals for infection and provides suitable medium for the proliferation of microbial organisms, so both of Gram positive and Gram-negative bacteria are known to cause wound sepsis. In the present study, the microbiological analysis reveals that Staph. aureus is the leading etiologic agent of wound infection with previous reports (Onche & Adedeji, 2004, Khorvash et al., 2008, Isibor et al., 2008 but is in contras to other studies which report Pseudo. aeruginosa as predominant organisms (Agnihotri et al., 2004, Anupurba et al., 2008. Staph. aureus is the most frequently isolated microorganism from body surface and this could easily be introduced into the wound either by the patients dressing materials or through the object that causes the injury. Microbiological investigations have noted that this organism is the single causative bacterium in approximately 25% to 69% of cutaneous abscess (Meislin et al., 1977, Brook & Finegold, 1981, and the same microorganism has also been recognized as the most frequent isolate in superficial infections seen in Hospital accident and Emergency Department. The second most frequent organism cultured in this study was Pseudo. aeruginosa (24.90%) followed by E. coli (14.94%) and Enterobacter spp. (9.96%). These results are in accordance with other studies (Agnihotri et al., 2004& Masaadeh & Jaran, 2008. This microorganism has, since the mid. Twentieth century, been held responsible for the majority of invasive wound infections in many hospitals worldwide (Olayinka et al., 2004& Rastegar et al., 2005. In general the increase rate of occurrence of Pseudo. aeruginosa is not unrelated with indiscriminate use of antibiotics without laboratory diagnosis and antibiotic sensitivity report. This single factor could eliminates thenormal flora and provide a non-competitive environment for Pseudo. aeruginosa. The resistant nature of this organism to antimicrobial agents, nutritional versatility and the difficulties encountered in maintaining proper hygienic standards especially among personal involved with wound dressing and general care of patients may have contributed to the high rate of Pseudo. aeruginosa infection (Oguntibeju & Rau, 2005).Antibiotic sensitivity patterns revealed that many of the isolates were resistant to commonly used antibiotics. The results indicated that there was a significantly high percentage resistance among Gram-negative bacilli to aminoglycosides like Gentamicin, Amikacin, Amoxicillin, Ciprofloxacin, Ceftazidime and Cefotaxime. This alarming trend was also seen for Staph. aureus and Pseudo. aeruginosa. A similar results of multidrug resistant Gram-positive and negative bacteria to various antibiotic routinely used has been reported from several studies (Howell-Jones et al., 2005;Basri, & Fan, 2005& Dhar et al., 2007. Since high antimicrobial resistance is probably promoted due to selective pressure exerted bacteria due to numerous reasons like non adherence to hospital antibiotic policy, and excessive and indiscriminate use of broad-spectrum antibiotics. These multi drug resistant strains establish themselves in the hospital environment in area like sinks, taps, railing mattress, toilets and thereby spread from one patient to another. In this study the results of the investigations show that the three extracts from Q. infectoria possess antimicrobial activities against tested microorganisms that are involved in causing wound infections at a concentration varied between 0.781 mg/ml and 25 mg/ml. These findings therefore support the use of this plant in the management of wound infection. Ethanolic extracts showed the strongest activity followed by methanolic extracts and aqueous extracts an indication that ethanol is a better extractant than the two other solvents used in this study and this may be due to the ability of the ethanol to extract a wide range of chemical constituent of the plant. Our finding was supported by other researches who reported that the crude powder of the galls of Q. infectoria was found to be active against Gram-positive and Gram-negative bacteria (Vorovuthikuchai et al., 2004;Makkar et al., 2006& Muskhazli et al., 2008. The inhibitory effects of gall nut may be due to the presence of some phytochemical components, and based on previous studies, Quercus species have been reported to contain high levels of tannins in both hydrolysable and condensed form which form irreversible complexes with proline-rich protein resulting in the inhibition of the cell protein synthesis (Hagermant & Butler, 1981). It can be concluded that the Q. infectoria extracts has beneficial effect as antiseptic and can use for the treatment of wound infection caused by pathogenic bacteria.