ANTIBACTERIAL ACTIVITY OF CERTAIN MEDICINAL PLANT AND THEIR ESSENTIAL OILS ON THE ISOLATED BACTERIA FROM UTI PATIENTS

Mohamed H. Mourad 1 ,Soheir Abdel-Rahman Salih 2 , Mahmoud M. Elaasser 1 , Nesreen A. Safwat 1 and Mostafa Y. Ibrahim 3 . 1. The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. 2. Department of Clinical pathology, Faculty of Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt. 3. Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Hot Water Extraction:-
Aqueous extracts were prepared according to the method of Li et al. (2006) with slight modifications. Briefly, 50 g of the powdered plant material was mixed with 200 mL of distilled water in a conical flask, which was boiled, and shaken for 30 minutes in boiling water bath. The resulting mixture was allowed to cool to room temperature before being filtered using Whatman ® No. 1 filter paper. Crude extracts were centrifuged at 4000 xg for 15 min. The water extracts were concentrated by heating in a water bath then filtered again using 0.45 μm aqua membrane nylon filter (Becton Dickinson ® Company) to obtain the sterile extracts. The concentrated extracts were kept in sterile glass bottle at -20 o C. Briefly, 50 g of the powdered plant samples were soaked in 200 ml of the 80% ethanol for three days, after which the extracts were filtered through Whatman ® No.1 filter paper. Solvent was evaporated with a rotary evaporator (Buchi ® Rotavapor R-124, Switzerland) and then filtered again using a 0.45μm membrane nylon filter (Becton Dickinson ® Company) to obtain the sterile extracts.
Extraction of Essential Oils Using Steam Distillation method:-Essential oils were isolated from all the plant materials by using Clevenger-type apparatus. Two hundred and fifty g from each plant were taken and placed into 2 L flask. Plant pieces were covered with 1.5 L of distilled water.Steam with essential oil vapors is condensed in thecondenser and is collected in a small round flat-bottom flask after 4-6 hours. The essential oil was separated using a reparatory funnel, dried underanhydrous sodium sulphate, transferred into a dark glass vials and stored at -20 o C until used (A.O.A.C, 1995).

Antibacterial Activity of Medicinal Plant Extracts:-
The antibacterial activity was carried out by agar disc diffusion assay and broth dilution assay. Dried extract was redissolved in the smallest possible volume of water or 20% Dimethyl sulfoxide (DMSO) to give stock solutions of high concentrations. Concentrations of aqueous and ethanolic extracts were recorded on a weight by volume (w/v) basis, while essential oil concentrations by volume/volume (v/v).

Disc Diffusion Assay:-
The Mueller-Hinton plates were inoculated with the bacterial suspension using a sterile swab to achieve a lawn growth. Sterile paper disks (6mm in diameter) (Whatman ® filter paper No.1) were impregnated with 20µl of medicinal plant extract solution in different concentrations (12.5%, 25%, 50%, 75%, and 100%) then placed on the inoculated agar surface. All plates were sealed with sterile laboratory bags to avoid evaporation of the test samples.
Plates were allowed to stand at room temperature for 60 min. to let the test plant materials diffuse into the agar, and afterwards, they were incubated at 37 o C for 24 hours. Results are determined by measuring the clear zone of inhibition (Hewitt and Vincent, 2003). Studies were performed in triplicate and mean value was calculated.

Broth Dilution Assay:-
The antimicrobial effects of the extracts of the selected medicinal plants against different MDRB were determined by the broth dilution method as described by Tepe et al. (2004). Minimum Inhibitory Concentration (MIC) is defined as the highest dilution or least concentration of the extracts that inhibit growth of organisms. To determine the MIC, two-fold Serial dilutions were prepared for each extract with sterile Mueller-Hinton broth. 0.5 ml of each bacterial suspension (1×10 6 CFU/ml) was inoculated in tubes with different concentrations of the extracts. Two controls were prepared, one containing MH broth and bacterial suspension serve as bacterial control, and one containing plant extracts in MH broth serve as negative control. Un-inoculated broth serve as blank, used to calibrate the spectrophotometer. The tubes were incubated at 37 o C for 24 h. Inhibition of bacterial growth was determined by measuring the absorbance at 600 nm. The measurements taken before and after incubation were compared and a difference of less than 0.05 indicates no microbial growth. The lowest concentration that had no microbial growth was determined to be the MIC. Bacterial cells of both treated and untreated bacterial cells were observed under Transmissions Electron Microscope  (TEM). The samples were prepared by standard protocol (Croft, 1999). Samples was fixed in 1% Glutaraldehde than washed in 0.1 M buffer, 1% Osmium tetraoxide was used for post-fixations and again washed with 0.1 M buffer. The samples were dehydrated in acetone, infiltrated and embedded in epoxy resin. Finally, the grids were dried in a desiccator and examined using TEM (JEOL 1010 Japan), for study biocidal action of essential oil and any morphological changes.

TEM observations of treated bacterial cells:-
Chromatographic analysis of essential oils composition:-Analysis of Essential Oil was done using GCMS to recognize the chemical composition of Thyme, Cinnamon, and Marjoram essential oils. The HP 5890 series II Gas Chromatograph interfaced to a 5973 Mass Selective Detector and controlled by HP Chemstation software (version b.02.05) was used. The chromatographic separation was achieved using a HP5-MS capillary column (30.0 cm x 25 mm x 0.25 mm). The column stationary phase comprised of 5:95% diphenyl: dimethylpolysiloxane blend. The operating GC condition was an initial oven temperature of

Results and Discussion:-
In the present report, among one thousand and six hundred urine samples screened; only 65% showed bacterial infection. However, 919 samples (88.4%) were sensitive to the tested antibiotics; 121 samples (11.6%) recognized as multi-drug resistant bacteria. These bacterial isolates were identified and differentiated by cultural, morphological, and biochemical analysis. The results also indicated that the most of urinary tract infection diseases were by Gram negative bacteria (102 isolates; 84.3 %). Escherichia coli was the most predominant organism causing UTI in this study (  In this study, antibacterial activities of the 15 medicinal plants were tested on five selected MDR bacterial clinical isolates: Escherichia coli, Klebsiella pneumoniae,Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis that exhibited the highest resistance pattern. In vitro antibacterial activity studies in the present study indicated that all plants essential oils and ethanolic or water extracts were found to be more effective at crude levels (100% concentration).
Out of 15 medicinal plants tested (Tables 2-16), cinnamon, thyme and marjoram showed maximum activity against all the bacterial isolates tested. Moderate effects were seen with clove, ginger, sage and rosemary. On the other hand, camel grass, celery, dill, echinacea, eucalyptus, fennel, fenugreek and parsley showed weak inhibition against tested clinical isolates.                        However, aqueous and organic extracts of dill seeds have exhibited potent antibacterial activity (Kaur and Arora, 2009).In the present study negligible inhibitory activity with aqueous extract was observed in some of the plants which may be due to loss of some active compounds during extraction process of the sample or there may be lack of solubility of active constituents in aqueous solution (Sampathkumar et al., 2008).

S. aureus
In addition, the type of solvent used to extract herbs and spices appeared to have a major impact on their antimicrobial activity. This is probably due to the fact that, although the solvents were removed from extracts by evaporation, and most of the components with antimicrobial properties are aromatic or saturated organic compounds which are generally more soluble in solvents such as ethanol or methanol The ethanolic extracts of all the plants have shown good antibacterial effect against the UTI isolates. The most effective antibacterial activity was recorded for Cinnamomum zeylanicum (Table 16) withmaximum effect observed against Escherichia coli (IZ value 26.8 mm) and least against Klebsiella pneumonia (IZ value 21.4 mm). This effect is in agreement with other researchers regarding the antibacterial effect against Escherichia coli; however there is a difference in the concentration of extract of cinnamon used in this study (Yuste and Fung, 2006).
In the present study, the alcoholic extracts of clove, ginger and thyme were the most effective than the aqueous extracts against Escherichia coli and Staphylococcus aureus isolates. These results are in agreement with that obtained by many authors (Ayoola et al., 2008; Al-Jiffri et al., 2011; Fuad et al., 2012).   This study also revealed that cinnamon essential oil showed the highest activity with MIC values ranging from 0.04 to 0.16mg/ml followed by thyme and marjoram essential oils with MIC values ranging from 0.625 to 2.5mg/ml (Table 17). However,the estimated minimal inhibitory concentrations of Cinnamomum zeylanicum are ranged from 0.21 to 0.63μl/ml (v/v) (Zainal-Abidin et al., 2013), 0.8 to 3.2 mg/ml (Prabuseenivasan et al., 2006).
According to the antibacterial assay done for screening purpose, Staphylococcus aureus was the most susceptible Gram-positive bacteria to all plant extracts, whereas Escherichia coli was the most susceptible Gram-negative microorganisms. On the contrary, the Gram-negative MDR Klebsiella pneumonia was the most resistant microorganisms.

Effect of Cinnamon Essential Oil on Ultrastructure of Bacterial Cells:-
The effect of cinnamon essential oils on bacterial cell structure was tested using transmation electron microscopy on Gram-negative tested bacteria Escherichia coli and Gram-positive tested bacteria Staphylococcus aureus.
The untreated Staphylococcus aureus appeared cocci that displayed normally dividing cells with sharp delineation between cell wall, cytoplasmic membrane and the cytoplasm (Fig. 3A&B). After incubation of the bacterial cells with cinnamon essential oil (at 0.04 mg/ml), dramatic cellular alterations became visible on electron microscopic image (Fig. 3C&D). The treated cells appeared oblong; edges become abnormal, triangle, or elongated. Cell wall disrupted and exhibited thickened in some parts and breakdown in other due to leakage of cytoplasm.
Escherichia coli appeared short rods in TEM micrograph of untreated cells and showed a continuous thin smooth cell wall, cell membrane and nuclear material (Fig. 4A&B). When subjected of Escherichia coli cells to cinnamon essential oil at MIC: 0.08 mg/ml, bacterial cells lysed rapidly, incapable of septum formation, so cells appeared as very long threads (Fig. 4C). Cytoplasm shrinked leaving cell wall, while other cells appeared metamorphosed, cytoplasm lost its even distribution and showed clumping of intracellular materials (Fig. 4C&D). Cell wall was lost smoothness and uniformity and leading to cell wall rupture and even strong damage in many areas with thickened appearance more pronounced at polar-regions (Fig. 4C&D).  Moreover, in the current study GC/MS Analysis of essential oils indicated that marjoram oil had nearly sixteen major components (Table 18; Fig. 5). The main components were: linalool (29.20%) trpenin-4-ol (20.04%) and γterpinen (11.89%). Cinnamon oils had fifteen components (Table 19; Fig. 6), and the major ones cinnamaldehyde (72.87%) and cinnamic acid (8.88%). However, thyme oil had 17 major components (Table 20; Fig. 7) includes Pcymen (29.15%), thymol (24.80%) and carvecol (22.69%).      The cinnamaldehyde being the major component of cinnamon bark oil is in agreement with those previously reported. Several studies have shown that cinnamon essential oil was very complex mixtures of compounds and many variations have been found in the chemical composition (Wang et al., 2009). The antibacterial activity   ofcinnamon was probably due to their major component,cinnamaldehyde and their constituents is also known to inhibitsbacterial acetyl-CoA carboxylase and responsible for majorantibacterial activity (Jantan et al., 2008;  Muthuswamy et al., 2008; Meades et al., 2011).
The ability of phenolic compounds to alter microbial cell permeability, thereby permitting the loss of macromolecules from the cell interior, could help explain some of the antimicrobial activity In conclusion, policies on the control of antibiotic usage have to be enforced due to higher resistance. In addition, the results of antibacterial assay in the current study revealed that essential oils and ethanol extracts of plant exhibited broad spectrum activity against tested isolates as compared to aqueous extract. Also, cinnamon essential oil showed the highest activity mainly due to effect of cinnamaldehyde on cell wall.