ANTIMICROBIAL ACTIVITY OF ALLIUM CEPA EXTRACTS AGAINST AEROMONASHYDROPHILA ISOLATED FROM DIARRHEA SAMPLES OF CHILDREN IN IRAQ

Ban mahdi shakir 1 , Sinaamahdi shakir 2 , Nadhee mahammood hussein 3 and khetam habeeb rasool 3 . 1. Imam Sadiq General Hospital, Babil Health Directorate /Ministry of Health /Iraq. 2. Teaching Laboratories Medical City/Ministry of Health /Iraq. 3. Department of Biology, College of Science, Al-Mustansiryah University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


64
Antibiotics resistant microorganism became a major clinical and public health problem during the lifetime of most people. This shift in susceptibility greatly affects our ability to successfully treat patients empirically. Plant derived products have been used for medicinal purposes for centuries. At present, it is estimated that about 80% of the world population rely on botanical preparations as medicines to meet their health needs. Herbs and spices are generally considered safe and proved to be effective against certain ailments. They were also extensively used, particularly, in many Asian, African and other countries. In recent years, in view of their beneficial effects, use of spices/herbs has been gradually increasing in developed countries also (3).
Search for new antimicrobials is very important in recent times, considering the escalating levels of antibiotic resistance among pathogenic bacteria. So this study aimed to isolation of Aeromonashydrophila from acute gastroenteritis in children, study the susceptibility of Aeromonashydrophila to defferent antibiotics and study the antimicrobial activity of Allium cepa (onion) extracts on Aeromonashydrophila by agar diffusion method.

Materials and methods:-
Collection of Samples:-Two hundred and seventy five watery diarrhea samples (clinical samples) were taken from children suffering from gastroenteritis collected from Welfare teaching hospital / Medical city. The samples were collected in sterile containers.

Isolation of Aeromonas:-
The clinical samples were inoculated in alkaline peptone water as enrichment medium and incubated for 24 h at 37ºC, one loopful of culture was inoculated on selective media (TCBS agar and MacConkey's agar plates) and incubated for 24 h at 37ºC (4).

Identification of Aeromonashydrophila:-
Morphological and biochemical tests were applied for the bacterial isolates according to Forbes et al. (5) and Ciprian (6).Aeromonashydrophilaisolates were also identified to species level by using API 20 E (Analytic Profile Index) system (Bio-Merieux, France), which is a standardized system for the identification of bacteria belong to Enterobacteriaceae family.

Antimicrobial Susceptibility Test:-
Kirby-Bauer method was used as described by Baron and Finegold (7) to carry out the antimicrobial susceptibility test. Bacterial culture for 18-24 h. was compared with the standard turbidity solution (McFarland), this approximately equals to (1.5x10 8 ) CFU/ml. A 0.1 ml of the culture was spreaded on the surface of Mueller-Hinton agar plates, left to dry for 15 minutes at room temperature. Thirteen antibiotics used in this study (table-1).
The antibiotic discs were placed on the surface of the medium and incubated at 37ºC for 24 h. Inhibition zones around the discs were measured according to National Committee for Clinical Laboratory Standards (NCCLS) (8).

Extraction of the plant materials(Onionextraction):-
Two types of onion bulbs (Allium cepa) white and purple skinned were purchased from local markets in Baghdad, Iraq (figure-1). The onions were washed with clean sterile distilled water and allowed to air dry for one hour. The outer covering of the onion were manually peeled off. The onion bulbs being separated were washed and extracted in the following ways:

Figure-1:-Allium cepa (Onion); A: white skinned and B: purple skinned Onion
Exactly 200g of fresh onion bulbs were blended into fine powder and soaked in 100ml of distilled water for 24hr. The pulp obtained was left in a clean, sterile glass container and shaken vigorously to allow for proper extraction and it was filtered using a sterile muslin cloth after which the extract was obtained, air-dried and stored below ambient temperature until required. Exactly 200g of fresh onion bulbs were blended and soaked in 100ml of hot water for 24hr., the resultant juice was extracted, air-dried and stored as in above. Exactly 200g of fresh onion bulbs were blended and soaked in 100mls of 95% ethanol for 24hr and the extract was obtained, air-dried and stored as in above (9).

Innoculum Preparation by Direct Colony Suspension Method;-
A small volume of sterile water was poured inside a test tube to which general colonies of the test organisms, taken directly from the plate were emulsified and the suspension was adjusted to match the 0.5 McFarland's standard this approximately equals to (1.5x10 8 ) CFU/ml, which has a similar appearance of an overnight broth culture by adding distilled water (9).

Antimicrobial activity of onion extracts against Aeromonashydrophila isolates:-
The sensitivity test ofAeromonashydrophila to the extracts of Allium cepa (onions) was carried out using the cupplate diffusion method. A glass dropper was used to add 0.02ml of the suspension to an already prepared medium. A sterile cotton swab was used to spread by streaking the organisms all over the surface of the medium and allowed to dry for about 5 minutes. Cups of 6mm in diameter were made in the agar using sterile corkborer.
Different dilutions of the plant extracts prepared in the order of 100µg/ml, 200µg/ml, 300µg/ml, 400µg/ml and 500µg/ml respectively were prepared in five different test tubes and placed in a test tube rack. Exactly 0.02ml of each concentration was introduced into each hole on the medium and was allowed to stand on the bench for about one hour for proper diffusion. It was thereafter incubated at 37°C for 24 hr. The sensitive bacteria grew everywhere except in areas around the holes in the medium. Then, the resulting inhibition zones obtained were measured in millimeters and recorded against the corresponding concentrations (9).

66
Results and Discussion:-Isolation and Identification of Aeromonashydrophila:-Two hundred and seventy five watery diarrhea samples were collected from patients suffering from gastroenteritis children fromless than 1 month to12 years of age.
In this study, alkaline peptone water was used as an enrichment medium for isolation of Aeromonas, Robinson et al. (10) and kayet al. (11) reported that this medium yielded 2.6 times the number of isolates compared to other media and was recommended for the isolation of Aeromonasfrom humans.
In case of cephalothin, 90% of the isolates were resistant to this antibiotic. The higher number of resistance exhibited by these bacteria was in good agreement with the result reported by Ram et al. (16).

67
An encouragement result was found with norfloxacin, none of the isolates showed resistance to this antibiotic. Norfloxacin should be considered the drug of choice in the treatment of severe diarrhea in patients (17). Also none of our isolates were resistant to tetracycline, nitrofurantion and chloramphenicol.
The results of the antibacterial activity of onion extracts against Aeromonashydrophila was listed in table-5 and figure-2.
Purple onion extracts did not show any inhibitory effect on the growth of Aeromonashydrophila isolates. The results were listed in table -6.The widest zones of inhibition was obtained with the 500 µg/ ml concentration of hotwater onion extract. These differences in the zones of inhibition may be directly related to the susceptibility of each isolates to the onions extracts.