Biological activity of 5-benzylidene thiobarbituric acid derivative against some bacteria isolated from burns infections

A total of fifty (50) samples were collected from burn infections from AL-Sader Medical City in Alnajaf Alashraf during the period from the 24th of January to the 4th of April . Different percentages of different bacteria were noted as follows , Pseudomonas aeruginosa was the highest percentage about 33% followed by Ps. florescence 8( 16 %) , both E.coli & Klebsiella Sp comprised 6 (12%) ,and Aeromonas sp was the lowest ratio comprising 2% . The most bacteria appeared resistance to antibiotics, Pseudomonas aeruginosa was found to be resistant to most of the therapeutic agents. In this paper a synthesis of 5-benzylidene thiobarbituric acid derivative have been described. The route of preparation involved the uses of thiobarbituric acid as starting material and treated with 4-dimethylaminobenzaldehyde compound to give required derivative this compound have been identified by a spectroscopic method like H-NMR, IR and CHNS analysis and also by measuring its melting point.A simple synthetic route for 5-(4- dimethylaminobenzylidene)thiobarbituric acid by the condensation reaction of 4-dimethylaminobenzaldehyde with thiobarbituric acid in water without catalyst is described. The results revealed that proteus sp. was affected by chemical model more than the other bacteria , increasing the concentration of the compound increases the inhibition zone .also bacteria Klebseilla sp. Pseudomonas aeruginosa. affected by compound model ,but bacteria Staphylococcus arueus was not affected by any concentration of compound. The aim of of this study was to determine the model of chemical compounds effect on the growth of some bacteria isolated from buruns in vitro.


Biological activity of 5-benzylidene thiobarbituric acid derivative against some bacteria isolated from burns infections
The most bacteria appeared resistance to antibiotics, Pseudomonas aeruginosa was found to be resistant to most of the therapeutic agents.
In this paper a synthesis of 5-benzylidene thiobarbituric acid derivative have been described. The route of preparation involved the uses of thiobarbituric acid as starting material and treated with 4-dimethylaminobenzaldehyde compound to give required derivative this compound have been identified by a spectroscopic method like H-NMR, IR and CHNS analysis and also by measuring its melting point.A simple synthetic route for 5-(4dimethylaminobenzylidene)thiobarbituric acid by the condensation reaction of 4-dimethylaminobenzaldehyde with thiobarbituric acid in water without catalyst is described.
The results revealed that proteus sp. was affected by chemical

Introduction
Burns are one of the most common and devastating forms of trauma. Patients with serious thermal injury require immediate specialized care in order to minimize morbidity and mortality. Data from the National Center for Injury Prevention and Control in the United States show that approximately 2 million fires are reported each year which result in 1.2 million people with burn injuries. Burn patients are ideal hosts for opportunistic infections. (1) The burn site remains relatively sterile during the first 24 hour; thereafter , colonization of the wound by gram negative bacteria is common. (2) Pseudomonas aeruginosa has emerged as a predominant member of the burn wound flora and in the absence of topical therapy is cultured from the burn injuries of 70% patients by the third week. ( (4) The surface of every burn wound is contaminated to some degree by bacteria. (5) Because of this, surface bacterial growth is routinely monitored in most centers to facilitate management and treatment. It has been found by many investigators that the distribution of various species of bacteria from burn wound surfaces is similar to that from blood specimens . (6) Active hydrogen compounds condense with aldehydes and ketones kown as Knoevenagel condensations. These aldol-like condensations usually are catalyzed with weak bases. Iminium ions are intermediates which from α,β-unsaturated compounds having structures corresponding to these formed by mixed aldol condensations followed by dehydration. These reactions are catalyzed by amines or buffer systems containing an amine and an acid are referred to as Knoevenagel condensations. (7) Cross aldoltype condensation of thiobarbituric acid with aromatic aldehydes using acetic acid as a catalyst is available for the preparation. (8) Thiobarbituric acid derivatives are known to possess antibacterial activity, (9) some are claimed to be sedatives, (10) and herbicides, (11) while some are classified as antiviral agents (12) 5-Arylidene thiobarbituric acids are widely used as precursors for the synthesis of bioactive derivatives (13) and its derivatives are also very important intermediates in organic reactions (14) Cross aldol-type condensation of thiobarbituric acid with aromatic aldehydes using acetic acid as a catalyst is available for the preparation. (8) In this paper, we describe a rapid and convenient method for the synthesis of 5-arylidene thiobarbituric acids under uncatalyzed conditions using ethanol as the solvent. It is interesting that the reaction easily occurs in water although the mechanism involves a net dehydration to the alcoholic intermediate obtained by nucleophilic attack of the active methylene neighboring to the carbonyl groups. (15)

Material and methods Sample collection
Clinical samples were collected from the burns unit of AL_Sader Medical City in Alnajaf Alashraf during the period from the 24 th of January to the 4 th of April. The swab samples were taken from infected sites of patients.

Isolation and identification
Collected samples were cultured on nutrient , blood and MacConkey's agars for identification . Non -lactose fermented colonies were selected and cultured onto blood agar , then incubated overnight at 37 °C for refreshment and for demonstration of their ability for blood hemolysis . Identification of samples: The identification of bacteria was performed by the following tests: Hemolysin production: A single colony of each isolate was streaked onto blood agar plate and incubated at 37 ºC for 24 -48 hours. Blood lysis around bacterial growth was recorded .

Synthesis of thiobarbituric acid
Thiobarbituric acid was prepared according to the literature. (16)

Synthesis of 5-benzylidene thiobarbituric acid derivative
A mixture of 4-dimethylaminobenzaldehyde (10 mmol) and thiobarbituric acid (10 mmol In this paper, we describe a rapid and convenient method for the synthesis of 5-arylidene thiobarbituric acids under uncatalyzed conditions using water as the solvent (Scheme 2).

Biological Activity Testing of Prepared Chemical Compounds
By using. (17) method for the test of biological activity of the prepared chemical compounds which includes the following steps: 1. Prepare bacterial suspension and compare with McFarland tube 0.5 .

Spread bacterial suspension on (Muller Hinton Agar)
homogeneously (0.1 ml) to cover the whole surface. Make holes in the agar by using 6 mm diameter cork piercing. 3. Prepare diluted solutions (5,10,15,20,)mg/ml for each compound at physiological pH(7). 4. Put the prepared solutions in holes to investigate their biological activity . 5. Incubate the petri-dishs at 37C. for 24 hours. 6. Measure the diameter of inhibition zone for each hole by the ruler to determine the effectiveness of each compound and compare with the standard limits of sensitivity of the same species of bacteria against antibiotics .

Statistical Analysis
The analysis of the results was according to the model testing process and the design was higher of accuracy and tested Dec incorporeal results by using a least significant difference(L.S.D.) below the level of probability P≥0.05 based. (18)
The results shown in Table(1) revealed that 50 samples(69%) gave positive bacterial culture whereas 25 (30.8%) showed no bacterial growth. Regarding skin swabs, were positive bacterial cultures consisting of single growth 35(83.6%), and mixed bacterial growth 9(16.4%). Meanwhile, no bacterial growth was found in 6 (14.1%) of skin swab cultures. The single and mixed bacterial growth results of skin swabs are shown in Table(1). These results agree with that obtained (26) who found that 86.5% of skin swabs were positive for bacterial growth. Also (27) reported that negative bacterial growth was found in approximately 18% of the cultures of skin swab.

3: Antibiotic Susceptibility
The susceptibility of isolates for common antibiotics was studied to determine the pattern of isolates resistance to various antibiotics depending on disk diffusion method. The antibiotics represented by, Trimethoprim , tetracyclines, Ciprofloxacin, and others.
The results showed that Ps. aeruginosa was the most resistant bacteria to all antibiotics also Klebseilla.sp, and staphlycoccus aureus but some isolates were sensitivity to Ciprofloxacin and Gentamicin , this result goes in agreement with the result (24,25) ; the results recorded that proteus , Aeromonas , Enterobacter and E. coli were less resistant than Ps. aeruginosa, Klebseilla.sp, these isolates were sensitive to Ampicillin,: Tetracyclin, Cefotaxime (28) ,this resistance is related to the genes carried on plasmid or chromosome (29)(30)(31) ,produce several extended spectrum β-laclamase enzymes which inhibit the action of β-lactam antibiotics, conferring bacteria the ability to persist under β-lactams treatment, as recorded. (25)  Ps. aeruginosa

4: Study of Biological Activity :
The results showed that Ps. aeruginosa, Klebseilla .sp and Proteus .sp , were sensitive to compound 5-benzylidene thiobarbituric acid derivative, and increasing the compound concentration increases the inhibition zone. but Staphylococcus arueus was found resistant in vitro. this different back to biological activity of this compound ,it has effecting groups working on bacteria metabolism, the compound model was capable to combine with chemical material in cell after that become complex inside the cell, the bacterial will become dehydrated and die .this result applicator with (25) used the prepare compound 2-[4-(dimethylamino)-3-[(4-methoxyphenyl) diazenyl]phenyl]-3-(aryl)-2-hydrobenzo[e]-1,3-oxazepine-4,7-dione) and also used prepare DBHBED found was effect to E.coli this compound effect to my be on metabilsim of this bacteria (25) .This might be due to the change in the genetic properties of the living cells. (32)  Klebseilla.sp