research

College of Education /Qurna                                                                                                                                       Department of Biology

Antibacterial effect of some extracts on the pseudomonas aeruginosa isolated from burn infections

By:

Fatima muhsen , Duaa Abd- Alrahman , Hura fadel

Supervised lecturer :

Dr. Kawakib I. Al-Zubaidy

1- Introduction

Burns provide a suitable site for bacterial multiplication and are more persistent richer sources of infection than surgical wounds, mainly because of the larger area involved and longer duration of patient stay in the hospital [1]. It has been estimated that about 75% of the mortality associated with burn injuries is related to sepsis especially in developing countries . Infection causes 50% to 60% of deaths in burn patients in spite of intensive therapy with antibiotics both topically as well as intravenous [2]. Immediately following injury, Gram-positive bacteria colonize the burn wound [3]. Following Gram-negative bacteria also rapidly colonize the burn wound surface in the first few days after injury [4].

Wound colonization by yeasts and fungi usually occurs later due to the use of broad spectrum antibiotic therapy [5]. Microorganisms routinely isolated from burn wounds include aerobic organisms like Staphylococcus aureus, Streptococcus pyogenes, E. coli, Klebsiella Spp., Proteus, Pseudomonas aeruginosa, anaerobic organisms like Bacteroides fragilis, and fungi like Aspergillus niger, Candida Spp and Zygomycetes. The efficacy of various topical antimicrobials in common use in modern burn centers is dynamic due to the ability of microorganisms to develop resistance rapidly. In order to accurately detect and track emerging trends in topical antimicrobial resistance in modern burn units, it is essential that standard reproducible methods be published for clinical implementation [6].

The continuous emergence of novel infections and the microorganisms developing resistance make already existing antibiotics less effective. In such scenarios, natural products which are a part of our daily diet serve as the best candidates for new antibacterial drug discovery (Sunil et al., 2012) [7].

 Coffee is the most importance source of caffeine (Minamisawa et al., 2004) [8].

 Caffeine in coffee depends on the extraction method and types of coffee include fresh or roasted beans. Caffeine (1,3,7-trimethylxanthine, C8H10N4O2) is a natural alkaloid or xanthine alkaloid found in coffee beans, tea leaves, cocoa beans, cola nuts and other plants It is one of the most widely used pharmacological substances in the world. Caffeine can be taken into the body by drinking coffee, tea, cocoa and cola and it is also used as component (Nawrot et al., 2003) [9].

 Natural coffee and tea contain some antimicrobial substances that could inhibit a variety of microorganism including pathogens (Pane et al., 2012[10] ; Chen et al., 2013) [11]. Caffeine was reported to inhibit mold (Suárez-Quiroz et al., 2004) [12]. and bacteria (Almeida et al., 2012) [13].

Pulses are of great importance to the health of individuals and communities. The nutritional value of these plants lies in the protein content which is often compromised due to the presence of certain anti nutritional fractions[14]. Lens

culinaris Medikus belonging to family Fabaceae (synonym Lens esculenta Moench.)

The genus name Lens is suggestive of the lens-like shape of the lentil seed.

Seeds are a good source of essential minerals such as calcium, iron, vitamin

B and constitute an important source of food in many countries. They are consumed as whole grains and reported to have high protein content, carbohydrates and fibers[15]. They are also claimed to have blood purifying property.Lentil paste is commonly used to get rid of old skin marks. It also treats various kidney and gastric ailments[16]. The seeds also exhibit antifungal properties[17].  High polyphenolic content present in the seed leads to excellent free radical-scavenging activity[18].

Therefore,  the current study aimed to fulfill the following objectives:

1.     Isolating of the bacterial isolates from  burn patients.

2.     Biochemical identification of bacterial strains.

3.     To prepare aqueous  extraction  of coffee and lentil seeds.

4.     Evaluating the sensitivity of these bacterial isolates toward  prepared extract by using of agar - well diffusion assay .

2- Materials and Methods

2.1 Sample collection

All samples were collected from burn  patients  admitted in the burn unit in Al-Fayha Hospital , surface swabs were taken during sample collection, tip of the swab was to be rolled on its side for one full rotation over 4 cm² of the part of the wound granulation tissue with the most obvious signs of infection and/or inflammation. The swabs were transported as soon as possible to the laboratory of  the biology department in Qurna education college . Swabs were transported in transport media in a well-sealed bottles. Afterwards, the samples were plated on culture media, according to Steer et al. [19].

2.2 Identification of pathogens

The bacterial isolates have been identified according to the below  laboratory steps:

1.     Swabs  was cultures  on  various  media; such as blood  agar, macConkey agar, nutrient agar, and mannitol salt agar. Cultural observation (color,  size,  and  colony  morphology)  were  observed  from  the incubated plates.

2.     During microscopic  examination  of  specimen, the slides  were  prepared  from  each  different  colonies observed  on  the  plates  and  Gram  staining  was  performed.  The  results  such  as  the  gram  positive  or  gram negative, motility, shape and arrangement of the bacteria were observed from this exam.

3.      Biochemical Examination

The selected  colonies have  been  subject to  biochemical examination such as  (  catalase test, coagulase , triple  sugar  iron test, indol production test, citrate utilization test(S.C)for verifying of the pathogens.

2.3Aqueous extraction of coffee and lentil

Coffee and lentil seed were purchased from local market.. A hundred gram of each seeds was crushed and sieved through screen mesh of cloth to get the fine powder. The powder was soaked in 200ml of  hot distilled water and was kept at a room temperature for 24 hours, and then it was filtered by using Whatman no. 1 filter paper. The filtrate was dried in a hot air oven at30 °C for three days to get rid of residual water. The residue was scraped, ground, weighed and stored in a

dried air tight container until it will be used [19].

2.4 Sensitivity test

The effect of extracts on the  bacterial isolates were carried out by agar well diffusion method.

2.5. Agar -well diffusion method

The available antibacterial in the plant extract are allowed to diffuse out into the medium and interact in a plate freshly cultured with the test of bacterial isolates. Muller Hinton agar plates were swabbed with a suspension of bacterial isolates, using sterile cotton swab then a sterile 4 mm cork borer was used to cut the wells at equidistance in each of the plates. 0.2 ml of each extract was added into each well and allowed to diffuse at room temperature for 20 minutes, then the plates were  incubated aerobically overnight at 37 ºC.  .The antibacterial activity was evaluated by measuring the diameter of zones of inhibition in millimeters [20].

3- References

1- Agnihotri N, Gupta V, Joshi RM (2004). Aerobic bacterial isolate from burn wound infections andtheir antibiograms - a five - year study. Burns 30: 241-243.

2- Arturson MG (1985).The pathophysiology of severe thermal injury. J Burn Care Rehabil 6:129-134.

3- Barret JP, Herndon DN (2003). Effects of burn wound excision on bacterial colonization and invasion. PlastReconstrSurg 111: 744-750.

4- Wysocki AB (2002). Evaluating and managing open skin wounds: colonization versus infection. AACN Clin Issues 13: 382-397.

5- Ramzy PI, Wolf SE, Irtun O, Hart DW, Thompson JC, et al. (2000). Gut epithelial apoptosis after severe burn: effects of gut hypoperfusion. J Am CollSurg 190: 281-287.

6- Altoparlak U, Erol S, Akcay MN, Celebi F, Kadanali A (2004). The time-related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients. Burns 30: 660-664.

7- Sunil Laxman Attimarad, Gaviraj N Ediga, Asif Abdulrahi-man Karigar, Ravindra Karadi, Nagesh Chandrashekhar and  Chandrashekara Shivanna. (2012). Screening, isolation and purification of antibacterial agents from marine actinomy-cetes. International Current Pharmaceutical Journal. 1(12): 394-402 .

8- Minamisawa, M., Yoshida, S. and Takai, N. (2004). Determination of biologically active substances in roasted coffees using a diode-array HPLC system. Analytical Science 20: 325-328.

9- Nawrot, P., Jprdan, S., Eastwood, J., Rotstein, J., Hugenholtz, A. and Feeley, M. (2003). Effects of caffeine on human health. Food Additives and Contaminants 20 (1): 1-30.

10- Pane, C., Celano, G., Villecco, D. and Zaccardelli, M.( 2012). Control of Botrytis cinerea, Alternaria alternata and Pyrenochaeta lycopersici on tomato with whey compost-tea applications. Crop Protection 38: 80-86.

11-Chen, J., Zhang, S. and Yang, X.( 2013). Control of brown rot on nectarines by tea polyphenol combined with tea saponin. Crop Protection 45: 29-35.

12- Suárez-Quiroz, M. L., González-Rios, O., Barel, M., Guyot, B., Schorr-Galindo, S. and Guiraud, J. P.(2004) . Effect of chemical and environmental factors on Aspergillus ochraceus growth and toxigenesis in green coffee. Food Microbiology 21 (6): 629-634.

13- Almeida, A. A. P., Naghetini, C. C., Santos, V. R., Antonio, A. G., Farah, A. and Glória, M. B. A. G( 2012). Influence of natural coffee compounds, coffee extracts and increased levels of caffeine on the inhibition of Streptococcus mutans. Food Research International 49 (1): 459-461.

14- Khandelwal S, Udipi SA, Ghugre P. Polyphenols and tannins in Indian pulses: Effect of soaking, germination and pressure cooking (2010) . Food Res Int; 43: 526-530.

15-Roy F, Boye JL, Simpson BK. Bioactive proteins and peptides in pulse crops: Pea, chickpea and lentil (2010). Food Res Int; 43:432-442.

16-Yadav SS, McNeil D, Stevenson P. Lentil: An Ancient Crop for Modern Times. Springer publications, (2007) : p. 58- 59.

17-Gupta VK, Kumria R, Garg M, Gupta M. Recent updates on free radicals scavenging Flavonoids: an overview( 2010) . Asian J Plant Sci; 9(3): 108-110.

18- Amarowicz R, Estrella I, Hernández RS, Troszynska A, Kosinska A, Pegg RB. Free radical-scavenging capacity, antioxidant activity, and phenolic composition of green lentil (Lens culinaris)( 2010). Food Chem; 121: 705-711.

19-Anonymous (2011). Henna http://www.en.wikipedia.com.Retrieved December 15th, 2011.

20-Christofilogiannisp, (2001). Current Inoculation Method In Mic Determination. Aquacultu, 196: 297-302.