Characterization of isolated bacteria from burn patients and its susceptibility against different antibiotics in district Swat

Burn is an injury to the skin or other body tissues caused by various agents like thermal injury, scalds, fire, electrical shock and chemical injury. The aims of current study were isolation, identification and sensitivity of isolated bacteria against antibiotics from burn infected patients of District Swat. A total of 50 swab samples were collected from 50 burn infected patients. On the basis of colony morphology and Biochemical tests, including Oxidase, Catalase, Coagulase, Indole, and Motility eight different bacterial isolates were identified, including Bacillus, Enterobacter, Epidermis, E.Coli, Klebseilla, Pseudomonas Aeruginosa, Proteus and Staphylococcus aureus. The frequency range were Bacillus (0.3%), Enterobacter 6.97%), Epidermis (6.97%), Proteus (9.30%), E.coli (11.62%), Klebsiella (13.95%), Staphylococcus aureus (20.93%) and Pseudomonas aeruginosa (30.23%). The maximum infections, i.e. 38% were reported in children’s age group 1-5 years. The infections reported in females were 65% while males 35%. A total of 12 antibiotics were tested in which levofloxacin and ciprofloxacin were found more effective. Moderate against Amikacin, Chloramphenicol and Gentamycin. Whereas Imipenem, Amoxicillin, Ceftriaxone were found less effective. Attention and concentration is need to avoid the infection in burn patients at distract swat. A further detail and comprehensive study is needed to avoid or reduce these infections.


Introduction
Burn is an injury to the skin or other body tissues caused by various agents like thermal injury, scalds, fire, electrical shock and chemical injury [1].Skin is the thin layer of tissue form the natural outer covering of body of a person or animal.It contains certain specific component of the innate immune system, which help to protect the host from pathogens.The skin has an immunological and neurosensory function.It may have other function like Vitamin D metabolism [2].According to literature the burn injuries are classified into three different categories.These categories are based on the depth of injuries.The first degree burn is surficial burn that affects the upper surface of skin.It appears as red spots of the skin and sensitive to touch for three to five days [3].Second degree are subcutinic and has effect on the under layer of the skin and causes blisters.In such injury the function of sweat glands is affected and may feel pain for six to eight weeks.Systemic burn injury is classified as third degree.In this class the injury extends to all layers of the skin, e.g.epidermis, dermis and hypodermis.A sever burn have many effect on the body and can penetrate deep skin layers, which causing muscle or tissue damage that effect every system of the body.It has effect on human immune system.The function of immune system is to protect cells from invasion by microorganisms.The body has three important immune defense systems, all of which are disrupted with burn injury [4].The response of immune system to burn injury is immediate, prolonged and or severe.The effect of burn in individuals is immune suppression, with increased susceptibility to fatal systemic burn wound or sepsis.The burn wound has a greater incidence of infections because in burn wound the skin barriers is destructed extensively and also alteration of the cellular and humoral immune responses.

Sample collection
The 50 sterile swabs samples were collected from burn sites aseptically at Saidu teaching hospital, Matta hospital, Khwazakhela Hospital and Kanju Burn Care Centre.Samples were collected from different body parts (hands, leg, chest, and abdomen) of burn patients and properly labeled with patients Name, Age, and Gender.The samples were taken to laboratory of Centre for Biotechnology and Microbiology at University of Swat for further processing.Media preparation Different media's were used for the growth of isolated agents such as Nutrient agar, Blood agar, and MacConkey's agar.The media was autoclaved for 15 minutes at 121°C.Then antifungal was added to inhibit fungal growth and poured in sterilized petri dishes under aseptic environment in laminar flow hood (LHF).Swab culture Swab samples were streaked on Nutrient agar media.After streaking plates were incubated for 48 hours on 37° C. Sub culture and pure culture Different Morphological colonies were appeared on Nutrient agar plates.To obtain pure culture subcultures of separated colonies were again sub cultured for 48 hours at 37°C.

Identification of bacteria
All cultures were examined for the growth and colony morphology.Gram staining and biochemical tests, e.g.oxidase, catalase, coagulase, indole, and motility were used for identification Antibiotic sensitivity test Antibiotic disk preparation For inoculums preparation a sterile distilled water ampoules were used.The inoculum of pathogenic bacteria were adjusted to 0.5 McFarland standards.In bacterial suspension a sterile cotton swab were dipped.In order to get a uniform growth of isolates, the isolated samples were streaked on Mueller Hinton agar with cotton swab smoothly.After 15 minutes of inoculation the antibiotic discs of known concentration were placed on the media surface with the help of disc dispenser.The list of antibiotics used in present research work is given in table 1.The plates were then incubated for 24 hours at 37°C in inverted position.The zones of inhibition were measured after 24 hours.

Antibiotic susceptibility test Disc diffusion Method
Antibiotic susceptibility test Disc diffusion technique [Kirby-Bauer method] was used to test antimicrobial sensitivity.Sterile swab was soaked in the test organism suspension and swab was streaked over the surface of Muller Hinton agar.The Petri-dish was allowed for 3-5 minutes to dry, by using sterile forceps the antimicrobial discs were placed evenly distributed on inoculated plate.The plates were incubated aerobically at 37°C for 24 hours.The zones of inhibition around each disc were measured in millimeters and the results were reported as sensitive, intermediate and or resistant.

Antimicrobial sensitivity Assay
The antibiotic sensitivity tests were performed for isolated bacteria, e.g.

Results
During current research work the patients were distributed on the basis of various categories given below.

Gender wise distribution of burn patients
The Gender wise distribution for all 50 Burn patients were made and found that 35% were male while the remaining 65 % were found female (Figure 1).

Burning source wise distribution of bacterial infection
The isolated bacteria were distributed on the basis of different source of burns.

Susceptibility of isolates against various antibiotics
During study the pseudomonas aeruginosa was associated with maximum number of patients, i.e. 26, followed by S. aureus, Klebsiella, Proteus, and E.coli 18, 12, 10 and 8 respectively while Enterococcus and epidermis both were associated with 6 patients (Table 3).The susceptibility of Pseudomonas aeruginosa against various antibiotics were determined and found 60% sensitive to levofloxacin (LEV) followed by TZP and AK 45% and 35% respectively, 10% was recorded against MP while no activity were shown against IPM and TGC each (Figure 2). Figure 3 revealed that klebsiella were found more sensitive against CIP, 55% and LEV 65%.Against AK, TZP, CPO recorded moderate while against E no activities were recorded.The bacteria proteus susceptibility were determined against various antibiotics and found susceptible against TZP (60%), LEV (70%) and CIP (65%).While against AK, CPO, SCF moderate, i.e. 45%, 35% and 40% respectively.Similarly CAZ, AMC showed no effect against proteus (Figure 4).In case of E.coli showed high resistance against Amoxicillin (AMC) 100% (Figure 5).The Staphylococcus aurous showed high resistance against Amoxicillin (AMC) 97.1% as compare to the other antibiotics (Figure 6).The bacteria Enterococcus showed 100% resistance to Erythromycin while less resistant to Ciprofloxacin (CIP) and Levofloxacin (LEV) recorded as 10% (Figure 7).Discussion Burn infection is a serious public health problem of Swat, Khyber Pakhtunkhwa.Burn injuries have body disinfection, lifelong difficulty even cause death [20].The burn infection explores the tissue for microorganisms of normal flora and contaminated environment.The virulence factor for infection including adhesion, nutrients and immune system of the patient.To prevent and treat burn infection numbers of antibiotics can be used to administrate directly as topical cream taken by orally or by injections [21].The results of current study showed 80% infection in burn patients.These results are in agreement with the study conducted by Mooney et al;2004, who  In present study the higher frequency were recorded for pseudomonas auregenousa 30.23% followed by staphylococcus aureus 20.93%, klebseilla 13.95%, E.coli 11.62% Proteus 9.30%, Enterobacter 6.97% and epidermis 6.97% (Figure 8).These results are in line with the results of [23]

13]. Materials and methods Study Area The
Propioni bacteria are gram positive anaerobic rod shaped bacteria.They are non-motile, not filamentous, catalase positive and are secondary agents of infected wounds and abscesses in humans and other animals.Gram negative bacteria are the most common and serious infectious source in burn patients.Among gram negative bacteria the important microbes are Pseudomonas aeruginosa, a rod shaped motile bacterium [17].It produces water soluble pigments which diffuse through the medium, e.g.Pyocyanin, Pyoverdine and Pyorubin [18].Klebsiella pneumonia is a non-motile, encapsulated, facultative anaerobic, lactose fermenting and rod shaped bacteria.It is catalase positive and oxidase negative.E.coli is rod shaped, facultative anaerobic, motile normal flora.It is Catalase positive and Oxidase negative [19].Proteusis motile, rod shaped bacteria.It is catalase and Urease positive and Oxidase negative [current study was carried out in laboratory of Centre for Biotechnology and Microbiology at University of Swat with the collaboration of Research and Clinical laboratory, Kanju.The study time was from January to August 2017.
The result indicates that six bacterial isolates, i.e.
The current study is in agreement with previous researcher.It is also in line with research work conducted by Barret et al;in 2002 [7].

.
The reasons for this high frequency of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsella may be that they are present abundantly in air, can survive in waste range of environmental conditions and showing resistance against a number of antibiotics.The present research work is also in conformations with the reports of [24].The lowest frequencies of our results were noted in Enterobacter and E.coli that was 6.97%each which is in agreement with [25].