Isolation, Identification and Antibiotic Susceptibility Pattern of Bacteria Isolated from Wounds of Patients Attending at Arsho Advanced Medical Laboratory

s: Wound infections cause clinical and societal consequences on the patients, but its bacteriological characteristicvaries with different factors. Therefore, effective treatment and management of wound infections in hospital and communitysetting will require detailed epidemiological knowledge of the infecting bacterial pathogens and their antibiogramunusual to theenvironment. Based on this information, we examined the frequency and antibiogram of bacterial pathogens isolated from woundinfection cases seen at ArshoAdvancedmedical laboratory over the study period. A total of 259 wound swabs/ and pus of different types of woundinfections from different anatomical sites were analyzed by standard bacteriological methods. Of the 259 clinical specimens analyzed, 177 (68.4%) yielded at least one bacterial pathogens, 6(2.3%) were polymicrobial, and 82(31.6%) yielded no bacterial growth. Overall, 20 different bacterial pathogens were identified 15 (75%) gram-negative bacteria isolates and 5(25%) gram-positive bacterial isolated. Staphylococcus aureus accounted formajority of the bacterial pathogens isolated, 86 (48.6% followed by E.coli20 (11.3%, and Citrobacterspp. 17(9.6%). The bacterial pathogens demonstrated high resistance to amoxicillin (79.7 %%), ampicillin (78.3%), and tetracycline (73.1%), in contrastto high sensitivity pattern observed with Meropinem (94.5%), Levofloxacin (87%), Amikacin (82.4%), and Ceftazidime (72.7%). Amikacin, meropenem and levofloxacin were the most effective drugs against the tested grampositive and -negative bacteria and should be considered in empirical antibiotic selection.


Introduction
Skin is one of our innate immunity that prevents infections of subcutaneous and systemic tissues physically and through the production of sweat and sebaceous secretions that provide protection by a virtue of their acid pH (3)(4)(5) and chemicals such asfatty acids and lysozyme that has antifungal property and dissolves bacterial cell respectively. Wound is abreach in the skin and the exposure ofsubcutaneous tissues following loss of skin integrity which provides a moist, warm, and conducive environment that is conducive to microbial colonization and proliferation [1].
Infection of the wound is the successful invasion, and proliferation by one or more organisms anywhere within the body's sterile tissues, sometimes resulting in pus formation [1]. Wounds can be classified as accidental, pathological or post-operative. Whatever the nature of the wound, infection is the attachment of microorganisms to host cells and they proliferate, colonize and become better placed to cause damage to the host tissues [2].
A wound can be considered infected if purulent material is observed without confirmation of a positive culture. The numbers of contaminants may not persist but specifically grow and divide and may become established, causing wound colonization or infection. Infection in a wound delays healing and may cause wound breakdown, herniation of the wound and complete wound dehiscence [3].
The control of wound infections has become more challenging due to widespread bacterial resistance to antibiotics and to a greater incidence of infections caused by methicillin-resistant S. aureus, polymicrobic flora and by fungi [4]. Wound infections have been a problem in the field of medicine for a long time. Apparent shift in the etiological agents of wound infection and the associated problem of antibiotic resistance amongst bacteria that cause wound infection from time to time and from one institution to another has initiated health institution to carry out continuous routine evaluation of would infection from the view point of their spectrum and drug susceptibility testing. The widespread uses of antibiotics, together with the length of time over which they have been available, have led to the emergence of resistant bacteria pathogens contributing to morbidity and mortality [5].
This study was aimed todetermine thefrequency and antibacterial susceptibilityof bacteria isolated from wound infectionsin patients referred to ArshoAdvanced medical laboratory, Addis Ababa, Ethiopia. As well as updates the clinicians in the variousantimicrobial alternatives available in thetreatment of wound infections.

General Objectives
To determine the frequency, bacterial outline and antibiotic susceptibility pattern of isolated from wound infection at Arsho advanced medical laboratory.

Specific Objectives
To assess the bacterial pathogens responsible for the wound infection To verify the antimicrobial resistance and sensitivity pattern of commonly isolated wound microbes

Study Site and Period
Wound samples were collected from the 259 patients on different days at the microbiology department of ArshoAdvanced Medical laboratory, Addis Ababa, Ethiopia fromJanuary to June 2016. The woundsamples were collected by using a sterile cotton swab, and then the swabs were transported with Amies transport media to the micro biology laboratory.

Sample Collection and Inoculation of Primary Isolation Culture Media
Up on admission to the study, wound specimens were collected aseptically from the study participants using sterile rayon tipped applicator stick swabs. Wound swabs collected from each patient was inoculated onto blood agar base to which 10% sheep blood is incorporated and macConkey agar. If a delay in culture was unavoidable wound swabs were transported to the laboratory by using Amies transport medium. Preparation and performance of evaluation of culture media were done as per the instruction of the manufacturer.

Bacterial Identification
Pure isolates of bacterial pathogen were preliminary characterized by colony morphology, hemolytic reaction on blood agar medium, gram-stain and catalase test. Identification of bacteria down to genus and/or species level was done by employing an array of routine biochemical tests such as DNASe, catalase, optochin, bacitracin, CAMP, bile esculine tests for gram positive bacteria and Indole production, H 2 S production, gas production, motility, urease, citrate utilization tests and fermentation of different carbohydrates for gram negative bacteria and signed by family member and/or adult guardian for participants under the age of 18 years.

Ethical Clearances
All ethical considerations and obligations were duly addressed and the study was conducted after the approval of the Internal Review Board (IRB) Arsho Advanced Medical Laboratory PLC. Informed written consent was obtained from participants before data collection. The respondent was given the right to refuse to take part in the study and to withdraw at any time during the study period. All the information obtained from the study subjects were coded to remain confidentially. When the participants were found to be positive for bacterial pathogen, they were informed by the hospital clinician and received proper treatment.

Discussions
Among the culture reports obtained, the most common organism was found to be Staphylococcus aureus (48.6%). Similarly, the previous studies done in various parts of India has shown a higher frequency of staphylococcus aureus (39%) isolates from pus culture reports [8]. The results obtained from Arsho advanced medical laboratory, Staphylococcus aureus was the predominant organism isolated from wound culture and showed high sensitivity to Meropenem, Levofloxacin, Amicacin, and Cefepime which was similar to the previous results of [9,10]. Wound infections serve as favorable medium for proliferation of microorganisms that are potentially pathogenic. In most wound infection studies, polymicrobial is a common phenomenon, in this study we reported a polymicrobial rate of 2.3% which is lower compared to 18.6% reported in a study in Ethiopia [11]. Similarly, relatively few numbers of bacterial pathogens were isolated. The reason for this few number recorded may be due to, (i). Quality of clinical specimens collected, (ii). Delay in the transportation of the clinical specimens from the branchesc/hospitals to the laboratory, (iii). Laboratory methods employed and (IV) possible preantimicrobial medication by the patients.
The frequency of bacterial isolation recorded in this study showed that gram-negative bacteria accounted for 75% as against 25% of gram positive bacteria. Similarly, in Nigeria gram negative bacteria accounted for 70% as against 305 gram positive bacteria [11].
In the breakdown of bacterial pathogens isolated, S.aureus isolates predominates, followed by E.coli, and Citrobacterspp, which is contrary to the pattern reported in some studies and varies with the frequency of isolation [12][13][14], but similar result was revealed by [15].
While other studies have reported pathogens like Pseudomonas aeruginosa, Klebsiellaspp and E.coli as leading pathogens in different wound infections and geographical locations [16,17].
Ageneral overview of the anti biogram of all the bacterial isolates indicated that both the Gram positive bacteriaand Gram negative bacteria had very high resistance levels. This situation raises serious concern. This suggests avery high resistance gene pool due to gross misuse and inappropriate usage of the antibacterial agents. The increase in the antibiotic resistance noticed in this study is in agreement with an earlier report by [18]. Where antibiotic mistreatment and high incidence of self medication with antibiotics were identifiedas being responsible for the selection of antibiotic resistant bacterial strains.

Limitation of the Study
It was not possible to include anaerobic bacteria due to unfortunate laboratory facilities constraints.

Conclusion
The findings of this study suggest that bacterial resistance in wound infections is becoming serious threat in the study area. Staphylococcus aureusis still the most frequently involved pathogen, showing high resistance rates of bacteria isolated from wounds followed by E.coli, Citrobacterspp, and the least occurring Edwardsiella spp and Morganella morgani. Amicacin and Meropinemare the best therapeutic options from the results (table 1) to treat Staphylococcal infections because of the lesser resistance caused by these organisms and also for Gram negative isolates.
Infections of the wound by these bacteria are one of the most common and important cause of morbidityand mortality in developing countries. The delay in recovery and subsequent increased length of hospital stayalso has economic consequences. The most commonly prescribed antibiotics in the facility were the penicillins, cephalosporins, and quinolones. The correct choice of antibiotics should be made only after antibiotic sensitivity testing.
Knowledge of the bacacterial pathogen of wound and the Antibiotic susceptibility pattern are important tools in the management of wound and are also useful in formulating balanced antibiotic policy.

Recommendations
This study recorded that Staphylococcus aureus and E.coliwere the most common organisms in wound infection. Meropinem and Levofloxacin were the most effective antimicrobial agents for this type of infections. Resistance due to inappropriate use of drugs is a common finding in our environment and medical staff & the community must be educated regarding the rational use of antibiotics. In future, the occurrence and drug susceptibility pattern of wound infections should be done by including anaerobic bacteria, fungus and other micro-organism those can be important causes of wound infections.