Study area and period
The study was conducted in Debre tabor town, which is located 666 km far from Addis Ababa, in South Gondar Zone of the Amhara Region. It is 100 km from Bahir Dar the capital city of Amhara regional state. In the town there are three Governmental Health Centers, 3 private clinics and one hospital. Based on figures from 2007 Central Statistical Agency of Ethiopia (CSA), Debre Tabor town has a total population of 55,596. The hospital serves as a teaching as well as patient-care providing center for the region. There are about 172 health professionals and 105 administration staff, who provide care for about 6600 outpatients per month. The hospital has a 91 bed capacity. Close to 192 patients visit the hospital every day. Debre Tabor Hospital has diabetic treatment centre which gives serves for about 2149 diabetic patients. The study
was conducted from September 20 to January 20, 2016.
Study design and populations
A hospital based cross-sectional study was conducted among those Diabetic patients who attend at Debre Tabor Hospital during study period who fulfill criteria was included and patient who had treatment with antibiotics in the last 14 days, unconscious patient, pregnant women, HIV positive patients and any patient who had treatment with steroid drug was excluded.
Sample size and sampling technique
The sampling technique was systematic sampling and the sample size (n) was calculated by taking highest prevalence of urethral tract infection (UTI) in previous study 17.8 % (15).
The expected margin of error (d) was 0.05 and the confidence interval (Zα/2) was 95%. Contingency for the unknown circumstance was 10%.
Data collection and laboratory methods
Socio-demographic and clinical data were collected from each study participant by using structured questionnaire by trained nurses.
Each diabetic patient was instructed how to collect a ‘clean-catch’ mid-stream urine specimen by laboratory personnel or staff. About 10 to 20 ml urine specimen was collected in a sterile screw-capped and wide-mouthed container. The container was labeled with unique sample number, date and time of collection. Following collection, it was delivered to the bacteriology laboratory of Debre Tabor University for culture and drug susceptibility testing.
Isolation and identification of bacteria
For the detection of pathogenic bacteria, collected urine specimens were inoculated onto cysteine lysine electrolyte deficient agar (CLED)(Oxoid, Ltd., Basingstoke, Hampshire, England) using calibrated loop (0.001ml).The inoculated plate was incubated in aerobic atmosphere at 37oC for 18 -24 hours (hrs). Then, the plate was inspected for growth. Significant bacteriuria was defined as colony count > 105cfu/ml; sub-culture on to maconkey and blood agar. The inoculated plate was incubated at 370c for 18-24 hrs. After obtaining pure colonies, further identification was done by using the standard microbiological technique, which includes gram stain, colony morphology and biochemical tests (Oxoid, LTD). Preliminary identification of bacteria was based on gram reaction, colony characteristics of the bacteria like hemolysis on blood agar and enzyme activities of the bacteria on different substrates.
Biochemical tests: Biochemical tests were performed on colonies from pure cultures for final identification of the isolates. Gram-negative rods were identified after performing a series of biochemical tests, which includes gas production, sugar fermentation, H2S production, indoleproduction, citrate utilization, lysine decarboxylase production, hydrolysis of urea and motility tests. The bacterial isolates were identified by combination of different biochemical tests. Gram-positive cocci were identified based on their gram reaction, catalase, coagulase, bacitracin and optochin test results (11).
Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was carried out on each identified organism by disc diffusion method on Muller Hinton agar (MHA) (11). After a pure culture was obtained, a loop full of bacteria was taken from a colony and was transferred to a tube containing 5 ml sterile normal saline (0.85 % NaCl) and mixed gently until it formed a homogenous suspension. The turbidity of suspension was determined in comparison with 0.5 Mac-Farland standard (11). A sterile swab was dipped in the broth suspension and excess suspension was removed by pressing the swab against the wall of the tube. The swab was used to distribute the bacteria suspension evenly over the entire surface of MHA (Oxoid). For antimicrobial testing of streptococci, 5% defibrnated sterile sheep blood was aseptically added to Mueller-Hinton medium. The inoculated plates were left at room temperature until dry. The antimicrobial impregnated disks were placed with sterile forceps on the agar surface at least 24 mm away from each other to avoid the overlapping zone of inhibition. After placed the disk the plate was allowed to stand for 30 minutes to dissolve the antibiotic in the media. Then, the plates were inverted and incubated at 37for 24 hrs and was read for the diameter of zone of inhibition. Grades of susceptibility pattern were recognized as sensitive, intermediate and resistant by comparison of zone of inhibition as indicated in the manufacturer’s guide (12).The antimicrobial agents tested were obtained from Oxoid in the following concentrations: For gram-negative ampicillin (AMP) (10μg), amoxicillin-clavulanic acid (AMC) (30μg), ceftriaxone (CRO) (30 μg), ciprofloxacin (CIP) (5μg), gentamycin (CN) (10μg), cotrimoxazole (25μg) and Tetracycline (TTC) (30 μg) and for gram-positive Ampicillin (AMP) (10μg), amoxicillin-clavulanic acid (AMC) (30μg), ceftriaxone (CRO) (30 μg), chloramphenicol (C) (30μg), ciprofloxacin (CIP) (5 μg), erythromycin (E) (15μg), gentamycin (CN) (10μg), penicillin (P) (10 IU), cotrimoxazole (25μg) and tetracycline (TTC) (30 μg) (10).
All specimens were collected and tested according to the standard operating procedure of each phase. The sterility of culture media was ensured by incubating 5% of each batch of the prepared media at 37oc for 24 hours. Performance of all prepared media was also checked by inoculating international standard-strains such as Escherichia coli (ATCC 25922), S. aureus (ATCC 25923). To standardize the inoculum density of bacterial suspension for the susceptibility test, 0.5 MacFarland standard was used (11). To ensure the accuracy of data, double data entry method was used.
Data were entered, clean and edited, using EPI info version 3.7 and were exported in to SPSS version 20 for data analysis. Frequencies and cross tabulations were used to summarize descriptive statistics. Tables were used for data presentation. Odd ratio and adjusted odds ratio were used in the analysis. Both bivariate and multiple logistic regression were employed to assess the association between outcome and explanatory variables. P-values < 0.05 were considered statistically significant