Urinary Tract Infection: Antibiotic Resistance Pro�les of Bacterial Etiologies and Associated Risk Factors in Diabetic Patients Attending Debre Tabor Hospital, Northwest Ethiopia

Background: Urinary tract infection is a major health problem especially in developing countries. Information about bacterial pathogens isolated from urinary tract infection in diabetic patients and their antimicrobial susceptibility patterns are limited in Ethiopia. Therefore, this study aimed at to isolate bacterial pathogens and their antimicrobial susceptibility patterns. Methods: A hospital based cross sectional study was conducted at Debre Tabor. Urine sample was inoculated on to cysteine lysine electrolyte de�cient (CLED) medium. Bacterial pathogens were identi�ed using standard bacteriological methods. The data were cleaned and entered in to SPSS version 20. P-Value less than 0.05 is considered statistically signi�cant. Result: A total of 250 study participants were included in the study. Of them, 28 (11.2%) bacterial pathogens were isolated. Gram positive bacteria were commonly isolated 19 (67.8%) than Gram negatives 9 (32.1%). The commonly isolated bacterial species were Staphylococcus aureus8 (28.6%),Escherichia coli5 (19.1%), Coagulase negative Staphylococci 8 (28.6%),Kelebsllapneumoniae4(14.3%), and Entrococcus(9.5%).Gram positive isolates were resistant to cotrimoxazole 10 (58.8%). Conclusion: Bacteriuria was signi�cantly associated with sex and type of diabetes. Multidrug resistance to two or more antibiotics was observed in 56.7% of bacterial isolates. Rational use of antimicrobial agent should be thought to prevent the emergency of multidrug resistance.


Introduction
Diabetes mellitus (DM) is a worldwide public health problem considering its high prevalence and extremely high economic and social costs.In developing countries, incidence of DM is becoming a serious public health problem (3).The risk of developing infection in diabetic patients is higher and urinary tract infection (UTI) is a major problem and urinary tract is the most common site for infection.
Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia resulting from impairments in insulin secretion, defects in insulin action, or both which in turn damage many of the body's systems and also cause several abnormalities of the host defense system that might increase the risk of certain infections (1).Based on pathogenic process diabetes mellitus have two broad categories.Type 1 diabetes is the result of complete or near-total insulin de ciency.Type 2 DM is a heterogeneous group of disorders characterized by variable degrees of insulin resistance, impaired insulin secretion, and increased glucose production (2).
Urinary tract infections (UTIs) are caused by growth of microorganisms anywhere in the urinary tract.It is usually due to bacteria from the digestive tract which climb the opening of the urethra and begin to multiply to cause infection (4).Urinary tract infections have been the major problem in the tropics and also a common problem seen in community as well as in hospitals (5) and the most common conditions causing individuals to seek medical care and results in a signi cant morbidity and high medical costs.It is occur in all age groups, in both genders and usually require urgent treatment (6).Globally the prevalence of UTI is high in healthy populations during the last 25 years and the morbidity and mortality due to UTI have remained static.It is most common particularly in females, about 10-20 % of women have UTI in their life and a signi cant number have recurrent infections at young age (7).In developing countries, UTIs are commonly encountered diseases by clinicians with an estimated annual global incidence of at least 250 million and also has been estimated that globally symptomatic UTIs result in as many as 7 million visits to out patient's clinics, 1 million visits to emergency departments and 100,000 hospitalizations annually (8).
Both gram positive and gram negative bacteria are implicated as common causes of UTI.Escherichia coli is the most common causative agent of UTIs in both DM patients.Other organisms reported include members of the family Enterobacteriaceae (i.e.Proteus, Klebsiella, Enterobacter and Citrobacter species), Pseudomonas species, Enterococcus species, streptococci, staphylococci and Candida albicans (9).The emergences of resistant bacterial strains in hospitals pose a continued challenge to treat and control the spread of infections.Moreover, the indiscriminate use of antibiotics often results in the increased resistance of urine pathogens to most commonly used antimicrobial drugs (10).Antimicrobial resistance is becoming an important public health problem in diabetic urinary tract infection.Information about the etiologies of UTI in diabetic patients is limited in Ethiopia.
For appropriate therapy, current knowledge of the organisms that cause UTI and their antibiotic susceptibility is mandatory.Since patterns of antibiotic resistance in a wide variety of pathogenic organisms may vary even over short periods and depend on type of isolation and on different environments, periodic evaluation of antibacterial activity is necessary (15).
Therefore, this study we aimed to assess the prevalence of UTI in diabetic patients as well as the type of microbiologically con rmed UTI bacterial pathogen and pattern of the antimicrobial drugs susceptibility in relation to diabetes mellitus patients

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 gures 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 ful ll 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 con dence 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.

Specimen collection
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 identi cation of bacteria
For the detection of pathogenic bacteria, collected urine specimens were inoculated onto cysteine lysine electrolyte de cient 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.Signi cant bacteriuria was de ned as colony count > 10 5 cfu/ml; sub-culture on to maconkey and blood agar.The inoculated plate was incubated at 37 0 c for 18-24 hrs.After obtaining pure colonies, further identi cation was done by using the standard microbiological technique, which includes gram stain, colony morphology and biochemical tests (Oxoid, LTD).Preliminary identi cation 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 nal identi cation of the isolates.Gram-negative rods were identi ed after performing a series of biochemical tests, which includes gas production, sugar fermentation, H 2 S production, indoleproduction, citrate utilization, lysine decarboxylase production, hydrolysis of urea and motility tests.The bacterial isolates were identi ed by combination of different biochemical tests.Gram-positive cocci were identi ed based on their gram reaction, catalase, coagulase, bacitracin and optochin test results (11).
Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was carried out on each identi ed 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% de brnated 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), cipro oxacin (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), cipro oxacin (CIP) (5 μg), erythromycin (E) (15μg), gentamycin (CN) (10μg), penicillin (P) (10 IU), cotrimoxazole (25μg) and tetracycline (TTC) (30 μg) (10).

Quality control
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 37 o c 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 analysis
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 signi cant

Socio-demographic and clinical characteristics of study participants
Of the total study participants, 250, included in the study, 133 (53.2%) were males and 117 (46.8%) were females.The age of the study participants ranged from 10 to 86 years for diabetic with a mean age of 39.8 +17.3 years.Among the diabetic patients 148 (59.2%) lived in rural areas, of whom 102 (40.8%) were farmers (Table 1).
Among the diabetic patients who were culture positive 5(17.9%) had symptomatic urinary tract infection and 23(82.1%)had asymptomatic urinary tract infection.Majority, 222 (88.8%) of the diabetic patients were asymptomatic and culture negative.
The relation between individual risk factors and bacteriuria among diabetic and non-diabetic patients were assessed using multivariate logistic regression.Statistically signi cant associations were observed between patient status (p=0.024) and sex (p=0.002) and urinary tract infection among diabetic and nondiabetic patients.Diabetic patients were 2.5 times as likely to develop UTI during the follow-up period as were non-diabetic patients (p = 0.026).
Statistically signi cant associations were observed between sex (p=0.002) and urinary tract infection among diabetic (Table 3).
Of the 8 (28.2%)Staphylococcus aureus isolates 5 (62.5%) showed multi-drug resistant strain while two of them was sensitive to all antimicrobial agents tested.The eight isolates of S. saprophyticus showed 5 (62.5%) of multi-drug resistant isolates.The overall multiple drug resistance patterns were 17 (56.7%).Among three isolates of Enterococci spp.only two were found to be resistant to one antibiotic.Of the ve Escherichia coli isolates one isolate showed multi-drug resistant and the other three were resistant to only one antibiotic (Table 6).

Discussion
In this study, the overall prevalence of signi cant bacteriuria (SB) in both symptomatic and asymptomatic diabetic patients was 11.2%.It was similar to the ndings of a study conducted in Addis Ababa (10.9%& 11.6%) (13,14).In this study the isolation rates of bacterial pathogens among diabetic patients were found lower than previous studies done in Gondar (17.8%) (10) and India (37%) (7).
In the present study the isolation rates of bacterial pathogens were (6%) and (16.3%) in male and female diabetic patients these ndings were consistent with the previous reports in Gondar (14%) (10) and (21.2%) (10) in males and females diabetic patients.The prevalence of UTI among female is high this may be due to low acidic pH of vaginal surface, poor hygienic conditions, short and wide urethra and proximity to anus (10).The present nding showed that the isolation rates of gram positive bacteria were found to be higher than the previous study done in Gondar (36.1%),Cameroon (41.2%) (15) and in Gondar (42.7%) (10).
In the present study, Staphylococcus aureus was the predominant isolate among diabetic patients.This nding was similar with a study in Nigeria (26.03%) (16) However, this nding is higher as compared with previous report from Gondar University Hospital (8.5%) (10).This discrepancy might be poor personal hygiene condition.
The isolation rate of S. saprophyticus (28.6%)in this study was consistent with a study nding from Gondar University Hospital (22%) (10).However, it is lower than a report from Cameroon (36.3%) ( 14).
This could be due to the appropriate use of antibiotics that may reduce the proliferation of S. saprophyticus bacteria or good personal hygiene practice.
The current study nding showed 33.4% of the isolates were gram negative bacteria which was lower than the previous report in Gondar (57.3%) (10).This inconsistency would be probably due to study design or sample size deference.On the other hand, this study was consistent with the previous report in Cameroon (45.1%) (15.) The second predominant bacterial isolate next to Staphylococcus aureus was Escherichia coli (Table 2) in diabetic patients.This ndings was higher than the study done in Libya (13%) (13).However, it was lower than a study ndings reported from Italy (54.1%) (19).In addition, the isolation rates of Kelebsilla pneumonia in the present study were (14.3%) that was similar to those reported from Cameroon (15.9%) (15) and Libya (13%) (13).
The nding of this study also showed that diabetic patients were more likely prone to UTIs than nondiabetic patients (10.9% vs. 4.7%).This could be due to diabetes affects many systems that protect against infection in general, and against urinary tract infections speci cally.
Urinary tract infection appears to be multi-factorial in subjects with diabetes and various diabetes-related risk factors.In this study being becoming female sex, and diabetic patient was four times more likely to be infected with bacteriuria [AOR: 4.011; 95% CI: 1.669-9.636](Table 3).Similar ndings have been reported in studies conducted elsewhere (10,18).In this study the susceptibility pattern of Staphylococcus aureus isolates demonstrated low and intermediate level of resistance to the commonly used antimicrobial agents (Table 4).
Most of the gram negative isolates (Table 5) were resistant to amoxicillin.This showed as sensitivity was decreased in contrast with the report from Gondar (59.6%) (10).Decreased susceptibility to this drug might be due to self-medication and indiscriminate use of antibiotics in the study area.
In this investigation, Escherichia coli isolates were 100% sensitive to cipro oxacin, gentamicin, chloramphenicol and ceftriaxone which was higher than the report from Addis Ababa (85%) (17).On the other hand, in the present study E .coliisolates were resistant to amoxicilin 100%thatwas also similar with a study done in Gondar: have shown increased the resistance of E .coli to amoxicillin -clavulanic acid is the result of hyper production of TEM-β lactamase, production of pencillinase resistant to inhibitors and production of cephalosporinase (10), ampicillin and tetracycline 40% (Table 5).
In the present study K. pneumonia isolates were 100% sensitive to chloramphenicol, gentamicin, nitrofurantoin, ceftriaxon and cotrimoxazole for diabetic patients.This result depicted similar nding with the report from Addis Ababa (17) and Gondar.However, inconsistent report was reported from Libya more than (20%) of Kelebsilla pneumonia isolates were resistant to cipro oxacin (13) (Table 5).This discrepancy could be study setting, geography, and population difference.
Multidrug resistance (MDR) to two or more drugs was observed in (53.6%) of the isolates in this study (Table 6).This was consistent with the previous report from Gondar (59.8%) (10).However, lower than the previous report from Addis Ababa (71.7%) (17).The lower multidrug resistance rate from the isolates could be rational use of antimicrobial agents.

Conclusion
In conclusion, signi cant bacteriuria was detected in 17.9% and 82.1% of symptomatic and asymptomatic diabetic patients, respectively.Based on this, UTI in asymptomatic diabetic patients should not be neglected and follow up studies are required to supplement the present ndings for appropriate management of asymptomatic UTI's in diabetic patients.As a complication of diabetes, UTI may be preventable with better glucose control and unnecessary use of antimicrobials.Gram-positive and gram-negative organisms were the commonest organisms isolated; among which Staphylococcus aureus and Escherichia coli were the principal urinary pathogen from gram-positive and gram-negative respectively.Statistically signi cant UTI was observed between sex differences.Most of the isolates showed intermediate to low level of resistance to one or more antimicrobials tested.This indicates that regular monitoring is required to establish reliable information about resistance pattern of urinary pathogens for optimal empirical therapy of diabetic patients with UTI.Nitrofurantoin, gentamicin and ceftriaxone were the choice of antimicrobial agents against gram positives and cipro oxacin, gentamicin and ceftriaxone were relatively sensitive for most of gram negative isolates.

Table 5 .
Antimicrobial resistance patterns of gram-negative bacteria isolated from urine of diabetic patients attending Debre Tabor Hospital,

7 )
Keys: R 0= No antibiotic Resistance R 1= Resistant to one antibiotic, R2=Resistant to two antibiotic, R3= resistant to three antibiotic, R4=Resistant to four antibiotic, R5=Resistant to ve and more than ve

Table 1 .
Socio demographic characteristics of diabetic patients attending Debre Tabor Hospital,

Table 2 .
Frequency and types of bacterial species isolated from symptomatic and asymptomatic Diabetic patients' urine culture attending Debre Tabor Hospital Northwest Ethiopia, Northwest Ethiopia

Table 3 .
Univariate and bivariate analysis of urinary tract infection among diabetic patients attending

Table 4 .
Antimicrobial resistance patterns of gram-positive bacteria isolated from urine of diabetic patients attending Debre Tabor Hospital, Northwest Ethiopia

Table 6 .
Multiple antimicrobial resistance patterns of bacterial isolates from urine of patients attending Debre Tabor Hospital, Northwest Ethiopia