Identification of E.coli from patients of urinary tract infection and its resistance in Dil-Chora hospital, Dire Dawa, Ethiopia

International Journal of Microbiology and Immunology Research Vol.4(2), pp. 015-020, April, 2015 Available online at http://www.apexjournal.org ISSN 2315-8743 2015 Apex Journal International Full Length Research Identification of E.coli from patients of urinary tract infection and its resistance in Dil-Chora hospital, Dire Dawa, Ethiopia Girum Faris 1, Ameha Kebede 2, and Genene Tefera 1 1 Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia 2 Haramaya University, College of Natural and Computational Sciences, Department of Biology, School of Graduate Studies, Haramaya, Ethiopia. Accepted 13 April, 2015; Published 25 April, 2015 A cross sectional study involving laboratory and questionnaire based survey method was conducted in Dire Dawa Dil Chora Hospital from February to May 2011 to determine the prevalence of E.coli in patients of Urinary tract infection (UTI), to see the association between risk factors and UTI and the antibiotic sensitivity patterns of and E.coli. In this study a total of 384 midstream urine samples were collected from 276(71.2%) females and 108(28.1%) males and examined in the hospital for UTI. Results showed that (53.1%) were proved to be positive for the presence of E.coli. The antimicrobial sensitivity study also showed that all isolates were resistant to ampicillin erythromycin (100%, 100%) and trimethoprim-sulphamethoxazole (70%) respectively. In contrast they weresensitive to chloramphenicol, norfloxacin and ciprofloxacin (33.3%, 30%, and 20%) respectively. Thus, on the basis of these results physicians may prescribe the appropriate drugs such as chloramphenicol, norfloxacin and ciprofloxacin and avoiding use of the antibiotics for which the pathogens had shown resistance. In addition to these, there should be a control of antibiotic distribution to prevent self-treatment, which leads to antibiotic resistance. Key words: E. coli, Prevalence, urinary tract infection, Antimicrobial resistance. INTRODUCTION Urinary tract infection (UTI) is a general term referring to an infection that occurs anywhere in the urinary tract (Hasan et al., 2006). Our urinary tract includes the organs that collect, store and release urine from our body. These organs are the kidneys which collect wastes and extra water from our blood to make urine, ureters carry the urine from our kidneys to bladder, bladder which stores urine and releases to urethra, and urethra carries the urine out of bladder during urination (Hasan et al., 2006; Stamm and Norrby, 2001). Ninety percent of UTI cases are caused by gramnegative bacteria while only 10% of the cases are *Corresponding author. Email: girumf@ibc.gov.et caused by gram positive bacteria (Lazarevic et al., 1986; Overturf, 2002). Escherichia coli are dominating the etiology of most cases of urinary tract infections (Conway et al., 2007). They are found in the normal flora of the intestinal tract, skin, and vagina. However, under some predisposing conditions they can multiply rapidly and are capable of adhering to uro-epithelial tissues and producing infection (Samara, 2001; Ghedira et al., 2004). UTIs are mostly caused by E. coli accounting for more than 70% of uncomplicated cases (Yuksel et al., 2006). At present the majority of pathogens isolated from urine are resistant to commonly used antibiotics (Norrby, 2007). The main factors and that are responsible for antibiotic resistance are extensive use and misuse of antimicrobial agents (Gales et al. 2000). To ensure the appropriate therapy, current knowledge

016 Int. J. Microbiol. Immunol. Res of the organism that causes UTI and their antibiotic susceptibility pattern is mandatory.thus the present study was made to assess (1) the overall prevalence of E. coli strains in patients complaining urinary tract infection (2) to study the association between E. coli infection UTI and potential risk factors (3) to determine the antibiotic resistance pattern of the isolated E. coli. MATERIALS AND METHODS Study area The study was conducted at Dil Chora Hospital, found in Dire Dawa Administrative Council.Dire Dawa lies between 9 28.1 N- 9 49.1 Nlongitudesand 41 o 38.1 E- 42 o 19.1 E latitudes; and it is 515 km east of the capital city Addis Ababa.Dil Chora Hospital was selected for this study as it had greater patient flow in the region. Study design A Hospital based cross sectional study was employed to determine the prevalence of Escherichia coli strains and antibiotic susceptibility. The study also involved questionnaire survey of the potential risk factors associated with UTI. Sample size was determined using 95% level of confidence, 5% precision and since there was no previous study done on the area, expected prevalence was taken as 50%.The inclusion criteria were all out patients and in patients of all age groups complaining about UTI, whereas in patients those who were taking antibiotics at the time of taking samples and children below one year were excluded. Dipstick screening technique and microscopy In the Laboratory of Dil Chora Hospital, dipstick technique was made by using Multisticks of Medi-Test combi 10 SGLleukocyte esterase and nitrite (Alper and Curry, 2005). The microscopic examination for white blood cells was done after centrifugation of samples (Alper and Curry, 2005). Isolation of italicize E.coli A loop full (0.001mL) of each urine sample was streaked on Blood agar plate and sub cultured on MacConkey agar plate for isolation E. coli. Pink colonies were taken from MacConkey agar plates based on colony morphology and coloration. Strain identification of E. coli was done at Addis Ababa (EBI) Ethiopian Biodiversity Institute microbial genetic resource laboratory.identification was carried out using standard operation protocols (SOP) for aerobic bacteria identification System of BIOLOG (ODCSISUG, 2008). Antimicrobial susceptibility test Susceptibility to antimicrobial agents was tested by using the disc diffusion method according to National Committee for Clinical Laboratory Standards using the following discs (NCCLS, 2001; Table 1). Data analysis The total prevalence of E. coli was calculated based on culture results using percentages. The quantitative data generated from questionnaire survey about the clinical findings and associations with positivity of E.coli strains was entered into a computer. Statistical software package (SPSS version 13.0) was used for statistical analysis. Ethical consideration Ethical approval was obtained from the ethical review committee of College of Health, Haramaya University and individual consent was made with the patient before taking samples. RESULTS Overall complaints of UTI by sex and age groups During the study period, a total of 384 inpatients and outpatients with complaints of urinary tract infection were examined of the 384 patients 276 (71.9%) were females and 108 (28.1%) were males. Number of female patients complaining about urinary tract infection exceeded that of the males by 168. The sex and age group distributions of patients are given in (Table 2).The age categorization showed that 233 (60.7%) of the patients with complaints of UTI were among the adult age group (15-24). From the result age may be one factor affecting the prevalence of UTI. The group that was affected next to adult age group was the young age group (between 25-64 years) which included comprises 116 (30.2%) of the patients (Table 2). In this age group, 69 (59.5%) were females and 47 (40.5) were males. The data showed that even amongst the young aged patients, UTI was more common in females than in males. This is consistent with other reports which revealed that UTI is more frequent in females than in males among youth (Akinyemi et al., 1997).

Faris et al 017 Table 1. Antibiotic disks used with their respective concentrations. No. Antibiotic disks Concentration 1 Trimthoprim-Sulphamethoxazole (OXOID,UK), 25µg 2 Ampicillin (OXOID, UK), 10µg 3 Erythromycin (BBL, France) 15µg 4 Ciprofloxacin (OXOID, UK), 5 µg 5 Chloramphenicol (OXOID, UK), 30µg 6 Norfloxacin (OXOID, UK), 30µg Table 2. The sex and age distribution of the study population (n =38). Age group Sex Female (%) Male (% ) Total (%) Children (under15 years) 0 0% 8 2.1% 8 2.1% Young (15-24 years) 69 18% 47 12.2% 116 30.2% Adult (25-64 years) 191 49.7% 42 10.9% 233 60.7% Old (above 65 years) 16 4.2% 11 2.9% 27 7% Total 276 71.9% 108 28.1% 384 100% Table 3. Bivariate and Multivariate logistic regression of risk factors for all UTI patients. E. coli infection Risk Factor Occurrence Positive Negative COR (95% CI) AOR (95%CI) Improper storage + 180 (88.2) 174 (96.6) 0.53 (0.304-0.935)* 0.54(0.304-0.977)* - 24 (11.8) 6 (3.4) 1 1 Improper emptying + 158(77.5) 46 (25.5) 1.16 (0.713-1.903) 1.46 (0.868-2.457) - 46 (22.5) 134 (74.5) 1 1 Infrequent voiding + 161 (78.9) 88 (49.9) 0.73 (0.458-1.177) 0.61 (0.379-1.012) - 43 (11.1) 92 (51.1) 1 1 Constipation + 172(76.7) 32 (17.8) 0.48 (0.294-0.797) * 0.44 (0.263-0.747)* - 32 (24.3) 148 (81.2) 1 1 *significant at p<0.05, COR = Crude Odds Ratio, AOR = Adjusted Odds Ratio, CI. = Confidence Interval and Numbers in bracket are percentage On the other hand, the prevalence of UTI and among children under-15 and oldpeople above-65 (old age group) was 2.1% and 7%, respectively. Of the total UTI cases, 16 (4.2%) and 11 (2.9%) of the old and people and 0% and 2.1% of the children were females and males, respectively. Prevalence of E. coli strains Of the total 384 examined urine specimens cultured 204, were positive for E.coli giving the overall prevalence of 53.1%.The reported prevalence in this study was found to and be higher than the reported prevalence from other parts of Ethiopia. Risk factors associated to UTI caused by E. coli The associations between potential risk factors and UTI infections in male and female patients were investigated (Table 3). Those patients who were not storing urine in their bladder for a long time were less likely to have UTI than those who were storing (AOR=0.54, CI=0.304-0.977, P=0.042). In a similar study done on risk factors of UTI in Pakistan, improper storage of urine in bladder was found to be one of the main causes of urinary tract infection, which produces a favorable environment for the growth of urinary tract pathogens (Ramzan et al., 2004). There is also significant association (AOR = 0.443, CI= 0.263-0.747, P = 0.002) was shown between constipation

018 Int. J. Microbiol. Immunol. Res Table 4. Risk factors associated with E. coli infection among the female UTI patients. Risk Factor E. coli infection Occurrence Positive Negative COR (95% C.I) AOR (95%C.I) Active sexuality + 120(77.9%) 67(54.5%) 2.89 (1.724.88) 2.4 (1.68-4.33) * - 34 (22.1%) 55(45.1%) 1 1 Hygiene + 125(81.2%) 53(42.6%) 5.7 (3.33-9.82) 5.02 (2.89-8.72)* - 29 (18.8%) 69(56.6%) 1 1 Use of diaphragm + 24 (13.9%) 16(13.1%) 1.07(0.53-2.15) 0.85 (0.39-1.87) - 130(86.1%) 106(86.9%) 1 1 *Significant at p<0.05 COR = Crude Odds Ratio, AOR = Adjusted Odds Ratio, C.I. = Confidence Interval. + = Present, - = Absent Table 5. The proportion of resistant, sensitive and susceptible E.coli isolates to six different antibiotics. Spp. (Total No) Sensitivity SXT (%) AMP (%) ER (%) CIP (%) CH (%) NOR (%) E.coli (60) S 10 (16.7) 0 (0) 0 46 (76.7) 38(63.3) 40 (66.7) I 8 (13.3) 0 (0) 0 2 (3.3) 4 (6.7) - R 42 (70) 60 (100) 60(100) 12 (20) 18 (30) 20 (33.3) SXT=Trimthoprim-Sulphamethoxazole, AMP= Ampicillin, ER= Erythromycin, CIP= Ciprofloxacin, C= Chloramphenicol, NOR= Norpholaxocin S = Sensitive, I = Intermediate, R = Resistance and contracting E. coli infection. Those patients who did not have constipation were less likely to have the risk of contracting E. coli infection UTI as compared to those who had constipation. The associations between E. coli infection and two potential risk factors exposing females were studied for the fact that the disease was more frequent in female patients than in male patients (Table 4). Significant difference (P<0.05) was observed on the prevalence E. coli infection between sexually active females. It was observed that those females who were active sexually or perform sex frequently were twice at risk of having E. coli infection UTI as compared to those who were not performing sex frequently (AOR=2.4, P = 0.00). Similarly females who were not keeping the hygiene of their genital organs regularly were found to be 5 times at risk of contracting UTI with E.coli infection than females who keeping the hygiene of their genital organs regularly (AOR=5.02). Antimicrobial susceptibility test The antibiotic susceptibility tests were made on a total of 60 isolates. The isolates were tested using a standard method of agar disk diffusion technique as recommended by the National Committee for Clinical Laboratory Standards (NCCLS, 2001). The overall percentage of resistance of E. coli isolates observed against the commonly prescribed antibioticswere 100%, 100%, 70%,33.3%, 30%, and 20% for Ampicillin, Erythromycin, Trimthoprim-sulphamethoxazole and Norfolaxacin, Chloramphenicol, and Ciprofloxacin, respectively. In this study E. coli isolates were highly resistant to Ampicillin (100%), Erythromycin (100%), and Trimthoprim-sulphamethoxazole (70%). The present result was comparable to earlier studies done in Mekele, Ampicillin (100%), Erythromycin (100%) and Trimthoprimsulphamethoxazole (81.8) (Zeamanuel, 2007), and in Jimma 100% for both Ampicillin and Erythromycin and 50% for Trimthoprim-sulphamethoxazole.The resistance of E.coli isolates in the present study was also higher than those reported from Turkey (Mujde et al., 2010) and India (Khan and Zaman, 2006), (36%, 56%, 15%) and (60%, 90%, 70%) respectively (Table 5). DISCUSSION In this study females presented the highest prevalence of the cases than males. In a similar study Stamm and Turck (1991) reported that women were the victims of UTI as compared to males.the higher number of females is due to close proximity of the female urethral meatus to the anus, shorter urethra, and frequent sexual intercourse. During sexual activity bacteria move from vagina to the urethra (Heffner and Gorelick, 2008). These have been reported as factors that contribute for the higher prevalence of UTI in women (Omoregie et al., 2008; Lipsky, 1990). In a similar study Stamm and Turck (1991) reported that UTI was a very common infection among the women. In fact they found out that women

Faris et al 019 were the victims of UTI as compared to males.several reports have also indicated that females were more prone to having UTIs than males (Kolawale et al., 2009), because the urethra is shorter in females than in males (Randrianirina et al., 2006). The very high prevalence recorded amongst the adult patients could be due to increased sexual activity in the age group (Mabta, 2007). This result shows that age may be one factor affecting the prevalence of UTI. The age categorization showed that the same pattern in that the infection was more in females than males. This is in agreement with other reports which indicated that UTI is more frequent in females than in males during adulthood (Burbige et al., 1984; Mbata, 2007; Asinobi et al., 2003). The prevalence of E. coli in Dil-Chora Hospital, Dire- Dawa, was found to be higher than the reported prevalence from other parts of Ethiopia (that is, Bahir Dar; Felege Hiwot Referral Hospital; Addis Ababa, Tikur Ambessa Specialized Hospital; Jimma, Jimma University Hospital;and Mekele, (Mekele Hospital). The higher prevalence in the study area may be directly related with the weather condition of the area. Geographical location may be the reason for the difference (Orrett, 2001). Dire Dawa is one of the hottest places in Ethiopia (DASA, 2010). In hot areas urine production becomes less and as a result more water is lost from the body through sweating. Concentration of urine leads to infrequent urination and provides the bacteria to have the opportunity to continue to multiply within the bladder and/or urethra without being washed away by urine. Conversely in cold places less sweating causes more dilute urine production, which washes away any multiplying bacteria (Cunnigham, 1990). In a similar study done on risk factors of UTI in Pakistan, improper storage of urine in bladder was found to be one of the main causes of urinary tract infection,which produces a favorable environment for the growth of urinary tract pathogens (Ramzan et al., 2004). In this study, most of the female patients were rural residents and thus the poor habit of hygiene keeping may contribute a lot to the multiplication of the bacteria and the resulting ascent to the urinary system.the higher prevalence of E coli strains in females may be due to its existence as a normal flora in the large intestine and vagina. Alternations in vaginal micro flora also play a critical role in encouraging vaginal colonization and this can lead to urinary tract infection (Hooton and Stamm, 1997). Conclusion Active sexuality and hygiene were significantly associated with an increase in E.coli infection UTI. The higher resistance against antimicrobials could be frequent prescription or free access of drugs. 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