Prevalence and antibiotic susceptibility of Uropathogens from cases of urinary tract infections (UTI) in Shashemene referral hospital, Ethiopia

Background Urinary tract infection (UTI) remains to be one of the most common infectious diseases diagnosed in developing countries. And a widespread use of antibiotics against uropathogens has led to the emergence of antibiotic resistant species. A laboratory based cross-sectional survey was conducted in Shashemene referral hospital to determine the prevalence and antibiotic susceptibility of uropathogens. Methods We have collected 384 clean catch mid-stream urine samples from all suspected UTI outpatients using sterile screw capped container. The urine samples were cultured and processed for subsequent uropathogens isolation. The isolated pure cultures were grown on BiOLOG Universal Growth agar (BUG) and identified using GEN III OmniLog® Plus ID System identification protocols. The identified species were then exposed to selected antibiotics to test for their susceptibility. Results The overall prevalence of urinary tract infection in the area was 90.1%. Most frequently isolated uropathogen in our study was Escherichia coli (39.3%). While, Staphylococcus species (20.2%), Leuconostoc species (11.4%), Raoultella terrigena/Klebsiella spp./ (8.4%), Salmonella typhimurium (6.3%), Dermacoccus nishinomiyaensis (6.3%), Citerobacter freundii (5.2%) and Issatchenkia orientalis/Candida krusei/ (2.7%) were the other isolates. We find that the relationship between uropathogens and some of UTI risk factors was statistically significant (P < 0.05). Gentamicin was the most effective drug against most of the isolates followed by chloramphenicol and nitrofurantoin. In contrast, amoxicillin, vancomycin and cephalexin were the antibiotics to which most of the isolates developed resistance. Conclusion Urinary tract infection was highly prevalent in the study area and all uropathogens isolated developed a resistance against mostly used antibiotics. Electronic supplementary material The online version of this article (10.1186/s12879-017-2911-x) contains supplementary material, which is available to authorized users.


Background
Urinary tract infection remains to be one of the most common infectious diseases diagnosed in outpatients [1]. It is most often caused due to bacteria, but may also include fungal and viral infections [2]. Gram-negative bacteria cause 90% of UTI cases while gram-positive bacteria cause only 10% of the cases. The most frequent isolated uropathogen is Escherichia coli, accounting for 65%-90% of urinary tract infections [3,4]. The relative frequency of uropathogens varies depending upon age, sex, catheterization, hospitalization and previous exposure of antimicrobials [5][6][7].
The emergence of antibiotic resistance in the management of UTIs is a serious public health issue. Particularly in the developing world where there is high level of poverty, illiteracy and poor hygienic practices, there is also high prevalence of fake and spurious drugs of questionable quality in circulation [4,8]. The easy availability in the community without prescription and low cost make the drugs subject to abuse [9]. With regards to resistance rates in Ethiopia, a report showed that high incidence of resistance to the commonly prescribed antibiotic agents was observed in some regions [6,10,11].
Even though, there are few published information concerning the etiology and resistance pattern of UTIs in some hospitals of Ethiopia [6,[10][11][12], there was no previous study and published information on UTI in the study area. This study was conducted in order to assess the prevalence of bacterial uropathogens and their in vitro susceptibility patterns to commonly used antibiotic agents amongst outpatients with complaints of UTI in Shashemene referral hospital.

Isolation of bacteria from urine samples and preservation
Urine dipstick test was done by using Multisticks of Medi-Test combi 10®SGL leukocyte esterase and nitrite [14]. The urine samples were also examined microscopically for pus cells and then inoculated on MacConkey agar and Blood agar media. Inoculated agar plates were incubated aerobically at 37°C for 24 to 48 h. The cultured plates were examined for growth and mixed colonies on a plate were re-inoculated further on blood agar and nutrient agar medium for growth of discrete colony. Gram staining was done for all isolates as per the standard procedures and the smears were examined microscopically for their morphology and staining reactions [15].
Isolates were streaked on BUG agar for further identification using standard operation protocols for aerobic bacterial identification in GEN III OmniLog® Plus ID System of BiOLOG [16].

Statistical methods
Our data were analyzed using SPSS for Windows, version 16.0 (SPSS, Inc., Chicago, Ill). Pearson Chi-square test was employed to test the existence of association between discrete variables. P-value of <0.05 was considered to indicate statistically significant differences. A binary logistic regression analysis was used to calculate odds ratio (OR); Crude Odds Ratio (COR) and Adjusted Odds Ratio (AOR) to ascertain the degree of association between risk factors and UTI.

Prevalence of urinary tract infection among outpatients in Shashemene referral hospital
We examined a total of 384 (Table 1) outpatients with complaints of urinary tract infection in Shashemene referral hospital and found 90.1% overall prevalence of UTI in the study area ( Table 2). The laboratory test results indicate that all samples 384 (100%) were positive for leukocyte esterase, while 88.5% were positive for nitrite and 11.5% were negative ( Table 2). On the basis of microscopy of urine, it was found that 90.1% of the samples were positive for both pyuria and bacteriuria ( Table 2). Of the total urine samples, 346 (90.1%) were positive and 38 (9.9%) were negative for the growth of different uropathogens on blood agar media (Additional file 2: Fig. S1.1c). On the other hand, 340  Fig. S1.1b). From the total patients with UTI compliant, 134 (34.89%) were in the old age group while 115 (29.94%) were in the young age group ( Table 2).

Clinical symptoms associated with urinary tract infection
Clinical symptoms of UTI are the result of a complex series of host pathogen interactions that could lead to bacterial invasion and persistence and ultimately to disease [19]. In this study, clinical symptoms were used in the diagnosis to determine the course of infections.

The prevalence of Uropathogens from urine samples of UTI positive patients
The relative prevalence of uropathogens isolated from mid-stream urine samples is shown in Table 3. Totally, 429 isolates of ten different kinds of uropathogens were identified from the urine samples. Of these, 417 (97.2%) belonged to bacteria while the rest, 12 (2.8%) were fungi.

Prevalence of uropathogens among UTI positive patients by sex
Our result shows, of the total positive patients for uropathogens, while 71.7% were female, 28.3% were male (Table 3). Statistical analysis revealed that there was a significant relationship between sex and the prevalence of uropathogens P = 0.041, X 2 = 4.192 and AOR = 2.396 ( Table 4).

Prevalence of uropathogens among UTI positive patients by place of residence
For all uropathogens isolate, the highest prevalence was observed in patients from the rural area (62.9%) than patients from the urban (37.1%) ( Table 3). Statistical analysis revealed that there is significant relation between place of residence and UTI causing microorganisms (X 2 = 13.089, P = 0.000, COR = 4.648) ( Table 5).
Prevalence of uropathogens among UTI positive patients by age group The highest prevalence of microbial isolates was observed in adult age group (69.69%) followed by the young (21.67%) ( Table 3) and it was statistically significant P = 0.022, X 2 = 5.235 and AOR = 3.404 (Table 4). This might be due to active sexuality of the age group [20,21]. However, it will require further investigations to validate.

Risk factors to urinary tract infection
We have considered various risk factors that might play a role in escalating UTI such as sex, age, spermicide or diaphragm use, catheter use, severe underlying illness, genital hygiene, frequent sex and improper urine storage.
Logistic regression analysis was used to calculate odds ratio (OR) to ascertain the degree of association between these risk factors and UTI. Both urine storage and place of residence had positive and statistically significant relationships with UTI (X 2 = 23.691, P = 0.000 COR = 0.121 and X 2 = 13.089, P = 0.000 COR = 4.648, respectively) ( Table 5).
Among risk factors pertinent to females, statistical analysis revealed that there was no significant relationship between use of diaphragm and the prevalence of   Table S1a and b). This might be due to the fact that most females were not using diaphragm.

Antibiotic susceptibility test
In-vitro antibiotic susceptibility tests were done on a total of 30 isolates using a standard method of agar disk diffusion technique following the National Committee for Clinical Laboratory Standards. Eleven antibiotic agents were used for the test (Amoxicillin, Chloramphenicol, Ciprofloxacin, Gentamicin, Nalidixic acid, Nitrofurantoin, Trimethprime-Sulfamethoxazole (TMP-SMX), Tetracycline, Vancomycin, and Cephalexin, Ceftriaxone (Additional file 2: Fig. S1.6a-c).

Discussion
We have shown that the overall prevalence of UTI was 90.1%. In accordance with [22,23], the prevalence of UTI is higher in females (63.3%) than males (26.8%) ( Table 2). This might be due to the anatomical differences of urogenital organs between the two sexes [24,25]. Prevalence difference has been also observed among various age groups. This difference suggests that age is one risk factor associated with UTI. The high incidence of UTI amongst the old age group could be due to genito-urinary atrophy and vaginal prolapse after menopause in female which in turn increases the risk of bacteriuria by increasing vaginal pH and decreasing vaginal Lactobacillus thereby allowing gram-negative bacteria to grow and act as uropathogens [26]. Moreover, it was indicated in another study [21,27] that UTI is the most common infection in elderly populations. The high prevalence recorded amongst young age group could be due to increased sexual activity in the age group [26].
Fever, dysuria, urgency, frequency, flank pain and suprapubic pain were the observed clinical symptoms in our study and is comparable with report of [28]. In contrast, dysuria and flank pain were symptoms statistically not significant. Even though statistically not significant, flank pain was the symptom in which positive cases were noted in 200 (57.8%) patients, next to supra-pubic pain and urgency.
Bacterial species were the more prevalent uropathogens compared to other groups of microbes. This result is in accordance with that reported by [2] which indicated that among different microorganisms causing UTIs, bacteria accounts for more than 95% and the rest may also include fungal and viral infections. Among the isolates, gram-negative bacteria, gram-positive bacteria and fungi constituted 59.2%, 38% and 2.7%, respectively. The highest prevalence of gram-negative bacteria in this study is in agreement with that reported by [29,30].
Moreover, the prevalence of E. coli (39.3%) in the current study is comparable with that reported from Nigeria, Zaria by [31], but higher than the reports of [32] from Brazil, [18] from Pakistan, and [33] from Mekele hospital, Ethiopia.
The prevalence of Staphylococcus epidermidis 12.1% in our study is also comparable with the study reported by [34] which was 13%. Similarly, the prevalence of other non-E.coli aerobic gram-negative rods is comparable with the study reported by [35], which was generally ranging from 5 to 10%.
Dermacoccus nishinomiyaensis (Micrococcus nishinomiyaensis) prevalence is 6.3%. According to the study conducted by [36], it was reported that Dermacoccus nishinomiyaensis is prevalent in urinary tract during microbial urethral stent colonization (MUSC). In similar study conducted by [37], urethral stents inserted during urinary tract infection were more frequently colonized (59%) by urophatogens compared to those placed in sterile urine (26%). Female sex and continuous stenting were significant risk factors for MUSC.
In agreement with [38], our study also showed that there was mixed bacterial species infection per a patient. Studies reported that mixed infections (poly-microbial) are more likely to occur in patients with underlying disorders that interfere with free urine flow. Moreover, it is frequent in those with indwelling catheter. The similarities and differences in the type and distribution of uropathogens may result from different environmental conditions and the prevailing practices in each country and region.
Furthermore, we assessed the relationship between various risk factors and UTI. Sex was one of the considered factors and the result indicated that UTI prevalence was higher in females than males for each isolate. Previously, [25,39,40] have shown that incidence of UTI was found to be higher in females than in males. This is probably due to multiple factors contributing to the problems among females. The first possible reason would be the anatomical feature of the female urethra, which is much shorter than males' urethra. The Shortness of the urethra, allow the pathogens easy access to the bladder during sexual intercourse. This in turn results in increased bacterial counts in the bladder after intercourse [41,42].
Statistical analysis showed that patients who were holding urine in their bladder for a long period of time had more probability of having UTI than those who were not holding. In a study done on risk factors of UTI in Pakistan, improper holding 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 [26,43].
Among the risk factors of UTI pertinent to females, active sexuality/frequent sex/ has a positive and statistically significant relationship with the prevalence of UTI (Additional file 3: Table S1a). Statistical analysis revealed that there was a significant difference between the prevalence of UTI in patients who were practicing frequent sexual activity and those who were not (Additional file 3: Table S1a and b). This indicates that those patients who were practicing frequent sexual activity would have more probability of having UTI than those who were not. This is consistent with the findings reported by many authors. They showed the incidence of UTI is higher in sexually active females causing 75-90% of bladder infections, [21,44,45].
Similarly, keeping genital hygiene has a positive and statistically significant relationship with the prevalence of UTI. Statistical analysis done using both univariate and multivariate logistic regression revealed that there was a significant difference in the prevalence of the UTI between females keeping their genital hygiene and those who were not (Additional file 3: Table S1a and b). This indicates that those patients who were not keeping their genital hygiene had more probability of contracting UTI than those who were keeping their genital hygiene. This could be attributed to multiple factors probably contributing to the increasing problem of infection among these females. One of such factors was most of the female patients were from rural areas, and they have poor hygienic practices. Poor hygienic practice results in direct fecal contamination of urinary tract from the anus in females. Consequently it provides easier access to the pathogens overgrowth and ascent to bladder [41].
The results also revealed that among eleven antibiotics used for susceptibility test, gentamicin was the most effective antibiotics 93.3% followed by chloramphenicol and nitrofurantoin. This might be due to the fact that gentamicin is offered in injection form and its unavailability in tablet form in the community, minimized the chance to abuse (Unpublished data).
We have shown there was multiple antibiotic resistances on many of the identified species. Thus, E. coli, L. cetreum and S. typhimurium were members resistant to more than five antibiotics while the rest of the isolates were resistant to three to five antibiotics. The development of higher resistance against the above-mentioned antimicrobials could be due to repeated use or prolonged exposure of uropathogens to the antibiotics [46]. Repeated use of antibiotics can damage peri-urethral flora, allowing uropathogens to colonize and subsequently infect the urinary tract. Hence, leaving clinicians with very few choices of drugs for the treatment of UTI. Moreover, this condition enables bacteria to exchange their genetic material through horizontal gene transfer resulting in resistant gene that confer resistance to a particular antibiotic [47].

Conclusions
Urinary tract infection is the most common problem throughout the world, particularly in developing countries. In addition, emergence of bacterial strains resistant to commonly used antibiotic agents is widespread phenomenon all over the world. From the results of our study, we concluded that, UTI is prevalent in the study area and the most frequently isolated uropathogen was E. coli followed by Staphylococcus spp. In addition, Leuconostoc species, Raoultella terrigena (Klebsiella spp), Salmonella typhimurium, Dermacoccus nishinomiyaensis, Citerobacter freundii and Issatchenkia orientalis were isolated. Female sex, poor hygienic practice of the rural residents, improper urine storage, frequent sex and lack of genital hygiene, were the major risk factors for the high prevalence of UTI. Gentamycin was the most effective antibiotic for the area followed by chloramphenicol and nitrofurantoin. In contrast, amoxicillin, vancomycin and cephalexin were the drugs to which the isolates developed resistance. Generally, as there was no previous study and published information on UTI in the study area, this study has provided baseline data on the prevalence, drug sensitivity, and some potential risk factors of UTI and is, therefore, of clinical and epidemiological significance.