The Bacterial Profile and Antimicrobial Susceptibility Patterns of Urinary Tract Infection Patients at Pawe General Hospital, Northwest Ethiopia

Urinary tract infection remains the most common infection widespread worldwide in both community and hospital settings. Rapidly increasing antibiotic resistance of uropathogens is resulting in limited treatment options. Thus, understanding the current uropathogens and their antimicrobial susceptibilities is essential for effective urinary tract infection treatment. The purpose of this study was to isolate, characterize, and determine the antimicrobial susceptibility patterns of bacterial pathogens associated with urinary tract infection at Pawe General Hospital in Northwest Ethiopia. A hospital-based cross-sectional study design was conducted from January to April, 2020, at Pawe General Hospital. Midstream urine specimens were collected from 141 individuals with suspected urinary tract infection for bacteriological identification and antimicrobial susceptibility testing. Among the 141 study participants, twenty-nine (20.6%) showed significant bacteriuria. Escherichia coli (42.6%) had the highest proportion of isolated uropathogen followed by Klebsiella spp. and Pseudomonas spp. each (10.7%); Proteus spp. (9.3%); coagulase negative staphylococci, Staphylococcus aureus, and Enterobacter spp. each (6.7%); Citrobacter spp. (4%); and Enterococcus faecalis and Streptococcus spp. each (1.3%). Outpatient isolates showed a resistance of 64% and 78.6% to amoxicillin-clavulanic acid and tetracycline, respectively. Inpatients showed 63.9% and 87.2% of resistance to cephalexin and tetracycline. It was also observed that all the isolates have a multiple antimicrobial resistance index greater than 0.20 except Citrobacter spp. (0.142) in inpatients. Even though in this locality, most isolates were sensitive to ceftriaxone, gentamicin, ciprofloxacin, nitrofurantoin, and norfloxacin, they are considered appropriate antimicrobials for empirical treatment of urinary tract bacterial infections. Periodic monitoring of etiology and drug susceptibility is highly recommended, along with health education on the transmission and causes of urinary tract infection.


Introduction
Urinary tract infections (UTIs) are infections caused by infectious agents (bacteria, fungi, virus, and parasites) present and propagate in any part (bladder � cystitis, kidney � pyelonephritis, urethra � urethritis, ureters � urethritis, and urine � bacteriuria) of the urinary tract [1]. e infection of the urinary tract can either be asymptomatic or symptomatic and can occur in uncomplicated (UTIs that occur in a normal genitourinary tract with no prior instrumentation) or complicated (infections that diagnosed in genitourinary tracts that have structural or functional abnormalities, including instrumentation such as indwelling urethral catheters, and are frequently asymptomatic) individuals [2]. Annually, one hundred fifty million people are diagnosed with urinary tract infections across the world and subsequently spend around six billion dollars on health care [1,2]. In low-income countries like Ethiopia, UTIs lead to treatment failures and long-term hospital complications, threaten our ability to perform modern medical procedures, impose a major economic burden on society, and finally result in too much morbidity and mortality [1][2][3].
Compared to other uropathogens, bacterial urinary tract infections are the most common and dangerous infections in humans and occur frequently in the community and hospital environments [1][2][3][4]. Escherichia coli, Klebsiella spp. Enterobacter spp., Proteus spp., Pseudomonas spp., Acinetobacter spp., Serratia spp., and Citrobacter spp. are Gram-negative bacteria with the most causative agents covering ninety percent of UTIs. Group B streptococci, Enterococcus spp., and Staphylococcus spp. are from the Gram-positive bacteria responsible for the remaining ten percent of UTI cases [3,4]. Of all, E. coli are the most common and frequently isolated uropathogens accounting for sixty-five up to ninety percent of the urinary tract bacterial infections [1][2][3][4][5][6]. Depending on the different factors like age, sex, catheterization, hospitalization, and previous exposure to antimicrobials; the relative frequency of both Gram-positive and Gram-negative bacterial pathogens may greatly vary.
Treatment and therapy for urinary tract infections is determined experimentally by the antimicrobial susceptibility testing. In Africa, however, it is observed that due to the high level of poverty, illiteracy, and poor hygienic practices, the easy availability and low cost of drugs, the high prevalence of fake and spurious drugs, the uncontrolled prescription and usage of antibiotics, and the lack of rapid laboratory facility for sensitivity test contributes to the emergence of resistant bacterial infections especially in uropathogens.
e resistant strains of bacteria can be accelerated and spread by the transfer of resistant genes among species and genera through horizontal gene transfer (transformation, transduction, or/and conjugation) with mobile genetic elements (plasmids, transposons, or/and bacteriophages). is process results in the development and dissemination of new antimicrobial resistance varieties and novel resistance mechanisms among uropathogens due to the presence of R-plasmids that mediate resistance genes against various classes of commonly used antimicrobial agents. Since antibiotic resistance rates of pathogenic bacteria may vary from country to country, regionally and locally, and can also change rapidly with time, as such, they need to be monitored and managed closely because of their public health implications and impacts [5,6].
Regarding the resistance rates in Ethiopia, different reports showed that a high incidence of resistance to the commonly used antimicrobial agents was observed [1,2,[4][5][6], even though there are few published information available concerning the etiology and resistance patterns of urinary tract bacterial infections in some hospitals of Ethiopia. To the best of our knowledge, there is no previous study and published information on UTIs in the study area.
us, this study was aimed to assess bacteriological profile and antimicrobial susceptibility patterns of symptomatic urinary tract infection among patients in Pawe General Hospital (PGH).

Study
Setting, Design, and Period. Hospital-based crosssectional study was conducted at Pawe General Hospital from January to April 2020. Pawe General Hospital is the mere public hospital found in Pawe town and provides various health services for routine cases. e hospital was chosen because it covers both rural and urban areas of the town. e town is found at a distance of 573 km from Addis Ababa which is the capital city of Ethiopia.

Sample Size Determination.
Single population proportion formula was used to determine the sample size.
Since there was no data in Ethiopia, the prevalence of UTI among both inpatients and outpatients was taken from Chad (32.7%) which was done by Kengne et al. [7] n � (1.96 erefore, a total of 141 UTI inpatient and outpatients were included in the study from the hospital.

Inclusion and Exclusion Criteria.
One hundred forty-one patients to whom a cytobacteriological examination of urine was prescribed and who had not received antimicrobials within the previous two weeks were eligible for inclusion due to the fact that the antibiotic must have inhibited or destroyed the pathogens. UTI patients who were not willing to participate were excluded from this study.

Sample Collection.
Clean catch mid-stream urine sample collection approach with aseptic measures was applied to minimize contamination of the sample [8]. A total of 141 fresh midstream urine samples were collected from both inpatient and outpatient UTI suspected individuals using sterile screw-capped universal container. e specimens were appropriately labeled and immediately processed after sampling.

Culturing and Identification of Isolates.
e collected urine samples were spread on cysteine lactose electrolytedeficient medium, MacConkey agar, and blood agar (Oxoid, UK) using L-shaped glass spreader and incubated aerobically at 37°C for 24 h. A significant bacteriuria was considered if urine culture yields ≥10 5 CFU/mL. All positive urine cultures with significant bacteriuria were further identified by their colony characteristics, Gram-staining reaction, and pattern of biochemical profiles using standard procedures. Enterobacteriaceae were identified by H 2 S production and carbohydrate utilization tests in TSI agar, motility test, urease test, and IMViC (indole, methyl red, Voges-Proskauer, and citrate utilization) tests. e Grampositive bacteria were identified using catalase and coagulase tests [9].
2.6. Standardization of the Inoculum. 0.5 McFarland standard was prepared by mixing 0.50 mL of a (1.175% w/v) dehydrate barium chloride (BaCl 2 .2H 2 O) solution with 99.50 mL of (1% v/v) sulfuric acid (H 2 SO 4 ) with constant stirring in a graduated cylinder. e turbidity standard solution was aliquoted into test tubes identical to those used to prepare the inoculum suspension. In addition, the absorbance of the prepared 0.5 McFarland standard solution was further confirmed to be 0.08 to 0.12 at 625 nm using spectrophotometry. To prevent evaporation, the tube containing a standard solution was tightly sealed and stored at room temperature. Before comparing with the bacterial suspension, the turbidity standard tube was vigorously mixed by the vortex mixer which makes a uniform turbid appearance [10].

Inoculum Preparation.
From an overnight culture, 3-5 morphologically identical bacterial colonies were suspended in 5 mL of normal saline (0.85%; 8.5g/L NaCl) vigorously mixed by the vortex mixer and comparing to that of 0.5 McFarland standards which are approximately equivalent to 1.5 × 10 8 CFU/mL. After adjusting the turbidity, a sterile cotton swab was dipped into the suspension and streaked over the entire surface of the prepared medium by rotating the plate in 60 o to ensure the even distribution of the inoculum [10].

Antimicrobial Susceptibility
Testing. Antimicrobial susceptibility testing was performed using Kirby-Bauer's disk diffusion method [11]. e antimicrobial agents were selected due to the physicians prescribed for treatment of UTIs in the study setting. e standard commercially available (Oxoid, UK) antibiotic discs, namely, amoxicillin/ clavulanic acid (AMC, 20/10 µg), cephalexin (CEPH, 30 µg), ceftriaxone (CRO, 30 µg), tetracycline (TE, 30 µg), gentamicin (CN, 10 µg), ciprofloxacin (CIP, 5 µg) nitrofurantoin (F, 300 µg), and norfloxacin (NOR, 10 µg) were used. ese discs were aseptically laid with proper spacing on the surface of the inoculated agar plates and then pressed firmly onto the agar with sterile forceps and incubated at 35-37°C for 18-24 hours. e diameter of inhibition around the discs was measured to the nearest millimeter and interpreted as sensitive (S), intermediate (I), or resistant (R) according to the annually published microbiological breakpoints of CLSI [12]. Bacterial isolates resistant to three or more antimicrobials belonging to the different structural classes were considered multidrug resistant (MDR).

Quality
Control. All materials, equipment, and procedures required for the study were adequately controlled. All specimens were immediately processed after collection. Only specimens which produced ≥10 5 CFU/mL of urine were considered significant for the study. E. coli (ATCC® 25922), and S. aureus (ATCC® 25923) standard reference strains were used for testing sterility and performance of culture media and antibiotic discs. Generally, CLSI 2020 guidelines were strictly followed.

MARI Determination.
e multiple antimicrobial resistance index (MARI) is the ratio between the number of antibiotics resisted by the pathogen (a) and the total number of antibiotics used in this study (b).

Ethical Considerations.
Ethical clearance was obtained from Pawe General Hospital Administration. A written informed consent was also obtained from each participant before collecting the data. All information obtained in the course of the study were kept confidential and used solely for the purpose of the study.

Results and Discussion
Urinary tract infections (UTIs) are one of the most common and serious infections of the human urinary system that occurred in the community and hospital environments.
UTIs are most often treated empirically and the antimicrobial agent selection criteria are determined on the basis of the pathogen type and its expected antimicrobial resistance patterns. Also, the management of UTIs has been jeopardized by the increase in the incidence of antimicrobial drug resistance. us, there is a need for periodic monitoring of the pathogens of UTIs and their antimicrobial susceptibility pattern in the locality [13].
In the present study, the details of culture characterization are shown in Table 1. From 141 samples collected in inpatient and outpatient settings, 29/141 (20.6%) samples were positive urine cultures, from which 75 bacterial isolates were obtained. E. coli (42.6%) was the most predominant bacterium isolated from urine, followed by Klebsiella spp. and Pseudomonas spp. each (10.7%); Proteus spp. (9.3%); coagulase negative staphylococci (CNS), S. aureus, and Enterobacter spp. each (6.7%); Citrobacter spp. (4%); E. faecalis and Streptococcus spp. each (1.3%), and this study was similar to the previous studies of Kibret and Abera [13] and Alemu et al. [14]. However, the observed high proportion of E. coli was varied with previously published studies conducted in Nigeria by Ekwealor and his colleagues [15] where Staphylococcus spp. was found to be the predominant urinary tract pathogen. is might be due to the variation of UTI-causing bacterial pathogens, differences in sample size, specimen collection technique, and study settings.
In the current study, the frequency of inpatient 47/75 (62.7%) uropathogens was greater than outpatients 28/75 (37.3%) ( Table 1). is is in line with the previous studies of Kengne et al. [7] that revealed bacteriuria was more present in inpatients (70.4%) compared to outpatients (29.6%). Tesfa et al. [16] also further confirmed that inpatients were two times more likely to have culture positive results than outpatients.
is might be due to the hospitalization, comorbidity, long-term antibiotic treatment, and immunocompromised conditions. In this study, Gramnegative bacterial isolates were more prevalent (80%) than Gram-positives (20%).
is finding is in agreement with studies done in Jimma, Jijiga, Dire Dawa, and Shashamane, where 80.9%, 76%, 73.1%, and 59.2% of the isolates were Gram-negatives [2,[17][18][19]. Differences in characterization and identification methods are known to influence the relative prevalence of bacteria, which makes comparison difficult. Bacterial etiologies of UTIs can also vary across regions and over time within a population.
is high level of resistance observed with amoxicillin-clavulanic acid and tetracycline might be due to the self-medication and irrational drug utilization habits of the communities for treating all kinds of bacterial infections.
MARI is a tool that indicates the spread of antimicrobial resistance in a given study population. In this study, 0.142-0.714 and 0.250-0.625 were the inpatient and outpatient MARI values of the isolates, respectively (Tables 4 and 5). Only Citrobacter spp. (0.142) gave MARI of <0.20 while others gave higher MARI. Similarly, [15] reported that only Streptococcus spp. (0.125) was < 0.20 MARI value. Any MARI greater than 0.20 implies that the strains of such bacteria originate from an environment where several antibiotics are used or misused [25]. is 6 Scientifica implies that a very large proportion of the bacterial isolates have been exposed to several antibiotics and thus have developed resistance to these antibiotics. Similar incidence was reported in the work of Ekwealor et al. [15] and Oli et al. [25] though not exactly with the same set of antibiotics.

Limitations of the Study.
e study did not include the patients' clinical data such as age, gender, and being catheterized and noncatheterized and other associated risk factors of the patients. Due to the COVID-19 pandemic, species level identification and the presence of genetic resistance determinants were not performed.

Conclusions
e overall prevalence of UTI in the study participants was 20.6% (29/141). In this study, E. coli, Klebsiella spp., Pseudomonas spp., and Proteus spp. were the most prevalent among the investigated uropathogens. Inpatients had a higher risk of developing bacterial infections compared to outpatients. Ceftriaxone, gentamicin, ciprofloxacin, nitrofurantoin, and norfloxacin are considered as appropriate antimicrobials for empirical treatment of UTIs in the study area. Greater than 0.2 MARI values of the uropathogens from urine samples of this study underscore the need for continuous monitoring of the etiology and antimicrobial susceptibility testing of bacteria, along with health education on the transmission and causes of urinary tract infection.

Data Availability
e data used to support the findings of this study are included within this article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
AG and AA designed the project, carried out the experiments, analyzed the data, and drafted and edited the manuscript. Both authors have read and approved the final manuscript.