Bacterial uropathogens and burden of antimicrobial resistance pattern in urine specimens referred to Ethiopian Public Health Institute

Background Urinary tract infections (UTIs) are the leading causes of morbidity in the general population, and is the second most common infectious disease after respiratory infections. Appropriate antibiotic therapy is essential to achieving good therapeutic results. Therefore, the purpose of this study was to investigate the profile of pathogens cultured from urinary tract infections and to determine their resistance profiles to commonly prescribed antibiotics. Method A cross-sectional study was carried out at the National Referral Laboratory of the Ethiopian Institute of Public Health from January 2017 to December 2018. All positive cultures were characterized by colony morphology, Gram stain, and standard biochemical tests. The antimicrobial susceptibility test of the isolate was performed using the Kirby- Bauer disk diffusion test on Muller-Hinton agar. In addition, bacterial identification, antimicrobial susceptibility testing and phenotypic detection of MDR were performed with VITEK 2 Compact according to the manufacturer’s instructions. Result Out of 1012 cultured urine specimens, 325 (32.1%) was showed significant bacteriuria. The overall prevalence of UTIs was 325(32.1%) and the highest prevalence rate was obtained from 21–30 years age group 73(22.5%). Among UTIs patients, 583(57.6%) were females and 429(42.4%) were males. The UTIs of 179 (55%) women is relatively higher than that of men 146 (45%). Among 325 isolates, Gram-negative bacteria (GNB) appeared more frequently 252 (51.7%) than Gram-positive bacteria 63 (19.4%). In GNB, E. coli 168(66.7%), Klebsiella species 32(12.7%), and Enterobacter species 13 (5.2%) were dominated isolates whereas in GPB accounted for coagulase-negative staphylococcus (CoNS) 33(52.4%), Enterococcus species 16(25.4%), and Staphylococcus aureus 10(15.9%). Major of the isolates showed high levels of antibiotic resistance to commonly prescribed antimicrobials. Imipenem, Amikacin, and Nitrofurantoin were the most sensitive antibiotics for Gram-negative isolates while Nitrofurantoin, clindamycin, and Gentamycin were effective against gram-positive uropathogens. Overall, 156/256(60.9%), 56/256(22.4%), 10/256(4%) of gram-negative isolates were MDR, XDR, and PDR respectively while among the GPB isolates, 34/63(53.1%), 10/63(15.8%), and 1/63(1.6%) were MDR, XDR, and PDR isolates respectively. Among the tested bacterial strains, 190/319 (59.5%) were MDR, 66/319 (20.7%) strains were XDR, and 11/319 (3.45%) were PDR isolated. Conclusion The prevalence of urinary tract infection was high, and Gram-negative organisms were the most common causes of UTIs in this study. It was found that the resistance to commonly used antibiotics is very high. Early detection and close monitoring of MDR, XDR, or even PDR bacterial strains must be started by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance that is now a global problem.

Introduction Urinary tract infections (UTIs) are infections caused by the presence, growth and spread of microorganisms in the urinary tract. It is usually caused by bacteria from the digestive tract climbing into the urethra opening and starting to multiply to cause an infection. It is one of the most common bacterial infections seen in clinical practice in developing countries [1]. Urinary tract infections are among the most common infections in humans, exceeded in frequency among ambulatory patients only by respiratory and gastrointestinal infections. It is believed to exist when pathogenic microorganisms are detected in the urine, urethra, bladder, kidneys, or prostate with or without the presence of specific symptoms [2].
The clinical presentations of UTIs depend on the part of the urinary tract affected, the causative pathogens, the severity of the infection, and the patient's ability to mount an immune response to it [3]. An infection in the bladder is called cystitis or a bladder infection. Infection of one or both kidneys is called pyelonephritis or a kidney infection. The ureters are tube-like structures that take urine from each kidney to the bladder are only rarely the site of infection. The urethra, that empties urine from the bladder to the outside and its infection is called urethritis [1,4].
Intestinal gram-negative bacteria are a serious urinary tract infection problem in many parts of the world. Symptomatic urinary tract infections (UTIs) have been estimated to occur with approximately seven million emergency department visits and 100,000 hospital admissions each year [1]. Urinary tract infections have become the most common nosocomial infection, accounting for 35% of nosocomial infections and the second leading cause of sepsis in hospitalized patients [5,6].
The causative agent of urinary tract infections may be of community or hospital origin. Community-based infections are caused by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus saprophyticus, or Enterococcus faecalis, while nosocomial infections are Escherichia coli, Pseudomonas aeruginosa, Proteus, Enterobacter, Serratia, or Enterococcus. [5]. Everyone is susceptible to UTIs; however, some traits increase exposure to uropathogens and others increase susceptibility to the development of symptoms after colonization.
Factors that increase exposure to uropathogens include the presence of urinary catheters and vaginal intercourse; markers of host susceptibility to the disease include age; Kind; and the presence of underlying conditions affecting the urinary tract, such as pregnancy, diabetes, or an enlarged prostate. Bacterial characteristics increase the ability of the agent to be transmitted, cause infection, and cause disease [3]. Recurrent infections are common and can lead to irreversible kidney damage, leading to renal hypertension and renal failure in severe cases [3].
UTI is the most common cause of nosocomial infection among hospitalized patients [6]. It is generally accepted that UTI can only be diagnosed based on a microscope and microbial culture. Urine culture is the gold standard for assessing infection, but in a majority of cases, therapy is initiated before having culture results. This can be rationalized in the sense that urine culture and susceptibility testing are costlier that than the costs of antibiotics in most countries [3].
In most cases, it is necessary to start treatment before the final microbiological results are available. The test strip/slide method is used in many centres only as a screening method, but cultures are required for the final diagnosis [7,8]. Region-specific surveillance studies that gather knowledge about UTIs and their resistance patterns can help clinicians choose the most appropriate empiric treatment. Antibacterial drugs are helpful in the management of bacterial infections. Although some bacteria have inherent resistance to even recently formulated antibacterial agents, the emergence of acquired antimicrobial resistance has been observed in most pathogens [9][10][11].
Antibiotic resistance has become a significant public health problem with grave consequences for the treatment of infections. Over the past decade, there has been an increasing prevalence of carriage and infection with multidrug-resistant organisms (MDROs). This ultimately affects both social and economic development. Multidrug resistance (MDR) has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans [12], poultry [13], cattle [14,15], and fish [16,17] that increase the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains [12][13][14]18]. Infections caused by MDR organisms have increased mortality compared to those caused by non-MDR bacteria. The problem is attributed to the misuse of antimicrobials, which create selective pressures that facilitate the emergence of resistant strains [10].
To prevent the problem of drug resistance, the World Health Organization (WHO) has put in place several interventions. These include the creation of a national task force, the development of indicators to monitor and assess the impact of antimicrobial resistance; and designing microbiological baselines capable of effectively coordinating the surveillance of antibiotic resistance among common pathogens [19,20]. Although these interventions appear to be well implemented in the developed countries, lack of resources has limited their implementation in many developing countries where treatment options also tend to be relatively limited.
Thus, although this is a global problem, antimicrobial resistance tends to be more important in developing countries than in developed countries [21]. Studies in the eastern part of Nepal, India, and Bangladesh have reported an increased resistance of urinary pathogens to commonly used antibiotics [22][23][24]. The magnitude of UTI-causing organisms and their sensitivity to antibiotics varies from region to region, so antibiotic resistance and sensitivity patterns need to be tested frequently. The causative agents and degree of resistance to the most commonly prescribed drugs used in the treatment of UTIs may also have changed over time. Therefore, continuous and periodic monitoring of the local prevalence of uropathogens and their susceptibility profile will be of public health importance to promote the appropriate use of existing antibiotics [3]. Therefore, this study was performed to determine the frequency and antibiotic susceptibility pattern of urinary tract pathogens received in the Ethiopian public health institute.

Study design and study period
A cross-sectional study was conducted from January 2017 to December 2018 in Addis Ababa, Ethiopia.

Study setting
The study was conducted at Ethiopian Public Health Institute, which is a national reference laboratory in Addis Ababa, Ethiopia.

Source population
All urine samples were referred to the Department of Clinical Microbiology of the Ethiopian Institute of Public Health for culture and sensitivity testing during the study period (January 2017 to December 2018).

Study population
All the urine samples obtained between January 2017 and December 2018 for urine culture and susceptibility testing from patients of all ages were included in this study.
Inclusion criteria. All data with complete information such as age, sex, number of patients, pathogens isolated, antibiotic resistances of isolates, were included in the study.
Exclusion criteria. Data with incomplete information was excluded from the study.

Sample size determination and sampling
A total of 1012 urine samples were collected between January 2017 and December 2018 at EPHI national microbiology referral Laboratory.

Ethical review
The ethical review was first obtained from the ethics and review committee of the Menelik II Health Science College and Ethiopian public health. Formal written letters have been distributed to the Department. Before collecting data, study participants were informed of the study and obtained their consent, and confidentiality was maintained by omitting their names and personal identifiers throughout the study. The research objectives and procedures were explained in writing (if they can read and write) or verbally (if they are illiterate), and the children and their parents or caregivers were asked if they would like the children to participate in the research. Parents or caregivers confirmed their children's willingness to participate in the study by signing an informed consent form. Confidentiality was strictly maintained during data processing and report writing.

Isolation, identification and characterization of bacteria causing UTIs
Isolation of pathogens. The pathogens have been isolated from urine sediments. The urine sample was shaken well to resuspend the organisms and 10 ml was decanted into a centrifuge tube. The tube was kept closed to avoid contamination [25]. The sample was centrifuged at 2000 rpm for 10 minutes. The entire sample was decanted, but 0.5 ml of sediment from the tube was suspended with a sterile metal loop. A loop of the sediment was inoculated into a tube with a medium [26].

Identification of pathogens
Cultural observation. Color, size, and colony morphology are observed from the incubated plates.
Microscopic examination of urine specimen. Slides were prepared from each different colony observed on the plates and Gram staining was performed. Results such as arrangement, gram reaction (gram-positive or gram-negative), and shape of bacteria are seen from the investigations [25,27]

Microbiological analysis of urine specimen
Uropathogens were identified by inoculating /streaking of urine samples on various selective and differential media such as CLED agar, blood agar, chocolate agar, MacConkey agar based on their colour morphology after an incubation time of 18-24 hours at 37˚C [26].

Biochemical examination
The selected colonies were subjected to culture-based biochemical examinations, microscopic and microbiological examinations (Oxoid Ltd, Basingstoke, UK) (carbohydrate utilization test, triple sugar iron agar test or Kligler iron agar), oxidase test, catalase test, nitrate reduction test, indole production test, methyl red test, Voges-Proskauer test, citrate recovery test, urease test) for pathogen detection [27,28].
Data analysis. The collected data was sorted in the prepared spreadsheet and entered into EpiData v4.6. Then, the data was entered into SPSSv.25 software, which was used for data analysis. The laboratory results are summarized and presented by age groups, sex and isolation types. The prevalence rate of the isolates, frequency, susceptibility patterns and other descriptive statistics were computed. Percentages and ratios were presented in the tables. P-value (p<0.05) was considered statistically significant.
Quality control. Data were collected from the EPHI Microbiology Laboratory for processing, analyzing and determination of prevalence uropathogens, and drug susceptibility profiles of uropathogens. The completeness, correctness, clarity and consistency of the data collected were checked before being included in the data entry form.

Demographic characteristics
Of the 1028 patient data recorded, 1012 laboratory test results were obtained and analyzed. Due to their incomplete information, 16(1.6%) of the participants' data were excluded from the study. The majority of the study participants were females, which accounts for 583 (57.6%). The mean age of the study participants was 40.17 with a standard deviation of ±18.57.

Discussion
Due to the high incidence of infection in the community and hospital environment, urinary tract infections have placed a huge burden on the health system [30]. Effective treatment of patients with bacterial urinary tract infections is usually based on the identification of the pathogen and the selection of effective antibiotics through continuous monitoring of the antimicrobial susceptibility pattern of urinary tract pathogens in specific areas [8]. The results of the current study provide insights into the antimicrobial resistance patterns in Ethiopia, which has limited antimicrobial resistance monitoring data available.
In this study, out of a total of 1012 urine samples, 325 (32.1%) had significant growth and had no bacterial growth in 687 (67.9%). The prevalence here is lower than in some other studies: 67.2% in Nigeria [31], 60% reported in Lafia, Nigeria [32], 35.5% in Jos Nigeria [33]. The reason for the lower relative prevalence in this study could be that the patient was treated with antibiotics before arriving at the institute for diagnosis and the pathogens were killed or inhibited. The prevalence of this study was higher than other studies, 22% in Ibadan, Nigeria [34] and 22.7%, 28.5% in Ethiopia [35,36]. Study design, target population, sample size, and geographic variation may cause these differences.
The highest age-specific prevalence was found in the 21-30-year-old age group 73 (22.5%), which is consistent with other studies, showing that sexually active groups, especially women, are more likely to develop urinary tract infections [35,36]. This study is consistent with the studies recorded in India [2] and Ethiopia [36].
This study shows that Gram-negative bacteria (61%) are more common than Gram-positive bacteria (19.3%). Similar findings have been reported in Ethiopia and elsewhere [37][38][39]. According to this study, E. coli is the main urinary tract pathogen, which is consistent with almost all similar investigations [35,36,40]. This is due to the existence of many virulence factors dedicated to colonization and invasion of the urinary epithelium. Bacterial urinary tract pathogens isolated from UTI patients indicate extremely high levels of single and multiple antimicrobial resistance to commonly used drugs.
Sixty-five isolates (85.5%) of Escherichia coli are sensitive to nitrofurantoin and the rate of resistance to the drug is 14.5%. This result is consistent with other studies [41,42]. Other  strains are sensitive to gentamicin and the drug resistance rate is 108 (69%). Compared with other studies and other studies from Bahir Dar, Ethiopia, this finding confers a higher sensitivity rate [37,39]. For trimethoprim/sulfamethoxazole resistance, our results also showed the resistance is much higher than previous studies [43].
In this study, the overall resistance rate of Gram-positive isolates was 51%, which is consistent with the studies documented in Ethiopia [44,46]. Among the isolated gram-positive bacteria, Staphylococcus aureus represents high drug resistance. This finding is consistent with studies conducted in Saudi Arabia [36], Ethiopia [35] and Iraq [31].
In this study, the estimated MDR isolates of GNB were 156(61%) which is higher than the study documented in Tigray [38], in Addis Ababa cancer patients [47]. In this study, 22.4% and 4% of the gram-negative isolates were XDR and PDR respectively, which is lower than the study conducted in Dessie [37], and in the Tigray region [38]. The reason for this observed high resistance may be due to the increasing irrational use of antibiotics, the transmission of resistance genes between people and people or /and animals to people, and consumption of animal products that treated antibiotics. Self-medication and non-compliance with medication and sales of the substandard drug may account for the rise in antibiotic resistance observed in this study. The clinical and financial burden to patients and healthcare providers for MDROs is challenging. Patients who are infected with MDROs often have an increased risk of prolonged illness and mortality. The cost of care for these patients can be more than double as  compared to those without MDRO infection. Regarding public health attention, MDROs are described as superbugs having very limited treatment options. These findings have important therapeutic implications for the treatment and management of urinary tract infections (UTIs), especially those caused by multidrug-resistant urinary tract pathogens. Clinicians should first be aware that UTI patients are more likely to be infected with common urinary tract pathogens or relatively rare isolated urinary tract pathogens. Second, the high rate of multidrug resistance observed in this study is a major problem in the treatment of urinary tract infections (UTIs), and a systematic approach is needed to reduce the rate of resistance to antibiotics or minimize the use of broad-spectrum antibiotics [48].
Finally, in the case of multidrug resistance, rapid diagnostic tests (point-of-care tests) for timely, targeted treatment are the top priority. There is also a need for a drug control system that optimizes drug use and allows for a personalized approach to proposed treatments [48].
We believe that urinary tract infection (UTI) is an accessible goal for the development of health education programs aimed at reducing the prevalence of diseases in the community and improving the quality of life of patients in low-and middle-income countries.

Limitation of the study
Our findings may not be inferred to specialized groups like HIV positive individuals or pregnant women and it was not possible to include anaerobic bacteria isolates due to lack of cultures media. Because of the lack of molecular methods and primes, antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test.

Conclusion
Levels of urinary tract infections are high, and Gram-negative bacteria are the most common cause of urinary tract infections. It is found that the resistance to commonly used antibiotics is very high. This shows that drug resistance is a deep-rooted problem in Ethiopia. Inadequate treatment regimens, insufficient patient adherence, and uncontrolled distribution and trade of drugs, as well as lack and poor quality of antibiotics, can also cause antibiotic resistance.
To this end, the incidence of urinary tract infections should be minimized and the susceptibility of specific pathogens to commonly used antibacterial agents should be continuously monitored. Therefore, based on this, there is a need to provide improved, adequate and affordable health services in the community, especially reproductive health services. Finally, a nationwide survey and study on antibiotic resistance are needed to assess this devastating nationwide situation and formulate control strategies.