Antimicrobial susceptibility of microorganisms causing Urinary Tract Infections in Saudi Arabia

Introduction: Urinary Tract Infections (UTIs) is one of the most common infections worldwide. UTIs remain a challenge to the healthcare system because of the emergence of antimicrobial resistance. The aim of this study is to report the most common UTI-causative organisms associated with the emergence of antimicrobial resistance in Saudi Arabia. Methodology: a retrospective cross sectional study of 1918 positive urine culture samples of both gender collected over 9 months (May 2015 to February 2016) from a major tertiary hospital in Riyadh, Saudi Arabia. Results: the median age of individuals involved in the study was 43 years, with males constituting 27.7% only of the population. Among cases deemed complicated (81.1%), common causes were diabetes, pregnancy, and immunocompromization, comprising 24.7%, 11.9%, and 10.8%, respectively. Escherichia coli (52%) was the most common uropathogen, followed by Klebsiella pneumoniae (15%), Pseudomonas aeruginosa (8%) Streptococcus agalactiae (Group B streptococcus) (7%), and Enterococcus faecalis (5%). Overall sensitivity studies showed the most highly resistant uropathogen was Escherichia coli (60%) followed by Klebsiella pneumoniae (16%), Pseudomonas aeruginosa (4%) Enterococcus faecalis (3%), and Enterobacter cloacae (2%). Concerning the first defense antibiotics prescribed for UTI, E. coli was most frequently resistant to Sulfamethoxazole/Trimethoprim (47%) followed by ciprofloxacin (34%). K. pneumoniae was most frequently resistant to Sulfamethoxazole/trimethoprim (35%) followed by cefuroxime (30%), while P. aeruginosa to ciprofloxacin (13%). Conclusion: Because of a high level of antimicrobial resistance amongst uropathogens in Saudi Arabia, the development of regional and national UTI guidelines is recommended.


Introduction
Urinary tract infections (UTI) are one of the most common bacterial infections in humans, both in the community and hospital settings [1]. Based on the location, UTIs are divided into three types; descending, ascending and complicated UTIs. Descending UTIs, also known as cystitis, are associated with polyuria, dysuria, and urgency. Ascending UTIs, also known as pyelonephritis, are associated with fever or flank pain in addition to urinary symptoms. Complicated UTIs are associated with comorbid conditions such as diabetes, pregnancy and manhood [2]. Worldwide, approximately 150 million people are diagnosed with UTIs, causing a heavy burden on the health financial system, with around 6 billion dollars in health care spending each year [3,4].
The most frequent causative microorganisms of UTI are mainly gram negative organisms such as E. coli, K. pneumoniae, Acinetobacter baumannii and P. aeruginosa. Furthermore, gram positive organisms such as Staphylococcus saprophyticus, Methicillinresistant Staphylococcus aureus (MRSA) and Enterococcus species can also cause UTI [5].
Bacteriological investigations of UTIs are not complete without an antibiotic sensitivity test of the isolate. Microorganisms causing UTIs vary in their susceptibility to antimicrobials from place to place and time to time [6]. Knowledge of the local bacterial etiology and susceptibility patterns is required to trace any change that might have occurred in time so that updated recommendations for optimal empirical therapy of UTIs can be made [7]. The Infectious Diseases Society of America (IDSA) published clinical practice guidelines on the treatment of women with acute uncomplicated cystitis and pyelonephritis [8]. Since then, antimicrobial resistance among uropathogens, and appreciation of the importance of the ecological adverse effects of antimicrobial therapy (collateral damage), has increased, newer agents and different durations of therapy have been studied, and clinical outcomes have increasingly been reported [2,9].
Antibiotic resistance among causative gramnegative bacilli is increasing and considered to be a challenge for clinicians because there are limited treatment options. Common examples of these organisms include AmpC β-lactamase and extendedspectrum β-lactamases (ESBLs)-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant (MDR) Pseudomonas aeruginosa [10,11]. Etiological agents of UTIs, and their susceptibility patterns vary between regions and geographical locations. Besides, the etiology and drug resistance change over time [12].
In addition, management of UTI in the era of antimicrobial resistance requires a systematic approach to diagnose the type of infection and to select the optimal dose, route, and duration of the antibiotic regimen. Unfortunately, standardized evidence-based clinical management is often lacking in general practice [13].
King Saud University Medical City (KSUMC) established its own Antimicrobial Stewardship Program (ASP). It was designed based on a thorough review of the literature, international guidelines, local antibiogram(s), and the consultation of external experts outside of the hospital as deemed necessary. It is to be used as guidance for all prescribing physicians at the health care facilities of KSUMC. Restricted antimicrobial agents will require proper approval of consultants of infectious diseases within 48 hours of initiating the prescription. Antimicrobial resistance may be associated with recurrent UTIs which hard to treat. Several studies examined the prevalence of multi-drug resistance organisms in UTIs [14][15][16].
Despite an increasing prevalence of antimicrobialresistant pathogens, the pattern of bacterial resistance has not been fully studied, especially in Saudi Arabia [17,18]. The aim of this study is characterize the etiology and antimicrobial susceptibility of microorganisms causing UTI among patients attending KSUMC, and report the most common causative organisms associated with the emergence of antimicrobial resistance by age, gender, ward, comorbidity, recurrence and immune status. The results of this study will be used to create a program to control antibiotic use and reduce the emergence of bacterial resistance

Study Setting
This study was conducted in a major tertiary hospital, the KSUMC (formerly known as King Khalid University Hospital), Riyadh, Saudi Arabia. This hospital receives referral services from nearby health centers, private hospitals, and clinics. Approval from the Institutional Review Board IRB of KKUH was obtained (protocol number 15/0391/IRB in October 29, 2015).

Study Design
A cross sectional retrospective study assessing all urine samples of patients who are suspected to have a UTI and sent to microbiology lab for culture and sensitivity, collected during the period from 16 May 2015 to the 21 February 2016. The demographic data of patients, clinical data including (patient setting, comorbidity, duration of treatment, recurrence and immune status), the organism isolated and the antimicrobial susceptibility profiles were collected from the Electronic Medical Record (EMR) using a standard data collection sheet.

Isolation and Identification of Bacterial Uropathogens
As the standard laboratory operation procedures show, clean-catch midstream morning urine specimens were collected using sterile wide mouth glass containers. Identification of organism and antimicrobial susceptibility testing were carried out by the MicroScan Walkaway 96 plus System (Beckman Coulter Diagnostics, Pasadena CA, United State of America) [19]. ESBL screening was carried out by both double disk and E-test according to the Clinical and Laboratory Standards Institute (CLSI), 2015 recommended guidelines [19]. A significant bacterium was considered if urine culture yield is ≥ 10 5 CFU/mL [20]. Negative urine samples were excluded from the study.

Antimicrobial susceptibility tests
Antibiotic susceptibility using the Kirby-Bauer method was carried out on Mueller-Hinton agar with commercially available disks (Oxoid, Hampshire, United Kingdom) in accordance with CLSI guidelines [19].
Descriptive analysis was carried out to describe the data. Mean and standard deviation was used to describe continuous variables. Frequency and percentage was used to describe the categorical variables. All statistical analyses were carried out using SPSS, v.22.0 (IBM Corp., Armonk, NY).

Results
During the study period the microbiology lab processed 23,342 urine isolates of which 1918 samples were positive for uropathogens. Among these positive isolates 1393 (73 %) belonged to females and 525 (27 %) belonged to males. Patients were categorized into 4 different age groups and the UTI rate was higher in patient who are older than 50 years old compared to other age groups. Demographics, comorbidities, risk factors, patient setting and type of UTI are displayed in Table 1 and were categorized in accordance to gender.
The most common microbial agent leading to UTI was E. coli 52% (n = 997) followed by K. pneumoniae 15% (n = 294), P. aeruginosa 8% (n = 149) S. agalactiae 7% (n = 138), and Enterococcus Fecalis 5% (n = 95) as shown in Figure 1. Moreover, these were the most common microorganism isolated in all agegroups. No significant difference was found between isolated organism and age of the patients. E. coli and S. agalactiae was more frequent in women, while K. pneumoniae and P. aeruginosa was more frequent in men. Forty-two percentage of patients affected by P. aeruginosa experienced a recurrent UTI, compared to seventeen and eighteen percent of patients with E. coli and K. pneumoniae respectively.   Being the most common causative UTI agents, E. coli showed highest resistance to ampicillin (65%), followed by sulfamethoxazole/trimethoprim (47%) and ciprofloxacin (34%), while K. pneumoniae showed highest resistance to ampicillin (97%) sulfamethoxazole/trimethoprim (35%) followed by cefuroxime (30%) and P. aeruginosa showed highest resistance to nitrofurantoin (40%) followed by cefotaxime (35%) ( Table 2). The gram positive organism Enterococcus faecium showed a high resistant (78%) to ampicillin, levofloxacin and ciprofloxacin, while Staphylococcus epidermidis had a high rate of resistance to cefotaxime (89%), amoxicillin/clavulanate (67%), levofloxacin (78%) and ciprofloxacin (78%).
ESBL producing uropathogens constituted 15% (n = 293). The most common ESBL producing uropathogens were E. coli, K. pneumoniae, Citrobacter amalonaticus, Enterobacter aerogenes and E. cloacae. ESBL-producing E. coli was isolated from urine culture in 226 of the cases and K. pneumoniae in 54 of the cases. The majority of ESBL-producing E. coli were resistance to ceftazidime, cefotaxime, levofloxacin and ciprofloxacin. However, they were highly susceptible to carbapenems, amikacin and piperacillin/tazobactam. On the other hand, the ESBL-producing K. pneumoniae showed highest resistance rate to piperacillin, cefotaxime and ceftazidime while ciprofloxacin and levofloxacin had a lower resistant rate compared to ESBL-producing E. coli.
Despite of lack of significant difference between isolated organisms across different ages, most of the causative uropathogens in our study were more frequent (43%) in patient older than 50 years compared to other age groups (< 18 years old with 21%; 18-24 years old with 5%; 25-49% with 31%) . These findings are different from an old study conducted by Eltahawy, who found that more than 50% of patients with bacteriuria were in the age group of 21-50, but consistent with the most recent studies who found that more than 50% uropathogens-causing bacteriuria are detected in patients older than 50 years old [22][23][24].
In our study, females represented 73% of all identified UTIs with E. coli and S. agalactiae being the most frequently isolated uropathogens, while K. pneumonia and P. aeruginosa were more frequent in males. In a study conducted by Almijalli, females were more prone for UTIs than males untill the age of 50 years, with E. coli being the most frequently isolated organism in females and males (50% and 28.5% respectively); however, K. pneumonia was more often found in males than females [25].
E.coli is considered the main causative organism of recurrent UTI, especially in women. It is estimated to be responsible for almost 80% of the recurrent cases [26]. Recurrent UTI in our study, which was identified if the patient has two or more positive urine cultures during the study period, was more associated with patients who were infected by P. aeruginosa (42%) more than E. coli (17%) or K. pneumonia (18%). The microbiological difference in the incidence of recurrent UTI between our study and published literature may be explained by the fact that our study identified the prevalence of causative and resistant uropathogens from laboratory sight. However, the high percentage of P. aeruginosa involvement in recurrent UTI raises the concern of its role in the recurrent UTI in future studies.
E. Coli & K. pneumonia resistant strains to sulfamethoxazole/trimethoprim are often associated with acquisition of the resistance genes such dfr, sul1 or sul2-gene, or presence of integron class I or II [27,28]. Cefuroxime resistance mechanisms in K. pneumonia may be realted to the release of ESBLs and downregulation of porins [28]. DNA gyrase mutations, enhanced efflux activity, decreased uptake of the drug were identified as resistant mechansims of E. coli towrads fluoroquinolones [29]. Gene mutation is considered the essential resistant-mechanism for P. aeruginosa against cephalosporin [28].
Similar results were found in a Saudi study conducted in a tertiary hospital to investigate the microbiological causes and antibiotic sensitivity of 116 outpatient urine samples [25]. In that study, E. coli showed 70.33% and 62.64% resistance to sulfamethoxazole/trimethoprim and ciprofloxacin, respectively, while K. pneumoniae showed 70% and 28% resistance to sulfamethoxazole/trimethoprim and ciprofloxacin, respectively.
The high incidence of resistant pathogens to the first-line therapy is contributed to several factors such as antibiotic misuse or using antibiotics without prescrition. Although, there is no well-defined study assesses the antbiotic utlilization in Saudi Arabia due to the poor practice of documentation and missing of definite diagnosis, a study conducted in a western region found that antibiotics prescription was accounted for 46% of all prescribed medications in the emergency department [30]. This is high percentage perceeds the World Health Orgnization (WHO) acceptable percentage (15-20%) of antibiotics prescription in the countries where an infectious disease is prevalent [31]. Self-medication with antbiotics is an essential dilemma in Saudi Arabia where 77.6% of the pharmacies are dispensing antibiotics without prescription, and almost 79% of the citizens purchased at least one antibiotic without a prescription [32,33].
Although emergence of antibiotic resistance may differ regionally and geographically, our results seem consistent with the global data of antibiotic resistance in urine isolates. E. coli that was isolated from 12 million urine samples from 2000 to 2010 in the United States showed accelerated rates of resistance towards ciprofloxacin and sulfamethoxazole/trimethoprim [34]. During this period, the incidence of E. coli resistance towards ciprofloxacin increased 5 fold from 3% to 17.1% while the resistance towards sulfamethoxazole/trimethoprim increased from 17.9% to 24.2%. Based on our results and previous studies in the region, nitrofurantoin has the best susceptibility profile to UTI caused by E. coli and K. pneumoniae; however, recommending nitrofurantoin as first line empirical therapy needs further research on the classification and severity of UTI, and microbiological data and antibiotic susceptibility based on the classification and severity [35][36][37].
The incidence of ESBL-producing Enteriobactercieae has increased dramatically in different countries [36][37][38][39]. The prevalence of ESBLproducing E.coli is higher than ESBL-producing K. pneumoniae. It is estimated that the prevalence of ESBL-producing E. coli is 11% in Europe and from 5.28 to 10.5 patients per 100,000 patients in the United States [35,36]. The prevalence of ESBL-producing E. coli has been increasing in different parts of Asia including Saudi Arabia [38]. Currently, Asia is known to have some deadly ESBL-producing pathogens such as Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase (NDM)-producing E. coli. Increasing trends in ESBL-producing pathogens, mostly E. coli and K. pneumoniae have been noted in Saudi Arabia. Some Studies have suggested that the prevalence of ESBL-producing Enterobacteriaceae has been increased from 4.8% in 2003 to 9.6% in 2004 in Saudi Arabia, of which ESBL producing E. coli accounts for 38.8% [39][40][41].
Our results are consistent with these local and global increasing trends in ESBL-producing pathogens. Of the 1918 urine isolates we collected during the period from May 2015 to February 2016, ESBLproducing uropathogens were identified in 15.2% (n = 293). The most common ESBL producing organism were E. coli (n = 226) and K. pneumoniae (n=54). Increasing trends in ESBL-producing pathogens has been noted globally. The incidence of ESBL-producing E. coli increased significantly in the United States from 5.28 patients per 100,000 patients in 2008 to 10.5 patients per 100,000 patients in 2014 [37,38]. A similar noticeable increase in the prevalence of ESBLproducing E. coli was seen in Spain from 8% in 2008 to 12% in 2010 [42].
The majority of ESBL-producing E. coli were resistant to third generation cephalosporins and fluoroquinolones, but highly susceptible to carbapenems amikacin, and piperacillin/tazobactam. Similar resistance patterns of ESBL-producing E. coli in urine isolates were identified in another study where urine-collected samples were resistant to third generation cephalosporins and fluoroquinolones in approximately 33% and 55% of the cases respectively. Samples were highly sensitive to Carbapenems, amikacin, and piperacillin/tazobactam in approximately 100%, 80%, and 91% respectively [43].

Conclusion
Our results further enhance research into the prevalence of classification and severity of UTIs, microbiological data and antibiotic susceptibility based on classification and severity. Also, the increasing prevalence of the most common uropathogens towards the most prescribed antibiotics, and the accelerated growth of ESBL-producing pathogens, should be thoroughly investigated because the use of inappropriate antibiotics may further worsen the situation. Although our study is a retrospective study focusing on laboratory reporting and healthcare provider documentation, it included high number of urine isolates and reports data that has not been reported in such numbers and manner locally. Our study investigated the most common causative and resistant uropathogens within a community teaching hospital. These findings highlight the importance of formulating local empirical guidelines and having antimicrobial stewardship programs to promote the ideal use of antibiotics.