Community-acquired urinary tract infections: causative agents and their resistance to antimicrobial drugs

Background/Aim. Urinary tract infections (UTIs) are among the most common infections in outpatients. The aim of this study was to define the causative agents of urinary tract infections and their resistance to antimicrobial drugs in the urban area of central Serbia, as well as to evaluate eventual differences associated with age and gender of the patients. Methods. This retrospective study analysed data taken from routine, consecutively collected urine cultures of outpatients with symptomatic UTIs, collected from the Department of Microbiology, Institute of Public Health in Kragujevac, Serbia, from January 2009 to December 2013. Results. There were 71,905 urine cultures, and 24,713 (34.37%) of them were positive for bacterial pathogens. The most common pathogen was Escherichia coli ( E. coli ) (56.56%), followed by Klebsiella spp. (16.20%), Proteus spp. (14.68%), Enterococcus spp. (5.29%) and Pseudomonas aeruginosa (3.74%). E. coli and Enterococcus spp. isolation rates were lower in males ≥ 60 years old (23.71% and 4.87%, respectively), while Klebsiella spp . was more prevalent in this group (32.06%). The most common causative agents isolated from 15–29 years old male patients were Enterococcus spp. and Pseudomonas aeruginosa (13.28% each). Among women, the isolation rate of E.coli was high in all age groups (around 70%). Proteus spp . was frequently isolated from females ≤ 14 years old (13.27%), while Klebsiella spp. was the most frequent in the oldest age female group (10.99%). Conclusion. Choice of antibiotics for treatment of UTIs should be governed not only by the local resistance patterns, but also by gender and age of patients. differences in frequency of urinary pathogens, emergence of new agents or susceptibility to antimicrobial drugs 9 . Two recent studies from Serbia 10 and and Herzegovina 11 support these recommendations, since isolated gram-negative causative agents of UTIs were highly resistant to beta-lactam antibiotics (> 25%), especially to ampicillin, amoxicillin and cephalosporins.


Introduction
Urinary tract infections (UTIs) are among the most common infections in outpatients. They are associated with a significant morbidity and mortality in general population and impose substantial financial burden to the society. About 150 million people worldwide are affected by UTIs every year, spending about 6 billion US dollars 1 . According to the 2007 National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey, UTIs are responsible for nearly 7 million office visits and 100,000 hospitalizations 2 . In Serbia, over 350,000 people is diagnosed with acute cystitis in primary care annually, and UTIs are the fourth leading cause of visits to general practitioners 3 .
Earlier studies indicated that 50-80% of uncomplicated UTIs are solely due to Escherichia coli (E. coli), while the remaining cases are caused by other Enterobacteriaceae (Proteus, Klebsiella, Enterobacter) together with Pseudomonas spp and gram-positive bacteria such as Enterococci, Streptococci and Staphylococci 4,5 . The host risk factors as well as the virulence of a pathogen determine clinical course of UTI. Well-known risk factors for UTIs are female gender (especially pregnancy), diabetes mellitus, spinal cord injuries, multiple sclerosis, anatomic abnormalities of the urinary tract, incontinence, urinary bladder catheterization and advanced age 6,7 .
According to the guidelines of the European Association of Urology (EAU), treatment of UTIs includes fosfomycin trometamol, pivmecillinam or nitrofurantoin as the first-line therapy; alternative therapy includes fluoroquinolones, cefpodoxime proxetil, and combination of sulfamethoxazole and trimethoprim, if the local resistance of E. coli to the latter is less than 20% 8 . However, local and regional adjustments of these recommendations are necessary, since there are significant local differences in frequency of urinary pathogens, emergence of new agents or susceptibility to antimicrobial drugs 9 . Two recent studies from Serbia 10 and Bosnia and Herzegovina 11 support these recommendations, since isolated gram-negative causative agents of UTIs were highly resistant to beta-lactam antibiotics (> 25%), especially to ampicillin, amoxicillin and cephalosporins.
The aim of this study was to define the causative agents of UTIs and their resistance to antimicrobial drugs in outpatients in the urban area of central Serbia, as well as to evaluate eventual differences associated with age and gender of the patients.

Methods
This retrospective study included data taken from routine, consecutively collected urine cultures of outpatients with symptomatic UTIs, collected from the Department of Microbiology, Institute of Public Health in Kragujevac, Serbia, from January 2009 to December 2013. For each outpatient, the following data were extracted: the date of the sample obtaining, age, gender, urine culture results, identification of the bacterial strain responsible for an UTI and results of the corresponding antimicrobial susceptibility test (AST).
The Department of Microbiology has internal quality control procedures and participates in the external program for quality assurance by The United Kingdom National External Quality Assessment Service (UK NEQAS) for Microbiology and by Institute of Public Health of Belgrade, Serbia. The Institute of Public Health in Kragujevac is the competent UTI diagnostic center for 6 municipalities of the Šumadija region with 240,000 inhabitants.
The study was approved by the Ethics Committee of the Clinical Centrе, Kragujevac, Serbia.
Before giving the urine sample, the outpatients received instructions for avoiding contamination with antimicrobials and for appropriate sampling technique, as a part of the routine procedure. The urine sample was collected early in the course of the disease, by midstream clean-catch technique after usual daily hygiene of genital area. The initial and the end portion of the micturition stream were discarded and the middle part was collected directly into a sterile recipient. In children up to two years of age urine samples were collected by collection bags taped to the skin surrounding the urethral orificium. Urine samples were transported to the laboratory and analyzed within the two hours after collection. When this procedure was not possible, urine samples were stored at 4°C and processed within the 24 hours after collection.
Identification of microorganisms was made by plating on chromogen coagulase positive staphylococci (CPS) agar (BioMerieux, France) and by incubation for 18-24 h at 35 ± 2 0 C.
The exclusion criteria were contamination (growth of two or more bacterial species) and negative samples [bacterial growth lower than 10 3 colony-forming units (CFU)/mL of urine]. The inclusion criterion was monomorphic bacterial growth higher than 10 5 CFU/mL of the culture. All isolates were subjected to antimicrobial susceptibility testing AST.
Primary analysis of collected data was made by descriptive statistics. The difference between females and males in the frequency of positive samples to each of the agents was analyzed by χ2-test. Statistical hypotheses were considered true if probability of null-hypothesis was less than 0.05. All calculations were performed by the statistical software SPSS (SPSS Inc, ver.18, Chicago, IL).

Results
During the study period, there were 71,905 urine cultures, and 24,713 (34.37%) of them were positive for bacterial pathogens. Generally, the most common pathogen was E. coli (56.56%), followed by Klebsiella spp (16.20%),  Table 2). The isolates frequency according to the age distribution of the patients is presented in Table 3. Female to male ratio was the highest in 15-29 years age group (F/M = 13.0) and the lowest in the oldest one (F/M = 1.5). There were significant gender differences in the isolation rates for four of the top five causative agents (the difference was not significant only for Enterococcus spp.): E. coli was isolated more frequently in females (11, (Table 2).
The data stratification according to both gender and age showed significant differences in regard to frequency of isolation between females and males throughout all age groups (  Table 3).
The pattern of resistance to antibiotics of main isolated uropathogens is shown in Table 4. Isolates of E. coli showed moderate degree of resistance to trimethoprim-sulfamethoxazole (40.1%), while resistance to fluoroquinolones was lower (32.6% ofloxacin and ciprofloxacin 26.1%), as well as the resistance to aminoglycosides (23.0% for gentamicin, and 6.1% for amikacin). Percentage of the isolates resistant to the 1st and 2nd generation of cephalosporins was the same, 32.1%, while that to the 3rd generation was 10.7%.
The most commonly isolated uropathogen from the gram-positive group, Enterococcus spp. showed a low grade of resistance to ampicillin (7.5%), and trimethoprimsulfamethoxazole (9.1%), but a high grade of resistance to tetracyclines (84.4%).

Discussion
Knowledge of the local or regional etiology of UTIs and antimicrobial resistance can be very useful as a guide for empirical therapy, because the frequency of pathogens and their features vary according to time and geographical area. As these infections are very common, adequate treatment have an important role in regard to the patients' health, development of antibiotic resistance and health care costs 1 . A large number of bacterial isolates included in this study (obtained from routine urine analyses), allowed stratification of data according to gender and age, and evaluation of association of these variables and UTI etiology, as well as determination of susceptibility of uropathogens to commonly prescribed antimicrobial drugs.
In our study, over 90% of all isolates were gramnegative pathogens. As it was expected, E. coli was the most frequent isolate (56.56%). It was also the most frequent uropathogen associated with the community-acquired UTIs (being implicated in more than a half of all the UTIs) in other studies 5,13 . E. coli generally belongs to normal flora of human colon and therefore may easily colonize the urinary tract. The other gram-negative pathogens found in this study were Klebsiella spp. Proteus spp. and although they were isolated in small percentages, they play substantial role in UTIs due to their pathogenicity and high resistance to antibiotics 14,15 .
In our study the obtained isolation rate of gram-positive bacteria was relatively low (6.72%) and among them, Enterococcus spp was responsible to 5.28% of UTIs. The other studies show similar results, confirming that these bacteria have minor role in UTIs 16 . However, true frequency is still unknown, since the studies published about the topic differ in design, sample size, inclusion and exclusion criteria and presentation style.
Women are more likely to experience UTIs than men. Nearly 70% of all isolates in our study were obtained from women. This could be explained by anatomical differences: the urethra is shorter and closer to the anal orifice in women than in men. Furthermore, women are more likely to get an infection after sexual activity or when using a diaphragm for birth control. Pregnancy and menopause also increase risk from UTIs 6 .
In our study, significant difference was also found in frequency of certain uropathogens in relation to gender: E. coli was isolated more frequently in females, whereas Klebsiella spp., Proteus spp. and Pseudomonas aeruginosa were more common in men, which is consistent with the results of other authors 4 . Previous studies have indicated that some uropathogens, especially Pseudomonas aeruginosa, were strongly associated with particular host characteristics, including male gender, recent antibiotic therapy, prior urinary tract procedures and neurogenic bladder 17 .
In our study significant differences in etiology of UTIs among different age groups were observed, too. Besides, frequencies of urinary pathogens were different across both age-and gender-stratified groups. E. coli, for example, was less prevalent in the oldest males (23.71%), but highly frequent in female patients from all age groups (approximately 70%). Klebsiella spp. was the most common in the oldest age group in both men and women (32.06% and 10.99%, respectively), and Proteus spp. frequency was highest in younger age groups of both males and females (35.64% and 13.27%, respectively). Age of the patients was linked to etiology of UTIs in several recent publications 18-20 : the study similar to our with the data stratification according to both age and gender showed lower E. coli isolation rate in both males ≥ 60 years old (52.2%) [Escherichia faecalis and Pseudomonas aeruginosa were frequent in this group (11.6% and 7.8%, resp.), and in those ≤ 14 years old (51.3%) (Proteus mirabilis was highly prevalent in this group: 21.2%]. On the other hand, Linhares et al. 21 in a ten-year study did not find differences in uropathogen isolation rates among age groups of patients with an UTI. However, when the age groups were stratified according to gender, the isolation rate increased with the age.
Proteus mirabilis is the most frequent uropathogen in boys 22 , which should be borne in mind when prescribing antimicrobial drugs to boys. On the other hand, the results of our study indicate that Proteus spp. is an important urinary pathogen in young females, in spite of its low frequency in the preadolescent female genital tract flora 23 .
The misuse of antibiotic drugs in medicine has led to an alarming increase of the microbial resistance 24 and the consequent spread of antibiotics-resistant strains is a serious public health problem. Approximately 15% of all communityprescribed antibiotics in the USA 25 and some European countries 26 are dispensed for UTIs. Prudent use of available antibiotics is the only option to delay the development of resistance 27 and the urological community has a responsibility to contribute to these efforts. Therefore, it is necessary to follow the guidelines of EAU in treatment of UTIs. Also, it must be noted that the recommended antibiotic for the first-line therapy pivmecillinam is not registered in Serbia and that the fosfomycin and nitrofurantoin are not frequently used, so it was not possible to draw some conclusions about their effectiveness in treatment of UTIs.
In our study, 40.1% of isolates of E. coli were resistant to trimethoprim-sulfamethoxazole, while the percentage of resistance to fluoroquinolones was lower (32.6% ofloxacin and ciprofloxacin 26.1%), but still relatively high and in line with other European countries 28,29 . This is probably due to extensive utilization of these antibiotics in treatment of community-acquired UTIs over the past decade in this region. Although values may vary among reports, resistance rate of recently community-isolated of E. coli to trimethoprim-sulfamethoxazole in Europe tends to be higher than 30% 30,31 .
According to the international Antimicrobial Resistance Epidemiological Survey on Cystitis (ARESC) conducted from 2003 to 2006, E. coli showed a high resistance to sulfonamides (29.4%) and to fluoroquinolone ciprofloxacin (8.1%) in nine European countries and in Brazil 32 , thus limiting use of these antibiotics in empirical therapy. It is necessary that entire community makes significant effort to maintain sensitivity of urinary pathogens to antibiotics which could be given for treatment of UTIs. What was encouraging from this study is relatively low level of resistance to second-line antibiotics for UTIs, aminoglycosides (gentamicin and amikacin, 23.0% and 6.1%, respectively) and third generation cephalosporins (cefotaxime and ceftriaxone, 10.7% both). However, some of these drugs do not exist in the oral form and are more expensive for the treatment of UTIs.
The isolates of Klebsiella spp. in our study showed a high degree of resistance to fluoroquinolones (64.4-66.6%), trimethoprim-sulfamethoxazole (69.1%) and second-and third-generation cephalosporins (57.7-72.5%), which is 2-3 times higher than in other recent European studies 21 . This result is worrisome due to a high proportion of UTIs caused by Klebsiella spp. in our community (16.2%).
The resistance rate to uropathogens Proteus spp. isolated in our study was generally high for all tested antibiotics. The resistance rate of this isolate to trimethoprimsulfamethoxazole is 74.7%, 79.5% to ampicillin, 63.9% to ofloxacin and 60.2% to ciprofloxacin, which is much higher than the rates shown in other similar studies 20 .

Conclusion
The results of our study are a useful tool for doctors who should prescribe antibiotics to patients with UTIs, as well as for regional health authorities who intend to formulate recommendations for rational antibiotic use and define standard treatment guidelines. When prescribing drugs for UTIs, Serbian physicians should be aware of a high resistance rate of urinary pathogens not only to semisynthetic penicillins and cephalosporins (> 30%), but also to fluoroquinolones (> 25%) and trimethoprimsulfamethoxazole (> 40%), and choose among the antibiotics with still low resistance rates. Choice of antibiotics for treatment of UTIs should be governed not only by the local resistance patterns, but also by gender and age of patients.