Antimicrobial Resistance among Community-Acquired Uropathogens in Mashhad, Iran

Background Antimicrobial resistance among community-acquired uropathogens is an emerging concern over the past decades that warrants a continuing reevaluation of the appropriateness of recommended empiric antimicrobial regimens for treatment of urinary tract infections (UTIs). Aims To describe the microbial spectrum and resistance profile of community-acquired uropathogens and predictors of isolation of resistant strains. Methods Between October 2017 and June 2019, individuals who visited the outpatient clinics for diagnosis of UTIs or screening of asymptomatic bacteriuria were included in the study if they were tested for urine culture in one of the three main medical diagnostic laboratories of Mashhad, Iran. The standard disk diffusion antimicrobial susceptibility testing was used, with the Clinical and Laboratory Standards Institute (CLSI) threshold cutoffs for susceptibility of isolated uropathogens. Results Three hundred thirty cases were included with a median age of 47 years. Two hundred seventy-six (83.6%) were female. The most common isolated uropathogens were Escherichia coli in 201 (60.9%) cases and Klebsiella species in 46 (13.9%) cases. E. coli isolates showed the highest rates of susceptibility to nitrofurantoin (89.3%), cefixime (75%), and gentamicin (72.4%). Exposure to antibiotics in the past 3 months was a predictor of resistance to ciprofloxacin (OR: 2.8, 95% CI: 1.33–6.28), and older age was a predictor of resistance to TMP-SMX (OR: 2.1, 95% CI: 1.07–3.97) among E. coli isolates. Conclusion. E. coli and Klebsiella species accounted for about two-thirds of community-acquired uropathogens. In regard to the high susceptibility rates, nitrofurantoin was identified as the first-choice agent for empiric treatment of community-acquired cystitis, while cefixime and gentamicin might be the second-choice alternatives. Ciprofloxacin and TMP-SMX, on the other hand, cannot be considered appropriate agents for empiric therapy of community-acquired UTIs, particularly in those who had exposure to antibiotics in the past 3 months and the elderly.


Introduction
Urinary tract infection (UTI) is the most common bacterial infection encountered in the ambulatory care setting [1], with a lifetime incidence of 50-60% in adult women [2]. It creates a significant social and personal burden, with a substantial number of medical visits, increased absenteeism, and negative impacts on quality of life [2]. Emergence of antimicrobial resistance among uropathogens, particularly those causing community-acquired UTIs, is one of the most important factors impacting the management of UTIs over the past decades [3].
In the appropriate clinical setting, the diagnosis of UTIs is typically made using both urinalysis and urine culture with susceptibility testing. Growth of bacteria on urine culture supports the diagnosis of UTIs, and susceptibility testing is considered essential to ensuring appropriate antimicrobial treatment [4]. However, empiric therapy is considered the standard management approach of acute community-acquired uncomplicated cystitis [1, 5, and 6]. e recommendation on management of uncomplicated cystitis is based on two important principles. First, the microbiological spectrum of acute uncomplicated UTIs is highly predictable, and second, the susceptibility pattern of these organisms is relatively predictable [7]. e appropriateness of the first-line agents for empiric therapy of UTIs is determined based on the local resistance rates of uropathogens. In this regard, thresholds have been suggested for the prevalence of resistance among uropathogens in a community above which some are not recommended; for an antibiotic to be considered a first-line empirical treatment for urinary tract infections, resistance should not exceed 10-20% in the most likely infecting strains [5]. us, local resistance surveillance of uropathogens and a continuing reevaluation of the appropriateness of the recommended empiric antimicrobial regimens are critical for informing empirical antimicrobial decisions [5,8]. Unfortunately, there is no antimicrobial resistance surveillance system for monitoring of community-acquired uropathogens in Iran. is results in lack of a regional protocol for the appropriate management of community-acquired UTIs in the country. In this study, we describe the microbial spectrum and resistance profile of community-acquired uropathogens as well as predictors of isolation of resistant uropathogens in Mashhad, Iran.

Methods
is is a cross-sectional study, and sampling was carried out using the census sampling method. e study was conducted between October 2017 and June 2019 in three main public medical diagnostic laboratories of Mashhad, Iran. Mashhad is the second largest city of Iran that is located in the northeast of the country. Individuals who visited the outpatient clinics for diagnosis of symptomatic UTIs or screening of asymptomatic bacteriuria were included consecutively if their urine samples were tested in one of the mentioned laboratories. ose with nonsignificant bacteriuria were excluded. Significant bacteriuria was defined as isolation of a single organism in quantitative counts ≥10 5 colony-forming units (CFU)/mL from a clean-catch voided urine specimen in asymptomatic patients [9] and ≥10 2 colony-forming units (CFU)/mL for symptomatic patients [10]. Information regarding age, sex, past medical history, history of exposure to antibiotics within the past 3 months, history of hospital admission within the past 3 months, pregnancy status, and indication for performing urine culture were recorded prospectively using a checklist. Isolated pathogens from urine samples and their antimicrobial susceptibility profile were also recorded.
All three clinical laboratories received midstream urine samples from each patient in a sterile container. e samples were analyzed within 2 h of collection. For this purpose, 10 μl of each urine sample was inoculated on routine culture media, i.e., blood agar and MacConkey agar, using a standard loop. After an overnight incubation at 35°C, the cultures were inspected for the presence of bacterial growth. Any mixed growth of bacterial colonies suggestive of contamination was excluded from further analysis. Finally, bacterial isolates were identified using conventional biochemical tests, and the Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing (AST). en, the AST results were interpreted according to the breakpoints defined by the Clinical and Laboratory Standards Institute (CLSI) for uropathogens.

Statistical Analysis.
Continuous data were described with mean and standard deviation and categorical variables with frequency and percentage. Two-sample t-test and Mann-Whitney test were used for continuous variables, whereas Fisher's exact test and chi-square tests were used for categorical variables, as appropriate. Significant predictors among the tested independent variables were analyzed by using the binary logistic regression test.

Ethical Approval.
e ethics committee of Mashhad University of Medical Sciences approved this study with the code of IR.MUMS.fm.REC.1395.542.
Univariate analysis showed that exposure to antibiotics in the past 3 months was a significant predictor of resistance to ciprofloxacin among uropathogenic E. coli isolates (OR: 2.89, 95% CI: 1.33-6.28;P value: 0.007) ( Table 3). Other tested variables, including different age groups (i.e., children, young adults, and the elderly), gender, diabetes mellitus, history of previous episodes of UTIs, and history of hospital admission in the past 3 months were not significantly associated with resistance to ciprofloxacin. e only predictor of resistance to TMP-SMX among uropathogenic E. coli isolates was older age (OR: 2.06, 95% CI: 1.07-3.97; P value: 0.032).

Discussion
Our study showed that community-acquired uropathogenic Escherichia coli isolates were highly resistant to trimethoprim-sulfamethoxazole (TMP-SMX) and ciprofloxacin, with resistance rates of 55% and 40%, respectively. According to the 2011 guideline of the IDSA on uncomplicated cystitis and pyelonephritis, local resistance rates of uropathogens that exceed 20% for TMP-SMX and 10% for fluoroquinolones make these antibiotics less appropriate choices for empirical treatment of uncomplicated UTIs [5]. Wide ranges of resistance rates to different antibiotics among uropathogens have been reported by previous studies on UTIs in Iran. Many of these studies lacked clear case definitions and did not determine whether the isolates were community-or hospital-acquired [13][14][15], or included a mixed population of community-and hospital-acquired isolates [16][17][18][19]. is limitation significantly decreases their practical value due to the different nature of uropathogens in the community and hospital settings [2]. Local resistance rates that are reported based on the antibiograms of hospitalacquired bacteria often reflect resistance rates among the uropathogens that were obtained from inpatients, or those with complicated or recurrent infections. us, they probably overestimate the rates of resistance among patients with uncomplicated UTIs [1]. ere are, however, several studies that mainly focused on community-acquired uropathogens in Iran and similarly reported high rates of resistance to TMP-SMX (32%-76%) [11,[20][21][22][23][24][25] and ciprofloxacin (32%-48%) [11,20,22,23] among E. coli strains. e high resistance rates among community-acquired uropathogenic E. coli strains in our study and most of the previous studies in Iran could be at least partly attributable to the high level of antibiotic consumption in the country, with 38.78 defined daily doses of antibiotic per 1000 people [12]. Recent or current antibiotic use has been reported as an important factor in the emergence of resistance among uropathogens [3,6,26]. e resistance rates of less than 10% to ciprofloxacin were reported by two studies from Iran that excluded cases with recent exposure to antibiotics [24,25]. Exposure to antibiotics in the past 3 months was also identified as a significant predictor of resistance to ciprofloxacin among uropathogenic E. coli strains (OR: 2.8) in our study. However, we did not investigate the type of antibiotics received by patients, nor their duration or dose before performing the urine culture, the factors that might affect the resistance pattern of isolated uropathogens.
Nitrofurantoin monohydrate/macrocrystals has been recommended as an appropriate choice for the treatment of acute uncomplicated cystitis due to minimal resistance and propensity for ecologic adverse effects of antimicrobial agents (i.e., selection for colonization or infection with multidrugresistant organisms-so-called "collateral damage") [5,27]. It was the most active antimicrobial agent with about 90%  susceptibility rate among E. coli isolates in our study. Similarly, most of the previous studies in Iran reported a high susceptibility rate of 87-97% to nitrofurantoin among community-acquired uropathogenic strains of E. coli [11, 24, and 25]. So, it is an appropriate antibiotic for empiric treatment of community-acquired uncomplicated cystitis in Iran. Cefixime and gentamicin may be considered the secondchoice alternatives, with susceptibility rates of 75% and 72%, respectively. Nevertheless, previous studies reported wide ranges of susceptibility rates among community-acquired uropathogenic strains of E. coli to these two antibiotics, with 56-80% susceptibility rates to cefixime [11,25] and 49-97% to gentamicin [11,[23][24][25]. us, conclusions regarding appropriateness of these antibiotics as empirical treatment for community-acquired uncomplicated UTIs in Iran cannot be drawn and it has to be determined in the future large-scale studies. In addition, cefixime and other cephalosporins are not generally considered as an optimal option for treatment of uncomplicated UTIs due to their propensities for collateral damage [5]. Amoxicillin, ampicillin, cephalexin, and tetracycline, on the other hand, were associated with highest resistance rates among uropathogenic strains of E. coli and Klebsiella, as compared to other classes of antibiotics. ese findings make these agents less appropriate antimicrobials for empiric therapy of community-acquired uncomplicated UTIs in the country.
Enterococci were the third most common uropathogens that were isolated in our study. Ampicillin and nitrofurantoin are considered among the best options for oral treatment of enterococcal cystitis, if susceptibility is documented [28]. All the uropathogenic Enterococci and more than 90% of them in our study were susceptible to nitrofurantoin and ampicillin, respectively. Similarly, high susceptibility rates of more than 95% to both nitrofurantoin and ampicillin were reported among community-acquired uropathogenic E. faecalis isolates in the study of Karimi et al. in Tehran, Iran [29].
us, these two antibiotics can be considered appropriate agents for empiric therapy of community-acquired enterococcal cystitis in Iran.
Our study had some limitations that should be pointed out. Although using a detailed checklist and recording the patients' information prospectively were methodological strengths compared to most of the previous studies in the country, we did not investigate the type of antibiotics received by patients, nor their duration or dose before performing the urine culture, the factors that might affect the resistance pattern of isolated uropathogens. Furthermore, a larger sample size of patients is needed for making more accurate estimation of resistance rates to different antimicrobials among uropathogens and to determine the individual-level predictors of resistance.

Conclusion
E. coli and Klebsiella species accounted for about two-thirds of community-acquired uropathogens in our study. In regard to the high susceptibility rates, nitrofurantoin was identified as the first-choice agent for empirical treatment of community-acquired uncomplicated cystitis, while cefixime and gentamicin might be second-choice alternatives that can also be appropriate in those with pyelonephritis. Ciprofloxacin and TMP-SMX, on the other hand, cannot be considered appropriate agents for empiric therapy of UTIs, particularly in those who had exposure to antibiotics in the past 3 months and the elderly. Data Availability e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.