An Epidemiological and Multidrug Resistance Study for E. coli Isolated from Urinary Tract Infection (Three Years of Study)

Uropathogenic E. coli (UPEC) is problematic and still the leading cause of urinary tract infections worldwide. It is developed resistance against most antibiotics. The investigation, surveillance system, and efficient strategy will facilitate selecting an appropriate treatment that could control the bacterial distribution. The present study aims to investigate the epidemiology and associated risk factors of uropathogenic E. coli and to study their antibiotic resistance patterns. 1585 midstream urine specimens were collected from symptomatic urinary tract infections (UTI) patients (225 males and 1360 females) admitted to Zakho emergency hospital, Zakho, Kurdistan Region, Iraq from January 2016 until the end of December 2018. Specimens were inoculated on blood and MacConkey plates and incubated at 37C for 24 hours. Uropathogenic E. coli was diagnosed based on gram staining, colony characteristics, and standard biochemical tests in accordance with local standards and guidelines. All isolates were screened for their antibiogram pattern using the disk diffusion method based on the Clinical and Laboratory Standards Institute guidelines. The results showed that out of 1585 urine specimens, 1026 (64.7%) were UTIs positive with a statistically higher rate in 2016 (83.6%) (P< 0.0001). The UTIs frequency in females was significantly higher than males (P< 0.0001). Generally, the uropathogenic E. coli represented 21.1% with the highest level in 2016 (22.9%). The uropathogenic E. coli rate was higher, statistically not significant, in females (21.4%) than males (18.5%) (P=0.4946). Additionally, through the three years of study, uropathogenic E. coli (UPEC) was in high frequency in February and May 2016. The female’s age group from 20 to 39 years was the most vulnerable (46%) form total infected females, while those from 70-74 years (1%) were the least susceptible in males and females. A high percentage (80.56 %) of multidrug resistance E. coli isolates was observed with high resistance against β-lactamase and macrolides antibiotics. However, higher sensitivity was towards imipenem and meropenem. In conclusion, the wrong and overuse of antibiotics will increase the resistance rate of E. coli. For this reason, proper use of available antibiotics is necessary. Also, the educational programs and periodic monitoring of antimicrobial susceptibility are essential for reducing the antibiotic resistance rate.

disturb the host cellular process 2 Furthermore, all these factors are significantly associated with the disruption of the affectivity of the most used and known antibiotics used to treat uropathogenic E. coli (UPEC) infections 3 . Uropathogenic E. coli is linked with 70 to 95% of the urinary tract infections (UTI) worldwide 3,4 . This bacterium can develop resistance against most discovered antibacterial therapy 5,6 . Unfortunately, antibiotic resistance is remarkably increased among UTI patients suffering from UPEC infections 6 . Applying a robust surveillance system to investigate the antibiotic resistance bacteria could help to eliminate and/or control the antibiotic resistance bacteria 7 . Investigation of the ability of pathogenic bacteria to resist the current antibacterial treatments as well as their geographic distribution will facilitate choosing an appropriate treatment with the antibacterial drugs and design an efficient strategy to apply the antibiotic to control the bacterial infection 8 . This investigation is highly demanded, especially in areas or cities with the absence of control antibacterial prescription. Thus, the present research aims to study the epidemiology of UTI infections caused by uropathogenic E. coli and to investigate the antibiotic resistance among the E. coli isolated from UTI patients.

Materials and Methods:
This study was conducted in the microbiology laboratories at Zakho emergency hospital in Zakho city, Kurdistan Region, Iraq, from January 2016 until the end of December 2018. A total number of 1585 patients of both genders (225 males and 1360 females) were recruited ( Table 1).
The patient's baseline data, such as gender, age (all ages were included) and the time of the year for the collected samples, were also recorded. All the included patients were symptomatic of UTI. Furthermore, frequency and percentages of UTIs and UPEC were evaluated along the three years of study as well as along the months of the indicated years of the study (2016, 2017, and 2018) ( Table 1, Fig.1 and 2). M and F represent males and females, respectively. The differences between males and females were significant. P value < 0.00001. All other differences were not significant. (a) The total patients there were admitted over three years of study (2016, 2017 and 2018). (b) The UTI confirmed patients, the percentage values were calculated from the tottal admitted patients. (c) The UTI infection caused by UPEC, the percentage values were calculated from the UTI confirmed patients.

Specimens' collection
Urine specimens were collected from all UTI patients under sterile conditions. Clean catch midstream urine was obtained from the patients to avoid contamination. The collected specimens were directly sent to the microbiology laboratory, which inoculated them on blood and MacConkey plates and incubated at 37 о C for 24 hours 9 .

Isolation and identification of E .coli
The culture that yielded bacterial growth (≥10 5 colonies) was considered positive, and it was as microbiological evidence of a UTI. For identification, the bacterial colonies were classified by gram staining and then the bacteria were identified based on colony characteristics and standard biochemical tests including indole test, methyl red test (M.R), Voges-Proskaur test (V.P), citrate utilization test, urease test. The identification of E. coli isolates was performed under local standards and guidelines 10 .

Antimicrobial susceptibility test
All uropathogenic E. coli isolates were screened for their antibiogram pattern. The sensitivity to commonly used antimicrobial agents from different groups was determined. A range of antibiotics that belong to different classes were tested ( Table 2). The susceptibility was investigated using the disk diffusion method on Mueller-Hinton agar. Each isolate was inoculated and incubated at 37 o C for 24 hours. The results were recorded on the basis of Clinical and Laboratory Standards Institute (CLSI) guidelines 2015 11 .

Statistical analysis
All the recorded data were statistically analyzed using the SPSS and GraphPad prism 8. Pvalue of <0.05 was considered significant.

Ethics Statement
The scientific committee approved this research and method of attaining consent of the Department of Sciences, University of Zakho, Kurdistan, Iraq.

Results:
Uropathogenic E. coli (UPEC) were isolated from urine specimens among patients admitted to Zakho hospital, Kurdistan region, Iraq over three years of study (2016, 2017 and 2018 Fig. 2, the results showed an apparent increase in the frequency of isolates in May and November (12% and 10%, respectively) of overall years of study (2016, 2017 and 2018). However, there were an obvious increase in frequency in February and May in 2016. The incidence of UPEC infection was also analyzed depending on the age group of the patients among all confirmed E. coli positive isolates. The prevalence of disease was shown to be increased from age five years and younger to reach the highest percentage of infection between 20 to 39 years old (98 patients, 47%), while the frequency of E. coli infection was decreased dramatically from patients aged 40 years and older to reach the lowest incidence in elderly patients70-74 years (2, 1%) (Fig. 3). The overall isolates were distributed according to a patient's age and gender that entered the hospital. The total numbers of female patients with UPEC were 196 patients over the three years (2016, 2017and 2018), while the total number of male patients with UPEC isolates were 20 patients that were admitted to the hospital through three years.
All confirmed E. coli isolates were analyzed for antimicrobial drug resistance depending on the availability of theses tested antimicrobial drugs in the hospital at the time of the test. Furthermore, all moderate sensitive results to antibacterial medications were excluded from the analyzed data. Disk diffusion antibiotic sensitivity test was used as a routine procedure to identify the resistance to available antimicrobial drugs. As detailed in Table  2, the analyzed E. coli showed a variant level of resistance value to the tested antimicrobial medicines that were chosen for antimicrobial sensitivity. This study revealed that 174 (80.56%) E. coli isolates were resistant to three or more antibiotic categories (the antibiotic categories and multidrug resistance were described in Table 2 and 3. These results indicate that these isolates were multidrug resistance 12 . High percentages of antimicrobial resistance E. coli have been observed against most members of β-lactamase antibiotics. More than 90% of tested bacteria were found in classically used antibiotics, including ampicillin, ampicillin/ cloxacillin, and cloxacillin, and relatively newly introduced medicines, including amoxicillin/ clavulanic acid, cephalexin, and ceftazidime. Alongside β-lactamase antibiotics, the tested UPEC was exhibited a high level of resistance (90% or more) to erythromycin and clindamycin (macrolide antibiotics) ( Table 2). The isolated UPEC also showed about 80-90% of resistance to another member of β-lactamase antibiotics, including amoxicillin, ceftazidime, ceftriaxone, cefixime, cefotaxime; and nalidixic acid. However, the high sensitivity of UPEC was observed for E. coli isolates subjected to both imipenem and meropenem. *The frequency and percentages were calculated separately for each years (2016, 2017 and 2018). The overall multidrug resistance was calculated based on the resistance of UPEC to three or more antibacterial categories (mentioned in Table 2). The isolates were considered as non-multidrug resistance when the resistance showed in two or less antibacterial category.

Discussion:
Urinary tract infections are inflammatory disorders caused by the abnormal growth of pathogens 14 . Annually, 150 million people worldwide are approximated to suffer from UTIs 15 . In the current study, the high rate of UTIs (64.7%) was identified in patients during the three years of the research with a higher frequency in 2016 (83.6%) ( Table 1). A similarly high rate of UTIs was reported in other studies locally and worldwide [16][17][18][19] . Additionally, among the total positive UTIs, UPEC during the three years was 21.1% with a higher level in 2016 (22.9%). Many factors associated with the spread and distribution of UTIs, for example, socioeconomic, demographic data, hospitalization, age, gender, marriage status, clinical features, behaviors, practices, genitourinary abnormalities, and seasonal variation 14,20 .
Furthermore, one factor of this high rate of the infection in 2016 could be attributed to the high number of people displaced from other Iraqi cities (such as Mosul city) toward Zakho city, which is aligned to Turkey border. This internal displacement occurred when ISIS attacked many cities in Iraq in 2016. This large number of people put pressure on the healthcare system and its institutions in the local area 21 . By the end of 2015, around 200,000 internally displaced persons (IDPs) were living in camps sheltering in Duhok governorate 22 . In the following years, 2017-2018, IDPs started gradually to return to their original areas, and the healthcare centers were increased and supported by different organizations and became more available for persons 23  This study showed a high incidence of UTIs and UPEC in females (Table 1). This incidence was in line with several pieces of research 24,25 . It revealed that about 50% of women experience at least one episode of UTI at some point in their lifetime 26 . It is supposed that women are at high risk for UTI compared to men due to their anatomical and physiological characteristics 27 . For example, the proximity between the genital tract and the urethra/anus in women is one potential reason that could facilitate the auto-transmission of the pathogens and lead to increasing the rate of UTIs in women 28 . Regarding the seasonal risk factor, in Iraq, the average temperature ranges from 48 o C in July and August to below freezing in January through a study that acquired temperature over 32 years (from 1980-2011) 29 . It was found that UPEC was at a higher rate during summertime (May) overall years of study (2016, 2017, and 2018) (Fig. 2a), and it was at a higher frequency in February and May 2016 (Fig.  2b). This finding was in agreement with other studies in Iraq and worldwide which found out that UPEC and most pathogens are seasonal diseases, with the majority of cases occurring in the summer months [30][31][32][33] . The primary associated risk factors for distributing UPEC in summer could contribute to different factors such as seasonal changes in human behavior such as eating locally prepared ice cream, travel, sexual activity, more consumption of water, and specific food contaminated with sewage, debris, and garbage 34  that explains this phenomenon could be the ability of E. coli to survive in the environment at higher temperatures 35 . Also, one possible risk factor may be as a result of seasonal disturbances in immune function that make the host more vulnerable to pathogens 36 . The incidence rates of UPEC were varied in different age groups (Fig. 3). It was observed that the highest rate of UPEC was prevalent among patients at the age of 20 to 39 years old, and the prevalence was reduced by increasing the age to reach the lowest rate at the age of 70-74 years.
These observations were consistent with several studies in Iraq 24,37,38 . Habeeb 39 and Pirko and Ali 37 demonstrated that UPEC occurred mostly in patients aged between 20 to 35 years. Furthermore, a study performed in Pakistan revealed that 90% of Uropathogens belonged to the age group of 16-30 years 40 . The possible causes of the increased infection in this age group could be that this group is the most active in the society regarding the work, consumption of unhygienic food and water in working places, and colleges along with an increased number of social gatherings 32,41 . Also, the sexual activities and hormonally induced changes in the vaginal flora of women could increase the UTI risks 40,42,43 .
Recently, the efficacy of antimicrobial treatment of pathogenic bacteria has decreased because of the emergence of pathogenic isolates that are multidrug-resistant 16,44,45 . This study revealed that about 80% of UPEC isolates were multidrug resistance. There were variable susceptibilities of the UPEC toward the tested antibiotics. Generally, high resistance was observed against ampicillin, ampicillin/ cloxacillin, and cloxacillin, amoxicillin/ clavulanic acid, cephalexin, erythromycin, and clindamycin. However, the high sensitivity of UPEC was observed for both imipenem and meropenem (Table 2). It has been noted that the meropenem was shown to be effective against other gram-negative bacteria 46,47 .
The permanent investigation of antibiotic prescription is crucially essential to eliminate antimicrobial resistance of high virulent UPEC strains 7 that has developed progressively the ability to resist antimicrobial drugs over the past decade 8 . E. coli is a normal flora of animals and humans. However, its virulence is reportedly associated with various fatal infections that could infect the different organs and responsible for causing multiple diseases 1 . The resistance to nalidixic acid and cotrimoxazole was shown at a high level. However, the UPEC showed high resistance to Nitrofurantoin 48 . Rise the resistance against the fluoroquinolones, in particular, ciprofloxacin, could be due to using this antimicrobial agent extensively. And this resistance seems to be more evident in developing countries than in European countries 4 . High resistance was shown against gentamicin in UPEC with biofilm formation strains.
Additionally, the previous study showed a high level of resistance to the cephalosporins, nalidixic acid, penicillins, and fluoroquinolones by E. coli isolated from UTI infections 8 . It is well established that wrong and overuse of these antibiotics will inevitably lead to selection pressure and increase the resistance rate 49,50 . Also, the mistaken diagnosis of diseases may lead to proscribing wrong antibiotics to patients leading to resistance to pathogens.

Conclusion:
It was clear that the UPEC is at high frequency among patients with a higher percentage in females in the age group of 20 to 39 years. There is a high prevalence of resistance of UPEC isolates towards various groups of used antibiotics for UTI treatment. The highest antibiotic sensitivity of E. coli is observed for imipenem and meropenem. The wrong and overuse of these antibiotics will inevitably lead to selection pressure and increase the resistance rate. Therefore, awareness and education programs are necessary for the right diagnosis of diseases, proper selection, and wise use of available antibiotics. Hence, periodic monitoring of antimicrobial susceptibility pattern of UPEC will help guide the judicious use of antimicrobial agents and will help in reducing the bacterial vigor of resistance.