Characterization of ESKAPE pathogens in urinary tract infections among Jordanian patients

Introduction: ESKAPE pathogens are a small group of pathogens of remarkable importance. The present study was carried out to determine the prevalence of ESKAPE pathogens in urinary tract infections (UTIs) and their antibiotic susceptibility patterns at the Jordan University of Science and Technology Health Center in Irbid, Jordan. Methodology: A one-year retrospective study was conducted from April 2021 to April 2022. A total of 444 samples of “clean-catch” (midstream) urine from outpatients were studied. Results: Our study showed that the vast majority of urinary tract infected patients were females (92%) compared to males (8%) and were most frequent in the age group 21-30 years old. The most associated co-morbidities with UTIs were hypertension followed by diabetes mellitus and hypothyroidism. ESKAPE pathogens were responsible for about 87.4% of the UTIs in this study, and all were identified in the urine samples except Acinetobacter baumannii . In this study, isolates were most sensitive to levofloxacin, ciprofloxacin, and third-generation cephalosporin's and least sensitive to doxycycline, amoxicillin, and clindamycin. Conclusions: This research work has shown that patients with UTI-associated ESKAPE pathogens in Jordan are at high risk of antibiotic resistance. To the best of our knowledge, this is the first study in the region that studies the association between ESKAPE pathogens and UTIs.


Introduction
The ESKAPE pathogens were reported for the first time in 2008 by Rice based on data collected from hospital surveillance studies and from the Infectious Diseases Society of America (IDSA) which emphasized a clique of multidrug-resistant bacteria (MDR).This faction is made up of six pathogens (both Gram-positive and Gram-negative species) [1].The word ESKAPE is consists of the first letter of each pathogen in the group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter/Escherichia coli species) [1,2].This acronym is used to indicate the capability of those pathogens of "escaping" the biocidal action of antibiotics [3,4].This small group of pathogens have exceptional importance as they are the leading cause of nosocomial infections in both developed and developing countries, and they have various paradigms in virulence, pathogenesis, mode of transmission, and multidrug resistance.Globally, multidrug resistance is one of the most important health challenges.It comes among the top three health threats and is typically instigated by high frequencies of using and/or prescribing of antibiotics, antibiotic misuse, patient self-medication, and exposure to infections in health settings [3,[5][6][7][8].MDR in ESKAPE pathogens is increasingly associated with both clinical and economic impact.It has a significant disease burden, high mortality and morbidity rates, high treatment and healthcare cost, prolonged admission times, high rates of treatment failure, limited treatment options, and an increase in diagnostic uncertainties [3,5,6,9,10].Other effects also include triggering life-threatening infections in critically ill and immunocompromised patients [11].Furthermore, ESKAPE pathogens can form biofilms making treatment even more challenging [12].In response to MDR's global health threats and diminishing antibiotic discovery and development pipeline, the World Health Organization (WHO) in 2017 announced a global priority list of the most important antibiotic-resistant bacteria to encourage research and development of new antibiotics.This list contains 12 bacteria and it includes the ESKAPE pathogens labelled with a "priority status" [8,13,14].
Urinary tract infections (UTIs) are among the most prevalent types of infections reported in patients visiting outpatient clinics and emergency rooms, or hospitalized patients.UTIs can be divided into lower UTIs that involve infections in the bladder and urethra or upper UTIs in the kidneys and ureters.UTIs have a wide range of variable symptoms, severity, causative pathogen and treatment plan.The European Association of Urology (EAU) guidelines have indicated the critical levels of midstream sample urine (MSU) to be in the range of 10 3 CFU/mL for uncomplicated UTIs and 10 5 CFU/mL in complicated cases [15,16].Treatment and management of UTIs must take into consideration various factors; the type of UTI (uncomplicated or complicated), the host factors, the severity of illness, and the association between causative pathogen and MDR [17].IDSA has standard treatment guidelines for the treatment of both upper and lower UTIs [18].One of the major challenges in UTIs treatment is UTI recurrence, either relapses or reinfection.UTI recurrence is defined as ≥ 3 UTIs per year or ≥ 2 UTIs per six months [19].
This study is a retrospective study aiming to examine patients diagnosed with UTIs by physicians to determine the prevalence of ESKAPE pathogens in UTIs among diagnosed patients.To the best of our knowledge, this is the first study locally and regionally to research the association between UTIs and ESKAPE pathogens.

Methodology
Four hundred and forty-four Jordanian patients diagnosed with UTIs at the clinics of Jordan University of Science & Technology (JUST) Health Centre, Irbid, Jordan were included in this retrospective study from April 2021 to April 2022.Medical files of all patients admitted for UTIs during the study period were extracted.Data forms were designed to collect and record demographic and clinical information of each patient.The Institutional Review Board (IRB) approved the study protocol under the reference number 13/1/2719.

Assessment of infectious pathogens and the sensitivity of antibiotics
A sample of "clean-catch" (midstream) urine was cultured in order to determine the microorganisms that cause UTIs.The antibiotic sensitivity of bacterial isolates was assessed using the Kirby-Bauer test for susceptibility.A standard chart was used to look up the zone sizes (in millimeters) and determine if they were "sensitive, resistant, or intermediate."The data of antibiotic susceptibility results were extracted for all patients from laboratory test results performed at the health center.

Statistical analysis
Data were obtained from JUST health centre registries and transferred to standard forms describing demographic/clinical characteristics, including gender, age, clinical symptoms, and comorbidities.Percentages were compared to demonstrate the trends of UTIs among the study group.Association between UTIs and potential risk factors was assessed with the percent prevalence as well as odds ratios (OR) and p values based on the results from bivariate logistic regression.Statistical significance was defined at p < 0.05.Pearson correlation (r) was calculated to describe the relationship between the characteristics of UTIs and antibiotic resistance.The data obtained were entered in Microsoft® Excel 2016 (Microsoft Corporation, USA) and analyzed using the Statistical Package SPSS (SPSS® Version 21.0, NY, USA).

Demographic data and clinical characteristics of patients with UTIs
This study assessed 444 patients with UTIs.92% of the studied patients were females and 8% were males (Figure 1).The age of the studied patients ranged from 1 to 72 years (Figure 2).Regarding clinical presentations, 343 patients (77.3%) presented multiple symptoms, including fever, dysuria, polyuria, lower back pain, and pelvic pain (Figure 3).More than half of the patients (55.4%) had no concomitant chronic diseases, while the rest suffered from commonly diagnosed illnesses such as hypertension (16.2%), diabetes mellitus (13.5%), and hypothyroidism (5.4%) (Figure 4).The statistically significant OR and p values for the risk factors were being a female (OR = 3.39; p = 0.001), and aged 21-30 years (OR = 1.42; p = 0.023).Suffering from multiple symptoms when presenting at the clinic was also a predictor of UTIs (OR = 2.46; p = 0.003).

Distribution of ESKAPE pathogens among patients with UTIs
Out of the total 444 patients; the frequency of Gram-negative bacteria causing the UTIs was 74.3%, compared to Gram-positive bacteria which caused 25.7% of the UTIs.Escherichia coli was responsible for more than 62% of the infections.Figure 5 shows the results of urine cultures.ESKAPE pathogens were responsible for about 87.4% of the UTIs in this study except Acinetobacter baumannii, which was not associated with any UTI cases in the study.

Risk factors for UTIs
The demographics and characteristics of UTIs, including bacterial isolates and sensitivity to the prescribed antibiotic were plotted to describe the relationship using Pearson's correlation.The analysis showed a strong correlation between gender and the risk of UTIs.Similarly, a strong correlation was noted between the type of bacterial isolate and the risk of UTIs.In addition, a weak correlation between the    bacterial isolates and sensitivity to the antibiotics were identified (Figure 6).

Discussion
This paper describes the association of ESKAPE pathogens and UTIs among 444 Jordanian patients, over the period of one year.To the best of our knowledge, this study is the first study to determine the association between ESKAPE pathogens and UTIs in Jordan and nearby regions.
UTIs are common in all age groups and genders, however they have a higher prevalence rate in younger females compared to males and a similar prevalence in both genders in the geriatric age group [20][21][22].This correlates to the findings of this study.According to our data, the vast majority of UTIs (92%) were in females (OR = 3.39; p = 0.001) compared to only 8% in males.Additionally, this is further corroborated by the statistical analysis performed in which a strong correlation was identified between gender and risk of UTI (0.7 < r <0.5).Various anatomical factors and behavioral practices like the short urethral length, the closeness of the urethral meatus to the anus, sexual intercourse, absence of prostatic secretions, pregnancy, easy contamination with gastrointestinal tract flora and fecal flora, incontinence, and bad toilet habits explain these findings [23][24][25][26].
According to studies, the prevalence and frequency of a symptomatic UTI in some form rises as people get older [15,17].The younger groups, between the ages of 21 and 30, and those between the ages of 31 and 40, are considered to be child-bearing.Members of these groups would therefore be sexually active, pregnant and exposed to contraception, whether it be spermicide, condoms, or hormonal birth control, and engaging in other related sexual behaviors like incomplete emptying, the use of lubricants, and delayed micturition after intercourse [20,27,28].Menopause and hormonal imbalances, catheterization and other medical procedures for institutionalized patients are additional risk factors that become important as the age groups get older [17;29].
In this study, more than three-quarters of the patients were suffering from multi-UTI symptoms including fever, dysuria, polyuria, lower-back pain, and pelvic pain, and these were the most common reported symptoms.This is comparable with other studies that reported these similar symptoms in UTI patients [26,30,31].However less than one-quarter of the patients suffered solely from dysuria.Even though it is statistically insignificant, 101 patients reported this symptom.It is noteworthy to mention though, that the presence of some or all these symptoms is not enough to diagnose UTIs, and it is imperative in this case to run a urine culture test to determine whether a type of pathogen is present and the appropriate medication needed according to the resistance and susceptibility profile of the pathogen [32,33].
In this study, around 55% of the patients did not suffer from any comorbidities.However, the remaining 45% have reported associated comorbidities including hypertension, diabetes mellitus, and hypothyroidism.This finding is similar to the comorbidities reported by Zavala-Cerna et al. [34].
Gram-negative bacteria cause almost 90% of all UTIs diagnosed, 65-90% of these cases are due to Escherichia coli [31,35,36].In this study, the highest prevalence was of Gram-negative bacteria (74.3%) of which 92.8% of the cases were attributed to Escherichia coli.On the other hand, Gram-positive bacteria were responsible for only 25.7% of the cases, half of which were due to Staphylococcus aureus.These findings are in agreement with other studies [37,38].
ESKAPE pathogens were responsible for about 87.4% of UTIs studied here.All pathogens in the list were identified in the urine samples except for Acinetobacter baumannii.In this study, a strong correlation was detected between the type of bacterial isolate and the risk of UTIs (0.5 < r <0.5), and this is indicated by the predominance of Escherichia coli (62.8%) in UTIs.In addition, Klebsiella pneumoniae (5.4%) was identified as the second most prevalent Gram-negative bacteria in this analysis.We also assessed the presence of Gram-positive bacteria in the cultures studied; Staphylococcus aureus (12.4%), followed by Group B Streptococci (9.5%) were the most predominant among Gram-positive microorganisms isolated from urine samples of patients with UTIs.Our findings are in line with data from Arab countries, including Saudi Arabia [39], Lebanon [40], and studies from various regions of the world [41,42].In Jordan a previous study described the clinical patterns among Jordanian patients with UTIs, however, the analysis was only limited to patients with positive Escherichia coli or Klebsiella pneumonia urine cultures [43] and did not include the rest of the pathogens from the ESKAPE pathogens group.This study performed an extended analysis to include all ESKAPE pathogens among patients with UTIs.
Based on our data, about two-thirds of the overall UTIs were caused by Escherichia coli.An understanding of the reasons behind the high prevalence of Escherichia coli in UTIs are given by the presence of uropathogenic Escherichia coli (UPEC).This pathogen includes Escherichia coli with a set of genes encoding for virulence factors allowing them to persist at the site of infection and successfully establish an infection [44].Moreover, fimbria, flagella, biofilm formations, and the external lipopolysaccharide in the bacterial Escherichia coli membrane and its capsule are additional virulence factors.These factors help bacterial cells in adhesion to the site of infection, finding new nutrient sources, escape the host's immune system, evade antibiotics and reach into the upper urinary tract [44][45][46][47].Our data analysis has also shown that Acinetobacter baumannii has not been associated with any UTI cases reported by the JUST health centre.This can be attributed to the fact that Acinetobacter baumannii is associated with UTIs when percutaneous nephrostomy tubes or urinary catheters are inserted in hospitalized patients.It has also been connected to patient's infrequent contact with the healthcare system.In our study, patients visiting the JUST health centre are treated as outpatients; thus, limited or no cases of UTIs caused by Acinetobacter baumannii would be expected [14,48].
In general, Gram-negative bacteria are highly resistant to vancomycin, and amoxicillin while having good sensitivity to cephalosporins.In addition, Gramnegative bacteria are intrinsically resistant to vancomycin.This is due to its bulky structure and incapability to penetrate the outer bacterial cell membrane [49].However, some bacteria developed a high resistance against some cephalosporins including cefuroxime (100%) and cefixime (87%) against Pseudomonas aeruginosa, as well as ceftriaxone (100%), and cefixime (100%) against Citrobacter freundii.Levofloxacin has shown a near 50% activity against Gram-negative bacteria, except for Citrobacter freundii which is completely resistant to it.Similarly, ciprofloxacin has been active in treating infections of Klebsiella pneumoniae, Proteus mirabilis and, to a lesser extent, Escherichia coli, with a complete resistant profile against Citrobacter freundiis, and Pseudomonas aeruginosa [37,[50][51][52][53][54][55].

Conclusions
Overall, the data indicated that ESKAPE pathogens were responsible for about 87.4% of UTIs in this study.In addition, the data indicated that the highest resistance rates were associated with doxycycline, amoxicillin, and clindamycin.While the lowest resistance rates were associated with levofloxacin and ciprofloxacin.This reflects the need for accurate and periodic assessment and surveillance data of ESKAPE pathogens prevalence as uropathogens and their susceptibly pattern.This continuous update in susceptibility profiles will directly affect the selection of empiric therapy for UTIs and aid in outlining and updating the primary healthcare guideline in the Jordanian health centers.This study will help in establishing a national policy regulating the use of antibiotics, notably against ESKAPE pathogens in patients with UTIs.

Figure 1 .
Figure 1.Gender distribution and its frequency in urinary tract infections.p-value and odds ratios (OR) are presented.

Figure 4 .
Figure 4. Urinary tract infections comorbidities and their frequencies.p-value and odds ratios (OR) are presented.

Figure 2 .
Figure 2. Age distribution and its frequency in urinary tract infections.p-value and odds ratios (OR) are presented.

Figure 3 .
Figure 3. Clinical presentations and their frequencies in urinary tract infections.pvalue and odds ratios (OR) are presented.

Figure 5 .
Figure 5. Distribution, frequency and characteristics of pathogens responsible for urinary tract infections (UTIs).In total Gram-positive bacteria are responsible for 114 (25.7%) of UTIs and Gram-negative bacteria are responsible for 330 (74.3%) of UTIs.

Figure 6 .
Figure 6.Correlation matrix for the urinary tract infections risk factors.