Opportunistic Pathogens in Patients with Urinary Tract Infection

Introduction : Urinary tract infections (UTIs) remain one of the most important problems of modern urology and medicine. Infections bring great discomfort and significantly reduce the quality of life. UTIs rank second after respiratory tract infections in outpatients. The most common pathogen of UTI are E.coli . The study of the etiology of UTI has great clinical and epidemiological importance in routine practice. Objective: To assess the etiological significance of pathogens in the occurrence of urinary tract infections in the Karaganda region of Kazakhstan. Methods: A total of 2378 patients presenting UTIs were enrolled and each provided a urine sample. The study was carried out in the Clinical Microbiology Laboratory MediTEC-NS between 2 January and 29 December 2018. Identification of isolated microorganisms was carried out on a WalkAway 96 Plus microbiological analyzer, Microscan model manufactured by Beckman Coulter (USA). Statistical Analysis was performed using the STATISTICA-6 package. were predominantly caused by ten opportunistic pathogens. The most common uropathogens with a frequency of 66.9% were E. coli -30.53%, S. epidermidis -20.16%, and Enterococcus spp. -16.21%. Frequently isolated pathogens included Klebsiella, S. haemolyticus spp., and Streptococcus spp . which amounted to 21.98%. The distribution within the patient group was equable and ranged from 6,67% to 8,15%. Etiologically significant pathogens included Enterobacter spp., Proteus spp., Acinetobacter spp., Pseudomonas spp. These bacteria accounted for 11.11%. The distribution within the group was again equable and ranged within 2,55% to 2,96%. 3 categories according to the frequency of detection. Common uropathogens were E.coli - 30,53% are followed by S.epidermidis -20,16% and Enterococcus spp. -16,21%. According to the results of our study, these microorganisms were included in category 1 with a high incidence rate 66,9%. The share of isolated pathogens in this group ranged from 16.9% to 30,53%.


Introduction
Urinary tract infections (UTIs) remain one of the most important problems of modern urology and medicine. Infections bring great discomfort and significantly reduce the quality of life. UTIs rank second after respiratory tract infections among outpatients. Every year, 150 million people around the world are diagnosed with UTI [1]; moreover, about 50% -60% of women have a UTI at least once in their lifetime [2] [3]. In the United States, about 7 million visits to a doctor with urinary tract infections are registered annually, more than 100 thousand patients are hospitalized, and annual costs exceed US $ 1.6 billion [4]. According to Qiao LD et al. [3] in China, UTIs comprise 50% of all nosocomial infections. It should be noted that about 50% of UTIs in children are not counted, suggesting a significant under-estimation of infection rates. [5]. Data analysis also showed that out of 448 216 hospital admissions in Kazakhstan (2014), 33 613 were associated with kidney and urinary tract infections [6]. According to numerous data from researchers, including Kazakhstan, the majority cases of UTI are caused by E. coli, with the bacteria accounting for over 80% and some cases nearly 90%. of uncomplicated UTIs. [7] [8] [9]. E. faecalis, C. albicans are also associated with hospital acquired UTIs in Kazakhstan [10,11]. Due to the fact that the ecological niche of pathogens, including uropathogens, cannot be the same for many years, and also taking into account modern diagnostic capabilities, it was interesting for us, from a scientific and practical point of view, to study the etiological significance of various opportunistic microorganisms in the occurrence of UTIs within our region.

Study area
The study was carried out in the Karaganda Clinical Microbiology Laboratory MediTEC-NS from January to December 2018.

Study design
A total of 2378 patients were enrolled and provided a urine sample. Inpatients and outpatients with clinical symptoms of UTI were included. The age range of the selected patients was from 15 to 80 years. The study participants were majorly females 1586 (66, 69%).

Sample collection and Processing
A total of 2378 patients were enrolled and provided a urine sample. Inpatients and outpatients with clinical symptoms of UTI were included. The age of the selected patients was from 15 to 80 years. The study participants were majorly females 1586 (66,69%). For each patient, midstream urine samples for bacterial culture were collected before treatment. Urine samples were collected in sterile containers with a urine needle and transported to the laboratory within two hours for culturing. The culture media were prepared on an automated ProfiClave system (Switzerland). The following (HiMedia, India) сulture media -nutrient agar, Columbia blood agar, MacConkey agar, Sabouraud agar with chloramphenicol, mannitol saline agar with yolk emulsion -were used for cultivation. Bacteria were incubated at T-37 C, fungi at T-20-22 C. Colonies were counted, and microscopic examination of their morphological tinctorial properties were observed. According to the classic definition, bacteriuria is caused by 10 5 colony-forming units in 1 mL [12] HootonTM et al [13] believe that true infection may occur in patients with lower colony-forming units in 1 mL in the presence of symptoms and urinary leukocytes. In this context, more than 10 3 colony-forming units in 1 mL of urine is associated with infection [14]. In our case, in the presence of leukocytes more than 7 per high power field, colony counts higher or equal to 10 3 (colony-forming units in 1 mL) were considered as positive culture test. Identification of the isolated microorganisms was carried out on a WalkAway 96 Plus microbiological analyzer, Microcsan model manufactured by Beckman Coulter (USA). The results of identification and sensitivity to antimicrobial drugs after 17-24 hours were taken into account on the analyzer display and were automatically transferred to the information system of the laboratory. Identification of bacteria of the genus Acinetobacter was carried out on the MicroScan® panels: Gram-negative bacteria on the NEG MIC 44 B1016-175 and Neg/Urine Combo 61 B1017-414; non-fermenting bacteria-on the NEG MIC 44 B1016-175; Gram-positive bacteria -on POS Combo 33 B1017-211, streptococci -on MICroSTREP 6 plus. These panels are designed to determine the sensitivity of aerobic and facultative anaerobic Gram-negative rod-shaped bacteria to antibacterial drugs and their identification to the species. The fungi were indicated on Sabouraud's agar medium with chloramphenicol. Presumptive identification on chromogenic Sabouraud's agar by colony color.

Statistical Analysis
A total of 2378 patients were enrolled and provided a urine sample. Inpatients and outpatients with clinical symptoms of UTI were included. The age of the selected patients ranged from 15 to 80 years. The study participants were majorly females 1586 (66, 69%). Statistical analysis was performed using the STATISTICA-6 package. The relative frequency (p) of the occurrence of an attribute was determined as follows: The attribute is defined as a specific characteristic or feature of a given subject p is calculated by sample, it reflects the population with some error: The confidence interval for the p is located within: and tαis the critical value of the bilateral t-criterion of the Student for a given α and (n-1) degrees of freedom. Table 1 shows the ratio of positive and negative urine culture test among investigated patients. From the subject total, 1177 patients tested positive for culture test and out of that 803 (68%) were females while 374 (32%) were males. 1201 tested as negative urine samples and out of that 398 (33,3%) cases the clinical pathogens were identified 10 1 or 10 2 (colony-forming units in 1 mL) and in 803 (66,7%) could not be detected.

patients with UTIs
Of the 1356 isolated opportunistic pathogens 1215 account for 90% of cases were of epidemic significance and 141 were considered as sporadic cases. Table 10 shows that the ten most common bacterial pathogens in patients with UTIs were ranked in 3 groups according to the frequency of occurrence.  16,21%. According to the results of our study, these microorganisms were included in category 1 with a high incidence rate 66,9%. The share of isolated pathogens in this group ranged from 16.9% to 30,53%.
The second category was frequently isolated including Klebsiella, S. haemolyticus spp., and Streptococcus spp. These bacteria amounted to 21.98%. The distribution within the group was equable and ranged from 6,67% to 8,15%.

Discussion
UTIs, a topical problem in urology and medicine in general, rank second after infections of the respiratory tract [15 ] [16 ]. The study of etiology UTIs in a particular region is of great diagnostic, prognostic and epidemiological significance. Out of 2378 urine samples, the pathogen was detected in 1177 (49, 5%) cases, and we considered 1201 (50,5%) as negative. According to our research, the sensitivity of the culture investigation is 49,5%. In fact, there are symptoms of the UTIs and leukocytes of more than 7 per high power field, hence urine cannot be sterile. Viable (or active) but nonculturable bacteria may also cause false-negative culture [17]. Numerous studies show that Gramnegative bacteria predominate in the UTIs [ 18] [ 19] [20 ]. Our results generally showed no clear difference in Gram-negative, at 690 (50,88%) and Gram-positive, at 630 (46,46%), recovery rates. Detailed information on the isolated pathogens is presented in tables 2-9. It should also be noted that the relatively small number of fungal isolates -36 (2,65%) in comparison with the bacteria, were isolated. We cannot conclude with confidence that these are regional features, since identification and indication of fungi was carried out by routine methods, while an automated modern system was used to identify bacteria.  [ 10]. According to our results, the role of gram-negative rods and of gram-negative non-fermenting bacteria in the occurrence of UTI is obvious and practically equivalent, we do not see a dominant microorganism in this group. The frequency of occurrence of Enterobacter spp. Proteus spp. Acinetobacter spp. Pseudomonas spp. did not differ significantly and varied from 2.55% to 2.96% (confidence interval 1.66 ; 3.91). It should also be noted that the frequency of occurrence of Acinetobacter slightly exceeds Pseudomonads, and Acinetobacter lwoffii prevails over other species of Acinetobacter.
They accounted for 11.11%. The distribution within the group was equable and ranged within 2,55% to 2,96%.

Author Contributions:
Conceptualization Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors have disclosed no conflict of interest.