MICROBIOLOGICAL PROFILE AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA ASSOCIATED WITH URINARY TRACT INFECTIONS IN UKRAINIAN ADULTS

. Microbiological profile and antimicrobial susceptibility of bacteria associated with urinary tract infections in

Problem of urinary tract infections (UTIs) is constantly growing in the world and also in Ukraine [1].UTIs have significant importance for public health and every year more than 150 million people are affected.From the economic point of view, its burden was estimated to be about $6 billion worldwide each year [2].The second to the respiratory tract infections, UTIs remain one of the most common infectious diseases.Furthermore, UTIs are among the most common causes of sepsis presenting in hospitals [3].The problem is escalated by the global growth of antimicrobial resistance (AMR).In 2019 in Ukraine, there were 8.4 and 31.5 thousand deaths attributable and directly associated with AMR, respectively [4].Pathophysiology of chronic UTIs is complicated by biofilm-formation, subsequent contributing to the resistance maintenance and dissemination [5].Treatment of UTIs implies empirical administration of antibacterial drugs, and the choice should consider the local data of antibiotic resistance patterns [6].
The aim of the study was to analyze the microbial profile of UTIs associated with urolithiases and to study susceptibility to antibiotics in its causative agents.

MATERIALS AND METHODS OF RESEARCH
The study was conducted in the cooperation of the department of surgery No. 1 and urology and the department of microbiology, virology, immunology, epidemiology and medico-biological physics and informatics of the Dnipro State Medical University from January 2023 to December 2023.Systematic sampling technique was employed.The inclusion criteria: patients with calcium urolithiasis complicated by urinary tract infections (UTI) and the absence of other chronic illnesses or pregnancy.Among these patients, there were 56 males and 72 females.The age of the patients ranged from 25 to 65 years, with an average age of 45 years.Patients enrolled in the study were trained to collect a midstream urine in the sterile container [7].From a patient only one sample was taken for microbiological study.
The research work was approved by the Bioethics Committee of the Dnipro State Medical University protocol No. 3, of 16.11.2022and was conducted in accordance with the written consent of the participants and in accordance with the principles of bioethics set forth in the Helsinki Declaration "Ethical Principles of Medical Research Involving Humans" and the "General Declaration on bioethics and human rights (UNESCO)".
The main method of the research was bacteriological.Collected urine extempore was inoculated on a set of growth mediums.For isolation of gram-negative non-fastidious bacteria, we used MacConkey agar; for selective isolation and fast identification of staphylococci, correspondent chromogenic agar was used; for selective isolation of fungi, we used Sabouraud dextrose agar with chloramphenicol.For non-selective cultivation, assessment of haemolytic activity and isolation of fastidious bacteria, blood agar with 5% sheep blood was used.The inoculated plates were incubated in thermostat for 24-48 h at 37°C aerobically.The quantitative evaluation of the bacteriuria was done using calibrated loop technique (10 μL) giving a count of bacteria and yeasts above a threshold of about 10 2 CFU/mL of urine.When bacteriuria comprised ≥10 4 CFU/mL, UTI was confirmed [7].
Antimicrobial susceptibility testing was conducted by serial microdilution assay in accordance with the EUCAST recommendations with supplements and updates [8, 9, 10].Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 were used for quality control.
The Microsoft Office Excel 2010 ® with customizations was used for statistical data processing (version 14.0.7265.5000,license No. 02278-581-24/Том XXIX/1 0218935-38456).Categorical variables are expressed as percentages and were compared by the χ2 twotailed test.Incidence proportions of pooled combinations and probabilities were calculated, which established the corresponding 95% confidence intervals and the p-values <0.05.The Kruskal-Wallis's test was used for multiplying comparisons of quantitative data; found differences further were assessed by the Mann-Whitney U-test.The results of the experiments were statistically significant at p˂0.05 [11].
The list of pathogens causing UTIs associated with urolithiasis in Ukrainian adults is given in the Figure 1.
Antimicrobial susceptibility testing was conducted for all bacteria isolated from patients with UTI and gave growth ≥10 4 CFU/mL.Antimicrobial sus-ceptibility of Enterobacterales was variable, and the best results were obtained for carbapenems, novel antibiotics (cefiderocol, ceftolozane-tazobactam and ceftazidime-avibactam), aminoglycosides and tigecycline.Patterns of antimicrobial susceptibility of Enterobacterales is demonstrated in the Figure 2.

Fig. 2. Antimicrobial susceptibility of enterobacteria associated with UTI in Ukrainian adults
Susceptibility to penicillins comprised 12.5-88.5% with the lowest results for ampicillin and temocillin and the highest indexes for clavulanic acid combinations.Susceptibility to cephalosporins was between 25.0-100.0%,with the best results for the III and IV generation cephalosporins and novel antibiotics.According to results of the antimicrobial susceptibility testing, there were 9 isolates with production of carbapenemases and resistant to all relevant β-lactam antibiotics.Also, such cultures demonstrated multidrug-resistance profile and remain non-susceptible to fluoroquinolones, aminoglycosides, fosfomycin and trimethoprim-sulfamethoxazole.All isolates were collected from patients already exposed to several courses of antimicrobial treatment.Multidrug-resistant K. oxytoca is demonstrated on the Figure 3.
Antimicrobial susceptibility of Enterobacterales to fluoroquinolones was found to be 50.0-60.3%.Suscep-tibility to aminoglycosides was 88.8-93.8%.Also, there were 88.8% isolates carrying sensitivity to fosfomycin.However, resistance to trimethoprim-sulfamethoxazole was high and comprised 71%.All tested isolates were susceptible to tigecycline and colistin.
All isolated Staphylococcus spp.were βlactamase producers according to the test with benzylpenicillin or ampicillin.Among six tested, one isolate of S. saprophyticus demonstrated methicillin-resistance.Susceptibility to screening with norfloxacin comprised 3/6, however, resistant isolates tested susceptible to other fluoroquinolones.Susceptibility to screening with erythromycin was 2/6.Similar poor results also were found for trimethoprim-sulfamethoxazole.Susceptibility to aminoglycosides and nitrofurantoin (S. saprophyticus) was found to be 100%.Antimicrobial susceptibility testing of Enterococcus spp.revealed that 8/12 tested isolates were non-susceptible to ampicillin, therefore, harboured resistance to ampicillin, amoxicillin, piperacillin with and without inhibitors.Such isolates also demonstrated non-susceptibility to gentamycin (indicates presence of high-level aminoglycosides resistance) and fluoroquinolones.All isolates tested susceptible to imipenem, vancomycin, tigecycline, linezolid and nitrofurantoin.
In this paper we discuss microbial spectrum of UTIs, and antimicrobial profile of bacteria related to infectious-inflammatory processes in the urinary tract with the background of urolithiasis.The main advantage of our study is sample size and systematic sampling approach of a chosen category of patients allowing us to evaluate microbial profile.Results describing the meaning of gram-negative bacteria is of special importance because depict the local profile of antimicrobial susceptibility.Our study provides important new insights into the implications of antimicrobial treatment in complicated UTIs.Here we provide data on susceptibility to the novel antibiotics, in particular, cefiderocol, ceftazidime-avibactam and ceftolozane-tazobactam in cultured uropathogens.Even though increasing resistance rates in urinary isolates have been already published [12,13,14], Ukrainian data still needs to be extended.Our study complements the knowledge regarding the global trends of antimicrobial resistance.
In our study women occupied bigger proportion in comparison to men.The features of the female anatomy of urinary system predisposes to higher prevalence among this category.In the analytic domestic study, it was also found that morbidity and prevalence of chronic cystitis among the female population of Ukraine is more then 3 times higher then in male and is constantly growing [1].Furthermore, the same figures on the age-sex UTIs rate were shown in the worldwide report and their data emphasize affection На умовах ліцензії CC BY 4.0 of low-and middle-income countries [15].In fact, the problem is not limited to the prevalence of pathological condition, but it is escalated by associated factors, in particular, antimicrobial resistance.Therefore, microbiological study is of special importance in chronic, recurrent and difficulty treated UTIs.
In our study, the main bacteria contributing to UTIs associated with urolithiasis were E. coli and K. oxytoca.These bacteria were equally distributed among men and women and, usually, they were isolated as monoculture, indicating the significance of the agents in infectious-inflammatory process.There were also some sex differences.Bacteria like S. saprophyticus, most Enterococcus and Candida fungi were found exactly in female samples.The meaning of C. albicans in pathogenesis of UTI is debatable, however, its presence in co-culture may either contribute to diseases severity or indicates local microbiota disturbances in adults with complicated UTI [16].
Antimicrobial resistance is of concern across the world and has been recognized as a rapidly growing challenge for the modern medical practice [1,4,17].In our study, there were Enterobacterales resistant to the most clinically relevant antibiotics, including carbapenems, fluoroquinolones and aminoglycosides.In the prospective multicentre cohort study, which participants were pregnant Ukrainian women with UTIs, significant proportion of the main causative agents of UTIs (E.coli, K. pneumoniae, P. mirabilis, P. aeruginosa) were also found to be multidrug resistant [18].Although here we didn`t study genetic background of antimicrobial resistance, according to tests with meropenem and/or imipenem, carbapenemase producers were revealed.Even though other isolates were susceptible to carbapenems, they expressed extended-spectrum β-lactamases and demonstrated resistance to the III generation cephalosporins.The problem is existence of resistance genes coded in plasmids and the risk of horizontal genes transfer in hospital and, sub-sequently, community sittings.Even more, simultaneous co-existence of other resistance mechanisms has also been documented [19,20,21].It was shown in armed conflict on Ukrainian territory, patients of the domestic military hospitals harboured the same bacterial clones as our civilians who have fled to Germany and other European countries since the beginning of the ongoing war [22,23,24,25].

CONCLUSIONS
1.There is an ongoing outbreak of multidrugresistant infections in Ukraine, and causative agents of urinary tract infections are among the most important contributors.
2. Antimicrobial resistance is emerging problem and several ways have been proposed to impede its growth.Chronic infections are known to be associated with biofilm-formation and approaches for their eradications may require administration of multi-targeted compounds or their combination.Availability of data on the local antimicrobial susceptibility profile may guide the informed decision making in etiotropic treatment, therefore, contribute to global efforts in rational drug use and fight the resistance escalation.