Monitoring of the Distribution of Antibiotic-Resistant Strains of Microorganisms-Causes of Infectious Processes of Urogenital Cystitis System of Patients

The purpose of the work was to conduct a comparative analysis of the quantitative and qualitative composition of the microbiota of the urogenital system in different segments of the population of Dnipro for the period of 1 January 31 August, 2020. Material and methods. To study the microbial landscape and determine the levels of resistance to antimicrobial drugs variants of microorganisms circulating in the urogenital tract, were examined 176 people of different ages and genders – patients of the Medical Diagnostic Center at the Dnieper Medical Academy (Dnipro) for the period of 1 January 31 August, 2020. Results and discussion. According to the results of the microbiological study, the fact of development of infectious processes was established in 86 people, which accounted for 32.31% of cases. When studying the microbial landscape of the reproductive system of the examined patients, there was the following spectrum of priority pathogens of urogenital infections: in children and adolescents: Klebsiella oxytoca, Haemophilus influenzae / H. parainfluenzae, Escherichia coli, Streptococcus agalactiae; in women: Candida albicans, Streptococcus agalactiae, Escherichia coli, Ureaplasma spp.; in men: Ureaplasma spp. and Escherichia coli. During the monitoring of the proliferation of antibiotic-resistant variants of microorganisms – pathogens of infectious processes, the highest levels of resistance in isolated clinical isolates in relation to the drugs of classes were established: fluoroquinolons – 29.16%, cephalosporins – 18.75% and carbopenems – 10.42% of cases. Conclusion. With the use of test systems “Femoflor” and “Androflor” the species spectrum of pathogens of dysbiotic conditions of the urogenital tract in 88 patients aged 17-75 years with the help of polymerase chain reaction in real time was investigated. The incidence of detection of conventional pathogenic and pathogenic microorganisms from the urogenital sphere of women and men and the ratio of aerobic and anaerobic microbiota in dysbiotic syndromes is shown. According to the results of the study, 60.97% of women and 76.59% of men had significant deviations toward reducing the titers of representatives of the symbiotic microbiota – bacteria of the genus Lactobacilllus on the background of increasing quantitative indicators of conventional pathogenic microorganisms Gardnerella vaginalis etc., Eubacterium spp., Ureaplasma spp., Candida spp., Anaerococcus spp., Bacteroides spp. etc., Enterobacterium spp., and the presence of pathogenic microorganisms Mycopasma genitalium, Trichomonas vaginalis, Chlamydia trachomatis, Ureaplasma parvum in significant titras.

The incidence of detection of conventional pathogenic and pathogenic microorganisms from the urogenital sphere of women and men and the ratio of aerobic and anaerobic microbiota in dysbiotic syndromes is shown. According to the results of the study, 60.97% of women and 76.59% of men had significant deviations toward reducing the titers of representatives of the symbiotic microbiota -bacteria of the genus Lactobacilllus on the background of increasing quantitative indicators of conventional pathogenic microorganisms Gardnerella vaginalis etc., Eubacterium spp., Ureaplasma spp., Candida spp., Anaerococcus spp., Bacteroides spp. etc., Enterobacterium spp., and the presence of pathogenic microorganisms Mycopasma genitalium, Trichomonas vaginalis, Chlamydia trachomatis, Ureaplasma parvum in significant titras.
Keywords: urogenital tract, microbiota, antibiotic resistance, real-time polymerase chain reaction Research relation to the programs, plans, and department themes. The work was carried out within the framework of the initiative topic "The biological basis of the functioning of the microbiocenoses of the environment and the human body", the State Registration Number is 0119U100097.
Introduction. In recent years, on the background of increasing urbanization, changes in lifestyle and living conditions, deteriorating environmental conditions, diet, hypovitaminosis and increasing the number of immunodeficiency conditions, the incidence of urogenital infections is growing [1]. Medical and socio-economic significance of inflammatory diseases of the urogenital tract of infectious origin due to global prevalence, a wide range of pathological conditions with a high frequency of residual manifestations, complications, reproductive or gestational dis-orders, the possibility of infection of newborns and the development of severe diseases in children [2]. The rapid growth of dysbiotic syndromes in the reproductive sphere remains a topical issue in scientific and medical practice. Biota imbalance is accompanied by immune and metabolic disorders, the severity of which can vary from asymptomatic carriers to severe clinical manifestations, which in turn contributes to the chronicity of the process, adverse effects on reproductive function and reduced quality of life [3][4][5][6][7][8]. Nowadays, numerous studies have shown that the etiological structure of urogenital infections is polymicrobial in nature and in some cases represented by associations of different species of microorganisms. The main issue is the timely detection of inflammation with the use of modern methods of laboratory diagnosis, the purpose of which is to prevent reproductive losses, opportunistic infections and dysbiosis [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. The method of polymerase chain reaction in real time allows to detect pathogens even at low concentrations, to conduct etiological diagnosis in the early stages, to assess the qualitative and quantitative content of urogenital biocenosis, to control the quality of the bioassay and the effectiveness of therapy. Of all the available methods, the most complete range of microorganisms is presented in the tests "Femoflor" and "Androflor" [3][4][5][6][7][8]. One of the main problems of the XXI century is still the development of resistance to antibiotics in microorganisms of the genitourinary tract. Irrational antibiotic therapy significantly changes the biological properties of microorganisms and promotes the formation of populations of antibiotic-resistant variants. Therefore, the trigger for the reduction of genital pathology is the creation of proper monitoring, an effective system of prevention and adequate treatment with chemotherapeutic drugs [3][4][5][6][7][8][10][11][12][13][14][15][16][17][18][19][20][21].
The purpose and objectives of the study. In view of the above, the purpose of the work was to conduct a comparative analysis of the quantitative and qualitative composition of the microbiota of the urogenital system in different segments of the population of Dnipro for the period of 1 January -31 August, 2020.
Material and methods. Studies of the composition of the microbiota on quality and quantitative indicators were performed by methods of bacteriological analysis, species identification of isolated clinical isolates of Enterobacteriaceae spp. and other gram-negative rods were determined using test kits "API 20E" ("BioMerieux", France), non-fermentable gram-negative rods of the genus Pseudomonas -for "API 20 NE" ("BioMerieux", France), staphylococci for "API Staph" ("BioMerieux", France), streptococci and enterococci according to" API 20 Strep" ("BioMerieux", France), other types of microorganisms -representatives of normal, opportunistic aerobic and anaerobic microbiota were performed according to the schemes of identification of bacteria by Bergie bacterium [22]. Levels of resistance to antibiotics were determined by disco-diffusion method. Sensitivity assessment of isolated strains of microorganisms was performed according to the criteria of CLSI / NCCLS [23].
The experiments used test systems "Femoflor-16" and "Androflor" in Real time mode, which allowed to obtain a complete quantitative characterization of the microbiome and to differentiate the states of physiological balance or dysbiosis. When isolating DNA used standard sets of reagents -RealBest DNA extraction 3, lysing solution (JSC "VectorBest", Russia), TEST-NK (LLC "NPO DNA-Technology", Russia). When determining the microbial landscape, a set of reagents was used to study the biocenosis of the urogenital tract in women and men by real-time PCR ("Femoflor-16" and "Androflor") and a detecting amplifier DT-96 / DT-322 manufactured by NPO DNA -Technology »(RF).
To obtain adequate results, only samples with a sufficient number of cells in a test tube with the analyzed sample and sufficient total bacterial mass were used. Samples in which the number of human DNA cells was more than 10 4 genome equivalents (GE) in the sample and the value of the total bacterial mass ranged from 10 6 to 10 9 GE / sample were considered. When the value of the control of material uptake (KVM) is less than 10 4 , the result of PCR analysis of biota is considered inaccurate, which requires re-capture of biomaterial.
Accounting and processing of results was performed automatically using the standard Microsoft Office Excel package. All examined patients signed an informative consent, the study was conducted in accordance with the Declaration of Helsinki of the World Medical Association, the Statute of the Ukrainian Association of Bioethics, standard provisions on ethics of the Ministry of Health of Ukraine №66 of February 13, 2006. Diagnosis of urogenital infections research methods, in accordance with the recommendations of the American Association of Microbiologists (ASM) and the European Association of Urologists (EAU) [24,25].
Results and discussion. To study the microbial landscape and determine the levels of resistance to antimicrobial drugs variants of microorganisms circulating in the urogenital tract, were examined 176 people of different ages and genders -patients of the Medical Diagnostic Center at the Dnieper Medical Academy (Dnipro) for the period of 1 January -31 August, 2020.
According to the results of a comprehensive study, the fact of the presence of an infectious process in 84 people was established, which was 32.31% of cases ( Table 1). To conduct a comparative analysis of the incidence of urogenital infections during the study period, patients of three groups were examined: 1) children and adolescents under 16 years (n = 59), 2) women (n = 63), 3) men (n = 54). Each group of patients was divided into age categories: 1) children and adolescents: I -children from 1 to 5 years (n = 17); II -from 6 to 10 years (n = 32); III -from 11 to 16 years (n = 10); 2) women: I -from 17 to 35 years (n = 34); II -from 36 to 55 years (n = 19); III -from 56 to 75 years (n = 10); 3) men: I -from 17 to 35 years (n = 22); II -from 36 to 55 years (n = 19); III -from 56 to 75 years (n = 13).
The examination of 54 men showed that 23 patients (27.38%) were infected with urogenital diseases, such as (  [32][33][34][35]. In order to determine the spectrum of pathogens of urogenital infections, the frequency of microbial agents isolated from patients of different categories was monitored. During the study period, bacteria of the following species were isolated: Escherichia coli -13.0% of cases, Streptococcus agalactiae -11.79%, Candida albicans -10.77%, Ureaplasma spp. - (Fig. 1).
As a result of the conducted research it is possible to define the priority activators which most often cause defeats of a urogenital tract of persons of different age categories and sex: Escherichia coli, Streptococcus agalactiae, Candida albicans and Ureaplasma spp.
The next stage of the study was to analyze the resistance of selected strains of uropathogenic microorganisms to antibacterial drugs, as the problem of antibiotic resistance, according to World Health Experts (WHO), the scale of the pandemic: the world has confidently entered the postantibiotic era. And taking into account the enormous damage caused by inadequate antibiotic therapy, researchers around the world agree that antibiotics should be preferred rather than broad-spectrum antibiotics, and if there is an effective alternative, it should be taken into account. Today, data on the etiological structure, susceptibility of pathogens to antibacterial drugs in the world differ, which indicates the need to study the regional features of antibiotic resistance. The increase in the number of patients with urogenital infections, the difficulties of therapy associated with antibiotic resistance, the complexity and ambiguity of many issues of the mechanism of the disease, the lack of clear criteria for diagnosis and impact of infection on the reproductive system, make this problem extremely relevant. The urgency of the problem of the spread of antibiotic-resistant strains of microorganisms -pathogens of urogenital tract infections reaches its incredible peak during treatment, which often leads to the development of recurrent infections and pathological changes in the tissues of the genitourinary tract. The presence or absence of complicating factors in patients with urogenital tract infections is one of the key parameters  [36][37][38].
According to the results of the research, 96 antibiotic-resistant variants from 195 isolated cultures of opportunistic and pathogenic microorganisms were identified.
The main mechanisms of resistance of classical bacteria to fluoroquinolones are connected with change of structure of two bacterial enzymes on which these antibiotics -DNA gyrase and topoisomerase IV which are responsible in a bacterial cell for the conformational changes of DNA necessary for its normal replication, and also from modification. targets as a result of mutations in the QRDR (Quinolone Resistance-Determing Region) of target genes, as well as with a decrease in the accumulation of drugs in the cell and the acquisition of determinants of resistance by horizontal gene transfer. Bacterial resistance to cephalosporin drugs is due to one of the mechanisms: 1) modification of peptidoglycan, which leads to a decrease in the effectiveness of the drug; 2) hydrolysis activation of the antibiotic by beta-lactamases; 3) violation of the permeability of the external structures of the microbial cell and the difficulty of binding of the antibiotic to the peptidoglycan. Resistance to carbapenems can be caused by various mechanisms, in particular: changes in the permeability of the out-er cell membrane, activation of efflux systems or the production of hydrolyzing carbapenems β-lactamases (carbapenemases) [39][40][41][42].
The obtained data correlate with the results of studies by Komolafe OO, Amyes S.G.B., Laxminarayan R., Malik B. et al. and Martinez J. L. et al., who noted the highest levels of resistance in clinical isolates of uropathogenic microorganisms to antibiotics of classes: fluoroquinolones -up to 35.0%, cephalosporins -up to 25.0% and carbopenems -up to 20.0% of cases [43][44][45][46][47].
Increasingly, scientists are noticing a rapid growth of dysbiotic disorders of the urogenital system in both women and men. Therefore, in today's conditions, urogenital imbalance is an urgent problem in clinical microbiology, given that not all the mechanisms of dysbiotic conditions are fully understood, there are no clear diagnostic criteria, difficulties in selecting effective therapy due to antibiotic resistance.
In the process of studying the state of the microbiota of the urogenital system of 41 women of different ages by real-time PCR, the fact of the presence of dysbiotic disorders in 25 (60.97%) patients was established.
Examination of 47 males in 36 (76.59%) patients revealed dysbiotic disorders in the urogenital tract.
It should be noted that the state of microbial coenosis of the reproductive system of women and men is influenced by a number of different environmental factors -environmental, climatic, sanitary and epidemiological, stress, sexual activity, drugs, invasive manipulations, surgery and more.
Dysbiotic conditions in the examined patients were established by quality and qualitative indicators of the presence of certain representatives of the microbiome of the urogenital system. As a result of real-time PCR analysis in the vast majority of patients revealed a significant decrease in titers of Lactobacillus -the main symbionts of the genital habitat of women from 0 to 10 6 CFU / ml at a rate of 10 7 -10 9 CFU / ml: 68.0% of cases (Fig. 3).
Among the representatives of the obligate anaerobic microbiota, the titers of which should not exceed the permissible values <10 4 -10 5 CFU / ml, significant deviations in the direction of increasing the quantitative indicators to 10 6 -10 9 CFU / ml: It should be noted that the bacteria of the species Ureaplasma spp., Gardnerella vaginalis etc. and   [52]. According to studies by Okodo M. et. al., who noted that with age the level of estrogen and the number of Lactobacillus decreases, which causes a gradual increase in pH to neutral, and this stimulates an increase in the number of yeasts and contamination with opportunistic and pathogenic biota agents [54].
For the male sexual sphere, the state of the normocenosis is provided by the bacteria of the species Lactobacillus spp. -10 4 CFU / ml and more, Staphylococcus spp., Streptococcus spp., Corynebacterium spp. in titers not more than 10 4 CFU / ml. More than half of the examined male patients showed deviations in normobiota in the direction of reducing the number of Lactobacillus spp. from 0 to 10 3 CFU / ml -77.77% of cases (Fig. 3).
Obligatory-anaerobic microorganisms with a high frequency were isolated in significant quantities 10 4 -10 9 CFU / ml at an acceptable rate of not more than 10 3 -10 4  Real pathogens were isolated in titers of 10 1 -10 3 CFU / ml from some patients. Their presence is not allowed at all reference values: Trichomonas vaginalis -19.44%; Chlamydia trachomatis and Ureaplasma parvum -11.11%; Neisseria gonorrhoae and Ureaplasma urealyticum -8.33%; Mycoplasma genitalium and Mycoplasma hominis -5.55% of cases. The detection rates of yeast-like fungi of the genus Candida significantly exceeded the norm (not more than 10 4 CFU / ml) to 10 5 CFU / ml in 2.77% of cases.
According to the results of real-time PCR studies using the test system "Androflor" showed significant changes in the microbiome of the male reproductive system due to excessive growth of opportunistic and pathogenic microorganisms against the background of reducing the number or complete elimination of symbiotic bacteria.
The results of the studies correlate with the data obtained by Okodo M. et al. and Kiessling A. A. et al., which noted significant deviations in the direction of increasing the quantitative indicators of obligate anaerobic (10 4 -10 9 CFU / ml) and facultative anaerobic (10 5 -10 9 CFU / ml) representatives, namely Anaerococcus spp. in 55.0-95.0%, Bacteroides spp. etc. in 40.0-90.0%, Peptostreptococcus spp. etc. in 45.0-85.0%, Eubacterium spp. in 55.0-80.0% patients [54]. Conclusion Prospects for further research. The data was obtained from continuous microbiological monitoring of infectious processes and dysbiotic conditions, as well as the spread of antibiotic-resistant variants of opportunistic and pathogenic microorganisms in the urogenital system in different segments of the population. This determines the need to improve and develop new diagnostic tools, corrective and therapeutic measures to improve the health of the population.