Annotation of the results obtained in 2021
Every year, researchers note an increasing number of mutations in the bacteria genomes that cause infectious diseases, which leads to the formation of more and more aggressive forms of pathogens. Female patients of all ages, regardless of ethnicity, social status, educational level and sexual orientation - all suffer from urinary tract infections (UTIs). Patients with infectious diseases of the urinary system have the highest risk of biofilm formation. The presence of resistant strains of pathogenic bacteria and the development of bacterial biofilms are major problems in the treatment of urinary tract infections. The increasing number of nosocomial bacterial strains in the hospital increases the postoperative period, the frequency of readmission, and the amount of antibacterial drugs used. In light of the increasing antibacterial resistance, the use of medical resources is dramatically increasing, which ultimately leads to an increase in treatment cost. Along with this, the selection of resistant strains brings to the fore both the rational use of antibacterial drugs and the search of alternative therapy methods. The emergence of a large number of bacterial strains resistant to various antibiotics and their rapid spread in the environment has led to an increase in scientific interest in bacteriophage therapy as an alternative method of treatment. Bacteriophages have been used to treat bacterial infections since the beginning of the last century, but their active use is limited mainly to some centers in Russia, Georgia, Poland, Belgium and Switzerland. Bacteriophages can be administered topically or orally mixed or as monotherapy; they can also be combined with antibiotics. With the advent of antibiotic resistance, clinical and scientific interest in phage therapy has gradually returned, and numerous studies have reported the relatively high efficacy of bacteriophages with relatively few (or no) side effects associated with treatment. The high percentage of urinary system infectious diseases prompted us to conduct our own study aimed at evaluating the developed drug based on bacteriophages in therapy of patients with chronic recurrent cystitis. The study included 75 patients with an average age of 50.6 years. The patients were divided into three groups in a 1 : 1 : 1 ratio. In the main group, a drug for intravesical administration based on bacteriophages was used, as well as rectal suppositories with bacteriophages; in the control group - only a drug for intravesical administration based on bacteriophages or rectal suppositories with bacteriophages. Before the administration of the studied drug, as well as after 14, 30 and 90 days of therapy, all patients underwent a blood test, a general urine test, a bacteriological urine test, and a polymerase chain reaction urine test to identify pathogens. Evaluation of the effectiveness included the analysis of the condition of patients in groups according to the dynamics of changes in the main clinical symptoms, data on the scale for assessing pelvic pain and urgency / frequency of urination and a validated scale for assessing symptoms of acute cystitis at the first and regular / follow-up visits, as well as the dynamics of changes in objective parameters of laboratory tests and possible changes in the sensitivity of uropathogens to antibacterial drugs. In all three groups, bacteriological examination of the urine showed a predominant growth of E. coli (≥10 * 4 CFU / ml). In the course of the study, a good tolerance to therapy was noted, as well as a decrease in clinical symptoms in all three groups of patients. The total score on the scale for assessing pelvic pain and urgency / frequency of urination (three groups of patients) at the first visit was 16.6 points, followed by a tendency to decrease to 10 points at the fourth visit. The total score of typical and differential symptoms on the scale of assessment of acute cystitis symptoms (three groups of patients) at the first visit was 7.3 and 1.16 points, respectively, while at the fourth visit, these indicators were 3.55 and 0.71 points, respectively. .The total assessment of the changes dynamics at the control visit according to the scale for assessing the symptoms of acute cystitis (three groups of patients) was 2.17 points at the second visit, at the fourth visit - 1.5 points. The total score of the quality of life on the scale for assessing the symptoms of acute cystitis (three groups of patients) at the first visit was 3.69 points, at the fourth visit - 1.5 points. The overall subjective effectiveness of the therapy (three groups of patients) was more than 80%, safety - 100%. None of the patients had adverse events and / or drug intolerance. A series of clinical and experimental studies have shown the sure efficacy and safety of intravesical and rectal use of the drug based on bacteriophages not only in complex therapy in patients with chronic recurrent cystitis, but also for the prevention of exacerbations, which undoubtedly contributes to its potential introduction into the routine practice of an urologist. The urgency of the problem solving the formation of biofilms in urinary tract infections is increasing, and therefore the obviousness of the use of bacteriophages is undeniable. The practical use of phages for removing biofilms in urinary tract infections is of particular concern. The efficiency of the destruction of biofilms with the help of phages, as studies of many authors have shown, can be significant, especially if phages are directed not at the medium, but directly at the biofilms, which is achieved with the intravesical application of bacteriophages in urinary tract infections. Obviously, at present, not everything is clear in the processes of biofilm formation, the formation and composition of extracellular polymeric substance, the interactions of bacteria with phages, especially since they occur on a microscopic scale. The interaction of phages with biofilms can be much more complex than we might imagine: biofilms can capture nonspecific phages or alter the activity of prophages found inside the corresponding bacterial lysogens. Biofilms usually consist of more than one species of bacteria, thereby complicating the conclusion about the presence of bacteria for phages locally within the biofilm. The key points in the processes of interaction of phages with biofilms also remain unclear: the biology and ecology of the phages themselves, the processes of modeling the growth of phage populations in semi-solid media. It is important to study the interaction of phages with biofilms and their penetration into biofilms, the enzymatic effect of phages on bacterial microcolonies, and the stages of destruction of biofilms by phages. The inability of the phage, in practice, to completely remove the host bacteria from the biofilm, either due to inaccessibility or due to the emergence of resistant strains, indicates that this method has not been well understood. However, the addition of phages to biofilms, where antagonistic interactions due to bacteriocin production already take place, can create an additional load and trigger the process of biofilm destruction. It is assumed that some phages in general can better adapt to growth in biofilms or in mixed cultures. This is also an area that needs to be studied in parallel with the action of phages on biofilms. Nevertheless, our research and similar studies should serve as a rational starting point for further understanding of the fundamental and applied aspects of the interaction of phage with biofilm, as well as for the development and implementation into clinical practice of more effective methods for destroying biofilms in urinary tract infections by means of phages. The obtained encouraging results contribute to the faster introduction of phage therapy into clinical practice. Verification of the UTI causative agent makes it possible to produce preparations containing phages that target bacteria, which makes these developments the most relevant in the era of antibiotic resistance. Phage therapy has the potential to combat antibiotic-resistant bacterial infections. Knowledge of the beneficial microbiota is critical to effective and reliable phage therapy strategies. Ideally, phage therapy should not target beneficial microorganisms. Unlike broad-spectrum antibiotics, phages can work in a narrowly targeted manner on a specific pathogen.

 

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Annotation of the results obtained in 2019
We conducted a study of microbial landscapes of clinical material obtained from urological patients. Urine, results before cystoscopy and 10 days after the manipulation were delivered to the laboratory within 2 hours. Universal culture media were used to isolate bacteria from urine in all groups of patients. The titer of bacteria in the urine is determined by the semi-quantitative dash method using a dipstrick-type test system. The obtained inoculated plates and test systems of the DeepStrik type were subsequently incubated in a normal atmosphere at 37 ° C for 22 hours. After morphological studies, the results of morphological studies were obtained. Diagnostically significant titer of pathogenic bacteria in 1 μl. the urine tested counted ≥10 / 3 CFU / ml (for primary pathogens of group I - E. coli and S. saprophyticus), 10/3 and 10/4 CFU / ml (for secondary pathogens of group II - P. mirabilis), group KES (bacteria of the genera Klebsiella spp., Enterobacter spp. And Serratia spp.), P. aeruginosa, P. vulgaris, S. Pneumoniae, etc.) When isolating them from men and women in a monoculture, respectively. After the appearance of pathogenic bacteria that determine the sensitivity of microorganisms to antibacterial drugs, thanks to the isolated Muller – Hinton cultural agents (agar thickness - 4 ± 0.5 mm). Microsoft Exceel 2010 Compute Program, which was used to make objective comparisons of results and a comprehensive assessment of the reliability of data differences. Bacteriological analysis of urine obtained before instrumental studies showed the absence of clinically significant titers of pathogenic bacteria in patient groups. 10 days after instrumental studies in 12 patients from the control group, the presence of bacteria from the groups of primary and secondary pathogens in titers 10/4 and 10/3 CFU / ml for E. coli and Enterobacter spp was shown in a microbiological urine study. respectively (for men) and the presence of bacteria from the group of primary pathogens in titers of 10/3 and 10/4 CFU / ml for E. coli and S. saprophyticus, respectively (for women). All patients (control group, 12/25) had subfebrile temperature, as well as moderate dysuria. Microbiological analysis allowed us to identify 12 strains of microorganisms, of which 8 strains of E. coli, 3 strains of Enterobacter spp. and 1 strain of S. saprophyticus. Of the total number of nosocomial E. coli strains, more than half (59%) were allocated to the association. In 9 patients from the control group, the development of symptomatic urinary infection requires systemic antibiotic therapy (3 patients received 3 doses of fosfomycin, 500 mg of levofloxacin in a single dose. Once in a period of up to 5 days). Sowing clinical material for solid nutrient media - URISELECT AGAR (BIO-RAD) - for the isolation and counting of microorganisms from the urinary tract; cardiac cerebral agar with 5% mutton blood (BHI) - to isolate bacteria Streptococcus, Corynebacterium, Arcanobacterium, Neisseria, Moraxella, Acinetobacter, Actinomyces; vitelline - salt agar with mannitol (for isolation and differentiation of staphylococci); differential diagnostic medium Endo or Levin (for the isolation of enterobacteria), Saburo medium with chloramphenicol (for the isolation of yeast and mycelial fungi); on BHI with 5% sheep blood and Chadler’s agar (for the isolation of Clostridium, Propionibacterium, Bacteroides, Fusobacterium, Prevotella, Porphyromonas affected by anaerobic bacteria). Inoculated solid nutrient media were incubated in an incubator at 37 ° C for 24-48 hours. To create anaerobic conditions, the Whitley A35 anaerobic apparatus is used, where the seeded nutrient medium is incubated at 37 ° C for 48 hours to 10 days. The viewing crops marked by morphology grew colonies, the presence of hemolysis, microorganisms in the form of monoculture or in associations. When associations were found in a dense nutrient medium, Industrial screenings of individual colonies on dense nutrient media were noted in order to obtain pure cultures. The obtained pure cultures of microorganisms, microscopy, their morphological and tinctorial features, as well as derivatives of ethanol / formic acid extraction according to the standard method, allow the use of mass spectrometry on a time-of-flight mass spectrometer (MALDI Biotyper, Bruker). As a result of the work, the microbial landscape of 25 clinical materials from urological patients was analyzed. The following pathogenic strains relevant in urology were isolated and identified: 1. Acinetobacter baumannii spp. 2. Enterococcus faecalis 3. Enterobacter cloacae spp. 4. Proteus vulgaris spp. 5. Proteus mirabilis spp. 6. Stenotrophomonas maltophilia 7. Klebsiella pneumonia 8. Klebsiella ozanae 9. Propionibacterium acne 10. Escherichia coli 11. Pseudomonas aeruginosa spp. 12. Staphylococcus aureus spp. 13. Staphylococcus epidermidis spp. 14. Staphylococcus warneri 15. Staphylococcus haemolyticus 16. Staphylococcus capitis 17. Staphylococcus caprae 18. Staphylococcus suсcinus 19. Streptococcus D (new modification Enterococcus faecium) 20. Streptococcus agalactiae The listed pathogens were used in further screening of promising phages. Initial screening of bacterial viruses made it possible to isolate 1-6 phages for each of the 20 types of pathogens. All bacteriophages formed transparent lysis spots on bacterial lawns upon titration using the agar layer method. The advantages of the gel form were also highlighted. The gel, which is part of the developed product, partially consists of water-soluble blanose and / or lidocaine hydrochloride and / or chlorhexidine hydrochloride. A series of experimental works revealed a number of unique properties of the developed gel substance: the gel does not liquefy when heated and does not leak after application, and the absence of alcohol in it will not cause dryness and irritation of the skin and mucous membranes of the urinary system

 

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Annotation of the results obtained in 2020
During the project implementation, the development and introduction of a new antibacterial and anesthetic drug in a gel form based on bacteriophages into clinical practice for the prevention of infection of the urinary system during medical and diagnostic procedures was done. Due to several types of viruses with lytic activity in relation to a specific pathogenic microorganism presence in the composition of the developed drug and it significantly differing one from each other in the specifics of interaction with a sensitive cell, the probability of phage-resistant strains of UTI pathogens formation is significantly reduced. The main tasks for 2020 were to assess the clinical efficacy and safety of the complex antibacterial anesthetic drug based on bacteriophages in a gel form usage in patients (of both sex) during various instrumental studies and therapeutic and diagnostic procedures, as well as the prevention of catheter-associated urinary tract infections. At the beginning of 2020, a previously started prospective study was continued, which included 275 patients (155 men and 120 women) who were treated at the urological clinic of MSMSU. The average age of male patients was 69.4 ± 1.3 years, and of female patients - 65.7 ± 1.9 years. A developed drug based on bacteriophages in a gel form was used in all patients during various instrumental studies, diagnostic and treatment procedures and surgical treatment, as well as for the prevention of catheter-associated urinary tract infections. In the control group (n = 275, 160 men and 115 women, respectively), a gel for local application based on chlorhexidine dihydrochloride and lidocaine hydrochloride was used. In all patients, the gel was injected intraurethrally after applying a disinfectant solution. 10 ml of the test drug was used for instillation in each case. The exposure time in all cases did not exceed 5 minutes. Within the framework of the study, we studied the effectiveness of the use of the developed antibacterial and anesthetic drug based on bacteriophages with the determination of primary points (presence of bacteriuria, analysis of the qualitative composition of causative urinary tract infections agents, the presence of urinary tract infection symptoms - subjectively according to complaints) and secondary points (assessment of the presence of fever 1 day after the manipulation (according to thermometry data) and the presence of pain before and after the manipulation using the visual analogue pain scale (VAS) The data of bacterial analysis of urine obtained before and after instrumental studies (diagnostic and / or treatment urethrocystoscopy , diagnostic ureterocystoscopy, transurethral operations), medical and diagnostic procedures (intraoperative installation of a urethral catheter). Analysis of bacteriological analysis of urine data from patients in the study and control groups did not reveal significant changes in comparison with the initial data. In all cases, there were no clinically significant titers of pathogenic bacteria in urine culture, while after the studies and manipulations, the number of pathogenic bacteria significantly prevailed in the control group (p > 0.04). The severity of pain syndrome (after 1 and 3 days according to telephone questionnaires) and the temperature index in the main group of patients undergoing outpatient treatment (patients who underwent cystoscopy) did not exceed the initial values. In the control group, an increase in body temperature was noted on average by 0.7 ° C (on the first day) and by 0.4 ° C (by the end of the third day); an increase in the total VAS score by an average of 1.1 ± 0.2 (in men) and 0.7 ± 0.3 (in women) with a gradual decrease by the end of 3 days. In 75% of cases in the control group, the use of painkillers was required on the first day after the procedure. The frequency of lower urinary tract symptoms events (as one of the manifestations of symptomatic catheter-associated infection) after removal of the urethral catheter was lower in the study group (p < 0.05). As a result of our own clinical and experimental studies, it was found that the gel composition of the developed drug provides reliable fixation of the drug on the mucous membrane of the urethra and provides an excellent optical view when performing transurethral procedures and manipulations. There were no dysuric phenomena in the main group. None of the patients had an allergic reaction and intolerance to the developed drug. An analysis of the scientific works carried out has shown that the rate of synthesis of new antibacterial drugs is inferior to the rate of development of antibiotic resistance, in this regard, the treatment and prevention of acute, chronic and recurrent forms of urinary tract infections is often ineffective. At the same time, the routine use of antibiotics can lead to an even greater increase in the number of antibiotic-resistant strains and the occurrence of adverse reactions. Our own data obtained in 2020 showed the efficacy and safety of a combined drug based on bacteriophages developed by joint efforts. Moreover, the fundamental difference between the developed complex antibacterial and analgesic drug in gel form based on bacteriophages from the existing ones is that it includes more than 60 types of bacteriophages (Appelman titers 10–8 - 10–9; that is, phages with proven high activity against opportunistic bacteria), which lyse the pathogens of the most common pathogenic bacteria, which, to a large extent, reduces the likelihood of changing the causative agent of urinary tract infections if present.

 

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