Bacteriophage Cocktail Specific to Pseudomonas Aeruginosa

Abstract

The invention relates to a bacteriophage cocktail formed by combining bacteriophages exhibiting lytic activity against P. aeruginosa isolates, which can be used as a therapeutic agent in the treatment of infections and as a biocontrol agent in hospital settings.

Description

1 TARIFF Bacteriophage Cocktail Specific to Pseudomonas Aeruginosa Technical Field to Which the Invention Relates The invention is intended for use against infections caused by Pseudomonas aeruginosa. This relates to the development of bacteriophage-based antimicrobial agents. More specifically... The invention describes a group of bacteriophages exhibiting lytic activity against P. aeruginosa isolates. formed by combining them, therapeutic agents in the treatment of infections and a bacteriophage cocktail that can be used as a biocontrol agent in hospital settings It is related to. 10 State of the Art Antibiotics are widely used in the treatment of bacterial infections. Together, antibiotic resistance is developing, particularly in hospital-acquired infections. This poses a significant problem in its control. Antimicrobial resistance rates are 15%. the increase in the effectiveness of current antibiotics in some bacterial infections the inability to demonstrate and the need for alternative antimicrobial approaches This is causing an increase, especially due to multidrug-resistant bacteria. It becomes more pronounced in infections and in the treatment of infections Evaluation of biological agents with different mechanisms of action is necessary. 20 Pseudomonas aeruginosa is frequently associated with hospital-acquired infections. associated with, adaptable to different environmental conditions and clinically resistant to multiple drugs It is one of the Gram-negative bacteria from which isolates can be found. Antibiotic treatment alternatives for P. aeruginosa infections where the previous ones were ineffective or insufficient The use of antimicrobial agents is therefore important. 25 Bacteriophages are viruses that infect bacteria and are widespread in the biosphere. This is the case. The recognition of bacterial host cells by bacteriophages is the subject of this. their ability to attach to cells and multiply within cells, giving them resistance against bacteria. This makes it possible to use it as an antimicrobial agent. Lytic properties 30 Bacteriophages, which are shown to infect host bacterial cells, It multiplies inside and causes the lysis of the bacterial cell. These characteristics Therefore, bacteriophages are used in the treatment of bacterial infections. Studies on this topic have been known for a long time, especially regarding antibiotic resistance. 2 With the rise of bacteria, the therapeutic use of bacteriophages has regained importance. It has been acquired and is caused by various Gram-negative bacteria, including P. aeruginosa. Research on phage therapy applications against infections. It has intensified. In terms of using bacteriophages as therapeutic agents, the phage should only be the target. It is not enough for it to be able to infect bacteria. Selected for therapeutic use. Bacteriophages have a lytic life cycle and a lysogenic life cycle. It should not exhibit associated undesirable characteristics. In addition, phage 10 genes in their genomes that are associated with pathogenicity, virulence, or antimicrobial resistance Its absence is important. Another important aspect from therapeutic use is... One characteristic is the host range of the phage. Bacteriophages target specific bacterial species or... Different phages infect the same bacteria because they infect specific strains belonging to particular species. It can exhibit varying levels of lytic activity against different isolates of its species. Therefore, phages with a wide host range or those with different host ranges 15 Using complementary phage combinations enhances therapeutic efficacy. It is important in this respect. There are also various commercial applications where bacteriophage preparations are used. It is known that in some countries, such as Georgia and Poland, commercial bacteriophage preparations are available. Although they exist, the widespread use of such preparations is limited. Different bacteria also products that combine phage mixtures that show activity against certain species Among them is the preparation known as Pyobacteriophage. Staphylococcus aureus, Streptococcus species, Escherichia coli, Pseudomonas A 25-member group of phages formed that are effective against different bacteria such as aeruginosa and Proteus species. It is known to contain a mixture. Such preparations are effective against different types of bacteria. by bringing together active bacteriophages within the same product The aim is to create a wide range of applications. However, there are many In mixtures prepared against numerous different types of bacteria, a specific type of bacteria is targeted It is possible that the diversity of phages targeting this species may remain limited. 30 The high host specificity of bacteriophages makes phage therapy suitable for both... This constitutes both one of its advantages and limitations. A bacteriophage selectively infecting a particular type or strain of bacteria 3 the ability to limit the effect on non-target microorganisms This can provide an advantage in terms of host specificity. Conversely, the same host specificity is unique to a single host. The bacteriophage preparation has the same efficacy against all clinical isolates of the target bacteria. This can lead to an inability to demonstrate [the virus]. Especially clinical isolates such as P. aeruginosa. A bacteriophage can have 5 different types of isolates on a particular bacterial isolate, which can vary among bacteria. The fact that it shows lytic activity suggests that it will show the same level of efficacy in other clinical isolates. This does not mean that the therapeutic efficacy of bacteriophages is not the same. In the evaluation, it is sufficient to identify the host only at the species level. not only that, but also evaluating their effectiveness on different clinical isolates. It is necessary. During the therapeutic use of a single bacteriophage, 10 Another problem encountered is the resistance of the target bacteria to the bacteriophage in question. It is the ability to develop. Bacterial populations are under selective pressure created by phages, prevents the phage from binding to the bacterial cell or causing infection. It can develop features, especially to target a specific type of bacteria. If only a single phage is used, the selection of phage-resistant bacteria is 15 This can reduce the effectiveness of the treatment. Therefore, using more than one bacteriophage together is recommended. its use is an approach to reduce the likelihood of phage resistance developing. It is known that different bacteria have different host recognition characteristics or are of the same bacterium. Phage cocktails are formed by combining phages that target surface structures. The resistance that a bacterium can develop against a single bacteriophage renders the entire treatment ineffective. The aim is to prevent this from happening. The effectiveness of phage cocktails depends on the number of bacteriophages contained within the cocktail. Next, let's look at the biological characteristics of these phages and their resistance to target bacterial isolates. It is related to activity. Combining different bacteriophages can theoretically increase activity by 25%. While it is possible to obtain a wide range of accommodation options, in the cocktail The phages involved showed sufficient lytic activity against the targeted clinical isolates. It is necessary. In multidrug-resistant clinical isolates, phage activity is not always the desired level. the possibility of not being at this level, in terms of the selection and combination of bacteriophages This constitutes a significant limitation. Therefore, phage 30 for therapeutic purposes. Bacteriophages to be used in preparations are tested on different clinical isolates. It is necessary to determine the activity spectra of phage diversity. increasing susceptibility that may arise within the targeted bacterial population 4 important in terms of meeting differences and reducing phage resistance It carries. Various conventional methods are used for isolating bacteriophages from environmental sources. Microbiological methods are known. Water, wastewater and similar environmental samples, 5 It is among the important sources in terms of bacteriophage diversity. Phage For isolation purposes, environmental samples are brought into contact with the target bacteria, and appropriate It is propagated under temperature and incubation conditions, and then centrifuged and Filtration processes are applied. Bacteriophages are found in the resulting suspensions. Methods such as spot tests are used to determine its presence. Spot 10 In the test method, a suspension potentially containing bacteriophages is used to target the bacteria. It is applied to the culture medium in which it is planted and, after incubation, in the application area. Inhibition of bacterial growth is an indicator of phage presence. is being evaluated. Purification of bacteriophages in samples where phage presence was determined and Double-layer agar, also known as double-layer agar, is used for enrichment purposes. This method is widely used. In this method, appropriately diluted phages are used. suspension, target bacteria suspension and soft agar are combined It is applied to solid culture medium. The phage plaques formed as a result of incubation are 20 They are being evaluated and purification processes are being carried out until a pure phage population is obtained. This process is repeatable. The phage suspensions obtained after purification are repeated. They are amplified, subjected to appropriate filtering processes, and phage titers are obtained. It is determined. Phage titer is usually measured in plaque-forming units / milliliter, in other words, It is expressed in PFU / ml. 25 Before bacteriophages are evaluated for therapeutic or biocontrol purposes It is necessary to characterize their biophysical properties. In this context, different Phage stability can be examined at different temperature and pH values. suspensions at different temperatures and pH conditions for specific periods of time 30 by measuring phage titers after incubation under these conditions Stability is determined. These analyses are related to the preparation and storage of bacteriophages. against environmental conditions to which they may be exposed during transportation and application This is important in terms of evaluating their durability. Biophysical stability. Insufficient phage titer leads to a decrease in the antimicrobial efficacy of the preparation. This can lead to a decrease. Transmission electrons are used to determine the morphological characteristics of bacteriophages. Imaging methods such as microscopy are used. Electron 5 Microscopy allows us to examine the size and morphological characteristics of phage particles. And classification can be made between different morphotypes. In addition To determine the host range of phages, different clinical samples of the target bacterial species were examined. Isolates can be used with reference strains of different bacterial species. Spot test Host range analyses performed using methods such as these reveal which bacteria the phage inhabits. This allows for the determination that it exhibits lytic activity on the host range. Knowing this information is important in selecting phages to be used for therapeutic purposes and in the use of multiple phages. It is an important criterion in creating cocktails in which they will be used together. To determine the multiplicity of infection characteristics of bacteriophages. (MOI), parameters such as latent period and burst size are used. 15 MOI represents the ratio of the number of bacteriophage particles to the number of host bacteria. and the appropriate phage-bacteria ratio that allows the phage to replicate effectively It is used in the determination of phages and bacteria at different MOI values. phages obtained after combining and incubating their suspensions The appropriate MOI value can be determined by comparing the titers of the phages. 20 In determining reproduction kinetics, a single-step growth curve is used. These analyses are used to determine the phage's binding to the host bacterium. latent period from adsorption to the release of new phage particles and expresses the approximate amount of phage particles released from an infected bacterial cell. The magnitude of the resulting explosion can be determined. 25 Determining the genomic characteristics of bacteriophages is also important for therapeutic use. This is a significant step. Advances in DNA sequencing technologies, particularly with bacteriophages... detailed analysis of their genomes and the genes carried by phages This has enabled their identification. 30 phages were evaluated for therapeutic purposes. lysogenic life cycle, associated with pathogenicity, virulence, or antimicrobial resistance. It is necessary to examine whether the genes are present or not. For this purpose, the genome... various bioinformatics analysis tools for annotation and comparison of sequences Genome annotation tools such as RASTTK and BLAST-based sequencing are used. 6 comparisons were made using tRNAscan-SE, BACPHLIP, ResFinder, VFDB, PathogenFinder, and Analytical methods like VIRIDIC characterize bacteriophage genomes from different perspectives. It is among the known approaches used in its implementation. In vitro 5 in evaluating the effectiveness of bacteriophages in infection treatment In addition to research, in vivo infection models are also utilized. There are various studies regarding the use of bacteriophages, particularly in wound and burn infections. Animal model studies exist. In known studies, burn wounds... single bacteriophages and phage cocktails against generated bacterial infections Therapeutic efficacy is compared and the effect of treatment on bacterial load and inflammatory response is evaluated. and its effects on parameters such as wound healing are being investigated. BALB / c Applications of single phage and phage cocktail in burn wound infections in mice studies on its evaluation, as well as with different bacterial pathogens. Studies on topical bacteriophage applications in infected wound models also It is located. 15 The application of bacteriophages is in the direct treatment of bacterial infections. not limited to, but also biocontrol applications aimed at reducing bacterial load. It includes organisms that can colonize in a hospital environment and adapt to environmental conditions. Selective 20 bacteriophages in the control of pathogens such as P. aeruginosa that can exhibit It is possible to take advantage of their antimicrobial properties. However, The narrow host range of bacteriophages also makes them suitable for biocontrol applications, targeting specific bacteria. This can lead to variability in effectiveness against different isolates. Therefore, bacteriophages are used in both therapeutic and biocontrol applications. To determine its effectiveness against target bacterial isolates and different host profiles. 25 Proper combination of the phages is important. In conclusion... In the current state of the technique, bacteriophages are used in the treatment of bacterial infections. Use as an antimicrobial agent, isolation of bacteriophages from environmental samples the production, purification and multiplication of phages, and the determination of host ranges of phages, Characterization of biophysical and morphological features, analysis of genome sequences 30 and combining different bacteriophages in the form of phage cocktails It is known that bacteriophages have high host specificity, which is relevant to different clinical settings. Their variable efficacy against isolates means that some phage preparations have limited host range. having a range and bacteria having resistance to a single bacteriophage 7 Its ability to develop is a key issue encountered in phage-based antimicrobial applications. This is among the limitations, especially for multidrug-resistant P. aeruginosa. In infections caused by these isolates, adequate lytic activity is observed against different clinical isolates. phages that can exhibit activity and reduce the likelihood of developing resistance to the phage. The technical need for determining combinations continues. 5 Brief Description and Objectives of the Invention The invention describes bacteriophages that exhibit a lytic effect on P. aeruginosa isolates. a combination of infections caused by the bacteria in question as a therapeutic agent in treatment and bacterial control in hospital settings 10 a bacteriophage cocktail for use as a biocontrol agent in its production It is explained. The aim of the invention is to develop an effective solution against infections caused by Pseudomonas aeruginosa. The goal is to provide therapeutic agents. The basic techniques that enable the achievement of this goal are 15. element; Bruynoghevirus / podo- showing lytic activity against P. aeruginosa isolates. like Pseudomonas aeruginosa bacteriophage Baskent_P1_112 (DSM 35944), Pbunavirus / myo-like Pseudomonas aeruginosa bacteriophage Baskent_P2_ICU (DSM 35945) and Pakpunavirus / myo-like Pseudomonas aeruginosa bacteriophage 20 formed by assembling Baskent_P3_3B (DSM 35946) bacteriophages It is a bacteriophage cocktail. One aim of the invention is to combat multidrug-resistant Pseudomonas aeruginosa isolates. The aim is to provide an alternative antimicrobial agent. This purpose is to target P. aeruginosa isolates. 25 DSM deposit numbers 35944, DSM 35945 and DSM 35946 showing counterlytic activity. by bringing bacteriophages together in the same cocktail This is being carried out. The combined use of these bacteriophages involves different P. The technical framework that enables the targeting of aeruginosa isolates It constitutes. One aim of the invention is to address phage resistance that may arise from the use of a single bacteriophage. The aim is to reduce the risk of development. The technical element that enables the achievement of this goal is... instead of a single bacteriophage, Baskent_P1_112 (DSM 35944), Baskent_P2_ICU (DSM three different lytic bacteriophages, namely 35945) and Baskent_P3_3B (DSM 35946). 8 It is used in combination. Thus, the target bacterium is targeted against a single phage. reducing the loss of effectiveness that may result from the development of resistance that is intended. Another aim of the invention is to detect 5 different isolates of Pseudomonas aeruginosa. The aim is to provide a bacteriophage preparation that can demonstrate efficacy. For this purpose, different phages Deposit numbers DSM 35944, DSM 35945 and DSM 35946 have the following characteristics. This is achieved by bringing together bacteriophages. Different bacteriophages The combination it forms may originate from the host range of a single phage. It helps to reduce the limitations. 10 Another aim of the invention is to create a system of bacteriophages suitable for therapeutic use. The goal is to provide the preparation. The technical element that enables the achievement of this goal is; Baskent_P1_112 (DSM 35944), Baskent_P2_ICU (DSM 35945) and Baskent_P3_3B (DSM 35946) bacteriophages exhibiting lytic characteristics and therapeutic use 15 It has suitable characteristics in terms of its compatibility with Pseudomonas aeruginosa. a bacteriophage preparation that supports the wound healing process in infected wounds The aim is to provide the best results in the group treated with the phage cocktail in an in vivo infected burn model. observation of a high wound closure rate and histopathological findings of epithelial abnormalities Increased regeneration and decreased inflammation make this technique effective. This supports the significant load of Pseudomonas aeruginosa in infected tissue. that enables its reduction and, in some cases, its reduction to an undetectable level. The goal is to administer a bacteriophage cocktail. Another aim of the invention is to use 25 for biocontrol against Pseudomonas aeruginosa. The aim is to provide a usable bacteriophage cocktail. This purpose is to use P. aeruginosa. DSM 35944, DSM 35945 and DSM 35946 deposits showing lytic activity This is achieved by using a cocktail of numbered bacteriophages. Thus, the presence of this bacterium, especially in a hospital setting, and A biocontrol approach is provided to reduce colonization. 30 The invention also provides a pharmaceutical component that can be adapted to different application requirements. The aim is to provide a bacteriophage composition. The bacteriophage cocktail is a liquid preparation. The phages are lyophilized and then recycled, provided that their biological activity is preserved. 9 Different pharmaceutical dosages containing dilutable or suitable excipients They are capable of being prepared in various formats. Explanation of the Figures Figure 1. Baskent_P3_3B genome map. 5 Figure 2. Baskent_P1_112 genome map. Figure 3. Baskent_P2_ICU genome map. Figure 4. CRP statistics graph. Explanation of References in Figures 10 100. Genes encoding hypothetical proteins. 101. Genes encoding proteins associated with phage assembly. 102. Genes that encode structural proteins. 103. Genes encoding proteins associated with lysis. 104. Proteins associated with DNA replication, metabolism, and regulation. 15 coding genes Detailed Description of the Invention The invention describes bacteriophages that exert a lytic effect on P. aeruginosa isolates. a combination of 20 infections caused by the bacteria in question as a therapeutic agent in treatment and bacterial control in hospital settings with a bacteriophage cocktail for use as a biocontrol agent in its provision It is related to the invention. The Pseudomonas aeruginosa bacteriophage cocktail that is the subject of the invention is P. aeruginosa. Three newly identified phages that showed lytic activity against isolates and It contains a pH 7.0 0.9% (w / v) NaCl solution as a carrier / diluent. These phages are 25 Baskent_P1_112 (DSM 35944), Baskent_P3_3B (DSM 35946) and respectively It is named Baskent_P2_ICU (DSM 35945). The phage cocktail described in the invention reduces the bacterial load and inflammation at the infection site. It is effective in reducing the level of inflammation and supports wound healing. (Source 30) The subject is phage cocktail, specifically targeting multidrug-resistant Pseudomonas aeruginosa. an alternative or complement to antibiotic treatment in the control of infections It has the potential to create an antimicrobial approach. In this context, phage cocktails, resistant P. aeruginosa for which current antibiotic treatments are insufficient In addition to being considered as a therapeutic agent in infections, as a biocontrol agent in the prevention and control of hospital-acquired infections It also has the potential to be used, especially on surgical wounds and burn tissues. bacterial colonization in areas favorable to P. aeruginosa colonization, such as Technical benefit in terms of reducing and supporting the eradication of the pathogen 5 This ensures that phages exhibit host specificity towards the target bacteria and Its ability to multiply in an environment containing susceptible bacterial cells, off-target limiting the effect on microorganisms and antimicrobial efficacy It offers an additional advantage in terms of being able to be maintained in the infection area. In addition, toxicity assessments revealed that the phage cocktail was effective against human tissue and 10 It does not show toxic effects on cells and in animal experiments... The absence of any adverse findings regarding toxicity indicates that the phage cocktail in question... This supports its usability in biomedical and therapeutic applications. When the technical effects are evaluated together, the developed phage cocktail is resistant to P. Phage therapy and biocontrol for the treatment and control of aeruginosa infections 15 It appears to offer a framework that can be used in the development of products. The genome sequence of the Baskent_P3_3B phage, which is the component of the cocktail that is the subject of the invention, was obtained from GenBank data. Access number at base PP766723, Baskent_P1_112 fam's PP766722, The phage in Baskent_P2_ICU is PP910332. Pseudomonas aeruginosa 20 The bacteriophage cocktail contained purified phages, each at a titer of ~108 PFU / ml. It contains phage lysates. The stated titer of approximately 10⁸ PFU / mL is obtained before cocktail preparation. first, express the titer of phage lysates prepared separately for each bacteriophage. Baskent_P1_112, Baskent_P3_3B and Baskent_P2_ICU phages each After one is adjusted to a titer of approximately 10⁸ PFU / mL, 25 are mixed together in equal volumes (1:1:1). By bringing them together, a faj cocktail is prepared. Theoretically, the cocktail prepared in this way... The total phage titer is also approximately 10⁸ PFU / mL. Equal volume mixture. Therefore, the individual concentration of each phage in the final cocktail is approximately The concentration is 3.3 × 10⁷ PFU / mL, and the total concentration of the three phages is approximately 1 × 10⁸ PFU / mL. Baskent_P1_112, Baskent_P3_3B and 30 are used within the scope of the invention. Each of the Baskent_P2_ICU bacteriophages was approximately 100% saturated before the cocktail was prepared. It is standardized to an infectious titer of 1×10⁸ PFU / mL. The standardized phage... 11 When suspension components are combined in a 1:1:1 volumetric ratio, the increase in volume is due to... Considering the dilution, the concentration of each phage in the final cocktail The total infectious phage titer formed by the three phages together is approximately 3.3 × 10⁷ PFU / mL. The concentration is approximately 1×10⁸ PFU / mL. Therapeutic bacteriophage preparations. In this regard, the literature is valid for all phages, infection types, and routes of administration, 5 There is no universally accepted fixed lower and upper PFU / mL limit. Therapeutic dose and titer depend on the density of the target bacteria, the adsorption characteristics of the phage, and... in vivo replication capacity, localization of infection, route of administration, pharmacokinetic / pharmacodynamic properties and available active phage in target tissue. This is determined based on factors such as concentration. Current EMA 10 The guidelines also state that the potency of the phage preparation is determined as an infectious titer, and It is expected to be expressed in PFU / mL, whereas all therapeutic No single mandatory titer range is defined for applications (15). However, the current scientific literature generally indicates high rates in therapeutic applications. This indicates that titered phage preparations are preferred. 15 for clinical application. In the evaluations, a level of approximately 10⁸ PFU / mL indicates rapid targeting of bacteria. It has been suggested as a reasonable phage density that could enable suppression; different clinical In applications, doses ranging from approximately 10⁶–10¹⁰ PFU / dose are used. It has been reported (16). In current evaluations of topical applications, it is frequently reported (16). While preparations in the range of 10⁷–10⁹ PFU / mL have been reported, some clinical applications are 20. In the protocols, a level of at least 10⁶–10⁷ PFU / mL is considered for therapeutic use. It has been considered as a starting point and 10⁸ PFU / mL in certain indications. It was used as the target titer (17). In this context, approximately within the scope of the invention The final phage cocktail with a total infectious titer of 1×10⁸ PFU / mL has been therapeutically studied in the literature. This is consistent with titer levels reported in phage applications. However, 1×10⁸ 25 The PFU / mL value is not considered an absolute therapeutic lower or upper limit, but rather falls within the scope of the invention. The standardized target final titer is defined. Use as needed. Depending on the form, target bacterial load and route of administration, the titer and / or of the preparation The total amount of PFU administered within the appropriate pharmaceutical formulation. Adjustable. 30 The multiplicity of infection (MOI) values ​​for the PAO1 strain are 100 for Baskent_P1_112. PFU / CFU, 10 PFU / CFU for Baskent_P3_3B and 1 PFU / CFU for Baskent_P2_ICU. It is of value. The bacteriophage cocktail created within the scope of the invention is Baskent_P1_112. 12 Baskent_P3_3B and Baskent_P2_ICU phages are combined in equal ratios (1:1:1). It is prepared by bringing them together. When preparing the cocktail, each phage suspension is prepared in equal proportions. a certain amount is added to the mixture, thus creating a cocktail of three bacteriophages. The volumetric contribution of each ingredient is kept equal. Accordingly, the total cocktail volume... approximately one-third Baskent_P1_112, one-third Baskent_P3_3B and one-third 5 Baskent_P2_ICU consists of phage suspension. It includes the latent periods of the phages and... The burst sizes were determined using "One-step growth curve" analysis and latent The periods were 15 minutes for Baskent_P1_112, 20 minutes for Baskent_P3_3B, and respectively. Baskent_P2_ICU's duration is 30 minutes. The explosion dimensions are similar to Baskent_P1_112's. 115 PFU / CFU, 288 PFU / CFU of Baskent_P3_3B phage and 10 PFU / CFU of Baskent_P2_ICU phage. The pH value is 123 PFU / CFU. The phages in the product can withstand temperatures up to 50°C and pH. They can maintain their stability in the pH range of 2-12. Therefore, this data indicates that phages... This shows that their cocktails are resistant to storage and transfer conditions. According to TEM analysis, the Baskent_P3_3B and Baskent_P1_112 phages belong to Myoviridae. Baskent_P2_ICU is a phage of the Podoviridae morphotype. 15 The BACPHLIP tool demonstrated that all three phages are lytic phages. In the phage genomes... any virulence, pathogenicity and antimicrobial resistance genes and lysogenic phages No genes are present. Thus, phages can be used as therapeutic phages. The phage cocktail is toxic after a series of filtration and ultracentrifugation processes. It has been purified from its components. In the invention, the possible toxic effects of the phage cocktail in vivo 20 The animals in this group were evaluated in a separate control group (G3). The phage preparation was applied to the animals without inducing bacterial infection. Survival and potential adverse effects were monitored throughout the study period. G3 In the group, 100% survival was observed, with mortality associated with phage application. no detection was made. Therefore, the study does not investigate classical cell culture-based cytotoxicity. 25 Although it does not include tests, an in vivo study in which the phage cocktail was administered alone The toxicity control group includes 100% survival in this group, and the dose used and No significant toxic effect associated with the phage preparation was observed under experimental conditions. This supports the use of phages to reduce potential endotoxin load. During the purification phase, the phage content is made safe as described in the literature. 30 The bacterial lysate was purified by ultracentrifugation in such a way as to bring it to a state where it is removed. Concentrated phage preparations were obtained (22). 13 Within the scope of the invention, Baskent_P1_112 (Φ1), Baskent_P2_ICU (Φ2) and The fundamental technical rationale for using Baskent_P3_3B (Φ3) phages together is that they are single Instead of being limited to the lytic spectrum of a single phage, they are complementary and partly By combining overlapping host ranges in a single preparation, a wider and more... The goal is to achieve a more reliable antibacterial coverage. The cocktail components are 5. The selection was not random, but from a wide range of clinical Pseudomonas aeruginosa isolates. Based on the range and EOP analyses performed on the panel The study was conducted on the host ranges of three phages in 111 carbapenem-resistant clinical P. Evaluated on the isolate aeruginosa. Baskent_P1_112 (Φ1) 42 / 111 isolate. (37.8%), Baskent_P2_ICU (Φ2) 42 / 111 isolate (37.8%) and Baskent_P3_3B (Φ3) 10 71 out of 111 isolates (63.9%) were lysed. In contrast, when three phages were used together... In the evaluation conducted with the cocktail, the cumulative accommodation coverage was 89.2%. Of the samples taken, only 12 / 111 isolates (10.8%) were found to be resistant to all three phages. Therefore, experimental data indicate that one of the key technical advantages offered by the cocktail is, a wider range of clinical isolates beyond the host reach of individual phages 15 This directly demonstrates that the group can be targeted. This result is particularly noteworthy. This is evident when compared to Φ3. It alone has the widest host range. The Φ3 component showed activity against 63.9% of clinical isolates, while the three phages... By combining complementary activity profiles, cumulative coverage reached 89.2%. In other words, the cocktail approach requires even the broadest spectrum of a single phage to reach 20%. This has enabled the targeting of additional clinical isolates that it could not cover. Therefore, three Using phages together not only increases the number of phages, but also affects different susceptibilities. the inclusion of clinical isolates with different profiles within the same formulation It is a functional combination that provides... In addition, EOP analysis, It is not only whether phages cause lysis in the spot test, but also their sensitivity in clinical tests. 25 Evaluation of the effectiveness of isolates in inducing productive infection. This has provided opportunities. In the study, EOP values ​​<0.001 were inefficient, and 0.001–<0.1 were low. The three phages were classified as having different activity levels: 0.1–<0.5 (medium) and ≥0.5 (high); Cocktail based on lytic profiles in overlapping clinical isolate groups It was created. In addition, all three phages were tested against the reference P. aeruginosa PAO1 strain at 30°C. It has demonstrated virulent activity. It also has multiple advantages in terms of limiting the development of resistance. The phage approach has an important technical rationale. It pits a single phage against another. bacterial resistance arises, for example, through the loss or modification of the phage receptor. It can emerge. In cocktails made up of complementary phages, one phage versus another. 14 Bacteria that lose their sensitivity become sensitive to the other phage or phages in the cocktail. It is possible for it to remain. Therefore, especially those using different receptors or Using phages that exhibit low cross-resistance together, compared to using a single phage. a rational approach that can delay or reduce the selection of resistant subpopulations It is accepted as a cocktail design strategy (18). As a result, three 5 in the invention The main experimentally demonstrated advantage of using phages together is that they are not used individually. The host coverage of phages, which were 37.8%, 37.8% and 63.9% respectively, complementary and Combining overlapping lytic activity profiles resulted in a cumulative clinical benefit of 89.2%. The goal is to achieve isolation coverage. This result indicates that the cocktail formulation is random. No, host-range and EOP 10 performed on a broad clinical isolate panel. This shows that it is a rational choice based on the analyses. Furthermore, multiple phages Its use, according to the literature, may lead to resistant bacterial substrates developing against a single phage. because populations can be targeted by other cocktail components It is an accepted strategy in terms of limiting the development of resistance (19). Within the scope of the meeting, a fishing area and city waste management were established in the Kızılırmak Kırıkkale settlement. from the Kalecik picnic areas located around the water center and the Kızılırmak River, and Wastewater from the Intensive Care Unit of Başkent University Hospital, 05.04.2023 - Between 17.05.2023 and 17.05.2023, water samples were taken regularly every 15 days. 20 different types of bacteria were collected using the previously described method for bacteriophage isolation. It was implemented by making modifications [1]. Both P. aureginosa clinical isolates and PAO1 is one of the common laboratory reference strains that models moderately virulent phenotypes. The strain was used. This was also to ensure that laboratory studies could be conducted according to standard procedures. PAO1 strain was used in all phages. It was cultured on blood agar medium the night before. The enriched PAO1 strain was adjusted to a concentration of 1 McFarland. The prepared 25 100 μl of bacterial suspension was taken and added to the water sample, and It was placed in incubation. After the incubation period was completed, the phage The suspension was centrifuged at a force of 8000g for 5 minutes and then filtered. The presence of phages in the collected water sample was analyzed using the spot test method. This was done using fresh bacterial colonies passaged the night before. (Inoculation 30) This was done. Later, a test was prepared to check for the presence of phages. Ten μl of each solution is dripped onto the inoculation line and left overnight at 37 °C. It was incubated. At the end of the incubation period, bacteria were found in the phage-added region. The absence of observed reproduction indicated the presence of an effective phage against this bacterium. [2]. For the purpose of purifying and enriching the phages, as seen in the spot test result... Agar was cut from the zones, diluted with TSB, and processed using the "Double-Layer" (DL) method. 5 It was applied. In the DL method, Luria Broth (LB) was placed in soft agar in a sterile glass tube. 100 μl of phage solution and 100 μl of PA01 suspension from each dilution, separately. added, then this mixture was poured into TSA petri dishes and left to solidify. [3]. The purification process takes an average of 5-20 days until pure phage plaques are seen on DL plaques. The process continued for 10 days. The purification process for preparing the phage solution was completed. The surfaces of the purified phage plates were then transferred to sterile falcons. Subsequently, 2 ml of PAO1 suspension at 1 McFarland concentration is combined with 5 ml of liquid medium. It was incubated overnight. The phage solution was centrifuged the next day. The presence of phages in the filtered solution was confirmed using a spot test, and Stored at +4 °C. Phage titer of the solution was calculated by DL method. 15 To calculate phage titers: Phage titer (PFU / ml) = Plaque number x 10 x 10 dilutions factor formula was used [4]. Stability of phages was measured at 4-60 °C and pH 2-12 Phages were investigated using the DL agar method by testing them at the specified values. and pH values ​​were incubated for 30 minutes, and the titers were obtained at the end of this process. It has been determined. 20 Phages were analyzed to determine their size, morphological characteristics, and genomics through a comprehensive analysis process. Their structures have been examined in detail. Carbon dating was used in the examination of their morphological structures. Coated copper grids were used and 10 μl of phage was dropped onto them. Afterwards, a 2% solution was applied. Counterstaining was performed with uranil acetate [5]. Grids were analyzed by TEM at 120 kV. 25 Clinical isolates of P. aeruginosa and Escherichia phages were used to determine the host range of the phages. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Proteus vulgaris ATCC 13315, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 The strains were used and a spot test was performed. Multiplicity of infection (MOI), optimum It indicates the ratio of the phage particle number to the host bacteria number. MOI 30 In the calculation, the PAO1 strain was prepared at 108 CFU / ml and the titers of the phages were 1010 and It is adjusted between 105. From the prepared phage suspensions (MOI = 0.001-100 200 μl (PFU / CFU) is taken and mixed with the same volume of PAO1 in a sterile glass tube and 3.5 It was incubated at 37°C for 4 hours. At the end of this period, the phage and bacteria mixture was added for 4 minutes. 16 Centrifugation was performed at a speed of 10,000g throughout. The resulting supernatant... TSA was added to the agar using the DL method, and titer counting was performed after this process. The protocol was used. At the end of incubation, the highest phage titer was determined at the specified ratio. It has given the optimal MOI value [6]. Growth curves of the isolated bacteriophages were obtained using methods applied in another related literature. The study was conducted by making some changes to the protocol [7]. 1 McFarland density 1 1 ml PAO1 was centrifuged for 4 min (10000g). 1 ml phage was added to the pellet remaining at the bottom. The suspension was added according to the MOI value. Bacteriophages were added to the host. To ensure adsorption to the cells, 10 and incubated at 150 rpm. Then again at 10000 rpm. The centrifugation process lasted 4 minutes. The pellet obtained at the end of centrifugation was 10 ml of liquid. It was added to the culture medium and placed in an incubator at 37°C for 0-65 minutes. 100 μl was taken every 5 minutes between each dose using the DL method, and at the end of 24 hours, each dose was analyzed. Phage titers corresponding to the time period were counted. Latent period and burst size 15 This was calculated using curve plots drawn for each phage. The protocol of Sambrook, J. and Russell was used for nucleic acid isolation [8]. 1 ml of phage suspension was centrifuged at 21000g for 120 minutes. Centrifuge Finally, after transferring 500 μl to a new tube and adding 1.25 μl of DNase I (20 mg / ml) to it... Then it was incubated at 37 °C for 30 minutes. The incubation period... Finally, in order, 1.25 μl proteinase K (20 mg / ml), 25 μl 10% SDS, 20 μl 0.5 M EDTA (pH 8) was added and the resulting mixture was placed in a heat block at 56 °C for 120 minutes. It was incubated. 500 μl of phenol chloroform isoamyl alcohol (25:24:1) was added and 3500 The mixture was centrifuged for 10 minutes at a time. At the end of centrifugation, the top 25% of the resulting mixture... 450 μl was taken from the phase and transferred to a clean Eppendorf tube. 40 μl in 1 / 10 volume 3M sodium acetate (NaOAc) and 400 μl of isopropyl alcohol are added and placed in ice for 30 minutes. It was incubated for 30 minutes. After being removed from the ice, it was heated at 15000g for 30 minutes. It was centrifuged. The resulting pellet was mixed with 70% ethanol. After washing, it was centrifuged at 12000 rpm for 10 minutes. Supernatant 30 After being removed, it was left to dry for a while in an Eppendorf tube incubator. After drying, 50 μl of nuclease-free water is added and stored at -20 °C. It has been preserved. 17 After a one-week acclimatization period for the application of the resulting phage cocktail The animals were divided into 5 groups, with 9 (8+1) mice in each group. Group 1: Control group (CG) in which burn wounds were induced but no infection was induced. Group 2: Burn wound and infection induced in an untreated control group (SHAM). It became infected after the thermal injury was created. Group 3: Burn wound 5 Toxicity created and treated with Phage cocktail without causing infection. Control group (CP); 100 µl dose of phage after thermal injury was induced. The cocktail was applied. Group 4: Burn wound and infection induced, phage cocktail. Patients receiving treatment (YEF) were infected after a thermal injury was created. Following infection, a phage cocktail was administered at a dose of 100 µl. The treatment was repeated every 12 hours. The dose was repeated. Group 5: Burn wound and infection induced, antibiotic Treatment-receiving group (YEA); those infected after thermal injury is created. In mice, cefepim

[10] was administered at a dose of 1 g per 10 ml every 12 hours following infection. It was repeated at the same dose. Mice were given a ketamine-xylazine mixture (50 mg / kg each, intramuscularly) prior to manipulation. Anesthesia was administered with (injected into) the burned area of ​​the animals. To avoid causing irritation, the area between the two scapulae was targeted. The targeted area... After the area was marked, it was shaved

[11] . A 1.8 x 1.8 cm brass plate was placed in the area. with the help of (the plate was heated between 255 °C and 260 °C and then cooled to 5-10 °C before application to the skin 20 (lost) Heat block reaching 250°C in contact with the shaved area for 5 seconds Burns were created by inflicting

[12] . Immediately after thermal injury, To prevent shock in the animals, all mice received fluid replacement. 0.5 ml sterile physiological saline and 5 mg / kg meloxicam administered intraperitoneally (ip). It has been applied

[13] . Infection in groups where the burn wound will be infected 25 To create it; a sample was taken beforehand from the caudal portion of the area where the burn wound was created. 0.9% isotonic sodium chloride prepared at a titer of 107 CFU / ml was injected subcutaneously with 100 µl of PAO1. It was injected using an insulin syringe. The antibiotic dosage was 60 mg / kg / 12 It was administered intramuscularly twice daily, with an hour's interval between doses. However... The prepared phage cocktail was administered intraperitoneally at a titer of 107 CFU / ml at 12-hour intervals for 30 days. has been implemented [9]. 18 To monitor tissue healing in the wound area, the wound site was examined daily throughout the experiment. Photographed with a digital camera, measured using a calibrator, and % wound The closure rate was calculated from the following equation

[13] . Wound closure percentage The formula is shown in Table 1. Furthermore, wound closure can also be distinguished macroscopically. It was photographed every day so that it could be documented. G4 5 where the Faj cocktail was applied. The highest wound closure rate was observed in the group with 16.6% on the 7th day, while the treatment No wound closure was observed in the uninfected G2 group. In parallel... In group G4, the bacterial load increased from 8.8 × 10⁶ CFU / g to an average of 10² CFU / g. It has regressed to that level and there is a significant decrease in biostatistical terms compared to G2. This has been shown (p=0.037). Histopathological examination revealed early wound healing. The highest epithelial regeneration during this period was observed in the G4 group. Significant reduction in inflammation and improvement in tissue integrity, phage its treatment controls the infection as well as the wound healing process This shows that he supports it. Table 1. 7-day wound measurements and wound closure percentages according to experimental groups. 15 When comparing phage therapy (G4) and cefepim therapy (G5), both treatments... wound healing compared to the untreated infected control group (G2) It has been observed to support this. However, the wound closure rate on day 7 was higher for phage 20. In the G4 group, which received treatment, 16.6% reported the virus, while in the G5 group, which received cefepim, this figure was 11.1%. This was determined and the highest wound closure was observed in the G4 group. This finding is consistent with other results. Bacterial load in group G4 was on average 10² CFU / g was detected in group G5 as 10³ CFU / g; also, three in group G4 The bacterial load in the animal has been completely eliminated. However, G4 to G5 25 The difference in bacterial load between them was not found to be statistically significant. 19 Histopathological evaluation revealed the highest epithelial regeneration in group G4. It has been observed that inflammation decreases in tissues treated with phage therapy, and tissue Healing has been determined in its entirety. Therefore, the wound closure rate is bacterial. When the burden and histopathological improvement are evaluated together, improvement is observed in both treatments. Although the findings are as follows, the parameters examined show more favor towards phage therapy. Significant findings have been obtained. In addition, the invention showed that in the G4 group treated with a phage cocktail, While bacterial load decreased to an average level of 10² CFU / g, tissue abnormalities were observed in three animals. No bacterial growth was detected in the culture, and the bacterial load was 0 CFU / g. This finding establishes the limits of detection for the culture method in the animals in question. It shows that microbiological eradication has been achieved within it. 10 To examine the inflammatory response using biochemical parameters; sacrifice Blood and tissue samples were taken from mice during those days. The animals were sacrificed. Cervical dislocation was performed. Ketamine-xylazine mixture was administered on days 3 and 7. The patient was anesthetized with (50 mg / kg each, administered intramuscularly). The patient was 15 years old when anesthesia was administered. Blood samples were taken from the animals into tubes for CRP measurement using an intracardiac method. When CRP results were evaluated on a group basis, the average CRP in Group 1 (G1) was... It was determined that the level decreased from 0.11 mg / L on day 3 to 0.06 mg / L on day 7. High CRP level indicates infection in G2 patients who have not received any treatment. It was detected as 0.35 mg / L in the group. In the G4 group treated with the phage cocktail, 20 The average CRP level was 0.20 mg / L on day 3 and 0.12 mg / L on day 7. has decreased. Comparison of CRP concentrations of the groups on days 3 and 7. A statistically significant difference was determined (Kruskal–Wallis H test, χ² = 7.600; (p = 0.0417). Using Dunn's post-hoc test and Holm–Bonferroni correction. In the multiple comparisons performed, the overall difference between groups was mainly G1 25 It was determined that this stemmed from a difference in CRP between G2 and G2. In contrast, G2, When G4 and G5 groups were evaluated against each other, CRP concentrations were compared. No statistically significant difference was found in this regard (Kruskal–Wallis H test, χ² = 3.00; p = 0.5000). The median CRP values ​​of these three groups were statistically significant. Similar results were found. However, CRP levels in group G2 were 30% higher than in group G4. and it has been observed that it is numerically higher compared to G5 groups, While the finding points to a biological trend, it is limited by the current sample size. It has not reached a statistically significant level. Therefore, CRP findings, especially Higher inflammatory response in untreated infected G2 group. In the G4 group, which was monitored and treated with phage therapy, CRP levels were measured from day 3 to day 7. quantitative tissue analysis in terms of the decrease in level from 0.20 mg / L to 0.12 mg / L It shows a trend consistent with the findings; however, CRP 5 between G2 and G4 It is not argued that the difference alone is statistically significant. To determine the bacterial load in the tissue, tissues from sacrificed mice were aseptically analyzed. Under these conditions, cross-sections will be taken at the border regions using sterile scissors and forceps. The tissue was removed in this manner. Half of the tissue obtained was weighed for quantitative culture and placed in sterile 10 It was homogenized in ceramic mortars and serial dilutions were made with TSB and TSA. 100 µl of culture medium was inoculated using the grid plate method, and the plates were stored at 37°C for 24 hours. The samples were incubated, and bacterial counts were performed at the end of the incubation period. Results This was calculated in CFU / ml. The infection load in the tissue was determined. Skin samples with lesions were taken after sacrifice at the end of the experiment. 15 It was detected in 10% buffered formalin solution for 24-48 hours. Histopathological examination was performed using a trinocular research microscope. The phage cocktail in the formulation described in the invention is available in liquid formulation as well as 20 in lyophilized form using suitable pharmaceutical excipients and stabilizers It can also be prepared. During lyophilization, the biological activity and titers of phages sucrose, trehalose, mannitol or similar suitable substances for preservation purposes. Cryoprotectants / stabilizers can be used. In addition, a phage cocktail can be used to target the area. Liquid solution / suspension depending on the route of administration and intended use, lyophilized powder, reconstituted lyophilized preparation, encapsulated or 25 microencapsulated preparation, gel / cream / hydrogel suitable for topical application or wound care. pharmaceutical systems and appropriate formulation development for application Following their studies, they were developed in the form of liquid or dry powder preparations for inhalation. It can be formulated. It is an auxiliary substance that can be used in the preparation of different pharmaceutical formulations. The type and quantity of ingredients, as well as the production, drying, and storage conditions, determine the composition of the cocktail (30). It can be optimized in a way that preserves the infectivity and titer of the phages found. This Therefore, the use of this phage cocktail is only possible with sterile 0.9% (w / v) NaCl. 21 Not limited to its liquid form, but also preserving the biological activity of phages. Provided that the conditions are met, it is possible to prepare it in different pharmaceutical dosage forms. The clinical P. aeruginosa isolates evaluated within the scope of the invention, in particular Clinical isolate exhibiting carbapenem resistance and various mechanisms associated with this resistance. It includes populations. In the relevant studies, carbapenem resistance was observed in only 5 not at the phenotypic level, but the mechanisms contributing to the development of resistance It is also seen that it has been characterized in detail in terms of clinical aspects. The examined clinical P. 33.7% of aeruginosa isolates had an active efflux pump, and 34.7% had an external efflux pump. A decrease in membrane proteins (OMP), ESBL in 21.4%, and AmpC β- in 15.3%. lactamase and metallo-β-lactamase (MBL) activity were determined in 33.7% of them. In addition, 10 It has been shown that resistance mechanisms can coexist in the same isolates; efflux MBL production in 30.3% of pump-positive isolates, MBL-positive isolates Reduced OMP expression was detected in 36.4% of MBL-positive isolates. 69.7% of the isolates and 70.6% of the isolates in which reduced OMP was detected also It has been reported that it forms a biofilm. The level of carbapenem resistance is also determined by MIC data as 15 This has been demonstrated. All P. aeruginosa isolates were analyzed according to CLSI breakpoints. It was found to be resistant to meropenem, and meropenem MIC values ​​were 8–>32 μg / mL. It has been reported that the isolates varied between these values. When EUCAST criteria were used, the isolates' values ​​were... 90.2% were classified as resistant to meropenem. Different isolates were observed within the same isolate group. Variable and multifaceted resistance profiles were also observed against anti-Pseudomonas β-lactams. It has been shown that this data indicates the presence of P. aeruginosa, the species targeted by the invention. a homogeneous bacterial population that is resistant to only a single antibiotic not limited to this group; carbapenem resistance, β-lactamase production, efflux pump its activity causes several effects such as changes in outer membrane permeability and biofilm formation. 25 This supports the antibiotic susceptibility of relevant clinical P. aeruginosa isolates. profiles, carbapenem resistance and the mechanisms associated with this resistance in detail Studies evaluating the phenotypic characteristics of these isolates have been published. Detailed data on resistance characteristics, MIC values, and resistance mechanisms. presented in the aforementioned publications (20,21). 30 The host specificity of the phages in question has been experimentally evaluated. This For this purpose, different Gram-negative and Gram-positive bacteria other than Pseudomonas aeruginosa 22 Bacterial species were also included in the host range analysis. This included Escherichia. coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Proteus vulgaris ATCC 13315, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 Reference strains were tested. In the analyses performed, Baskent_P1_112, The tested heterologous reference 5 of Baskent_P2_ICU and Baskent_P3_3B phages It was determined that the strains did not show lytic activity. In contrast, the phages did not exhibit lytic activity against P. aeruginosa. The phages in question demonstrated lytic activity against the tested strains. Within the microorganism panel, it has a host profile specific to P. aeruginosa. experimentally supports this. The subject of the invention is the Baskent_P1_112, Baskent_P2_ICU and Baskent_P3_3B phages. Host interval conducted to assess host specificity In the analyses, Escherichia coli ATCC 25922, other than Pseudomonas aeruginosa, Klebsiella pneumoniae ATCC 700603, Proteus vulgaris ATCC 13315, Enterococcus 15 of the reference strains faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 No lytic activity was observed in either of them. Thus, both Gram-negative and Gram-negative bacteria were studied. A negative result in a test panel containing different types of bacteria indicates... The subject phages tested within the panel of microorganisms was P. aeruginosa. This experimentally supports host specificity. Antibiotic susceptibility / resistance of clinical P. aeruginosa isolates used in the invention 20 Their profiles have been determined, and the efficacy of phages on these clinical isolates has also been investigated. The results were evaluated. The findings showed that the phages in the cocktail were resistant to antibiotics. It has been shown that it can also demonstrate lytic activity against resistant clinical isolates. This finding suggests that the mechanism of action of phages differs from that of antibiotics. Therefore, antibiotic resistance necessarily eliminates phage susceptibility. 25 Experimental evidence supports the fact that it does not remove it. Industrial Applicability of the Invention The invention describes a group of bacteriophages exhibiting lytic activity against P. aeruginosa isolates. formed by combining 30 therapeutic agents in the treatment of infections a bacteriophage cocktail that can be used as a biocontrol agent in hospital settings It is related to and applicable to industry. 23 The invention is not limited to the above descriptions, and a person skilled in the field can easily make further discoveries. It can demonstrate different applications of the invention. These are the claims and demands of the invention. It should be evaluated within the scope of the protection granted. 10 20 30 24 REFERANSLAR 1. Lin N-T, Chiou P-Y, Chang K-C, Chen L-K, Lai M-J. Isolation and characterization of ΦAB2: A novel bacteriophage of Acinetobacter baumannii. Research in Microbiology. 2010 May;161(4):308–14. doi:10.1016 / j.resmic.2010.03.007 5 2. Kasman LM, Porter LD. Bacteriophages. In: Statpearls Publishing.2021. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK493185 / 3. Santos SB, Carvalho CM, Sillankorva S, Nicolau A, Ferreira EC, Azeredo J. The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique. BMC Microbiology. 2009 Jul 23;9(1). 10 doi:10.1186 / 1471-2180-9-148 4. Titering-phagesdb. https: / / phagesdb.org / protocols / 69 / Accessed June 10, 2022. 5. Yang Z, Liu X, Shi Y, Yin S, Shen W, Chen J, et al. Characterization and genome annotation of a newly detected bacteriophage infecting multidrug-15 resistant Acinetobacter baumannii. Arch Virol. 2019 Jun;164(6):1527-1533. DOI: 10.1007 / s00705-019-04213-0 6. Tan CW, Rukayadi Y, Hasan H, Abdul-Mutalib N-A, Jambari NN, Hara H, et al. Isolation and characterization of six vibrio parahaemolyticus lytic bacteriophages from seafood samples. Frontiers in Microbiology. 2021 Mar 20 10;12. doi:10.3389 / fmicb.2021.616548 7. Bolger-Munro M, Cheung K, Fang A, Wang L. T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age. J Exper Microbiol and Immunol. 2013;17:115-119. 8. Sambrook J, Russel DW. 2001– Rapid isolation of yeast DNA. In: Sambrook 25 J, Russell DW (Eds) Molecular Cloning: A Laboratory Manual, 2nd Edn, Cold Spring Harbor Laboratory, New York, pp 631–632. 9. Chadha P, Katare OP, Chhibber S. In vivo efficacy of single phage versus phage cocktail in resolving burn wound infection in BALB / C mice. Microbial Pathogenesis. 2016 Oct;99:68–77. doi:10.1016 / j.micpath.2016.08.001 30 10. O’Connor A, Lopez MJ, Patel P, Eranki AP. Cefepime. 2024; Treasure Island (FL): StatPearls Publishing; 2024 Jan–. PMID: 31194389 Available from: https: / / pubmed.ncbi.nlm.nih.gov / 31194389 / 11. Dale RM, Schnell G, Wong JP. Therapeutic efficacy of “Nubiotics” against burn wound infection by Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. 2004 Aug;48(8):2918–23. doi:10.1128 / aac.48.8.2918- 2923.2004 12. Santelis, Santiago J & Abali, Ayse & Ozgun, Gonca & Ozdemir, Binnaz 5 Handan & Tutuncu, Neslihan & Haberal, Mehmet. (2022). Impact of Severe Burns on Pancreatic Islets: An Experimental Model in Rats. 2. 23-28. 13. Fayez MS, Hakim TA, Agwa MM, Abdelmoteleb M, Aly RG, Montaser NN, et al. Topically applied bacteriophage to control multi-drug resistant Klebsiella pneumoniae infected wound in a rat model. Antibiotics. 2021 Aug 10 27;10(9):1048. doi:10.3390 / antibiotics10091048 14. Nabil, N. M., Tawakol, M. M., Samir, A., Hassan, H. M., & Elsayed, M. M. (2024). Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens. BMC microbiology, 24(1), 338. 15 15. EMA. Guideline on quality aspects of phage therapy medicinal products, EMA / CHMP / BWP / 1 / 2024, Committee for Medicinal Products for Human Use, Netherlands 16. Suh, G. A., Lodise, T. P., Tamma, P. D., Knisely, J. M., Alexander, J., Aslam, S., ... & Antibacterial Resistance Leadership Group. (2022). Considerations 20 for the use of phage therapy in clinical practice. Antimicrobial agents and chemotherapy, 66(3), e02071-21. 17. Sandhu, J. S., & Parida, A. (2026). Spot-on phage therapy: stable formulations, smarter dosing for topical phage application. Frontiers in Cellular and Infection Microbiology, 16, 1697070. 25 18. Holger, D., Kebriaei, R., Morrisette, T., Lev, K., Alexander, J., & Rybak, M. (2021). Clinical pharmacology of bacteriophage therapy: a focus on multidrug-resistant Pseudomonas aeruginosa infections. Antibiotics, 10(5), 556. 19. Yang, Y., Shen, W., Zhong, Q., Chen, Q., He, X., Baker, J. L., ... & Le, S. 30 (2020). Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa. Frontiers in microbiology, 11, 327. 26 20. Mirza, H. C., Hortaç, E., Koçak, A. A., Demirkaya, M. H., Yayla, B., Güçlü, A. Ü., & Başustaoğlu, A. (2020). In vitro activity of ceftolozane–tazobactam and ceftazidime–avibactam against clinical isolates of meropenem-non- susceptible Pseudomonas aeruginosa: A two-centre study. Journal of Global Antimicrobial Resistance, 20, 334-338. 5 21. Üsküdar-Güçlü A, Mirza HC, Ünlü S. 2022. Co-existence of multiple resistance mechanisms in clinical isolates of carbapenem-resistant Pseudomonas aeruginosa. FLORA;27(1):45-54. doi: 10.5578 / flora.20229905 22. Bourdin G, Schmitt B, Marvin Guy L, Germond J-E, Zuber S, Michot L, et al. 10 Amplification and purification of T4-like escherichia coli phages for phage therapy: From laboratory to pilot scale. Applied and Environmental Microbiology. 2014 Feb 15;80(4):1469–76. doi:10.1128 / aem.03357-13 20 30

Claims

27 REQUESTS 1. It is a Pseudomonas aeruginosa bacteriophage cocktail, characterized by: • Baskent_P1_112 (DSM 35944), • Baskent_P2_ICU (DSM 35945) and • Baskent_P3_3B (DSM 35946) 5 It contains bacteriophages.

2. According to Claim 1, it is a bacteriophage cocktail, characterized by its carrier and / or diluent. It contains a 0.9% (w / v) NaCl solution with a pH of 7.

0.

3. A bacteriophage cocktail according to claim 1 or 2, with the characteristic; Baskent_P1_112, Baskent_P2_ICU and Baskent_P3_3B bacteriophages were mixed at a volumetric ratio of 1:1:

1. It is found in that proportion.

4. It is a bacteriophage cocktail according to any of the previous requirements, and its characteristic is; bacteriophages Baskent_P1_112, Baskent_P2_ICU and Baskent_P3_3B each at a concentration of approximately 3.3 × 10⁷ PFU / mL in the final cocktail It is to be found. 15 5. A bacteriophage cocktail according to any of the previous requirements, and its characteristic is; The total infectious phage titer of the cocktail should be approximately 1×10⁸ PFU / mL.

6. It is a bacteriophage cocktail according to any of the previous requirements, and its characteristic is; bacteriophages Baskent_P1_112, Baskent_P2_ICU and Baskent_P3_3B They are lytic bacteriophages. 20 7. It is a bacteriophage cocktail according to any of the previous requirements, and its characteristic is; liquid solution or suspension, lyophilized powder, reconstituted lyophilized in the form of a preparation, encapsulated preparation, or microencapsulated preparation. It is the fact that.

8. A bacteriophage cocktail according to any of claims 1-6, with the following characteristics: gel, 25 It is available in cream or hydrogel form.

9. A bacteriophage cocktail according to any of claims 1-6, with the characteristic of being a wound treatment. It is in the form of a pharmaceutical system for its application.

10. A bacteriophage cocktail according to any of claims 1-6, and its characteristic is: It should be in the form of a liquid preparation or a dry powder preparation for inhalation. 30 11. It is a bacteriophage cocktail according to claim 7, and its characteristics are; sucrose, trehalose and at least one of the mannitols as a cryoprotectant and / or stabilizer It includes. 28 12. Request 1 for use in Pseudomonas aeruginosa infections. A pharmaceutical containing a bacteriophage cocktail according to any of the 11 criteria. composition.

13. Associated with carbapenem-resistant Pseudomonas aeruginosa clinical isolates A pharmaceutical according to Claim 12 for use in infections. 5 composition.

14. In wound or burn infections associated with Pseudomonas aeruginosa A pharmaceutical composition according to Claim 12 for use. 15 25

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