A method for preparing bacteriophage for treating lung infection caused by multidrug-resistant bacteria

Abstract

The present invention discloses a method for preparing bacteriophages for treating lung infections caused by multidrug-resistant bacteria, comprising the following steps: collecting samples, extracting bacteriophages from samples and performing experimental analysis on them; 1. Extracting bacteriophages from samples and performing experimental analysis on them; 2. Performing project result analysis on the extracted bacteriophages; and phage result file description: It is concluded from multiple results whether the extracted bacteriophages can treat lung infections caused by multidrug-resistant bacteria. In summary, bacteriophage therapy for lung infections caused by multidrug-resistant bacteria has been deeply studied in in vitro experiments, animal experiments and clinical trials. Although bacteriophage therapy for bacterial multidrug-resistant lung infections still faces many challenges, with the continuous advancement of bacteriophage therapy technology, the current difficulties such as production difficulties, toxin removal, insufficient clinical trials, storage difficulties and lack of hospital bacteriophage libraries will be gradually overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophage therapy, and in particular to a method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection. Background Art

[0002] Respiratory infections are the third leading cause of death and disease worldwide, accounting for more than 10% of all disability-adjusted life years (DALYs), and more than 4 million people die each year from acute respiratory infections. With the abuse of antibiotics and bacterial mutations, bacterial resistance is becoming increasingly serious. Pulmonary infections caused by multidrug-resistant bacteria are mainly caused by Gram-negative bacilli, such as Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Resistance to Staphylococcus aureus among Gram-positive bacteria is also common, and the resistance rates of these clinical isolates are still on the rise. Overall, the current status of multidrug-resistant lung infections is not optimistic. The high incidence of drug-resistant bacteria has brought huge health and economic burdens, given that antibiotics have unfavorable factors such as long research and development cycles, high difficulty, and high costs.

[0003] Bacteriophages are viruses that can infect bacteria. They are abundant in nature and highly specific to host cells, and can kill bacteria with precision. Most phages are tailed phages, which are generally composed of a protein capsid that encapsulates genetic material and a tail. A small number of phages have shorter tails or no tails. According to classification, there are also phages such as small isometric ssDNA viruses, filamentous phages, and small ssRNA phages. Bacteriophages were used as antibacterials when they were first discovered, but were abandoned after the emergence of antibiotics. They continue to be used in large quantities in clinical practice, but lack effective records. At present, there is a lack of strong evidence for randomized controlled trials of phage therapy, which has hindered its establishment as an approved antibacterial treatment method. People's interest in phage therapy has re-emerged, and a large number of case reports have been published on phage treatment of lung infections, and many clinical trials are also underway. Pathogen resistance to antibiotics does not affect the bactericidal ability of phages against pathogens, so phages are often used as adjuvant or alternative therapies for multidrug-resistant or pan-resistant bacterial infections. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing bacteriophage for treating lung infection caused by multidrug-resistant bacteria.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection comprises the following steps:

[0007] S1: Sample collection: After the laboratory receives the samples containing bacteriophages, it will carry out a strict sample quality control process on the received samples, and qualified samples will enter the formal experimental process;

[0008] S2: Extract the bacteriophages from the sample and perform experimental analysis on them.

[0009] (1) Phage DNA extraction;

[0010] (2) Nucleic acid quality inspection;

[0011] (3) DNA fragmentation;

[0012] (4) Library quality control;

[0013] (5) Sequencing on a machine.

[0014] S3: Extract the bacteriophages from the sample and perform experimental analysis on them. Process 2:

[0015] (1) Phage DNA extraction;

[0016] (2) Accounting quality inspection;

[0017] (3) RNA reverse transcription;

[0018] (4) DNA fragmentation;

[0019] (5) Library construction;

[0020] (6) Library quality control;

[0021] (7) Sequencing on a machine.

[0022] S4: Analyze the project results of the extracted phages;

[0023] S5: Phage result file description: From multiple results, it can be concluded whether the extracted phage can treat multidrug-resistant bacterial lung infections.

[0024] Preferably, the analysis of the phage project results in S4 includes sequencing data filtering and statistics, genome assembly, genome annotation, gene function annotation, virulence factor analysis and drug resistance analysis.

[0025] Preferably, the sequencing data filtering and statistics: In this project, phage nucleic acid was extracted and library constructed for 3 samples, and DNBSEQ-T7 was used for sequencing to ensure the reliability of subsequent information analysis results. Fastp[1] was used to filter and quality control the obtained raw sequencing data, cut the adapter, remove low-quality reads and reads with a high N ratio, and obtain clean reads.

[0026] Preferably, the genome assembly: use metaSPAdes software to perform de novo assembly on clean reads, select different k-mer lengths for testing and obtain the best assembly result, and then use bwa software to align the clean reads to the assembled genome sequence to calculate the coverage.

[0027] Preferably, the genome annotation: Prokka is used to annotate the coding genes and tRNA of the genome, and then blastp is used to compare the protein sequence with the NR library to obtain sequence information of each gene with high similarity in the NR library.

[0028] Preferably, the gene function annotation: uses eggNOG-mapper to perform functional annotation on genes, which contains annotation information from COG, GO, KEGG, CAZy, BiGG and PFAM databases.

[0029] Preferably, the virulence factor analysis: using blast to perform homology comparison between the gene sequence and the VFDB database to predict virulence genes.

[0030] Preferably, the drug resistance analysis: uses resfinder to perform analysis to predict whether there are genes with drug resistance on the genome.

[0031] In summary, due to the adoption of the above technology, the beneficial effects of the present invention are:

[0032] Compared with existing technologies, the main advantages of phage therapy are:

[0033] (1) Short R&D cycle: Bacteriophages are widely distributed in nature, with large numbers and abundant populations. Therefore, they are easy to obtain in the natural environment and the isolation method is relatively simple. It only takes a few months to collect phages from sewage and extract a new phage. Compared with the development of new antibiotics, which takes ten or even more than ten years, the R&D cycle is shorter. Therefore, it has great potential in the treatment of lung infections caused by multidrug-resistant bacteria, providing a simpler and more cost-effective way to treat bacterial infections.

[0034] (2) High specificity: Phages are highly specific to target bacteria, that is, they only infect specific host bacteria. The lytic phages commonly used in treatment recognize specific receptors on the surface of bacteria to adsorb and inject the genetic material in the capsid. The phage then uses the host's metabolic mechanism to replicate and translate its own genetic material and proteins, assemble and reproduce progeny phages, and cause the host bacteria to lyse and die. Therefore, during the treatment process, phages can selectively infect pathogenic bacteria without interfering with the host's normal flora. This selectivity and specificity reduces the adverse effects of traditional antimicrobial treatment on the patient's microbial community, and is more advantageous than antibiotics;

[0035] (3) Great potential for resisting bacterial resistance: When phage preparations are used for antibacterial treatment, as bacteria mutate, it is easy for them to develop resistance to phages. However, in this process, phages are prone to evolutionary trade-offs with resistant bacteria, that is, the evolution of bacterial resistance to phage attacks changes the efflux pump mechanism, resulting in increased sensitivity to drugs in several antibiotic classes. Multiple research results have shown that phages, as targeted antibacterial agents, can make multidrug-resistant bacteria evolve in a direction that is more sensitive to traditional antibiotics. Therefore, phage preparations can also be used in combination with conventional antibiotics to achieve bactericidal purposes. This approach helps provide a new treatment option to solve the current problem of increasing antibiotic resistance.

[0036] (4) High safety: Phage therapy is considered a safer alternative to antibiotic treatment because of its low incidence of adverse reactions and side effects. Antibiotic-related adverse drug events are relatively common, with 20% of patients experiencing such events. Phages are naturally occurring microorganisms that survive by infecting host bacteria and are harmless to eukaryotic cells such as humans, animals, and plants. To date, there have been very few serious adverse reactions caused by the use of phages, supporting their use in the treatment of drug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The clean date base ratio distribution diagram and the clean date base quality distribution diagram proposed in the present invention;

[0038] Figure 2 This is a distribution comparison diagram of the cleanreads proposed in the present invention aligned to the genome. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0040] In the description of the present invention, it is necessary to understand that the terms indicating orientation or positional relationship are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0041] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances of the specification.

[0042] The present invention provides Figure 1 A method for preparing bacteriophage for treating multi-drug resistant bacterial lung infection is shown, comprising the following steps:

[0043] S1: Sample collection: After the laboratory receives the samples containing bacteriophages, it will carry out a strict sample quality control process on the received samples, and qualified samples will enter the formal experimental process;

[0044] S2: Extract the bacteriophages from the sample and perform experimental analysis on them.

[0045] (1) Phage DNA extraction;

[0046] (2) Nucleic acid quality inspection;

[0047] (3) DNA fragmentation;

[0048] (4) Library quality control;

[0049] (5) Sequencing on a machine.

[0050] S3: Extract the bacteriophages from the sample and perform experimental analysis on them. Process 2:

[0051] (1) Phage DNA extraction;

[0052] (2) Accounting quality inspection;

[0053] (3) RNA reverse transcription;

[0054] (4) DNA fragmentation;

[0055] (5) Library construction;

[0056] (6) Library quality control;

[0057] (7) Sequencing on a machine.

[0058] S4: Analyze the project results of the extracted phages:

[0059] (1) Including sequencing data filtering and statistics: In this project, phage nucleic acid was extracted and library constructed for 3 samples. Sequencing was performed using DNBSEQ-T7 to ensure the reliability of subsequent information analysis results. Fastp[1] was used to filter and quality control the obtained raw sequencing data, cut the adapter, remove low-quality reads and reads with a high N ratio, and obtain clean reads;

[0060] Sequencing data filtering and statistics table:

[0061] Table 1 Sequencing data quality control table

[0062]

[0063] (2) Genome assembly: metaSPAdes software was used to perform de novo assembly on clean reads. Different k-mer lengths were selected for testing and the best assembly result was obtained. Then, bwa software was used to align clean reads to the assembled genome sequence to calculate coverage.

[0064] Genome assembly quality statistics table:

[0065] Table 2 Genome assembly quality statistics

[0066]

[0067] (3) Genome annotation: Prokka was used to annotate the coding genes and tRNA of the genome, and then blastp was used to compare the protein sequence with the NR library to obtain the sequence information of each gene with high similarity in the NR library;

[0068] (4) Gene function annotation: eggNOG-mapper was used to perform functional annotation of genes, which included annotation information from the COG, GO, KEGG, CAZy, BiGG, and PFAM databases;

[0069] (5) Virulence factor analysis: blast was used to compare the gene sequence with the VFDB database to predict virulence genes;

[0070] Virulence gene statistics table:

[0071] Table 3 Virulence gene statistics

[0072]

[0073] (6) Drug resistance analysis: Use resfinder to analyze and predict whether there are genes with drug resistance in the genome;

[0074] Table 4 Result file description

[0075]

[0076] S5: Phage result file description: From multiple results, it can be concluded whether the extracted phage can treat multidrug-resistant bacterial lung infections.

[0077] Single bacteriophage therapy for multidrug-resistant lung infections:

[0078] (1) In vitro and animal experiments

[0079] At present, researchers mainly use mouse lung infection models to evaluate the effect of phage therapy. Most animal experiments use intranasal aerosol inhalation of phage preparations to treat rodent lung bacterial infections, proving that it has good efficacy against lung infections caused by multiple drug-resistant bacteria [28-32]. Phages are foreign microorganisms with immunogenicity. After entering the body, they will activate the adaptive immune system and innate immune system of the human body and will be attacked and cleared by the immune system. Therefore, it is crucial to effectively transport phages to the site of infection by pathogenic bacteria and maintain their activity. At the same time, nasal aerosol inhalation and intraperitoneal injection of a single phage preparation were used to treat mice with Burkholderia cepacia lung infection to find a more effective phage therapy route. Mice were treated with 200 μL intravenous injection or 2.5 mL of phage aerosol inhalation for at least 10 minutes. The results showed that three days was the best treatment time for mice. The peritoneal injection method was not effective, while the aerosol inhalation method had a killing effect on bacteria in mice and was better than nasal instillation. The results confirmed the effectiveness of this treatment route. However, the treatment of phages by aerosol inhalation has the characteristics of high equipment cost, complex preparation storage and transportation conditions, and difficulty in promotion. In addition, experimental studies have shown that the infectivity of phages caused by aerosol inhalation is reduced. This may be because the phages are subjected to strong pressure during aerosolization, which damages the phage structure and leads to a decrease in the titer of the phage preparation [34-35]. Therefore, in response to these problems in the phage treatment approach, researchers have made further improvements. Pranav P

[36] used poly (lactic acid-co-glycolic acid) to load phages and used endotracheal intubation to infuse them into the lungs of mice. Fluorescence imaging technology showed that this method successfully delivered phages to the entire lung. After that, mice with Pseudomonas aeruginosa lung infection inhaled phage powder. After 24 hours, the bacteria and phages in the mouse lungs were cultured and counted. The results showed that the number of bacteria in the mouse lungs was reduced by one order of magnitude (P = 0.0203), indicating that nasal inhalation of phage-carrying powder has a good therapeutic effect on Pseudomonas aeruginosa lung infection in wild-type mice. Hak-Kim Chan et al.

[37] also tried to use a mixture of phage and antibiotic powder for nasal inhalation therapy to reduce the difficulty and cost of phage therapy. The feasibility of this method was confirmed by inhaling phage powder in a mouse model infected with Pseudomonas aeruginosa.

[0080] The host's own immune system will also interact with the phage that enters the body, which may affect the activity and therapeutic effect of the phage. Roach et al.

[38] used phage preparation inhalation therapy to treat mice with normal immunity, myd888 deficiency, lymphocyte deficiency, and neutrophil deficiency caused by Pseudomonas aeruginosa lung infection. They found that some mice with immune deficiency could not be treated by phage, indicating that the immune function in the host is crucial to the phage's killing of bacteria. Later, Abeer M Abd El-Aziz et al.

[39] used a mouse model of acute Pseudomonas aeruginosa lung infection to explore the therapeutic effect of phage MMI-Ps1 on Pseudomonas aeruginosa lung infection. For the first time, the test showed that the phage can cooperate with complement to eliminate serum-resistant Pseudomonas strains, which once again confirmed the above view.

[0081] Clinical trials of phage therapy have been tried for patients who have failed to respond to all antibiotics, and have shown good efficacy without side effects. However, clinical trials are subject to varying degrees of restrictions because they must follow strict ethical standards and respect the wishes of patients. Therefore, current research is mainly focused on in vitro and animal experiments. Through in-depth research on phage in vitro and animal experiments, it is hoped that the appropriate treatment pathways, treatment time, and interaction with the immune system of phages can be more accurately determined to improve the efficacy and safety of phage therapy in future large-scale clinical trials.

[0082] (2) Clinical trials

[0083] Clinical trials of phage therapy are usually conducted in patients with multidrug-resistant bacterial infections or those who have not responded well to antibiotic treatment. Therapeutic phage preparations are currently commercialized for clinical use only in Georgia and Russia. Elsewhere in the world, their use is limited to humanitarian treatment of individual patients or clinical trials in accordance with the guidance of Article 37 of the Declaration of Helsinki

[40]

[40] . Therefore, as a new approach to combat multidrug-resistant bacteria, clinical trials of phage therapy for multidrug-resistant lung infections are currently reported in the form of case reports. However, phage preparations have shown good clinical effects in clinical practice, and many studies have shown that phages are effective as a synergistic or alternative strategy to antibiotics [41-42].

[0084] In the cases of phage treatment of multidrug-resistant lung infections reported so far, phage preparations are usually used together with traditional antibiotics during treatment. During the treatment process, it was observed that phage preparations may cause multidrug-resistant bacteria to become resistant to antibiotics again. In 2023, Li Linlin et al.

[43] treated a 40-year-old patient with progressive interstitial lung disease, severe pulmonary Pseudomonas aeruginosa infection, and type I respiratory failure by aerosol inhalation of phage preparations. Each time, the phage was diluted in 10 mL of normal saline to a titer of 108 PFU / mL. Treatment was given once every 4 hours for four days, for a total of three courses. After the treatment, the bacterial load in the patient's sputum was measured. As a result, the number of bacteria in the patient's body decreased and the symptoms improved. Six months after discharge, the patient underwent lung transplantation and recovered well during the follow-up visit without any lung infection. Although the patient relapsed after the second course of treatment, the relapsed bacteria still showed sensitivity to several antibiotics and phiYY, providing hope for subsequent treatment. Bradley et al.

[44] successfully treated an 11-year-old patient with multidrug-resistant Achromobacter cystic fibrosis using intravenous phage preparations. Two highly active purified phages were injected every 8 hours, combined with 14 days of piperacillin / tazobactam to treat CF exacerbations. During treatment, sputum and blood were collected from the patient for metagenomic analysis, and sputum analysis was performed in a follow-up visit 1 month after the end of treatment. The results showed that the patient tolerated phage treatment well without adverse reactions. After the end of treatment, the number of sputum bacteria decreased and lung function gradually stabilized. During treatment, a sputum isolate was found to have become sensitive to antibiotics again. At present, there is a lack of results from large-scale clinical trials of phage therapy, and its safety and drug resistance still need to be further evaluated, and coping strategies need to be explored.

[0085] (3) In vitro and animal experiments

[0086] Phages are highly specific to host bacteria, which makes it difficult for a single phage to cope with the complex situation of multiple types of bacterial lung infections and makes it easy for bacteria to develop resistance to a single phage

[45] . To address this challenge, researchers have tried to use the phage cocktail approach. A large number of studies have reported the feasibility of phage cocktail therapy and demonstrated its wider host range and potential to reduce the occurrence of bacterial resistance [46-49].

[0087] E. Saussereau et al.

[50] studied the effectiveness of phage cocktails against Pseudomonas aeruginosa in 58 sputum samples from patients with cystic fibrosis (CF) collected from three different hospitals. In the study, 20ul of a mixture of 10 phages collected at a concentration of 108 PFU / ML was mixed with pathogens (sputum) in complex biological samples, a control group was set up, and three replicates were set up in each treatment group, and cultured in a 42°C constant temperature incubator for 24 hours. The results showed that in the absence of phages, the total number of bacteria increased during the culture period (p<0.001). However, the addition of phages led to a significant decrease in the level of Pseudomonas aeruginosa strains (p=0.024), indicating that the phage mixture has a good bactericidal effect, its antibacterial spectrum is relatively broad, and regardless of the patient's clinical characteristics, the phages will infect bacterial hosts in the sputum environment. In addition to in vitro experiments, the researchers used a mouse pneumonia model to evaluate the efficacy of the newly discovered different phage cocktails [51-52], and the results all showed good efficacy, and the number of bacteria in the mouse lungs was significantly reduced after treatment. However, considering the differences in physiological structure and drug dosage between mice and humans, it is necessary to further verify and use animal models that are closer to human physiological conditions for experiments. After confirming the antibacterial effect of phage cocktail preparations in animal experiments on mice, Antoine Guillon et al.

[53] continued to use a swine pneumonia model that is more similar to the human anatomical structure. In this experiment, a cocktail preparation made of five phages was used to mechanically inhale the pig model of Pseudomonas aeruginosa lung infection. Inhalation phage treatment was performed 2 hours and 11 hours after the piglets were infected with Pseudomonas aeruginosa. 6mL of 1.1x1010PFU / mL phage cocktail preparations were inhaled, and the nebulization time was 14.1±1.9 minutes each time. The number of Pseudomonas aeruginosa and the infectious phage titer in respiratory samples (lung tissue and BAL), blood and urine were measured. The results showed that the bacterial load in the lung lesions of animals treated with phages was reduced by 1.5 logs compared with untreated piglets (p<0.001), and the bacteria in BAL were significantly reduced, proving that inhaled phage therapy can inhibit PA pneumonia with significant efficacy. This result is of great value in converting preclinical research results into clinical trials, and provides an important reference for evaluating the safety and feasibility of clinical trials.

[0088] (4) Clinical trials

[0089] In 2019, Wu Nannan et al.

[54] used phage cocktail therapy (φAb124, φAb121) for intranasal inhalation treatment of secondary Acinetobacter baumannii infection in four critically ill COVID-19 patients. Eventually, two patients were successfully discharged from the hospital, and the remaining patient died of respiratory failure one month after discharge. In another patient, CRAB was eliminated from the body, but he subsequently developed carbapenem-resistant Klebsiella pneumoniae (CRKP) infection and died of respiratory failure 10 days after phage treatment. LAW et al.

[55] reported the successful use of phage-assisted treatment in a 26-year-old cystic fibrosis (CF) patient with multidrug-resistant (MDR) Pseudomonas aeruginosa pneumonia who was awaiting lung transplantation. The patient was given an intravenous cocktail of four phages, with 5 mL of 4×109 PFU / mL phage preparation used each time. After 8 weeks of treatment, the patient's symptoms improved. The patient did not experience recurrence of Pseudomonas pneumonia or exacerbation of CF within 100 days after the end of treatment, and successfully underwent bilateral lung transplantation 9 months later. Aslam et al.

[56] reported the experience of using phage therapy to treat respiratory tract infections in three lung transplant recipients. Among them, the phage cocktail was used to successfully treat two patients with non-CF bronchiectasis and post-lung transplant course, and complicated with refractory resistant Pseudomonas aeruginosa lung infection. The patients received a 4-week course of intravenous (iv) AB-PA01 treatment. Pseudomonas aeruginosa no longer grew in the patient's body within 60 days after the completion of phage therapy. Although the target pathogen was not eradicated, subsequent strains showed improved antibiotic resistance.

[0090] Several phage research centers have issued recruitment notices for patients with drug-resistant bacterial infections or lower respiratory tract infections who cannot be controlled by long-term high-dose antibiotic combination therapy or who cannot tolerate the toxic side effects of antibiotics, attempting to use phage therapy on them, but no results have been reported so far. Phage therapy has not established a standardized management system and has not become a routine treatment for bacterial infections. Although it has shown a sterilization effect in vitro and in animal models, animal experiments are different from the clinical conditions of patients, and further clinical trials are needed to verify its safety and stability. Published literature tends to report successful treatment cases, and no trials have been reported that failed to improve clinical outcomes

[57] . Therefore, more clinical trials are still needed to explore the potential effects and impacts of phage therapy in various situations. These trials should cover a wide range of clinical cases and fully evaluate the adverse reactions and limiting factors that may occur during treatment, so as to more fully understand the clinical application prospects and limitations of phage cocktail therapy.

[0091] Recent preclinical and clinical research evidence confirms the potential of phage therapy in treating lung infections caused by bacteria that are extensively resistant to antibiotics. An increasing number of clinical trials have enhanced the safety of phage therapy, and no serious adverse events due to phage therapy have been reported, proving its safety.

[0092] Phage therapy technology is constantly developing, and research on phage derivatives such as endolysins, lytic enzymes, and engineered phages has also been further deepened. Phage derivatives generally refer to related functional proteins of phages, such as lytic enzymes and depolymerases

[58] . Studies have shown that phage derivatives, such as lytic enzymes, have a wider antibacterial spectrum, and some clinical trials have been conducted to confirm their safety in treatment

[59] . In order to prevent bacteria from developing resistance to phages and enhance the infection ability of phages, the genome of phages can be modified and edited through genetic engineering technology

[60] . It has been reported that phages have been genetically modified and have been verified in vitro and in mouse models, with significant bactericidal effects

[61] . The continuous development of these technologies and methods has promoted the advancement of phage therapy technology, making phage therapy a treatment method that is gradually known to the general public and improving its recognition in the medical community. With the deepening of understanding of phages and the continuous improvement of related technologies, the application prospects of phages in the medical field are becoming more and more broad. Phages will become a powerful tool for dealing with multi-drug resistant lung infections, and even play an important role in the treatment of other infectious diseases. Therefore, the application of phages will gradually expand to more fields and industries, bringing positive and far-reaching impacts to human health.

[0093] Although phages have shown potential effectiveness in the treatment of multidrug-resistant lung infections and other infectious diseases, their widespread application in clinical practice still faces considerable challenges. First, phages are highly specific in infecting bacteria and have a narrow antibacterial spectrum. Therefore, cocktail preparations containing multiple phages are usually required to expand the antibacterial range, which also increases the difficulty and cost of production

[20] . Second, some phages carry virulence genes. When phages enter host bacteria, virulence genes can be continuously expressed during the process of bacterial lysis, thereby producing virulence factors that have adverse effects on the human body. The removal of virulence factors still requires more research

[21] . Third, phage therapy is still an exploratory therapy before its efficacy is clearly demonstrated in future large-scale randomized controlled trials. Further clinical research is needed. Although there are many animal trials on phage therapy, clinical trials on humans are still relatively insufficient. In most regions except for Eastern European countries such as Russia and Poland, phages still lack effective regulatory standards and have not obtained authorization and licenses from government departments. Fourth, there is a lack of phage libraries in Chinese hospitals. The construction of phage libraries is the foundation of phage therapy research

[62] . However, there are few hospitals in my country that have established phage libraries. Currently, the established phage libraries are mainly concentrated in economically developed cities such as Shanghai and Guangdong, which limits the promotion and application of phage therapy. Hospitals at all levels in China should establish phage libraries for different bacterial strains. They need to systematically collect, identify and preserve various phages to meet the treatment needs of different infections.

[0094] In summary, phage therapy for lung infections caused by multidrug-resistant bacteria has been studied in depth in in vitro experiments, animal experiments, and clinical trials. Although phage therapy for multidrug-resistant lung infections still faces many challenges, with the continuous advancement of phage therapy technology, the current difficulties such as production difficulties, toxin removal, insufficient clinical trials, storage difficulties, and lack of hospital phage libraries will be gradually overcome. Therefore, phages have broad prospects in the treatment of multidrug-resistant lung infections.

Claims

1. A method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection, characterized in that: The following steps are involved: S1: Sample collection: After the laboratory receives the samples containing bacteriophages, it will carry out a strict sample quality control process on the received samples, and qualified samples will enter the formal experimental process; S2: Extract the bacteriophages from the sample and perform experimental analysis on them. (1) Phage DNA extraction; (2) Nucleic acid quality inspection; (3) DNA fragmentation; (4) Library quality control; (5) Sequencing on a machine. S3: Extract the bacteriophages from the sample and perform experimental analysis on them. Process 2: (1) Phage DNA extraction; (2) Accounting quality inspection; (3) RNA reverse transcription; (4) DNA fragmentation; (5) Library construction; (6) Library quality control; (7) Sequencing on a machine. S4: Analyze the project results of the extracted phages; S5: Phage result file description: From multiple results, it can be concluded whether the extracted phage can treat multidrug-resistant bacterial lung infections.

2. The method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection according to claim 1, characterized in that: The analysis of the phage project results in S4 includes sequencing data filtering and statistics, genome assembly, genome annotation, gene function annotation, virulence factor analysis and drug resistance analysis.

3. The method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection according to claim 2, characterized in that: The sequencing data filtering and statistics: phage nucleic acid was extracted and library constructed for 3 samples in total, and DNBSEQ-T7 was used for sequencing to ensure the reliability of the subsequent information analysis results. Fastp[1] was used to filter and quality control the obtained raw sequencing data, cut the adapter, remove low-quality reads and reads with a high N ratio, and obtain clean reads.

4. The method for preparing bacteriophage for treating multidrug-resistant bacterial lung infection according to claim 2, characterized in that: The genome assembly: metaSPAdes software was used to perform de novo assembly on clean reads, and different k-mer lengths were selected for testing and the best assembly result was obtained. Then, bwa software was used to align clean reads to the assembled genome sequence for statistical coverage.

5. The method for preparing bacteriophage for treating multi-drug resistant bacterial lung infection according to claim 2, characterized in that: The genome annotation: Prokka was used to annotate the coding genes and tRNA of the genome, and then blastp was used to compare the protein sequence with the NR library to obtain the sequence information of each gene with high similarity in the NR library.

6. The method for preparing bacteriophage for treating multi-drug resistant bacterial lung infection according to claim 2, characterized in that: The gene function annotation: eggNOG-mapper was used to perform functional annotation of genes, which contained annotation information from COG, GO, KEGG, CAZy, BiGG and PFAM databases.

7. The method for preparing bacteriophage for treating multi-drug resistant bacterial lung infection according to claim 2, characterized in that: The virulence factor analysis: the gene sequence was homologously compared with the VFDB database using blast to predict virulence genes.

8. The method for preparing bacteriophage for treating multi-drug resistant bacterial lung infection according to claim 2, characterized in that: The drug resistance analysis: resfinder is used to perform analysis to predict whether there are genes with drug resistance on the genome.

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