A new type of Acinetobacter baumannii phage and its application

Abstract

The embodiments of the present application relate to a new type of Acinetobacter baumannii phage and its application, which belongs to the field of microbial technology. The new Acinetobacter baumannii phage of the embodiment of the present application is named bacteriophage HD01, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on September 23, 2024, with a deposit number of GDMCC No: 65174-B1, and a taxonomic name: Acinetobacter baumanniiphage. The bacteriophage HD01 provided in the embodiment of the present application can not only enrich the germplasm resource bank of Acinetobacter baumannii phage, but also provide an optional basic material for the biological control of Acinetobacter baumannii; bacteriophage HD01 has good temperature tolerance and acid-base tolerance; in medical environments where drug-resistant bacteria are prevalent and common disinfectants are ineffective, bacteriophage HD01 has a high safety factor and a wide range of applications in the preparation of phage disinfectants or phage preparations for medical device surfaces, hospital environments and other environments, and has good application prospects.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of microbial technology, and in particular to a novel Acinetobacter baumannii phage and its application. Background Art

[0002] Acinetobacter baumannii (Ab) is a non-fermenting, oxidase-negative, catalase-positive Gram-negative bacterium that is widely found in water, soil and hospital environments in nature. It is a major conditional pathogen of hospital-acquired infection, which can cause respiratory tract, urinary tract and wound infections in hospitalized patients, and is also a pathogen that causes burn infections. The bacterium is widely distributed in the hospital environment and can survive for a long time, which can easily cause infection in critically ill patients. It is often isolated from specimens such as blood, urine, pus and respiratory secretions of infected patients. As one of the pathogens of ESKAPE (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter), Acinetobacter baumannii is identified as a global threat because it can quickly acquire multidrug resistance. Nowadays, Acinetobacter baumannii is resistant to most common clinical antibiotics. Therefore, we urgently need to develop new antibiotics and other alternative therapies to face the serious threat posed by clinical Acinetobacter baumannii infections.

[0003] As a unique biological entity, bacteriophages play an important role in the fields of microbiology and biotechnology. As a virus, it specifically infects and replicates in bacterial and archaeal cells, and completes its life cycle by injecting its genome into the host cytoplasm. Bacteriophages can specifically infect and lyse host bacteria, including a variety of drug-resistant strains. This ability is based on the specific recognition mechanism formed by long-term evolution between phages and bacteria. Therefore, phage therapy, as an alternative or auxiliary therapy, is highly expected to be used to combat drug-resistant bacterial infections. The application of phages in dealing with clinical drug-resistant strains has become a research hotspot in the biomedical field in recent years.

[0004] Phages have been used to treat and prevent bacterial diseases in the early days. In 1919, phages were first licensed for the treatment of human diseases. In recent years, due to the prevalence of multi-antibiotic resistant strains, the research and application of phages have once again attracted much attention. For example, phage display technology, phage cocktails, and phage-assisted continuous evolution technology, although they have not yet been widely used in clinical practice, research on phage treatment of Acinetobacter baumannii infection has shown its feasibility and advantages. Most of the reported treatments of Acinetobacter baumannii phages are mainly based on mice as animal models. Studies have shown that Acinetobacter baumannii phages can effectively control the infection of Acinetobacter baumannii, and the treatment effect is significant, indicating that phages are expected to become new antibacterial agents for the treatment of Acinetobacter baumannii infection. However, before being applied to clinical treatment, the mechanism of phage therapy has not been fully explored, and phage therapy still needs to overcome many difficulties. Summary of the invention

[0005] In view of this, the embodiments of the present application provide a novel Acinetobacter baumannii phage and its application. The present application isolates and identifies a phage that can strongly lyse Acinetobacter baumannii, and studies its biological characteristics and genomic information, in order to lay a foundation for the practical application of phages, which is of great significance for understanding phage therapy and overcoming the limitations of phage therapy, and can effectively overcome the defects of the above-mentioned prior art.

[0006] The first aspect of the embodiment of the present application provides a new Acinetobacter baumannii phage, named phage HD01, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 23, 2024, with the deposit number GDMCC No: 65174-B1, and the taxonomic name: Acinetobacter baumanniiphage.

[0007] In some embodiments that may include the above embodiments, the phage HD01 is a long-tailed phage with a polyhedral head; the titer number level is ≥10 10 The growth latent period is 80 minutes, the rapid growth period is 80 to 180 minutes, the temperature stability range is 4°C to 37°C, and the pH stability range is 6 to 9.

[0008] In some embodiments, which may include the above embodiments, the sequence of the genomic DNA of bacteriophage HD01 is shown as SEQ ID NOs: 1-7.

[0009] The second aspect of the embodiments of the present application also provides a drug or biological preparation for treating infectious diseases caused by Acinetobacter baumannii, including the above-mentioned novel Acinetobacter baumannii phage.

[0010] In some embodiments, which may include the above embodiments, the drug or biologic contains bacteriophage HD01 as the only active ingredient.

[0011] In some embodiments, which may include the above embodiments, the Acinetobacter baumannii includes Acinetobacter baumannii ATCC 17978.

[0012] The third aspect of the present application also provides the use of the novel Acinetobacter baumannii phage described above in drugs or biological preparations for treating Acinetobacter baumannii infectious diseases, such as lysate, phage pharmaceutical preparations, compositions, and combinations of phages and antibiotics.

[0013] The fourth aspect of the present application also provides the use of the novel Acinetobacter baumannii phage described above in the preparation of phage disinfectants, phage inhibitors or phage sprays for sterilization and disinfection of medical equipment environments, animal breeding environments and other environments.

[0014] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0015] 1. This application uses Acinetobacter baumannii ATCC 17978 as the host, samples are taken from the stream water near the hospital, and phage HD01 is screened out. This phage can efficiently lyse Acinetobacter baumannii ATCC 17978, providing a phage source for the hospital to treat Acinetobacter baumannii infection;

[0016] 2. Bacteriophage HD01 can not only enrich the germplasm resource bank of Acinetobacter baumannii phage, but also provide an alternative basic material for biological control of Acinetobacter baumannii;

[0017] 3. Bacteriophage HD01 has good temperature tolerance and acid-base tolerance; and bacteriophage HD01 can show obvious lysis effect on Acinetobacter baumannii within 7 hours, showing good therapeutic effect and application prospects;

[0018] 4. The whole genome detection of bacteriophage HD01 showed that it had no virulence genes, which ensured the safety and reliability of the phage preparation during use. The open reading frame encoding endolysin was found in the protein function annotation results, which ensured the effectiveness of the phage preparation;

[0019] 5. In medical environments where drug-resistant bacteria are prevalent and common disinfectants are ineffective, bacteriophage HD01 has a high safety factor and a wide range of applications in the preparation of bacteriophage disinfectants or bacteriophage preparations for medical device surfaces, hospital environments and other environments, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The phage plaque image of Acinetobacter baumannii phage HD01 of the present application example;

[0022] Figure 2 The genome map of Acinetobacter baumannii phage HD01 of the present application example;

[0023] Figure 3 This is the biological evolutionary tree analysis of Acinetobacter baumannii phage HD01 in the embodiment of the present application;

[0024] Figure 4 This is a transmission electron micrograph of the Acinetobacter baumannii phage HD01 of the present application example;

[0025] Figure 5 This is the lysis kinetics curve of Acinetobacter baumannii phage HD01 of the present application example;

[0026] Figure 6 This is the optimal infection multiplicity diagram of Acinetobacter baumannii phage HD01 according to the embodiment of the present application;

[0027] Figure 7 This is the one-step growth curve of Acinetobacter baumannii phage HD01 of the present application example;

[0028] Figure 8 This is a temperature stability diagram of Acinetobacter baumannii phage HD01 according to an embodiment of the present application;

[0029] Fig. 9 This is a pH stability graph of Acinetobacter baumannii phage HD01 according to an example of the present application. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0031] Unless otherwise specified, the experimental methods used in the examples of this application are all conventional methods.

[0032] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0033] The Acinetobacter baumannii phage isolated in the example of the present application is named phage HD01 and has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on September 23, 2024. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with a storage number of GDMCC No: 65174-B1 and a taxonomic name: Acinetobacterbaumanniiphage.

[0034] Example

[0035] 1. Preparation of culture medium

[0036] (1) LB solid medium: add 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, and 15 g agar powder to 1 L ultrapure water, fully dissolve and sterilize at high temperature and high pressure for later use.

[0037] (2) LB liquid culture medium: add 5 g yeast powder, 10 g trypsin, and 10 g sodium chloride to 1 L ultrapure water, fully dissolve, and sterilize at high temperature and high pressure for later use.

[0038] 2. Isolation and purification of Acinetobacter baumannii phage

[0039] Acinetobacter baumannii ATCC 17978 was used as the host bacteria. A single colony was inoculated into LB liquid culture medium and cultured at 37°C until the logarithmic phase. The stream water next to the hospital was centrifuged at 4000rpm for 5 minutes, the supernatant was filtered with a 0.22μm filter membrane, the treated water was mixed with the logarithmic phase host bacteria, and cultured at 37°C overnight. After the culture was completed, the mixed solution was centrifuged at 4000rpm for 5 minutes, the supernatant was filtered with a 0.22μm sterile filter, and the filtrate was obtained as the phage stock solution. The logarithmic phase bacterial solution was mixed with the phage stock solution, and then the mixed solution was quickly mixed with the LB solid culture medium cooled to an appropriate temperature, and the LB solid double-layer plate was poured and cultured at 37°C overnight. If a clear and bright plaque is formed, it proves that the phage has been successfully isolated; otherwise, there is no plaque.

[0040] If plaques appear on the plate, pick a single, large and transparent plaque and inoculate it into the host bacterial solution in the logarithmic phase. After cultivation, dilute it and purify it again using the double-layer plate method for 3-4 times until plaques of uniform size and shape are formed. This is the purified phage. Pick a single plaque and inoculate it into the host bacterial solution in the logarithmic growth phase, expand the culture, mix the phage bacterial solution with 60% glycerol in equal amounts to obtain the phage preservation solution (final concentration 30% glycerol), and store it at -80°C for later use. The results are as follows Figure 1 The phage can form clear plaques on the double-layer plate with no halo around and clear and regular edges. It is a baumannii phage.

[0041] 3. Extraction and sequencing of bacteriophage HD01 genome

[0042] Transfer the phage culture solution treated with 0.5% chloroform to a centrifuge tube, centrifuge at 8000g for 10min, and remove the precipitated bacterial debris. Add 5μLRNaseA and 10μLDNase I to 10mL supernatant, mix thoroughly and incubate at 37℃ for 30min. Add 4mL phage precipitation solution to the supernatant, shake until dissolved, and ice bath for 1h or overnight at 4℃. Centrifuge at 10000g for 20min at 4℃ and discard the supernatant. Add 1mL SM buffer to fully wash the tube wall and precipitate, transfer to a new centrifuge tube, add 40μL phage lysis solution, and incubate at 68℃ for 15min. Add an equal volume of protein removal solution, mix gently, centrifuge at 12000g for 5min, take the supernatant to a new centrifuge tube, add an equal volume of pre-cooled phage rinse solution, mix gently, incubate at -20℃ for 1h, centrifuge at 12000g for 10min at 4℃, and discard the supernatant. Add an appropriate amount of 70% ethanol solution and mix well. Centrifuge at 8000g for 8 min at 4°C, discard the supernatant, and repeat the washing step once. Dry the DNA naturally at room temperature, add an appropriate amount of TE buffer, and store at -20°C.

[0043] The extracted whole genome DNA samples of bacteriophage HD01 were transported to Shanghai Painosen Biotechnology Co., Ltd. for sequencing. The sequencing method was the second-generation sequencing technology based on the Illumina Miseq high-throughput sequencing platform. The specific steps are: the extracted total phage DNA was amplified and deeply sequenced using Illumina sequencing technology, and the Illumina PE library was constructed. The obtained reads were first assembled from scratch using A5-MiSeq and SPAdes to remove the sequencing data of the adapter sequence and construct contigs; the sequences were extracted according to the sequencing depth of the spliced ​​sequences, and the sequences with high sequencing depth were blastn-aligned with the NT library on NCBI, the viral genome sequences of each splicing result were selected, and gene function annotations were performed. BRIG was used to construct a gene circle map for HD01. The results are as follows: Figure 2The DH01 genome size is 56791bp (the sequence of Acinetobacter baumannii phage HD01 is as shown in SEQ ID NO: 1-7, the entire sequence is SEQ ID NO: 1-7 connected in sequence, and since the sequence is too long, it is divided into 7 segments including SEQ ID NO: 1-7), the GC content is 41.47%, and the genome does not contain antibiotic resistance genes and virulence factors. In addition, phage HD01 has a total of 81 protein-coding genes, including 40 hypothetical proteins and 41 known functional proteins.

[0044] 4. Phage tree analysis of bacteriophage HD01

[0045] The genome of phage HD01 was uploaded through VipTree and the proteomic evolutionary tree was constructed using the default settings. In addition, based on the tBLASTx results, VipTree was used to generate the genome of phage HD01 and selected similarity scores greater than 0.05 (S G >0.05) to construct a proteome evolutionary tree. The results are as follows Figure 3 The results showed that phage HD01 was closely related to phage vB_AcoS-R7M of Alteromonas and phage MiCath of Pseudomonas, but distantly related to other phages. From the perspective of evolutionary distance, it was a new species of phage.

[0046] 5. Observation of morphological characteristics of bacteriophage HD01

[0047] The morphology of bacteriophages was observed by uranyl acetate staining. 10 μL of bacteriophage sample was placed on a carbon-coated copper grid for absorption for 10 min, then stained with uranyl acetate (PH 6.5, 3%) for 3 min, and observed and photographed using a transmission electron microscope. Figure 4 As shown, the phage HD01 is a long-tailed phage with a polyhedral head with a head diameter of approximately 64 nm and a tail length of approximately 138 nm.

[0048] VI. Lysis Kinetics of Bacteriophage HD01

[0049] Pick a single plaque and inoculate it at OD 600 The enriched solution was centrifuged at 4000 rpm for 5 min and filtered through a 0.22 μm pore size sterile filter to obtain the phage HD01 filtrate. The overnight cultured Acinetobacter baumannii solution was diluted to its OD 600 The ratio of 0.1 was 0.1, and the bacterial solution of Acinetobacter baumannii and the HD01 phage solution were added to a 96-well plate. The bacterial solution of Acinetobacter baumannii was used as a control, and the LB medium was used as a blank. Each well was repeated at least 3 times, and the kinetics was measured for 20 hours. The results are shown in Figure 5 After 24 hours of culture, the OD of the bacterial solution containing HD01 was600 The OD value was significantly lower than that of the bacterial solution containing only Acinetobacter baumannii within 8 hours. 600 , which indicates that bacteriophage HD01 can effectively inhibit the growth of host bacteria.

[0050] VII. Determination of the titer of bacteriophage HD01

[0051] Pick a single plaque HD01 and inoculate it at OD 600 The enriched solution was centrifuged at 4000rpm for 5 minutes and filtered with a 0.22μm pore size sterile filter to obtain the phage HD01 filtrate. The HD01 filtrate was diluted 10 times with LB medium, and the logarithmic phase Acinetobacter baumannii solution was mixed with the HD01 phage solution of each dilution in equal proportions, and then mixed with the solid medium and poured into LB double plates, placed at 37℃ for culture, and the titer was calculated according to the following formula. After calculation, the titer of HD01 was 8×10 11 PFU / mL.

[0052] Titer (PFU / mL) = number of plaques × dilution factor × 10.

[0053] 8. Determination of the optimal MOI of bacteriophage HD01

[0054] A single colony of Acinetobacter baumannii was inoculated into LB liquid medium and cultured at 37°C until the logarithmic phase. It was diluted 10 times in a gradient manner, and 100 μL of the Acinetobacter baumannii dilution was plated for overnight culture, and the number of colonies on the plate was calculated. Acinetobacter baumannii was cultured until OD 600 is 0.1, at which point the concentration of Acinetobacter baumannii is 1×10 8 CFU / mL, HD01 was diluted to the corresponding concentration according to the infection multiplicity MOI of 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001, and the HD01 dilution and the Acinetobacter baumannii dilution were mixed in LB in equal proportions and cultured at 37°C for 5 hours. The culture solution was centrifuged and filtered to obtain the filtrate. After appropriate dilution, the phage titer was determined using the double-layer plate method. The highest titer ratio was used as the optimal phage infection multiplicity. Repeat 3 times. The results are as follows Figure 6 , HD01 phage titer dilution was 1×10 7 PFU / mL, the number of Acinetobacter baumannii bacteria is 1×10 8 CFU / mL, the optimal infection multiplicity MOI of HD01 is 0.1, at which the phage titer is 1×10 9 PFU / mL.

[0055] IX. One-step growth curve of bacteriophage

[0056] A single colony of Acinetobacter baumannii was inoculated into LB and cultured at 37°C until the logarithmic phase. HD01 and Acinetobacter baumannii were diluted and mixed in equal proportions according to the optimal multiplicity of infection MOI of 0.1 and allowed to stand for 2 minutes to allow the phage to fully adsorb to the bacterial surface. The mixture was centrifuged at 4000rpm for 5 minutes, and the phage supernatant that failed to adsorb was removed. 1mL of LB was added to wash and resuspended twice. 1mL of the resuspension was added to LB and gently pipetted to mix. The mixture was cultured at 37°C with shaking and timing. Samples were taken at different time points. After appropriate dilution, the phage titer was determined using the double-layer plate method. A one-step growth curve was drawn with titer as the ordinate and culture time as the abscissa, and the results were repeated 3 times. Figure 7 As shown, the incubation period of bacteriophage HD01 was 80 min, the burst period was 100 min, and then it entered a stable period.

[0057] 10. Effect of temperature on phage stability

[0058] The phage solution with determined titer was added to a centrifuge tube and incubated at 4°C, 37°C, 40°C, 50°C, 60°C, 70°C and 80°C for 1 hour. After appropriate gradient dilution, the phage titer was determined by the double-layer plate method. With temperature as the horizontal axis and phage titer as the vertical axis, GraphPad Prism9 was used to draw a curve of the effect of temperature on phage stability. Repeat 3 times. The results are as follows Figure 8 The optimum temperature of HD01 is 4℃~37℃. Low temperature has little effect on the phage titer. When the temperature is higher than 40℃, the phage titer is significantly reduced.

[0059] XI. Effect of pH on phage stability

[0060] Use 1 mol / L HCl and 0.1 mol / L NaOH to adjust the LB liquid culture medium to 11 different pH values, namely 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0. The phage with a determined titer was mixed with LB of different pH values, incubated at 37°C for 1 hour, and the phage titer was determined using the double-layer plate method after appropriate gradient dilution. GraphPad Prism 9 was used to draw a curve of the effect of temperature on phage stability, with pH as the horizontal axis and phage titer as the vertical axis. Repeat 3 times. The results are as follows: Fig. 9 HD01 can maintain good activity at pH 6-9 and has a wide tolerance range.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A novel Acinetobacter baumannii phage, characterized in that: The name is bacteriophage HD01, which was deposited in Guangdong Microbiological Culture Collection Center on September 23, 2024, with the deposit number GDMCC No: 65174-B1, and the taxonomic name is: Acinetobacter baumannii phage; Phage HD01 is a long-tailed phage with a polyhedral head; the titer level is ≥10 10 , the growth latent period is 80 min, the rapid growth period is 80-180 min, the temperature stability range is 4 ℃-37 ℃, and the pH stability range is 6-9; The sequence of the genomic DNA of bacteriophage HD01 is shown in SEQ ID NOs: 1-7.

2. A drug or biological preparation for treating infectious diseases caused by Acinetobacter baumannii, comprising a novel Acinetobacter baumannii phage according to claim 1.

3. The drug or biological preparation according to claim 2, characterized in that: The drug or biological preparation uses bacteriophage HD01 as the only active ingredient.

4. The drug or biological agent according to claim 2, characterized in that: The Acinetobacter baumannii includes Acinetobacter baumannii ATCC 17978.

5. Use of the novel Acinetobacter baumannii phage according to claim 1 in the preparation of a medicine or biological preparation for treating Acinetobacter baumannii infectious diseases.

6. Use of the novel Acinetobacter baumannii phage according to claim 1 in the preparation of a phage disinfectant, a phage inhibitor or a phage spray.

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