Bacteriophage and use thereof

Abstract

A bacteriophage, the accession number of which is GDMCC No: 64029-B1. When allicin and the bacteriophage are used in combination, the two have the effect of synergistically resisting Pseudomonas aeruginosa, and the dosages of the two substances during the combined use are all greatly reduced.

Description

A bacteriophage and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and mainly relates to bacteriophages and applications thereof. Background Art

[0002] The continuous discovery and development of antibiotics has led to a significant decrease in the morbidity and mortality of diseases caused by bacterial infections, which is recognized as one of the most important advances in the history of modern medicine. However, with the widespread use of antibiotics in clinical practice, bacterial resistance has rapidly increased, and multidrug-resistant strains have rapidly increased, posing a huge crisis to global health. Among them, Pseudomonas aeruginosa (PA) is one of the most common pathogens causing refractory lower respiratory tract infections. Currently, antibiotics commonly used to treat PA infections include carbapenems, β-lactams, aminoglycosides, etc. However, in recent years, the emergence of antibiotic resistance and multidrug-resistant strains of PA has been widely reported, which makes the treatment of lower respiratory tract infections caused by PA increasingly challenging. For this reason, PA has been identified by the World Health Organization (WHO) as one of the "urgent priority" pathogens. [1] Therefore, there is an urgent need to develop alternative treatment strategies to antibiotics and apply them rationally in clinical practice.

[0003] Natural antibacterial activity such as allicin has attracted much attention in recent years as a method to combat bacterial resistance. Allicin is a natural compound mainly found in garlic. It is produced by the action of enzymes on a sulfide called allyl sulfide (Alliin) in garlic after it is damaged or cut. It has broad-spectrum antibacterial properties. [2] , showing significant inhibitory effects against Gram-positive and Gram-negative bacteria, as well as fungi. Furthermore, allicin boasts advantages such as a simple structure, easy accessibility, significant biological activity, and resistance to drug resistance. Its antibacterial mechanism, distinct from that of antibiotics, gives allicin a natural advantage in treating infections caused by antibiotic-resistant pathogens. We separately determined the minimum inhibitory concentration (MIC) of allicin against Pseudomonas aeruginosa, and the result was 1.25 mg / mL.

[0004] Finding new antimicrobial agents, reducing the use of antibiotics, and alleviating the pressure caused by the abuse of antibiotics have become problems that are being faced in existing technologies.

[0005] Summary of the Invention

[0006] We discovered a bacteriophage that, when combined with allicin, has a synergistic effect against Pseudomonas aeruginosa, and the dosage of both substances is significantly reduced. Furthermore, in the face of drug-resistant bacteria caused by the current overuse of antibiotics, this combination not only effectively combats infection but also reduces antibiotic use to a certain extent, alleviating the pressure caused by overuse.

[0007] The invention provides a bacteriophage, and the preservation number of the bacteriophage is GDMCC No: 64029-B1.

[0008] The present invention also provides an application of a bacteriophage combined with allicin in a Pseudomonas aeruginosa inhibitor, characterized in that: the inhibitor uses 1 / 1000MIC allicin and 1x10 2 pfu / mL phage combination.

[0009] The bacteriophage provided by the present invention has a broad-spectrum antibacterial effect and can be used in the preparation of bactericides.

[0010] Beneficial effects of the present invention:

[0011] 1. Allicin has no negative effect on the potency of the phage provided by the present invention. When allicin and the phage provided by the present invention are used in combination, they show a significant synergistic effect against Pseudomonas aeruginosa. When used in combination, compared with single use, the amount of phage used is reduced by 1 order of magnitude, and the amount of allicin used is reduced by 3 orders of magnitude.

[0012] 2. The combined use of allicin and the bacteriophage provided by the present invention effectively reduces the dosage of allicin and bacteriophage, while avoiding the large-scale use of antibiotics and reducing the pressure caused by the abuse of antibiotics.

[0013] 3. The bacteriophage provided by the present invention has a broad-spectrum antibacterial effect when used alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1 is a diagram showing the determination results of the minimum inhibitory concentration of allicin in a specific embodiment of the present invention;

[0015] FIG2 is a graph showing the determination results of the minimum inhibitory concentration of bacteriophage in a specific embodiment of the present invention;

[0016] FIG3 is a graph showing the test results of the effect of allicin on bacteriophage in a specific embodiment of the present invention;

[0017] FIG4 is the test data of allicin used alone in the combined effect test in a specific embodiment of the present invention;

[0018] FIG5 is the test data of phage alone in the combined effect test in a specific embodiment of the present invention;

[0019] FIG6 is the test data of the combined use of allicin and bacteriophage in the combined use effect test in a specific embodiment of the present invention.

[0020] Preservation Information

[0021] Bacteriophage Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 is deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, deposit number GDMCC No: 64029-B1, deposit date is November 16, 2023. DETAILED DESCRIPTION

[0022] Reagents: Distilled water, LB medium (Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L, pH adjusted to 7.0 with NaOH), agar powder, MH broth (Beef extract powder 2 g / L, Soluble starch 1.5 g / L, Casein acids hydrolysate 17.5 g / L, pH adjusted to 7.4 ± 0.2 with NaOH)

[0023] Biological materials: Pseudomonas aeruginosa bacterial suspension, Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 (isolated from a mixture of river water and sewage collected in Guangzhou City, Guangdong Province in July 2023).

[0024] Experimental equipment: bacterial constant temperature incubator, biological constant temperature incubation shaker, clean bench, biological safety cabinet, spectrophotometer, high pressure sterilizer, refrigerator, balance, centrifuge.

[0025] Experimental methods:

[0026] 1. Isolation of bacteriophage

[0027] Collect sewage or river water samples, centrifuge at low speed, and filter the supernatant through a 0.22μm filter to obtain a sample solution. Co-culture 10mL of the sample solution with 5mL of triple-strength nutrient broth (LB) and 1mL of the target host bacteria overnight at 37°C in a shaker. After low-speed centrifugation of the co-culture solution, filter the supernatant through a 0.22μm filter to obtain a lysate. Mix 100μL of the host bacteria with 0.7% semi-solid LB agar and evenly spread it on a Petri dish. Spot 5μL of the lysate on the semi-solid surface of a dried Petri dish. Place the dish in a 37°C incubator for 8 hours or overnight. If a clear zone is observed at the spotting location, phage has been isolated.

[0028] 2. Classification and Identification of Bacteriophages

[0029] The genome sequencing of the isolated bacteriophage Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 was performed by MegaGene, and the results are shown in Table 1-1. According to the standards of the International Committee on Taxonomy of Viruses (ICTV), when the genome sequence similarity of two bacteriophages is less than 95%, they should be classified as different species. [3] To assess the classification of phage S1002, we first used NCBI BLASTn [4] Homology analysis of its entire genome was performed. The results showed that Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 shared the highest sequence similarity of 98.04% with Pseudomonas phage KPP25 (Genbank ID: NC_024123) (coverage: 96%, see Tables 1-2 for details). To further analyze the possible classification of Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002, we further used Stretcher, Matcher, Water, and SSEARCH2SEQ from the EMBOSS suite, as well as the ClustalW algorithm from MEGA software, to perform a global pairwise sequence alignment of all known phages in the Kochitakasuvirus genus and S1002. Notably, the sequence similarity values ​​obtained by these methods were lower compared to BLASTn. This may be attributed to the fact that BLASTn primarily focuses on local sequence alignments, which may overestimate the true global similarity. For example, considering the local alignment similarity and coverage between Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 and KPP25, the true global similarity is 94.11%, which exceeds the 5% difference threshold for species classification by ICTV. [5] According to our double sequence alignment results, the genome homology of S1002 phage ranges from 44.9% (with Pseudomonas phage R18, using the EMBOSS Water method, see Table 1 for details) to 94.76% (with Pseudomonas phage KPP25, using the ClustalW method in MEGA, see Table 1 for details). Referring to the classification guidelines of BAVS, viruses with nucleotide sequence similarity exceeding 50% in a population can be classified into the same genus. [3]Given that the genome of S1002 shares 50%–95% similarity with other phages in the genus Kochitakasuvirus, Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 was confirmed to represent a new species of the genus Kochitakasuvirus according to the taxonomic guidelines of BAVS.

[0030] Table 1-2 Pairwise comparison of the genome of Pseudomonas phage A1002 with all full genomes of Kochitakasuvirus genus members using different tools

[0031] 3. Determination of phage titer

[0032] The phage solution was diluted to a certain extent. 100 μL of host bacteria was mixed with 100 μL of the diluted phage and incubated at 37°C for 15 min. The incubation solution was mixed with 0.7% semi-solid LB agar and evenly spread on top of 1.5% solid LB agar pre-spread on the culture dish. The dish was placed in a 37°C constant temperature incubator and cultured for 8 h or overnight. After incubation, several translucent plaques were visible on the culture dish. The best ones were those with 30-300 plaques in the field of view. The titer was calculated as (10 × number of plaques) / dilution.

[0033] 4. Determination of the antibacterial effect of phage combined with allicin

[0034] 1. Reagents, Materials, and Instruments

[0035] The reagents, materials, and instruments used in the determination of the antibacterial effect of the combination of phage and allicin are shown in Table 2.

[0036] Table 2 Materials, reagents and instruments required for the experiment

[0037] 2. Determination method (broth microdilution method)

[0038] Allicin was diluted to 7.5 mg / mL, 3.75 mg / mL, 0.375 mg / mL, 0.0375 mg / mL, 0.00375 mg / mL, and 0.000375 mg / mL, respectively; phage was diluted to 3×104 pfu / mL, 3×103 pfu / mL, 3×102 pfu / mL, and 3×101 pfu / mL, respectively; and host bacteria was diluted to 3×105 cfu / mL. 50 μL each of allicin, phage, and host bacteria were added to each well of a 96-well plate, and the OD600 value was measured. After incubation in a shaking incubator at 37°C and 220 rpm overnight, the OD600 value was measured again. The absorbance change was calculated to determine the inhibitory effect on the bacteria.

[0039] 3. Preliminary Experimental Results

[0040] (1) Determination of the minimum inhibitory concentration of allicin

[0041] After experiments, the experimental test results are shown in Figure 1, and the minimum inhibitory concentration (MIC) of allicin was measured to be 1.25 mg / mL.

[0042] (2) Determination of the minimum inhibitory concentration of phage

[0043] After the experiment, the experimental results are shown in Figure 2. The minimum inhibitory concentration (MIC) of the phage was 1×10 3 pfu / mL.

[0044] (3) Effect of allicin on bacteriophage

[0045] Take phage A1002 and dilute it to 10 -8 , take 100 μL of phage and mix it with 100 μL of allicin, and take 100 μL of phage and mix it with 100 μL of LB as the control group, repeat three times in parallel, incubate at 37℃ for 3 hours, and then spread the plate to determine the titer.

[0046] In this example, a paired sample t-test was used to determine whether allicin had an effect on the phage titer. The results are shown in FIG3 , where LB+P represents the control group, A+P represents the experimental group, and ns represents no significant difference. There was no difference between the groups without and with allicin at the 0.05 significance level, indicating that allicin had no negative effect on the phage titer.

[0047] (4) Phage coverage of different host bacteria

[0048] Refer to the phage isolation method to obtain lysis solution, take phage, use 100 μL host bacteria mixed with 0.7% semi-solid LB agar and evenly spread it on the culture dish; each host bacteria is inoculated in a different culture dish, and 5 μL of lysis solution is dripped on the semi-solid surface of each dried culture dish. The culture dish is placed in a 37°C constant temperature incubator for 8 hours or overnight. If a transparent circle or plaque is observed at the dripping position, it means that the phage has infected the corresponding host bacteria.

[0049] As shown in Table 3, the coverage rate of this phage against host bacteria tested for drug resistance was 75.81%, and the total coverage rate was 80.00%.

[0050] Table 3 Drug resistance and coverage test data of Pseudomonas aeruginosa phage vB_PeaM_GZMU_A1002 against various host bacteria

[0051] 4. Combined Data

[0052] (1) Allicin alone

[0053] In the combined experiment, the antibacterial effect of various concentrations of allicin (i.e. allicin alone) was calculated and analyzed when the phage concentration was 0 pfu / mL.

[0054] As shown in Figure 4, at the lowest allicin concentration of 1.25 mg / mL, the OD600 was approximately 0, achieving an antibacterial effect. Therefore, the MIC of allicin alone was 1.25 mg / mL, consistent with previous experimental results.

[0055] (2) Phage alone

[0056] In the combined experiment, the antibacterial effects of phages at various concentrations (i.e., phages used alone) were calculated and analyzed when the allicin concentration was 0 μg / mL.

[0057] As shown in Figure 5, when the lowest phage concentration was 1×10 3 OD was measured at pfu / mL 600 The MIC of phage alone is 1×10 3 pfu / mL, which is consistent with the previous experimental results.

[0058] (3) Blank control

[0059] In the combined experiment, only MH culture medium and bacterial solution were added to the blank control group.

[0060] Table 4 OD600 changes in the blank control group

[0061] Group 1 was parallel control 1, group 2 was parallel control 2, and group 3 was parallel control 3. The bacteria in the blank control group all grew, proving that the bacterial solution was not contaminated.

[0062] (4) Combination of allicin and bacteriophage

[0063] Using the microbroth dilution method, allicin at different concentrations was mixed with phage and added into a 96-well plate to measure the OD value. 600 After overnight incubation at 37℃ and 220rpm, measure OD again. 600 Values, draw a chart.

[0064] According to Figure 6, at 1 / 1000MIC allicin and 1x10 2 pfu / mL phage at OD 600 The optimal concentration for combined use is 0.2-0.3, and the well is clear and free of precipitation when observed with the naked eye.

[0065] According to the previous experiments and the data of single use, it can be seen that the MIC of allicin when used alone is 1.25 mg / mL and the MIC of bacteriophage is 1×10 3 pfu / mL. The combined effect was determined based on the graded inhibitory concentration (FIC) index. [6] :FIC=MIC(Group A combined) / MIC(Group A alone)+MIC(Group B combined) / MIC(Group B alone). This experiment shows that the FIC of allicin combined with phage is (1 / 1000) / 1+(1×10 2 ) / (1×10 3 )=0.101<0.5, so allicin and bacteriophage have synergistic effect.

[0066] From Figure 6, it can be determined that the combined dose range for synergistic effect is: ≥1.25μg / mL allicin and ≥1×10 2 The best dosage combination was 1.25 μg / mL allicin and 1×10 2 pfu / mL phage.

[0067] FIC index interpretation standard: when the FIC index is less than 0.5, the two drugs have a synergistic effect; when the FIC index is 0.5-1, the two drugs have an additive effect; when the FIC index is greater than 1 and less than 2, the two drugs have an unrelated effect; when the FIC index is greater than 2, the two drugs have an antagonistic effect.

[0068] References:

[0069] [1]Cassandra W.The drug-resistant bacteria that pose the greatest health threats[J].Nature,2017.543:15.

[0070] [2]Ankri,Mirelman D.Antimicrobial properties of allicin from garlic. Microbes and Infection,1999.1(2),125–129.

[0071] [3]Adriaenssens E,Brister JR.How to Name and Classify Your Phage:An Informal Guide.Viruses.2017 Apr 3;9(4):70.

[0072] [4]Johnson,M.;Zaretskaya,I.;Raytselis,Y.;Merezhuk,Y.;McGinnis,S.;Madden,T.L.NCBI BLAST:A better web inter face.Nucleic Acids Res.2008,36,5–9.

[0073] [5]de Melo ACC,da Mata Gomes A,Melo FL,Ardisson-Araújo DMP,de Vargas APC,Ely VL,Kitajima EW,Ribeiro BM,Wolff JLC.Characterization of a bacteriophage with broad host range against strainsof Pseudomonas aeruginosa isolated from domestic animals.BMCMicrobiol.2019 Jun 17;19(1):134.

[0074] [6]Patil SV,Hajare AL,Patankar M,Krishnaprasad K.In VitroFractional Inhibitory Concentration(FIC)Study of Cefixime andAzithromycin Fixed Dose Combination(FDC)Against RespiratoryClinical Isolates.J Clin Diagn Res.2015 Feb;9(2):DC13-5.

Claims

1. A bacteriophage, whose deposit number is GDMCC No: 64029-B1.

2. The use of the bacteriophage combined with allicin in a Pseudomonas aeruginosa inhibitor as claimed in claim 1, characterized in that: The inhibitor used ≥1.25μg / mL allicin and ≥1×10 2 pfu / mL phage combination.

3. The use of the bacteriophage combined with allicin in a Pseudomonas aeruginosa inhibitor as claimed in claim 1, characterized in that: The inhibitor used 1 / 1000MIC allicin and 1x10 2 pfu / mL phage combination.

4. Use of the bacteriophage as claimed in claim 1 in an antibacterial agent.

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