CN114015661A - Culture medium and method for improving titer of phage - Google Patents
Culture medium and method for improving titer of phage Download PDFInfo
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- CN114015661A CN114015661A CN202111521551.9A CN202111521551A CN114015661A CN 114015661 A CN114015661 A CN 114015661A CN 202111521551 A CN202111521551 A CN 202111521551A CN 114015661 A CN114015661 A CN 114015661A
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- 238000000034 method Methods 0.000 title claims abstract description 10
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- 239000002609 medium Substances 0.000 claims abstract description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 9
- 239000000600 sorbitol Substances 0.000 claims abstract description 9
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- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 8
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- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 claims abstract description 7
- 229960002413 ferric citrate Drugs 0.000 claims abstract description 7
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims abstract description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- 239000011670 zinc gluconate Substances 0.000 claims abstract description 7
- 235000011478 zinc gluconate Nutrition 0.000 claims abstract description 7
- 229960000306 zinc gluconate Drugs 0.000 claims abstract description 7
- 239000001888 Peptone Substances 0.000 claims abstract description 6
- 108010080698 Peptones Proteins 0.000 claims abstract description 6
- 235000015278 beef Nutrition 0.000 claims abstract description 6
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000000284 extract Substances 0.000 claims abstract description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 6
- 235000019319 peptone Nutrition 0.000 claims abstract description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 239000012138 yeast extract Substances 0.000 claims abstract description 6
- 238000009630 liquid culture Methods 0.000 claims description 10
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- 241000588724 Escherichia coli Species 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
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- 241000186361 Actinobacteria <class> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
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- 101710146873 Receptor-binding protein Proteins 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
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- 239000007864 aqueous solution Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00051—Methods of production or purification of viral material
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a culture medium and a method for bacteriophage. A medium for increasing phage titer, comprising per 1000mL of liquid medium: 5.0g of peptone, 3.0g of beef extract, 2.0g of yeast extract powder, 5.0g of sodium chloride, 1.5g +/-0.12 g of oligosaccharide, 0.18g +/-0.015 g of ferric citrate, 0.39g +/-0.02 g of magnesium sulfate, 0.55g +/-0.05 g of calcium chloride, 2.9g +/-0.23 g of zinc gluconate, 0.54g +/-0.03 g of sorbitol, 0.018g +/-0.002 g of disodium hydrogen phosphate and 0.0036g +/-0.0004 g of potassium dihydrogen phosphate. The culture medium provided by the invention can improve the titer of the phage, and the phage produced by fermentation by using the culture medium has the advantages of easy storage at normal temperature or low temperature, long storage period, slow titer reduction and the like, greatly reduces the storage and transportation cost of the phage, and is beneficial to industrial production and application of the phage.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a culture medium and a method for bacteriophage.
Background
With the widespread use of antibiotics, the emergence and prevalence of drug-resistant bacterial strains (including multi-drug-resistant strains) has become a serious challenge to the efficacy of antibiotic therapy. In recent years some "superbacteria" have emerged, which are insensitive to almost all antibiotics, resulting in human infection with such bacteria with little drug availability, and a great need for new anti-infective weapons to replace antibiotics has arisen. In this context, humans are again looking for phage therapy. The research on phage therapy in the scientific community has entered a new phase. Scientists in various countries have begun to look for phages for various pathogenic bacteria, and a large number of phages have been discovered. With the continuous update of sequencing technology, the bacteriophage is easier to be identified, the new bacteriophage genome sequence is continuously increased, and the new bacteriophage family is continuously grown.
The bacteriophage is a natural enemy of microbes such as bacteria, fungi, actinomycetes and the like, widely exists in nature, is large in quantity and various in variety, and is an ideal natural antibacterial agent and antibiotic substitute at present.
Currently, the basic and applied research range for bacteriophages is wide, involving various types of in vitro infection control models and various infectious diseases. Not only aims at preventing and treating human diseases, but also relates to the elimination of pathogenic bacteria in food and feed and the prevention and control of bacterial biofilms. The overall results of the study show that the effect of the treatment is generally positive as long as the phage used is sensitive to the infecting strain. In recent years, phage therapy research has been supported and encouraged by national policies, and a series of products have entered the market
With the recognition of the advantages of phage therapy, phage therapy is widely recognized, and more research is focused on the development of phage products, and the industrialization of phage is undoubtedly the booster of phage therapy. With the continuous marketing of phage products at home and abroad, more and more phage products are prepared to be applied to industries such as food, medicine and the like.
The phage products play an increasingly important role in the food and drug industry, the demand of the phage is higher and higher along with the promotion of the phage industrialization, and the phage multiplication is the key of the phage industrialization. Exploring a high-efficiency phage fermentation process is also an important measure for reducing the cost of phage products. At present, the research on the phage mainly focuses on the aspects of biological characteristics, genomics and proteomics, and few researches on the preparation conditions of phage multiplication production fermentation parameters, large-scale production and the like are carried out.
Disclosure of Invention
The invention aims to provide a phage culture medium, which is beneficial to industrial application of phage by optimizing components of the culture medium to obtain a phage culture with higher titer.
The technical scheme adopted by the invention is as follows: a medium for increasing phage titer, comprising per 1000mL of liquid medium: 5.0g of peptone, 3.0g of beef extract, 2.0g of yeast extract powder, 5.0g of sodium chloride, 1.5g +/-0.12 g of oligosaccharide, 0.18g +/-0.015 g of ferric citrate, 0.39g +/-0.02 g of magnesium sulfate, 0.55g +/-0.05 g of calcium chloride, 2.9g +/-0.23 g of zinc gluconate, 0.54g +/-0.03 g of sorbitol, 0.018g +/-0.002 g of disodium hydrogen phosphate and 0.0036g +/-0.0004 g of potassium dihydrogen phosphate.
The liquid culture medium uses water as a solvent, and the pH value is 7.5.
The invention also provides a method for improving the titer of the phage, which comprises the following steps:
inoculating the phage and its corresponding host bacteria into the above culture medium, and culturing at 37 deg.C by shaking.
The invention adds oligosaccharide, sorbitol and calcium chloride (CaCl) with different concentrations into the fermentation liquor of common culture medium2) Magnesium sulfate (MgSO)4) Zinc gluconate (C)12H22O14Zn), ferric citrate, etc. to optimize the phage culture condition. Ca2 +、Mg2+The divalent cations not only have the effects of stabilizing the DNA spiral structure of the bacteriophage and improving the adsorption rate of the bacteriophage to host bacteria, but also enhance the activities of bacteriophage lyase and receptor binding protein and improve the lytic force of the bacteriophage to the host bacteria. Oligosaccharide and sorbitol effects: enhancement of microorganismsThe utilization rate of the carbon source is promoted, and the metabolism of host bacteria and phage is promoted. Sorbitol can also complex metal cations, improving the utilization rate of the phage to the cations.
The culture medium for improving the titer of the phage provided by the invention can improve the titer of the phage, and the phage produced by fermentation by using the culture medium has the advantages of easy storage at normal temperature or low temperature, long storage period, slow titer reduction and the like, so that the storage and transportation cost of the phage is greatly reduced, and the culture medium is favorable for industrial production and application of the phage.
Detailed Description
In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to specific examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The host bacteria used in the invention are escherichia coli standard strain EC-ATCC 25922, salmonella enteritidis standard strain SA-ATCC 14028 and klebsiella pneumoniae KP-ATCC 10031. The standard strain was purchased from the Guangdong province culture Collection of microorganisms. The phages corresponding to the above-mentioned phages are RDP-EC-20123 (preservation number: CGMCC NO.22486, patent application number: 202110894071.0), RDP-SA-20032 (preservation number: CGMCC NO.21994, patent application number: 202110540869.5) and RDP-KP-20004 (preservation number: CGMCC NO.21407, patent application number: 202011599621.8). The three phages are gifted by Rieke Union (Qingdao) bioengineering Co.
Example 1 this example provides a medium for increasing phage titer, which comprises 5.0g of peptone, 3.0g of beef extract, 2.0g of yeast extract, 5.0g of sodium chloride, 1.5g of oligosaccharide, 0.18g of ferric citrate, 0.39g of magnesium sulfate, 0.55g of calcium chloride, 2.9g of zinc gluconate, 0.54g of sorbitol, 0.018g of disodium hydrogen phosphate, and 0.0036g of potassium dihydrogen phosphate per 1000mL of aqueous solution.
The components are dissolved in water and stirred uniformly, the pH value is adjusted to 7.5, and the culture medium is prepared after autoclaving. The culture medium can be a liquid culture medium, or agar powder is added to prepare a solid plate culture medium.
To investigate the effect of the media provided by the present invention on the growth of phages and their hosts, the present invention provides the validation, comparative experiments described in examples 2-5.
Example 2 Effect of the Medium provided by the invention on host growth
5.0g of peptone, 3.0g of beef extract, 2.0g of yeast extract powder, 5.0g of sodium chloride, 1.5g of oligosaccharide, 0.18g of ferric citrate, 0.39g of magnesium sulfate, 0.55g of calcium chloride, 2.9g of zinc gluconate, 0.54g of sorbitol, 0.018g of disodium hydrogen phosphate and 0.0036g of potassium dihydrogen phosphate are respectively weighed and dissolved in 1L of water, the pH value is adjusted to 7.5, and the mixture is sterilized to prepare a liquid culture medium. Meanwhile, 1L of liquid culture medium is additionally prepared, 15g of agar powder is added, high-pressure steam sterilization is carried out for 20min, and after the temperature is reduced to 50 ℃, the liquid culture medium is poured into a flat plate to prepare the culture medium solid plate for later use.
Respectively inoculating 1 strain of Escherichia coli EC-ATCC 25922, 1 strain of Salmonella SA-ATCC 14028 and 1 strain of Klebsiella KP-ATCC 10031 into 100mL of the culture medium, simultaneously inoculating each group into 100mL of common commercial LB culture medium, and culturing at 37 ℃ and 180r/min for 12 h.
And respectively counting the pathogenic bacteria cultured by different fermentation liquids by using different solid culture media. The results are shown in Table 1.
TABLE 1 enumeration of pathogens cultured on different media with different fermentation broths
The results show that the difference of the colony numbers of the pathogenic bacteria cultured by the liquid culture medium and the LB liquid culture medium is not large, and the colony counting of the same fermentation liquid by the solid culture medium and the LB solid culture medium is also small, which indicates that the culture medium has no promotion effect or inhibition effect on the growth and the propagation of the host bacteria.
Example 3 Effect of the liquid, solid Medium of the invention on phage titer
The experiment is carried out in groups, and 1 colibacillus phage, 1 salmonella phage and 1 klebsiella phage RDP-EC-20123, RDP-SA-20032 and RDP-KP-20004 are used. The corresponding hosts EC-ATCC 25922, SA-ATCC 14028 and KP-ATCC 10031 are inoculated in 100mL of liquid culture medium provided by the invention, each group is simultaneously inoculated in common LB culture medium on the market, and the culture is carried out for 8h under the conditions of 37 ℃ and 180 r/min.
Each group takes different solid culture mediums as the lower layer and semi-solid culture medium as the upper layer, and the double-layer plate method is adopted to measure the titer of the phage. The results are shown in Table 2.
TABLE 2 Effect of different media on phage titer
The results show that the phage titer of the liquid culture medium fermentation broth is obviously higher than that of the LB culture medium fermentation broth, which indicates that the phage titer can be obviously improved by using the culture medium as the fermentation broth.
EXAMPLE 4 Effect of the Medium of the invention on the fermentation in scale-up
Preparing 300L of the culture medium of the invention in a 500L fermentation tank, 1500.0g of peptone, 900.0g of beef extract, 600.0g of yeast extract powder, 1500.0g of sodium chloride, 450.0g of oligosaccharide, 54.0g of ferric citrate, 117.0g of magnesium sulfate, 165.0g of calcium chloride, 870.g of zinc gluconate, 162.0g of sorbitol, 5.5g of disodium hydrogen phosphate, 1.08g of potassium dihydrogen phosphate and 30L of water, adjusting the pH value to 7.5, and autoclaving at 120 ℃.
The phages RDP-EC-20123, RDP-SA-20032 and RDP-KP-20004 and the corresponding hosts EC-ATCC 25922, SA-ATCC 14028 and KP-ATCC 10031 are respectively inoculated into 500L of the culture medium fermentor and LB culture medium fermentor of the invention according to the optimal multiplicity of infection of 0.1.
TABLE 3 Effect of different media on phage amplification fermentation
The results show that the bacteriophage titer of the culture medium is obviously higher than that of an LB culture medium when the culture medium is compared with the LB culture medium, so that the bacteriophage titer can be obviously improved when the culture medium is used for fermentation broth amplification culture, and the culture medium is suitable for amplification culture of bacteriophage.
Example Effect of Long-term storage at 54 ℃ on the titer of phages cultured in the Medium of the invention
According to the preparation method of the culture medium, the phages RDP-EC-20123, RDP-SA-20032, RDP-KP-20004 and the corresponding hosts EC-ATCC 25922, SA-ATCC 14028 and KP-ATCC 10031 are inoculated into 100mL of the culture medium according to a certain proportion, each group is simultaneously inoculated into a common LB culture medium in the market, and the culture is carried out for 8 hours at 37 ℃ under the condition of 180 r/min.
And (3) measuring the titer of the phage fermentation liquid by a double-plate method. The phage fermentation liquid is stored in a refrigerator at 4 ℃ after being centrifugally filtered and sterilized. And measuring the titer of the phage again after 60 days, and verifying the influence of fermentation liquor of different culture media on the preservation titer of the phage. The results are shown in Table 4.
TABLE comparison of the titer of phage fermentation broths after 60 days storage at 44 deg.C
From the above results, it can be seen that the phage fermentation broth prepared by the culture medium of the present invention has higher titer than the phage fermentation broth prepared by the common LB culture medium after being preserved for 60 days at 4 ℃; after 60 days of storage, the titer of the phage prepared in LB medium decreased more rapidly. This indicates that the phages prepared with the medium of the invention are more suitable for long-term preservation of phages.
Example 6 Effect of Long-term storage at Normal temperature on the titer of phage cultured in the Medium of the present invention
According to the preparation method of the culture medium, the phages RDP-EC-20123, RDP-SA-20032, RDP-KP-20004 and the corresponding hosts EC-ATCC 25922, SA-ATCC 14028 and KP-ATCC 10031 are inoculated into 100mL of the culture medium according to a certain proportion, each group is simultaneously inoculated into a common LB culture medium in the market, and the culture is carried out for 8 hours under the conditions of 37 ℃ and 180 r/min.
And (3) measuring the titer of the phage fermentation liquid by a double-plate method. And centrifugally filtering and sterilizing the phage fermentation liquid, and storing the phage fermentation liquid in a cool and dry place at room temperature. And measuring the titer of the phage again after 60 days, and verifying the influence of fermentation liquor of different culture media on the preservation titer of the phage. The results are shown in Table 5.
TABLE 5 comparison of the titer of phage fermentation broths after 60 days storage at room temperature
From the results, the titer of the phage fermentation liquid prepared by the culture medium is higher after the phage fermentation liquid is stored for 60 days at normal temperature, and the titer of the phage fermentation liquid prepared by the LB culture medium is reduced more quickly. This indicates that the phages prepared with the medium of the invention are more suitable for long-term preservation of phages. Moreover, the requirement on the storage condition is not high, and the low-temperature or conventional storage can be carried out for a long time.
According to the invention, different media are added into the culture medium to improve the titer of the phage, and the method has important significance for phage industrial production.
Claims (3)
1. A culture medium for improving the titer of a bacteriophage is characterized in that: every 1000mL of liquid culture medium contains: 5.0g of peptone, 3.0g of beef extract, 2.0g of yeast extract powder, 5.0g of sodium chloride, 1.5g +/-0.12 g of oligosaccharide, 0.18g +/-0.015 g of ferric citrate, 0.39g +/-0.02 g of magnesium sulfate, 0.55g +/-0.05 g of calcium chloride, 2.9g +/-0.23 g of zinc gluconate, 0.54g +/-0.03 g of sorbitol, 0.018g +/-0.002 g of disodium hydrogen phosphate and 0.0036g +/-0.0004 g of potassium dihydrogen phosphate.
2. The phage titer enhancing medium of claim 1, wherein: the liquid culture medium uses water as a solvent, and the pH value is 7.5.
3. A method for improving the titer of a bacteriophage is characterized in that: inoculating the phage and its corresponding host bacteria into the culture medium of claim 1, culturing at 37 deg.C, and shake culturing.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774345A (en) * | 2022-04-26 | 2022-07-22 | 青岛润达生物科技有限公司 | Method for reducing virulence of bacteria |
| CN115261337A (en) * | 2022-07-29 | 2022-11-01 | 青岛润达生物科技有限公司 | Culture medium and method suitable for culturing and screening vibrio phage |
| CN117487767A (en) * | 2023-11-30 | 2024-02-02 | 青岛润达生物科技有限公司 | Method for improving phage outbreak by using low-concentration antibiotics and application |
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| EP1041141A1 (en) * | 1999-03-30 | 2000-10-04 | Council Of Scientific And Industrial Research | A novel bacteriophage, a process for the isolation thereof and a universal growth medium useful in the process thereof |
| CN106085967A (en) * | 2016-06-14 | 2016-11-09 | 苏州埃瑞特生物技术有限公司 | A kind of production method of high-titer phage |
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| CN113151194A (en) * | 2021-05-18 | 2021-07-23 | 瑞科盟(青岛)生物工程有限公司 | Salmonella bacteriophage resistant to antiviral drugs and application thereof |
| CN113230215A (en) * | 2021-05-21 | 2021-08-10 | 中国科学院深圳先进技术研究院 | Phage freeze-dried powder preparation and preparation method, preservation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114774345B (en) * | 2022-04-26 | 2024-05-10 | 青岛润达生物科技有限公司 | Method for reducing bacterial virulence |
| CN115261337A (en) * | 2022-07-29 | 2022-11-01 | 青岛润达生物科技有限公司 | Culture medium and method suitable for culturing and screening vibrio phage |
| CN117487767A (en) * | 2023-11-30 | 2024-02-02 | 青岛润达生物科技有限公司 | Method for improving phage outbreak by using low-concentration antibiotics and application |
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