CN117442705A - Application of fingolimod combined polymyxin in preparation of anti-klebsiella pneumoniae drugs - Google Patents
Application of fingolimod combined polymyxin in preparation of anti-klebsiella pneumoniae drugs Download PDFInfo
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- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention discloses an application of fingolimod combined polymyxin in preparation of anti-klebsiella pneumoniae medicines. The fingolimod combined with the polymyxin has remarkable inhibitory activity on the polymyxin-resistant klebsiella pneumoniae, damages the cell membrane structure of the klebsiella pneumoniae, causes intracellular material leakage and further causes bacterial death, can be used for preparing antibacterial medicines for resisting the polymyxin-resistant klebsiella pneumoniae, and further can be used for preparing medicines for treating pneumonia or liver abscess of human beings or animals caused by the polymyxin-resistant klebsiella pneumoniae infection.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of fingolimod combined polymyxin in preparation of anti-klebsiella pneumoniae medicines.
Background
Klebsiella pneumoniae infection (Klebsiella pneumoniae infection, KPI) is a generic term for a group of diseases caused by klebsiella pneumoniae infection in humans and animals, and its clinical symptoms often cause diseases such as pneumonia, urinary tract infection, septicemia, endocarditis operation wound or other wound infection. Klebsiella pneumoniae (Klebsiella pneumoniae, KP) was found in the earliest 1884 as a gram-negative bacterium, enterobacter. The strain is widely distributed in the nature, is commonly parasitic on the skin, respiratory tract, intestinal tract and the like of people and animals, and is a conditional pathogenic bacteria for people and animals. Over the past several decades, with the widespread use of antibiotics, classical klebsiella pneumoniae strains have developed a degree of resistance to antibiotics, creating a great threat to public health safety for humans. In the veterinary field, klebsiella pneumoniae can infect animals such as pigs, cows and sheep, and after infection, the animals can have symptoms such as dyspnea, cough, fever, inappetence and the like, so that the economic loss of animal husbandry is caused, and the welfare and the breeding environment of the animals can be negatively influenced.
Fingolimod (Fingolimod), an engineered compound of myriocin extracted from Cordyceps sinensis, is the first FDA approved drug for the treatment of relapsing-remitting multiple sclerosis. Fingolimod has CAS number 162359-55-9 and molecular formula C 19 H 33 NO 2 The chemical structural formula is as follows:
fingolimod is reported to have various biological activities including treatment of relapsing remitting multiple sclerosis, resistance to carbapenem-resistant escherichia coli, resistance to gram-positive bacterial biofilms, and the like. Although the literature is widely studied for the biological activity of fingolimod, there is no report on the activity of fingolimod in combination with polymyxin against klebsiella pneumoniae.
Disclosure of Invention
The invention aims to provide an application of fingolimod in preparing an antibacterial drug for resisting klebsiella pneumoniae, and aims to solve the problem that the antibacterial effect of polymyxin is poor due to drug resistance of klebsiella pneumoniae strains to antibiotics.
The invention discloses application of fingolimod combined polymyxin in preparation of antibacterial drugs for resisting klebsiella pneumoniae.
Preferably, the klebsiella pneumoniae comprises a polymyxin-resistant klebsiella pneumoniae.
The invention further discloses application of the fingolimod combined polymyxin in preparation of medicines for treating human or animal diseases caused by klebsiella pneumoniae infection.
Preferably, the klebsiella pneumoniae is a polymyxin-resistant klebsiella pneumoniae.
Preferably, the disease comprises pneumonia, liver abscess.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the fingolimod combined polymyxin has remarkable inhibitory activity on polymyxin-resistant klebsiella pneumoniae (Klebsiella pneumoniae), damages the cell membrane structure of the klebsiella pneumoniae, causes intracellular substance leakage and further causes bacterial death, can be used for preparing antibacterial drugs for resisting the polymyxin-resistant klebsiella pneumoniae, further can be used for preparing drugs for treating human or animal pneumonia or liver abscess caused by polymyxin-resistant klebsiella pneumoniae infection, and has definite and remarkable curative effect and no obvious side effect.
Drawings
FIG. 1 is a graph showing the growth of Klebsiella pneumoniae resistant to polymyxin in an embodiment of the invention under inhibition by an antibacterial agent (fingolimod in combination with polymyxin);
FIG. 2 is a graph showing the sterilization curve of Klebsiella pneumoniae resistant to polymyxin in an embodiment of the invention after treatment with an antimicrobial agent (fingolimod in combination with polymyxin);
FIG. 3 is an SEM image of bacterial morphology of Klebsiella pneumoniae resistant to polymyxin in an embodiment of the invention after treatment with an antibacterial agent (fingolimod in combination with polymyxin);
FIG. 4 shows the results of an antibacterial activity test in mice infected with Klebsiella pneumoniae Balb/C in the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1: in vitro antibacterial Activity test of Klebsiella pneumoniae
1. Experimental materials and apparatus
Strains: klebsiella pneumoniae ATCC700603 commercially available from ATCC company in the united states; the clinical klebsiella pneumoniae was isolated from clinical samples (KP 2105, KP2134, KP2109, KP2135, KP2138, KP2107, KP2102, KP2108, KP 2125).
Reagent: MH liquid medium and MH agar medium were purchased from guangdong cyca biotechnology limited; fingolimod and polymyxin were purchased from Shanghai leaf Biotechnology Inc.
Test instrument: the system comprises an ultra-clean workbench, an autoclave, a constant temperature incubator, an electronic analytical balance, a Mitsubishi turbidimeter, a pipettor and a Direct-Q3 ultrapure water system.
2. Test method
2.1, preparing a liquid medicine: preparing liquid medicine with the concentration of 1280 mug/mL by using distilled water for fingolimod as fingolimod mother liquor; the polymyxin is respectively prepared into liquid medicine with the concentration of 2560 mug/mL by distilled water to be used as a polymyxin mother solution.
2.2, preparing bacterial liquid: the recovered klebsiella pneumoniae is streaked and inoculated to an MH agar culture plate for culturing for 24 hours, single bacterial colony of the klebsiella pneumoniae is picked by an inoculating loop, and inoculated to a sterilizing test tube containing about 5mL of MH liquid culture medium, and the culture is carried out for 18-24 hours at the temperature of 37 ℃ by a constant-temperature shaking incubator. The bacterial liquid concentration was measured with a turbidimeter to give an absorbance=0.5 McF. Diluting the bacterial liquid 100 times with MH liquid culture medium to make the concentration of bacterial liquid about 1×10 6 CFU/mL was used as bacterial suspension for later use.
2.3, minimum Inhibitory Concentration (MIC) determination: in a 96-well plate, 180. Mu.L of liquid medium was added to column 1, and 100. Mu.L of liquid medium was added to columns 2 to 11. Subsequently, 20. Mu.L of polymyxin was added to column 1 at a concentration of 2560. Mu.g/mL, and 100. Mu.L was aspirated into well 2 after air-blow mixing, and so on. After the dilution was completed, different concentrations of fingolimod (fingolimod mother liquor diluted) were added to each well, 10 μl each. Finally, adding the concentration of 1X 10 to each well 6 The CFU/mL bacterial suspension was 90. Mu.L, and the polymyxin concentrations in each column were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. Mu.g/mL, respectively, with final fingolimod concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5. Mu.g/mL. And simultaneously setting a blank control group and a positive control group.
And placing the 96-well plates into a constant temperature incubator, culturing at 37 ℃ for 24 hours, observing results, and visually observing under a dark background, wherein the mixture in each well of the 96-well plates is clear, and the minimum concentration formed by aseptic dropping of the bottom of the well is the MIC value of the compound. The above test was repeated 2 times.
3. Test results
The fingolimod combined polymyxin has remarkable in vitro inhibition activity on klebsiella pneumoniae, and MIC values on commercial klebsiella pneumoniae ATCC700603 and clinical klebsiella pneumoniae are shown in the following table.
TABLE 1 determination of minimum inhibitory concentration of fingolimod in combination with polymyxin on klebsiella pneumoniae
Example 2: growth curve measurement of polymyxin-resistant klebsiella pneumoniae
1. Test materials
Strains: the clinical klebsiella pneumoniae (KP 2108) is obtained by separating clinical samples.
Reagent: MH liquid medium and MH agar medium were purchased from guangdong cyca biotechnology limited; fingolimod and polymyxin were purchased from Shanghai leaf Biotechnology Inc.
Test instrument: an ultra-clean workbench, an autoclave, a constant temperature incubator, an electronic analytical balance, a cell densitometer, a pipettor and a Direct-Q3 ultra-pure water system.
2. Test method
2.1, preparing a liquid medicine: the fingolimod and the polymyxin are respectively prepared into liquid medicine with the concentration of 1280 mu g/mL and 2560 mu g/mL by distilled water for standby.
2.2, preparing bacterial liquid: the recovered klebsiella pneumoniae is streaked and inoculated to an MH agar culture plate for culturing for 24 hours, single bacterial colony of the klebsiella pneumoniae is picked by an inoculating loop, and inoculated to a sterilizing test tube containing about 5mL of MH liquid culture medium, and the culture is carried out for 18-24 hours at the temperature of 37 ℃ by a constant-temperature shaking incubator. Cell densitometer measures bacterial concentration to make OD 600 Absorption value=0.1, and the concentration of the bacterial liquid is about 1×10 6 CFU/mL was used as bacterial suspension for later use.
2.3, determination of growth curve:
taking 5 sterilization test tubes, respectively sucking 5mL of the bacterial suspension, placing the bacterial suspension into 4 test tubes, adding corresponding volumes of fingolimod and polymyxin liquid medicine by using a liquid transfer device, wherein the concentration in each test tube is fingolimod (64 mug/mL), polymyxin (128 mug/mL), fingolimod and polymyxin (8 mug/mL and 1 mug/mL), no liquid medicine is added, and the 5 th test tube only contains blank MH liquid culture medium. Mixing, and culturing at 37deg.C. 200 mu L of bacterial liquid is respectively sucked from each test tube at 0h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h and 24h, and the concentration of the bacterial liquid is tested by a cell densitometer.
3. Test results
As shown in FIG. 1, when fingolimod or polymyxin was added alone to the bacterial liquid, the bacterial growth could not be inhibited. After 8 mug/mL and 1 mug/mL of fingolimod and polymyxin are added, the growth of bacteria can be obviously inhibited, and the result shows that fingolimod and polymyxin can obviously inhibit the growth of polymyxin-resistant klebsiella pneumoniae.
Example 3: sterilization curve determination of fingolimod synergistic polymyxin on polymyxin-resistant klebsiella pneumoniae
1. Test materials
Strains: klebsiella pneumoniae (KP 2108) is isolated from clinical samples.
Reagent: MH liquid medium and MH agar medium were purchased from guangdong cyca biotechnology limited; fingolimod and polymyxin were purchased from Shanghai leaf Biotechnology Inc.
Test instrument: an ultra-clean workbench, an autoclave, a constant temperature incubator, an electronic analytical balance, a cell densitometer, a pipettor and a Direct-Q3 ultra-pure water system.
2. Test method
2.1 preparation of liquid medicine: the fingolimod and polymyxin are prepared into liquid medicine with the concentration of 1280 and 2560 mug/mL by distilled water for standby.
2.2 preparation of bacterial liquid: streaking and inoculating the recovered klebsiella pneumoniae to an MH agar culture plate for culturing for 24 hours, picking single colony of the klebsiella pneumoniae by an inoculating loop, inoculating the single colony of the klebsiella pneumoniae into a sterilizing test tube containing about 5mL of MH liquid culture medium, and culturing for 18-24 hours at the constant temperature of a shaking incubator at 37 ℃. Cell densitometer measures bacterial concentration to make OD 600 Absorption value=0.1, and the concentration of the bacterial liquid is about 1×10 7 CFU/mL was used as bacterial suspension for later use.
2.3 growth curve determination:
taking sterilized test tubes, respectively sucking 5mL of the bacterial suspension, placing the bacterial suspension into the test tubes, adding corresponding volumes of fingolimod and polymyxin liquid medicine by using a pipettor, wherein the concentration of each test tube is fingolimod (16 mug/mL), polymyxin and fingolimod (4 mug/mL and 16 mug/mL), and no liquid medicine is added. Mixing, and shaking culture at constant temperatureCulturing in a incubator at 37 ℃. 200 mu L of bacterial liquid is sucked from each test tube at 0h, 1h, 2h, 4h, 6h, 8h, 10h and 24h respectively, and is serially diluted to 10 by MH liquid culture medium 9 Multiple times. Then, 100. Mu.L of bacterial solutions with different dilution factors are sucked and evenly spread on an MH agar culture plate, and colony counting is carried out after 24h of culture at 37 ℃. And a blank control group is set.
3 test results
As shown in FIG. 2, when fingolimod or polymyxin alone was added to the bacterial liquid, the growth of bacteria could not be inhibited within 24 hours. After 4 mug/mL and 16 mug/mL of polymyxin and fingolimod are added, the sterilizing effect is remarkable, and the number of viable bacteria can be increased from 10 within 8 hours 8 Down to 10 2 Compared with polymyxin, the composition exhibits a more excellent bactericidal effect.
Example 4: cytotoxicity test
1. Test materials
Ccaco-2 cells, shanghai cell bank of China academy of sciences; CCK-8 cell viability detection kit, sigma reagent company; a carbon dioxide incubator, membert, germany; the enzyme-labeled instrument, sieimer's Feier in U.S.; 96-well plates, MEM medium, fetal bovine serum, optional amino acids, were purchased from Sieimer, USA.
2. Test method
Taking Caco-2 cells in logarithmic growth phase, and adjusting cell density to 1×10 5 cell/mL density was seeded in sterile 96-well plates, 100. Mu.L of cell suspension was added to each well, and at 37℃5% CO 2 After 24 hours of incubation, the original medium was discarded, and 100. Mu.L of different concentrations of fingolimod solution (2, 4, 8, 16, 32, 64. Mu.g/mL) was added and 6 duplicate wells were set for each concentration. After culturing the cells for 12 hours, 10 mu L of CCK-8 solution is added into each hole, and the cells are put into a cell culture box for continuous culture for 1-2 hours, and then an OD value at 450nm is measured by an enzyme-labeled instrument. Based on the OD value brought into the calculation formula, the half-maximal inhibitory concentration (IC 50 ) Values.
3. Test results
Based on the measurement results, ji Suande Fingolimod IC for Caco-2 cells 50 Is 37.51 mug/mL. The result isIt is suggested that the fingolimod combined with polymyxin has no obvious cytotoxicity to Caco-2 cells at an effective concentration, and has higher safety in clinical use in future.
Example 5: structural destructive SEM detection of polymyxin-resistant klebsiella pneumoniae
1. Test materials
Strains: klebsiella pneumoniae (KP 2108) is isolated from clinical samples.
Reagent: MH liquid medium and MH agar medium were purchased from guangdong cyca biotechnology limited; fingolimod and polymyxin were purchased from Shanghai leaf Biotechnology Inc.; the electron microscope fixing solution, 0.1M and 0.01M PBS were purchased from Wohai Weibull biotechnology Co., ltd; absolute ethanol, isoamyl acetate was purchased from national pharmaceutical group chemical company, inc; osmium acids were purchased from Beijing Bolid Biotech Co.
Test instrument: ultra-clean bench, autoclave, incubator, electronic analytical balance, cell densitometer, pipettor, direct-Q3 ultra-pure water system, critical point dryer, ion sputtering instrument, scanning electron microscope.
2. Test method
2.1, preparing a liquid medicine: the fingolimod and the polymyxin are respectively prepared into liquid medicine with the concentration of 1280 mu g/mL and 2560 mu g/mL by distilled water for standby.
2.2, preparing bacterial liquid: the recovered klebsiella pneumoniae is streaked and inoculated to an MH agar culture plate for culturing for 24 hours, single bacterial colony of the klebsiella pneumoniae is picked by an inoculating loop, and inoculated to a sterilizing test tube containing about 5mL of MH liquid culture medium, and the culture is carried out for 18-24 hours at the temperature of 37 ℃ by a constant-temperature shaking incubator. Cell densitometer measures bacterial concentration to make OD 600 Absorption value=0.5, and the concentration of the bacterial liquid is about 1×10 8 CFU/mL was used as bacterial suspension for later use.
2.3, SEM sample preparation:
taking 4 sterilization test tubes, respectively sucking 5ml of the bacterial suspension, placing the bacterial suspension into 3 test tubes, adding corresponding volumes of fingolimod and polymyxin liquid medicine into the test tubes by using a pipette, and the concentration in each test tube. Fingolimod (32. Mu.g/mL), polymyxin (128. Mu.g/mL), fingolimod and polymyxin (32. Mu.g/mL and 2. Mu.g/mL), and test tube 5 were treated without adding any drug solution, respectively. Mixing, and culturing in a constant temperature shaking incubator at 37deg.C for 1 hr. After centrifugation at 4500g at room temperature for 10min and rinsing 3 times with 0.01M PBS, the mixture was fixed with an electron microscope fixing solution at room temperature in the absence of light for 2h. The immobilized samples were rinsed 3 times with 0.1M PBS. After fixation with 1% osmium acid at room temperature for 2h in the dark, rinsing 3 times with 0.1M PBS. 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol is dehydrated for 15min each time, and finally isoamyl acetate is used for 15min. And (3) placing the dehydrated sample in a critical point dryer for drying, and then placing the dried sample on an ion sputtering instrument for metal spraying. Finally, observing the acquired images under a scanning electron microscope.
3. Test results
As shown in fig. 3, when only fingolimod or polymyxin is added into the bacterial liquid, the bacterial liquid has no obvious effect on the form of the polymyxin-resistant klebsiella pneumoniae, but after the fingolimod and the polymyxin are combined, the cell form can be obviously destroyed, and the cell structure collapses.
Example 6: in vivo antibacterial activity test of Fingolimod combined with polymyxin on Klebsiella pneumoniae Balb/C mice
1. Test materials
Reagent: MH liquid medium and MH agar medium were purchased from guangdong cyca biotechnology limited; fingolimod and polymyxin were purchased from Shanghai leaf Biotechnology Inc.; 0.01M PBS was purchased from Whansai Weibull biotechnology Co.
Experimental animals: balb/C mice, available from St Bei Fu (Beijing) Biotechnology Co., ltd.
2. Test method
The fingolimod and the polymyxin are respectively prepared into liquid medicines with different concentrations by PBS according to dosage, and the liquid medicines are uniformly mixed by vortex and stored at 4 ℃ for standby.
40 Balb/C mice were randomly divided into 5 groups of 80, each of the normal group, model group, polymyxin (8 mg/kg) group, fingolimod (16 mg/kg) group, polymyxin (2 mg/kg) in combination with fingolimod (4 mg/kg) group.
Will be repeatedThe threo klebsiella pneumoniae is streaked and inoculated to an MH agar culture plate for culturing for 24 hours, single bacterial colony of the klebsiella pneumoniae is picked by an inoculating loop, and inoculated to a sterilizing test tube containing about 5mL of MH liquid culture medium, and the culture is carried out for 18-24 hours at the temperature of 37 ℃ by a constant-temperature shaking incubator. Cell densitometer measures bacterial concentration to make OD 600 Absorption value=0.5, and the concentration of the bacterial liquid is about 6×10 8 CFU/mL was used as bacterial suspension for later use. And (3) drip-irrigating 50 mu L of the bacterial liquid in the nasal cavity of each Balb/C mouse except the normal group, and establishing a Balb/C mouse experimental model infected by klebsiella pneumoniae. After 2h of infection, the liquid medicine was injected separately, and the normal group was injected with equal volume of PBS 1 time per day for 5 consecutive days. The survival of Balb/C mice was observed and recorded every 24h for 5 days following infection.
3. Test results
The Balb/C mice infected with the control group have different degrees of symptoms of difficulty in erecting hair, bowed back and opening eyes after being infected with klebsiella pneumoniae for 24 hours, and the weight is obviously reduced. By day 2 post infection, severely symptomatically mice died. Balb/C mice in the infected control group begin to die at day 2 after infection, with a survival rate of 12.5% after 5 days. The survival rate of the polymyxin group was 25% and that of the fingolimod group was 12.5%. The survival rate of the polymyxin combined with the fingolimod group is 87.5%, which is significantly higher than that of the polymyxin and fingolimod alone group (P < 0.01). Normal groups did not die within 5 d. The specific results are shown in FIG. 4.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (5)
1. The application of fingolimod combined with polymyxin in preparing antibacterial drugs for resisting klebsiella pneumoniae.
2. The use of claim 1, wherein the klebsiella pneumoniae comprises a polymyxin-resistant klebsiella pneumoniae.
3. Use of fingolimod in combination with polymyxin for the preparation of a medicament for the treatment of human or animal diseases caused by klebsiella pneumoniae infection.
4. The use according to claim 3, wherein the klebsiella pneumoniae is a polymyxin-resistant klebsiella pneumoniae.
5. The use according to claim 3, wherein the disease comprises pneumonia, liver abscess.
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