CN116688090A - Drug composition of sensitization resistant bacteria and application thereof - Google Patents
Drug composition of sensitization resistant bacteria and application thereof Download PDFInfo
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- CN116688090A CN116688090A CN202310860143.9A CN202310860143A CN116688090A CN 116688090 A CN116688090 A CN 116688090A CN 202310860143 A CN202310860143 A CN 202310860143A CN 116688090 A CN116688090 A CN 116688090A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 56
- 239000003814 drug Substances 0.000 title claims abstract description 43
- 229940079593 drug Drugs 0.000 title claims abstract description 41
- 239000000203 mixture Substances 0.000 title claims abstract description 16
- 206010070834 Sensitisation Diseases 0.000 title claims abstract description 14
- 230000008313 sensitization Effects 0.000 title claims abstract description 14
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 74
- 108010024636 Glutathione Proteins 0.000 claims abstract description 70
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- 229960002260 meropenem Drugs 0.000 claims abstract description 59
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- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 18
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- HHXMXAQDOUCLDN-RXMQYKEDSA-N penem Chemical compound S1C=CN2C(=O)C[C@H]21 HHXMXAQDOUCLDN-RXMQYKEDSA-N 0.000 description 1
- 150000002960 penicillins Chemical class 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/06—Tripeptides
- A61K38/063—Glutathione
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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Abstract
The invention relates to a drug combination of sensitization resistant bacteria and application thereof, comprising carbapenem antibiotics and reduced glutathione; the mass ratio of the carbapenem antibiotics to the reduced glutathione in the composition is 1:1-390. According to the invention, the sensitivity of NDM positive drug-resistant bacteria to meropenem can be effectively recovered by the reduced glutathione through a micro chessboard dilution method, a time sterilization curve and in-vivo infection and treatment evaluation tests, and the effectiveness of the combination of the two in-vivo and in-vitro is systematically evaluated. The reduced glutathione is proved to be capable of preparing a preparation for restoring the sensitivity of NDM positive drug-resistant bacteria to carbapenem antibiotics. Therefore, the composition composed of the reduced glutathione and the meropenem provides a new technical idea for treating infectious diseases caused by NDM positive drug-resistant bacteria and provides a new research direction for application of the reduced glutathione.
Description
Technical Field
The invention relates to a pharmaceutical composition of sensitized drug-resistant bacteria and application thereof, belonging to the technical field of biological medicine.
Background
Beta-lactam antibiotics (beta-lactam antibiotic) are broad spectrum antibiotics, including penicillins and their derivatives, cephalosporins, monoamide rings, carbapenems, and penem enzyme sensitizers, etc. The chemical structure of the beta-lactam antibiotics has a beta-lactam ring, and is the most widely used type of the existing antibiotics. The antibiotic has the advantages of strong bactericidal activity, low toxicity, wide application and good clinical curative effect. With the widespread use of beta-lactam antibiotics, more and more bacteria are caused to develop resistance mediated by beta-lactamase.
Meropenem is a typical carbapenem antibacterial drug, which is the "last line of defense" for clinical treatment of severe infections with multiple resistant gram-negative bacteria such as ultra-broad spectrum beta-lactamase, but it also makes gram-negative bacteria resistant to carbapenems, triggering outbreaks of global antibiotic resistance crisis. Month 8 of 2010, journal of willow is reporting a new deli-beta-lactamase-1 (NDM-1), called "superbacteria", that hydrolyzes carbapenems. NDM-1 is the most widely-affected, most severely compromised metallo beta-lactamase discovered in recent years and exhibits high resistance to almost all antibiotics. The resistance gene encoding NDM-1 (bla NDM-1 ) Is located on plasmid, can be independently replicated outside chromosome, and can be horizontally transferred between different strains to make original pairAntibiotic-sensitive strains acquire resistance and are expressed in different bacteria. NDM-carrying strains hydrolyze almost all β -lactam antibiotics, and NDM renders bacteria resistant to almost all available β -lactam antibiotics, resulting in broad resistance of the host bacteria and further in poor clinical outcome of carbapenem antibacterial drug treatment. Therefore, finding a substance that can effectively restore the sensitivity of NDM-positive bacteria to carbapenem antibiotics is a problem that has been urgently needed to be solved by researchers.
Disclosure of Invention
The first object of the present invention is to provide a pharmaceutical composition of sensitized drug-resistant bacteria, which solves the problem of serious drug resistance of carbapenem antibiotics in the prior art.
A second object of the present invention is to provide the use of a pharmaceutical composition of sensitized drug-resistant bacteria for preparing a drug-resistant bacteria sensitizer, to solve the above-mentioned problems.
In order to achieve the above purpose, the technical scheme of the pharmaceutical composition of the sensitization resistant bacteria in the invention is as follows:
a pharmaceutical composition of sensitization resistant bacteria, comprising carbapenem antibiotics and reduced glutathione; the mass ratio of the carbapenem antibiotics to the reduced glutathione in the composition is 1:1-390.
The beneficial effects of the technical scheme are that: the inventor performs screening from compounds approved to be marketed and used for non-antibacterial purposes in the early stage, and discovers that the reduced glutathione of the compounds approved for treating liver diseases and heavy metal antidotes can effectively restore the sensitivity of NDM positive bacteria to carbapenem antibiotics meropenem.
Specifically, reduced Glutathione (glutethione), an alias: G-SH, chemical name N- (N-L-gamma-glutamyl-L-cysteinyl) glycine, molecular formula C 10 H 17 N 3 O 6 S, molecular weight 307.33. White powder, melting point 192-195 ℃, which is soluble in water, diluted alcohol, liquid ammonia and methylformamide, but insoluble in alcohol, ether and acetone, having the following structural formula:
as a further improvement, the mass ratio of carbapenem antibiotics to reduced glutathione in the composition is 1:1-195.
As a further improvement, the carbapenem antibiotic is meropenem.
The beneficial effects of the technical scheme are that: meropenem is a typical carbapenem antibacterial drug, has good antibacterial effect and low toxicity, and has the advantages of good clinical effect and wide application.
Specifically, meropenem has the formula: c (C) 17 H 25 N 3 O 5 S, the molecular weight is as follows: 383.5.
in order to achieve the above purpose, the application of the drug combination of the sensitization resistant bacteria in the preparation of the drug resistant bacteria sensitization agent comprises the following technical scheme:
the application of a drug combination of sensitization resistant bacteria in preparing sensitization resistant bacteria sensitization agent.
The beneficial effects of the technical scheme are that: experiments prove that the combined use of the carbapenem antibiotics meropenem and the reduced glutathione can effectively recover the in-vivo and in-vitro antibacterial activity of the meropenem on drug-resistant bacteria and can effectively relieve the problem of severe drug resistance of beta-lactam antibiotics in the prior art. Meanwhile, the reduced glutathione has no biotoxicity, has the effects of protecting organisms and expelling toxin, has wide medical application and is suitable for being popularized and used in a large range.
As a further improvement, the drug-resistant bacteria are metallo-beta-lactamase positive bacteria among gram-negative pathogenic bacteria.
As a further improvement, the metallo-beta-lactamase is metallo-beta-lactamase 5 (NDM-5).
The beneficial effects of the technical scheme are that: NDM-5 is the most widely distributed metal-beta-lactamase with broad-spectrum hydrolytic activity, and NDM-5 positive bacteria can be used for more comprehensively explaining that reduced glutathione can effectively recover the sensitivity of meropenem to drug-resistant bacteria, and has quite broad-spectrum.
The beneficial effects of the invention are as follows:
1. the invention discovers for the first time that the combined application of the reduced glutathione and the meropenem can be used for treating drug-resistant bacteria infectious diseases, and belongs to the new application of the reduced glutathione.
2. The invention clarifies that the reduced glutathione can restore the sensibility of drug-resistant bacteria to meropenem, systematically evaluates the effectiveness of the combination of the reduced glutathione and meropenem in vitro and in vivo, is beneficial to developing a novel antibiotic synergist and solves the problem of severe drug resistance of the existing beta-lactam antibiotics.
Drawings
FIG. 1 is a time sterilization curve (MEM, meropenem (16. Mu.g/mL), L-GSH, reduced glutathione (1.56 mg/mL or 3.12 mg/mL), MEM+L-GSH, meropenem (16. Mu.g/mL) +reduced glutathione (1.56 mg/mL or 3.12 mg/mL)) for the combination of meropenem and reduced glutathione in example 6 of the present invention;
FIG. 2 shows the effectiveness of meropenem (10 mg/kg) and reduced glutathione (10 mg/kg) alone and in combination in the treatment of a model of infection with drug resistant bacteria of the larvae of Chilo suppressalis in example 7 of the present invention;
FIG. 3 shows changes in the in vivo bacterial load of the larvae of Chilo suppressalis at 24 hours in example 8 of the present invention (PBS group, MEM group (10 mg/kg body weight), MEM+L-GSH group (10 mg/kg+10mg/kg body weight));
FIG. 4 shows the change in the in vivo bacterial load of the drug-resistant bacteria of the larva of Chilo suppressalis at 48 hours in example 8 of the present invention (MEM is meropenem group (10 mg/kg body weight), MEM+L-GSH is meropenem and reduced glutathione group (10 mg/kg+10mg/kg body weight)).
Detailed Description
The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; the present embodiments are merely exemplary and do not limit the scope of the invention in any way, and modifications and substitutions may be made in the details and form of the invention without departing from the spirit and scope of the invention, which are intended to fall within the scope of the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.
The following examples and test examples will briefly describe some of the biological materials, test reagents, test equipment, etc., as follows:
strains:
the NDM-producing positive drug-resistant escherichia coli E.coli 1911034 is an isolated NDM-5 positive escherichia coli. The full length of the E.coli 1911034 gene was amplified by PCR and sequenced, with 100.0% homology to the NDM-5 positive bacteria in NCBI Genbank.
Biochemical reagent:
reduced Glutathione (GLPBIO), cat No.: GC12203-5g, meropenem (microphone).
Culture medium:
coli LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0;
MHB broth: beef powder 6.0g/L, soluble starch 1.5g/L and acid hydrolyzed casein 17.5g/L.
The tests in the following examples were all set up in triplicate and the results averaged.
The preparation method of the mother solution of the reduced glutathione and the antibiotics comprises the following steps:
reduced glutathione mother liquor (50 mg/mL): 5.0g of reduced glutathione was weighed and dissolved in 1mL of sterile water.
Meropenem mother liquor (5120 μg/mL): 51.2mg of meropenem is weighed out and dissolved in 10mL of sterile water.
1. Specific embodiment of drug combination of sensitization resistant bacteria
Example 1 composition of reduced glutathione and carbapenem antibiotic 1
The carbapenem antibiotic in the composition 1 of this example is meropenem, and the mass ratio of reduced glutathione to meropenem is 48.75:1.
example 2 composition of reduced glutathione and carbapenem antibiotic 2
The carbapenem antibiotic in the composition 2 of this example is meropenem, and the mass ratio of reduced glutathione to meropenem is 97.5:1.
example 3 composition 3 of reduced glutathione and carbapenem antibiotic
The carbapenem antibiotic in the composition 3 of this example is meropenem, and the mass ratio of reduced glutathione to meropenem is 195:1.
example 4 composition of reduced glutathione and carbapenem antibiotic 4
The carbapenem antibiotic in the composition 4 of this example is meropenem, and the mass ratio of reduced glutathione to meropenem is 390:1.
2. specific examples of application of pharmaceutical compositions of sensitized drug-resistant bacteria
Example 5 determination of minimum inhibitory concentration
The embodiment adopts a chessboard drug sensitivity analysis method to evaluate the synergistic antibacterial activity of meropenem and reduced glutathione on drug-resistant bacteria, and the specific implementation operation is as follows:
1. test procedure
Taking positive drug-resistant escherichia coli E.coli 1911034 for producing NDM according to the following ratio of 1:100 is inoculated into 3mL LB culture medium, the temperature is 37 ℃, shaking culture is carried out at 180rpm for about 10 hours until bacteria are in the late phase of logarithmic phase (the OD value of bacterial liquid is between 0.2 and 0.6), and the bacterial liquid is diluted ten-thousand times by MHB broth culture medium. Dissolving reduced glutathione to 50mg/mL with water, and diluting reduced glutathione (50 mg/mL,25mg/mL,12.5mg/mL,6.25mg/mL,3.12mg/mL,1.56mg/mL,0.78 mg/mL) with a bacterial liquid multiple ratio after ten thousand times dilution in a 2mL centrifuge tube; meropenem was taken and dissolved in water to 5120 μg/mL. Taking a 96-well plate, adding 10 mu L of meropenem (5120 mu g/mL) and 90 mu L of bacteria liquid diluted by ten thousand times into a first column, adding 50 mu L of bacteria liquid diluted by ten thousand times into a 2 nd-11 th column, and sucking out 50 mu L of bacteria liquid diluted by ten thousand times from the first column to 11 columns for discarding. Reduced glutathione (50 mg/mL,25mg/mL,12.5mg/mL,6.25mg/mL,3.12mg/mL,1.56mg/mL,0.78 mg/mL) diluted with a double ratio was then added to lines 1-7 of the 96-well plate in this order. After incubation of 96-well plates at 37℃for 24h, FIC results were read.
The bacteriostatic concentration FIC index was calculated according to the following formula:
fic=mic (a combination)/MIC (a alone) +mic (B combination)/MIC (B alone); wherein A is meropenem and B is reduced glutathione;
FIC index interpretation criteria: when the FIC index is less than 0.5, the two medicines are synergistic; when the FIC index is 0.5-1, the two medicines are added; when the FIC index is greater than 1 and less than 2, the two drugs are unrelated; when the FIC index is greater than 2, the two drugs are antagonism.
2. Test results
The test results are shown in Table 1, and the FIC value of the combined use of meropenem and reduced glutathione is 0.0936, which shows that the meropenem and the reduced glutathione have remarkable synergistic antibacterial activity.
TABLE 1 MIC value and FIC value of meropenem and reduced glutathione for NDM-5 Positive E.coli
In summary, the minimum inhibitory concentration test results show that the MIC value of the reduced glutathione is increased by one order of magnitude compared with that of the meropenem alone, which indicates that the reduced glutathione alone has no inhibitory effect, and the combination of the reduced glutathione and the meropenem can reduce the MIC value of the meropenem on NDM positive escherichia coli by 32 times. The FIC index shows that the combination of the reduced glutathione and the meropenem has obvious synergistic effect on the strain for sensitization of the NDM.
Example 6 time-sterilization Curve test
1. Test procedure
Taking E.coli 1911034 as a test strain, picking single colonies from a pure culture plate of the NDM-positive E.coli, culturing overnight at 37 ℃ in 3mL LB broth, and taking activated NDM-5-positive E.coli 1 the next day: 100 were inoculated into 30mL of LB medium.
Setting a blank control group without antibiotics; 16 μg/mL meropenem group; 1.56mg/mL reduced glutathione; 3.12mg/mL reduced glutathione; 16 μg/mL meropenem and 1.56mg/mL reduced glutathione combination; 16 μg/mL meropenem and 3.12mg/mL reduced glutathione combination. The test group and the control group are cultivated at 37 ℃ under the condition of the same bacterial liquid concentration, 1mL of bacterial culture liquid is taken out from each group for gradient dilution in agar culture medium at 0, 4, 8, 12, 16, 20 and 24 hours, colony count is carried out after cultivation for 18-24 hours at 37 ℃, the logarithm of the colony count is taken as an ordinate, the cultivation time is taken as an abscissa, and a time sterilization curve is drawn.
2. Test results
The test results are shown in fig. 1, and the time sterilization curve in the graph can be known: the number of the drug-resistant bacteria is basically unchanged when the meropenem or the reduced glutathione is singly used, which indicates that the meropenem or the reduced glutathione has almost no effect on the drug-resistant bacteria; compared with meropenem or reduced glutathione alone, the combination of meropenem and reduced glutathione significantly reduces the number of drug-resistant bacteria, and the number of drug-resistant bacteria is reduced by more than 8log 10 CFU/mL indicates that the synergistic effect between meropenem and reduced glutathione can obviously sensitize the growth of NDM positive escherichia coli.
EXAMPLE 7 Meropenem and reduced glutathione combination treatment of Chilo suppressalis larva bacterial infection
In the embodiment, a drug-resistant bacterial infection model is firstly constructed, then meropenem and reduced glutathione are used in combination, and further the treatment effect of the combination is evaluated, and the specific implementation operation is as follows:
1. test procedure
1) Preparation of drug-resistant bacterial liquid
Taking NDM-producing positive drug-resistant escherichia coli E.coli 1911034 as a test strain, picking single bacterial colonies from a pure culture plate of the NDM-positive escherichia coli, culturing overnight at 37 ℃ in 3mL LB broth culture medium, and taking activated NDM-positive escherichia coli 1 the next day: 100 were inoculated into 30mL of LB medium.The concentration of the bacterial liquid grown to the logarithmic phase is 1.5X10 8 CFU/mL, washed 3 times with PBS solution.
2) Test grouping and injection:
40 larvae of Chilo suppressalis with a weight of 300mg are randomly divided into a Control group, a meropenem treatment group, a reduced glutathione treatment group and a meropenem+reduced glutathione synergistic treatment group, wherein each group contains 10 larvae. The following treatments were respectively carried out: control group: injecting 10 mu L of escherichia coli suspension into the left lower foot of the larva of the Chilo suppressalis, and injecting 10 mu LPBS buffer into the right lower foot after 1 h; meropenem treatment group: injecting 10 mu L of escherichia coli suspension into the left lower foot of the larva of the Chilo suppressalis, and injecting 10 mu L of meropenem solution (10 mg/kg body weight) into the right lower foot after 1 h; reduced glutathione treatment group: injecting 10 mu L of escherichia coli suspension into the left lower foot of the larva of the Chilo suppressalis, and injecting 10 mu L of reduced glutathione solution (10 mg/kg body weight) into the right lower foot after 1 h; synergistic treatment group: the lower left foot of the larvae of Chilo suppressalis was injected with 10. Mu.L of E.coli suspension, and after 1h the lower right foot was injected with 10. Mu.L of a mixed solution of meropenem (10 mg/kg body weight) and reduced glutathione (10 mg/kg body weight).
3) Culturing:
after the administration, the larvae are placed in culture dishes of corresponding groups, placed in a constant temperature incubator at 37 ℃ for culture, taken out and observed every 24 hours until the fifth day, and the death number of the larvae at each 24 hours is recorded.
2. Test results
The test results are shown in fig. 2, which shows that: all death occurred in the observed period of the larvae of Chilo suppressalis in the PBS treatment group, which indicates that the construction of the drug-resistant bacterial infection model was successful. The survival rate of the meropenem single treatment group is 25%, the survival rate of the reduced glutathione single treatment group is lower than 20%, and the survival rate of the meropenem and reduced glutathione combined treatment group reaches 91%, which shows that the synergistic effect of the meropenem and the reduced glutathione obviously improves the survival rate of the larvae of the Chilo suppressalis.
Example 8 detection of bacterial load in Chilo suppressalis
In this example, E.coli 1911034 was used to inject bacterial load into larvae of Chilo suppressalis at a sub-lethal dose, and the larvae were infected with the same, and the larvae were divided into PBS group, meropenem group (10 mg/kg body weight), meropenem group and reduced glutathione group (10 mg/kg+10mg/kg body weight) after infection. The specific implementation operation is as follows:
1. test procedure
1) Collecting larva samples
Sampling once at 24h from the culture dish placed in the incubator, randomly selecting 8 live larvae from each group (8 dead larvae are randomly selected if the death rate of each group of larvae in the culture dish reaches more than 70% at the first sampling, and the group is not considered at the second sampling); samples were taken once at 48h, from which 8 live larvae were randomly selected for each group. The selected larva of Chilo suppressalis is placed in a 2mL EP tube containing 75% alcohol for soaking for 30min, and then placed in a clean 9cm empty culture dish, so that the larva is naturally dried in an ultra-clean workbench.
2) Larva grinding treatment
A2 mL EP tube was placed in a small steel ball at the bottom, and dried worms were placed therein, and 1mL of PBS solution was added to each 2mL EP tube. Placed in a tissue refiner and ground at 300HZ for 180s.
3) Sample dilution
20. Mu.L of homogenized tissue was taken in 180. Mu.L of PBS solution at 1:10 ratio serial dilutions 6 gradients.
4) Drip plate
Sterile LB agar plates prepared in advance were taken, and each plate was divided into six sections according to dilution. 10. Mu.L (3 replicates) of each dilution was placed in culture dishes of the corresponding gradient. After the bacterial liquid is fully dried and absorbed, the bacterial liquid is inverted in a 37 ℃ incubator for 16 to 18 hours of incubation.
5) Counting
After the incubation time, the plates were removed, the colony count of each zone was counted and visually observed. The colony count was averaged over three plates at the same dilution and the colony count per unit mass was calculated according to the formula.
Bacterial load per g of larvae (CFU/g) = (average colony count x dilution)/weight of larvae/1 mL PBS.
2. Test results
The mortality of the PBS group bodies in 24 hours is more than 70%, so the bacterial load of the PBS group is not measured after 48 hours. The in vivo bacterial load of the meropenem and the reduced glutathione group in 24 hours is obviously lower than that of the PBS group and the meropenem group, and the obvious statistical difference is provided, which shows that the reduced glutathione can improve the sensitivity of the drug-resistant bacteria to the drugs in vivo, and the specific result is shown in figure 3. After 48 hours of single administration, the inter-group bacterial load of the large wax moth infection has obvious statistical difference, which indicates that the sensitization effect of the medicine has durability, and the specific result is shown in figure 4.
The last explanation is: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (6)
1. A pharmaceutical composition of sensitization resistant bacteria, which is characterized in that: including carbapenem antibiotics and reduced glutathione; the mass ratio of the carbapenem antibiotics to the reduced glutathione in the composition is 1:1-390.
2. The pharmaceutical composition of sensitized and resistant bacteria according to claim 1, wherein: the mass ratio of the carbapenem antibiotics to the reduced glutathione in the composition is 1:1-195.
3. The pharmaceutical composition of sensitized and resistant bacteria according to claim 1 or 2, characterized in that: the carbapenem antibiotic is meropenem.
4. Use of a pharmaceutical composition of a sensitized drug-resistant bacterium according to any one of claims 1 to 3 for the preparation of a drug-resistant bacterium sensitizer.
5. The use of the pharmaceutical composition of sensitized and resistant bacteria according to claim 4 for preparing a drug resistant bacteria sensitizer, characterized in that: the drug-resistant bacteria are metal-beta-lactamase positive bacteria in gram-negative pathogenic bacteria.
6. The use of the pharmaceutical composition of sensitized and resistant bacteria according to claim 5 for preparing a drug resistant bacteria sensitizer, characterized in that: the metallo-beta-lactamase is metallo-beta-lactamase 5.
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