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Application of glutamine in preparation of drug for inhibiting escherichia coli from generating gene mutation

A technology of Escherichia coli and glutamine, applied in the field of medicine, to reduce the probability of mutation, reduce the survival rate, and slow down the effect of drug resistance gene mutation

Pending Publication Date: 2020-10-16
GUANGDONG LITAI PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technical solution is mainly to improve the bactericidal efficiency of existing antibiotics. There is no relevant research report on how to inhibit bacterial gene mutations and slow down bacterial drug resistance.

Method used

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  • Application of glutamine in preparation of drug for inhibiting escherichia coli from generating gene mutation
  • Application of glutamine in preparation of drug for inhibiting escherichia coli from generating gene mutation
  • Application of glutamine in preparation of drug for inhibiting escherichia coli from generating gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The in vitro passage experiment of embodiment 1 clinical sensitive bacterium and clinical drug-resistant bacterium

[0036] 1.1 Sample preparation

[0037] Pick clinically isolated Escherichia coli sensitive bacteria K12, S13, S14 and drug-resistant bacteria Y9, Y17, Y23 single clones (initial strain WT), inoculate them in LB liquid medium, culture at 37°C, 200rpm for 16h, and centrifuge at 8000g for 5min , collect the bacteria, and then suspend and wash the bacteria with sterile physiological saline, repeating three times. Resuspend the prepared bacteria with saline to OD 600 0.2, aliquot 5mL in a test tube for later use.

[0038] 1.2 Passage of clinically sensitive bacteria and clinical drug-resistant bacteria

[0039] Divide each strain into 2 groups, one group is ampicillin single-use group (AMP, abbreviated as A), the concentration of added antibiotics is K12: 0.003125mg / mL, S13: 0.003125mg / mL, S14: 0.0125mg / mL, Y9: 0.8mg / mL, Y17: 0.8mg / mL, Y23: 0.8mg / mL; the o...

Embodiment 2

[0040] Example 2 Sequence Analysis of Main Genes of Glutamine Metabolism Pathway and Purine Metabolism Pathway of Subcultured Bacteria

[0041]Glutamine metabolism ( carA, carB, gdhA, glnA), purine metabolism (purF, purB, purD, purE, purK, purH, purM, purN, purT, yfbR, rihB), dual regulatory system (cpxA, cpxR) and outer membrane antibiotic entry channel proteins (ompF) pathway main drug resistance gene sequence determination. The sequencing primers used are listed in Table 2.

[0042] Table 2 Sequencing Primers

[0043]

[0044]

[0045] The sequence obtained after sequencing was analyzed by BLAST, and compared with the gene sequence of the corresponding bacterial initial strain, it was found that: for the K12 strain, 1 to 3 missense mutations were found in the genes such as carA, purB, purK, purH, purM, and purT in K12-30A point, while K12-30AG only found missense mutations in purK and purH ( figure 1 A) For Y9 strain, 1 to 3 missense mutation sites were found in c...

Embodiment 3

[0046] Example 3 In Vitro Passage Strain Drug Resistance Research

[0047] 3.1. Determination of minimum inhibitory concentration

[0048] Get 6 kinds of bacteria (K12, S13, S14, Y9, Y17, Y23) according to the 2 kinds of passage ways of embodiment 1 (antibiotics are used alone or antibiotics and glutamine are used in combination) to obtain the 0th, 5th, 10th, 15th, 20, 25, and 30 subcultured bacteria were used to measure the minimum inhibitory concentration of ampicillin by the micro-broth dilution method, and the specific methods were as follows:

[0049] The subcultured bacteria of each generation were inoculated in LB liquid medium, and cultured at 37°C and 200rpm for 16h; respectively transferred to 5mL LB medium and cultured to OD 600 0.5, diluted 10 times; ampicillin was added to the 1st to 11th columns in a sterile 96-well plate, 100 μL per well, and no antibiotics were added to the 12th column as a control; 10 μL of bacterial solution (approx. 10 5 CFU bacteria, tha...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of glutamine in preparation of a drug for inhibiting escherichia coli from generating gene mutation.According to the application, the glutamine and ampicillin are combined for use, and after multiple passages, the escherichia coli can be inhibited from generating drug-resistant gene mutation, so that mutation probability of drug-resistant genes is remarkably reduced, the increase times of bacteria on the lowest inhibitory concentration of the ampicillin after passage is reduced, and the survivalrate is reduced. Animal experiments also prove that passage bacteria added with the glutamine and the ampicillin are easier to remove by the body, so that the effect of slowing down the drug-resistant gene mutation of the escherichia coli on the ampicillin is achieved.

Description

technical field [0001] The invention belongs to the technical field of medicine. More specifically, it relates to the application of glutamine in the preparation of drugs for inhibiting Escherichia coli from producing gene mutations. Background technique [0002] Gene mutation is a natural phenomenon, which is the result of biological evolution over thousands of years, among which the gene mutation of microorganisms is more common. Some pathogenic bacteria, such as Escherichia coli, will produce gene mutations during the growth and reproduction process. Under the selection pressure of antibiotics, bacteria that can continue to grow after mutations are screened out and reproduce advantageously. The inhibitory effect on bacteria is gradually weakened, and even superbugs are produced, making it more difficult to treat bacterial infections. In addition, in the cultivation of experimental bacteria, after the bacteria contaminated by antibiotics have gene mutations, their genoty...

Claims

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Application Information

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IPC IPC(8): A61K31/198A61K31/43A61P31/04
CPCA61K31/198A61K31/43A61P31/04A61K2300/00Y02A50/30
Inventor 彭宣宪陈东松李惠方建华赵贤亮田妙容彭博罗庆发
Owner GUANGDONG LITAI PHARM CO LTD
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