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Establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models

A levofloxacin and sequential model technology, applied in the biological field, can solve the problems that the order of quinolone-resistant drug target gene mutations cannot exclude its own random mutations, and cannot explain the problem, so as to achieve the effect of eliminating its own random mutations

Inactive Publication Date: 2014-04-30
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] Although some studies have used quinolones to induce Shigella, only one strain of Shigella was induced to form a single induction system. The sequence of mutations in the quinolone-resistant target genes gyrA and parC cannot rule out the influence of their own random mutations, and The accuracy of the mutation timing cannot be explained from the consistency and statistical probability, and the problem cannot be explained

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  • Establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models
  • Establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models
  • Establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models

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Embodiment 1

[0073]Embodiment 1: The establishment of levofloxacin-induced Shigella drug-resistant gene mutation time series model: 1. To identify, preserve Shigella sonnei, carry out cefazolin, cefotaxime, ampicillin, gentamicin, chloramphenicol Antibiotic susceptibility test of 12 kinds of antibiotics, including tetracycline, cotrimoxazole, nalidixic acid, pipemidic acid, ciprofloxacin, norfloxacin and levofloxacin, with the quality control strain as the control, measuring with ruler including drug susceptibility The diameter of the inner antibacterial ring of the paper is limited to the limit of no obvious bacterial growth with the naked eye as the edge of the antibacterial ring, and the guidelines established by the National Committee for Clinical Laboratory Standards (NCCLS) are used to determine the sensitivity of the bacteria , Moderately sensitive or resistant. Through the above drug sensitivity test on the identified and preserved Shigella sonnei, it was verified that it was sensi...

Embodiment 2

[0100] Example 2: Establishment of a time-series model of levofloxacin-induced Shigella drug-resistant gene mutation: the method steps are the same as in Example 1, wherein the bacterial solid medium is LB plate, the bacterial liquid medium is LB liquid culture, and the temperature in step (4) is 32°C Bacteria were cultured with shaking until OD600=1 in the logarithmic phase. Take a certain amount of bacterial liquid in step (4) to 5 tubes of LB liquid medium with a levofloxacin content of 0.03125 μg / ml for another generation, and then take a certain amount of bacterial liquid to LB liquid medium with a levofloxacin concentration of 0.0625 to pass two tubes. Generation, before the drug concentration reached the levofloxacin sensitive and intermediate critical value, the concentration of levofloxacin was doubled in turn to culture the bacteria, and when the drug concentration reached the levofloxacin sensitive and intermediate critical value, the concentration of levofloxacin wa...

Embodiment 3

[0104] Example 3 Establishment of a time-series model of levofloxacin-induced Shigella drug-resistant gene mutation: the method steps are the same as in Example 1, wherein the bacterial solid medium is LB plate, the bacterial liquid medium is LB liquid culture, and in step (4) shake at 39°C Bacteria were cultured and grown to log phase OD600=0.6. Take a certain amount of bacterial liquid in step (4) to 10 tubes of LB liquid medium with a levofloxacin content of 0.03125 μg / ml for another generation, and then take a certain amount of bacterial liquid to LB liquid medium with a levofloxacin concentration of 0.0625 for two passages. Generation, before the drug concentration reached the levofloxacin sensitive and intermediate critical value, the concentration of levofloxacin was doubled in turn to culture the bacteria, and when the drug concentration reached the levofloxacin sensitive and intermediate critical value, the concentration of levofloxacin was successively increased by...

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Abstract

The invention relates to establishment of levofloxacin-induced Shigella drug-resistance gene mutation time sequence models. The establishment method comprises the following main steps: 1, carrying out drug sensitive tests of 12 antibiotics such as levofloxacin and the like on Shigella, and selecting sensitive strains to carry out an induction experiment; 2, detecting MIC0 of levofloxacin on the primary induction strain; 3, inoculating the single colony into a levofloxacin bacterial liquid culture medium to culture; and 4, taking the bacterial solution in the step 3, transferring to the levofloxacin bacterial liquid culture medium to passage the one generation, taking the obtained bacterial solution, transferring to a levofloxacin liquid culture medium to passage the two generations, sequentially carrying out levofloxacin concentration multiplication culture of bacterial before the drug concentration achieves the sensitive and medium critical value, sequentially carrying out levofloxacin concentration increase culture after the drug concentration achieves the sensitive and medium critical value, carrying out induction culture on the strain for 2 days at the concentration when the induced anti-bacterial drug concentration achieves 2, 4 and 8 mug / ml, carrying out induction culture on the well-growing single colony to obtain the induced Shigella drug-resistance spectral pattern, and carrying out mutation analysis on the induced drug-resistance spectral pattern strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for constructing a quinolone-resistant Shigella model in vitro, in particular to a method for establishing a multi-group Shigella drug-resistant model induced by quinolones. Background technique [0002] The emergence and spread of bacterial resistance is largely a challenge to anti-infection treatment. Taking Shigella as an example, many studies have shown that drug-resistant strains frequently appear in Shigella, and the drug-resistant development speed is fast, and the resistance is high. Due to the characteristics of wide range of drugs, high drug resistance rate, and different drug resistance spectrum of different bacterial types, quinolones are currently the first choice for the treatment of bacillary dysentery. The curative effect of bacillary dysentery was once improved. However, in the past 40 years, quinolone antibiotics have been widely used, and the drug is also...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N1/20C12Q1/68C12R1/01
Inventor 段广才王颖芳杨海燕宋春花马楠张卫东
Owner ZHENGZHOU UNIV
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