Preparation method for inducing bacteria to form small colony variants

A technology of small colonies and variants, applied in the field of microorganisms, can solve the problems of easy coverage by wild strains, large workload of SCVs, and difficulty in separation and purification.

Inactive Publication Date: 2022-03-01
TARIM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the purpose of the present invention is to provide a method for simply inducing bacteria to form SCVs in the laboratory, which has a high induction success rate, and solves the problem of inducing SCVs with a large workload and being easily covered by wild strains, making it difficult to separate and purify

Method used

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  • Preparation method for inducing bacteria to form small colony variants
  • Preparation method for inducing bacteria to form small colony variants
  • Preparation method for inducing bacteria to form small colony variants

Examples

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Effect test

Embodiment 1

[0037] The K-B disc method provided by the invention induces the test method of Escherichia coli to form SCVs, uses following reagent:

[0038] Sterile filter paper discs containing 30 μg, 60 μg, 120 μg and 240 μg of kanamycin, respectively;

[0039] Sterile filter paper discs containing 5 μg, 10 μg, 20 μg and 40 μg of gentamycin respectively;

[0040] Trypsin Soy Agar (TSA), Trypsin Soy Broth (TSB) and Columbia Blood Agar (referred to as blood plate), Nutrient Agar (NA).

[0041]The main components of trypticase soybean agar medium: casein trypsin hydrolyzate 15.0g / L, soybean powder papain hydrolyzate 5.0g / L, sodium chloride 5.0g / L, agar 15.0g / L, pH value 7.3±0.2 .

[0042] The main components of trypticase soybean broth medium: casein trypsin hydrolyzate 15.0g / L, soybean powder papain hydrolyzate 5.0g / L, sodium chloride 5.0g / L, pH value 7.3±0.2.

[0043] Main components of Columbia blood agar medium: Add 5-10% sterile sheep whole blood on the basis of TSA.

[0044] Main ...

Embodiment 2

[0056] Specific gene detection of Escherichia coli and its SCVs

[0057] Using the standard strain of Escherichia coli ATCC 25922 as a positive control, Escherichia coli and its SCVs were detected for the amplification of specific gene phoA. The amplified product was 720 bp in size and a single bright band was positive for the amplification. This experiment uses 25μL PCR amplification system: dd H 2 O (20.35 μL), Easy Taq enzyme (0.15 μL), Easy Taq buffer (2.5 μL), dNTP (0.5 μL), 0.5 μL of upstream and downstream primers (see Table 1 for primer sequences), and 0.5 μL of DNA template. The conditions for PCR amplification of phoA gene were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 50 s, annealing at 55°C for 50 s, a total of 30 cycles, extension at 72°C for 1 min, and final extension at 72°C for 7 min.

[0058] Table 1 Primer sequences of phoA gene

[0059]

[0060]

[0061] figure 2 with Figure 7 Electropherograms for the detection of Escherichia...

Embodiment 3

[0063] Biochemical test, growth curve determination and auxotrophic test of Escherichia coli and its SCVs

[0064] 1. With Escherichia coli standard strain ATCC 25922 and sterile normal saline as positive and blank controls respectively, use micro biochemical identification tubes to carry out sugar fermentation experiments on Escherichia coli and its SCVs (lactose, sorbitol, mannitol, glucose and trisaccharide iron ) and catalase test, nitrate reduction test and acetate utilization test.

[0065] Among them, in the acetate utilization test, the wild strains all utilized acetate, while the induced strains did not utilize acetate (results see image 3), the wild strain and its SCVs all fermented and utilized glucose and lactose, and the results are shown in Table 2. image 3 Among them, A is the acetate utilization test, if the strain to be tested can utilize acetate, it will appear blue. If the strain to be tested cannot utilize acetate, it will not change color or be light y...

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Abstract

The invention relates to a preparation method for simply inducing bacteria to form SCVs in a laboratory, the induction success rate is high, and the problems that the SCVs induction workload is large, and the SCVs are easily covered by wild strains and are difficult to separate and purify are solved. The method can greatly simplify the induction and purification procedures of the bacteria SCVs and improve the induction screening efficiency. According to the preparation method disclosed by the invention, the continuous drug gradient concentration taking the filter paper as the center is formed on the solid culture medium, and various antibiotic filter paper can be placed on the same solid culture medium, so that the purpose of inducing and screening the same strain by using various antibiotics at the same time is achieved, and the working efficiency is greatly improved. The resistance of SCVs to antibacterial drugs is increased, so that the selection difficulty of clinical antibacterial drugs is improved. The escherichia coli SCVs are successfully separated and identified, a biological material is provided for research on SCVs related diseases, and a basis is also provided for diagnosis, treatment and prevention of escherichia coli SCVs related diseases.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a preparation method for inducing bacteria to form small colony variants. Background technique [0002] Small colony variants (Small Colony Variants, SCVs) are a special form of bacteria in response to environmental stress or host stress. SCVs are part of the normal growth cycle of bacteria. They are selected to become highly dynamic or static under harsh living conditions, and show resistance to multiple antibiotics. They are an important way to persist in the host, allowing bacteria to hide in host cells. It avoids antibacterial drugs and host immune response and causes persistent recurrent infection, which brings great difficulties to clinical treatment. Even SCVs hidden in host cells regain their virulence after antibiotic treatment ends or when the host immune response decreases, rapidly growing and killing host cells. [0003] Usually bacterial SCVs have different ph...

Claims

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Application Information

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IPC IPC(8): C12N1/36C12N1/20C12N15/11C12Q1/689C12Q1/10C12R1/19
CPCC12N1/36C12N1/20C12Q1/689
Inventor 陈伟吴自豪池昊明蔡依龙吴静
Owner TARIM UNIV
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