High throughput screening method for antimicrobial compounds and application of high throughput screening method

A screening method and compound technology, applied in the field of veterinary microbiology and veterinary pharmacy, to achieve the effect of short detection period and high sensitivity

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, effective antibacterial drugs have not been reported with this Actinobacillus pleuropneumoniae

Method used

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  • High throughput screening method for antimicrobial compounds and application of high throughput screening method
  • High throughput screening method for antimicrobial compounds and application of high throughput screening method
  • High throughput screening method for antimicrobial compounds and application of high throughput screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Establishment of high-throughput screening method for antibacterial compounds

[0055] The present invention uses Actinobacillus pleuropneumoniae HB0503 (in this specification, the applicant simply refers to it as the HB0503 strain or the target strain, both of which are the same strain) as the screening strain. The bacterial strain was isolated in Hubei Province, China by the applicant in 2005 (Kang M, Zhou R, Liu L, Langford PR, Chen H.Analysis of an Actinobacillus pleuropneumoniae multi-resistance plasmamid, pHB0503.Plasmid, 2009, 61 (2): 135-9), the strain was resistant to six antimicrobial drugs including sulfamethazine, gentamicin, chloramphenicol, streptomycin, ampicillin and kanamycin. The strain can be cultured and survived normally in TSA or TSB medium (purchased from Difco Laboratories, USA) containing 10% filtered bovine serum and 10 μg / ml NAD (nicotinamide adenine dinucleotide).

[0056] 1. Determination of the linear range

[0057] Inoculate a...

Embodiment 2

[0064] Example 2. Antibacterial activity of eight compounds

[0065] 1. Using the LOPAC compound library to screen antibacterial compounds

[0066] The Library of Pharmacologically Active Compounds (LOPAC) used in this example was purchased from Sigma, containing 1280 compounds, dissolved in DMSO at a storage concentration of 10 mM, and stored at -80°C for future use.

[0067] Firstly, 1280 compounds in the compound library were diluted to final concentrations of 50 μM, 25 μM, and 12.5 μM, respectively, and the antibacterial activity was screened by OD value method, and the antibacterial activity of the compound on the target bacteria HB0503 strain was calculated when the concentration of the compound was 50 μM rate (see Figure 5In A), the formula for calculating the bacteriostatic rate is: bacteriostatic rate=(bacterial control value-value after compound action) / bacterial control value. On the basis of the first screening, 89 compounds with antibacterial activity ≥ 30% wer...

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Abstract

The invention belongs to the technical field of veterinary microbiology and veterinary pharmacy and particularly relates to a high throughput screening method for antimicrobial compounds and the application of the high throughput screening method. Actinobacillus pleuropneumoniae isolates HB0503 resisting six antibiotics are taken as target bacteria; tetracycline is taken as positive control; an opaque 96-well plate is taken as a screening carrier; the number of live bacteria is measured through the chemiluminescence; the antibacterial effect of the antibacterial compound is identified. According to the quality determination criterion of the high throughput screening method, the bacterial turbidimetry is compared with the chemiluminescence, and the chemiluminescence is determined as the optimal solution. Eight compounds are indentified to have inhibitory activity to drug-resistant actinobacillus pleuropneumoniae strains, wherein four of the eight compounds has relatively good bacteriostatic activity to haemophilus parasuis, escherichia coli, streptococcus suis type 2and staphylococcus aureus.

Description

technical field [0001] The invention belongs to the technical field of veterinary microbiology and veterinary medicine. It specifically relates to a high-throughput screening method and application of antibacterial compounds. The invention screens and obtains some compounds with antibacterial activity. Background technique [0002] With the abuse of antibiotics, the phenomenon of bacterial drug resistance is becoming more and more serious. In recent years, the development speed of bacterial drug resistance has exceeded the speed of research and development of new drugs (David ML.Discovery research: the scientific challenge of finding new antibiotics.J Antimicrob Chemother, 2011, 66 (9): 1941-1944), multi-drug resistance is increasingly serious (Donadio S, Carrano L, Brandi L, Serina S, Soffientini A, Raimondi E, Montanini N, Sosio M, Gualerzi CO.Targets and assays for discovering novel antibacterial agents. J Biotechnol, 2002, 99: 175-185; Walsh FM, Amyes SG. Microbiology a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12R1/01
Inventor 周锐黎璐宋云峰吴新娟
Owner HUAZHONG AGRI UNIV
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