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A plasmid mediating recipient bacteria for decreased sensibility to quinolones antibacterial drugs

A quinolone and antibacterial technology, applied in the field of microbiology, can solve the problems of decreased sensitivity, quinolone drug resistance, clinical drug resistance and other problems

Inactive Publication Date: 2011-08-17
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of clinical drug resistance, especially the problem of quinolone drug resistance and for the research and development of new quinolone antibacterial drugs, to provide a method that can mediate receptor bacteria sensitive to quinolone antibacterial drugs. sex-reduced plasmid

Method used

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  • A plasmid mediating recipient bacteria for decreased sensibility to quinolones antibacterial drugs
  • A plasmid mediating recipient bacteria for decreased sensibility to quinolones antibacterial drugs
  • A plasmid mediating recipient bacteria for decreased sensibility to quinolones antibacterial drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning and screening of quinolone resistance genes from clinically isolated Gram-negative bacilli

[0044] Step 1. Plasmid-mediated screening of unknown quinolone-resistant gene clinical strains

[0045] Collect clinical isolates of Gram-negative bacilli with reduced sensitivity to quinolones, use PCR to detect known drug-resistant genes qnrA, qnrB, and qnrS, and retain known drug-resistant gene-negative strains for follow-up research;

[0046] Step 2: Acquisition of plasmids containing unknown quinolone resistance genes

[0047] The known drug-resistant gene-negative gram-negative bacilli clinical isolates obtained in step 1 were used as donor bacteria, and E.coliJ53Az R The conjugation experiment was carried out for the recipient bacteria (for specific methods, refer to the literature Antimicrob. Agents Chemother. 2003, 47: 2242-2248.). Obtained from the clinically isolated Proteus mirabilis No. 06-489 (preserved in the General Microbiology Center of China Committe...

Embodiment 2

[0053] Sequence analysis confirmed the new drug resistance gene qnrC

[0054] Step 1 In order to clarify that quinolone resistance is mediated by the qnrC gene, excluding the influence of qnrC upstream and downstream genes, qnrC was cloned again, and a fragment containing only the full sequence of qnrC was amplified with primers Primer1 and Pimer3 and cloned into pMD19( Takara, Dalian, China), and transformed into DH5α, the above-mentioned ampicillin screening plate was used to screen the transformant, and the transformant containing the recombinant plasmid also had increased drug resistance to ciprofloxacin. However, the fragment amplified with primers Primer2 and Primer3 without the complete sequence of qnrC was cloned into pMD19, and the transformants screened by ampicillin screening plate had no increased drug resistance to ciprofloxacin although they contained recombinant plasmids , further verified that quinolone resistance was caused by qnrC.

[0055] Step 2 is to furt...

Embodiment 3

[0057] Establish the PCR method for detecting qnrC gene according to the sequence of the present invention

[0058] Step 1. Primer Design

[0059] Input the nucleotide sequence of the present invention into oligo software for primer design, select primers from the primer sequences screened by the software, the expected product size is 395bp, and the primer sequences are as follows:

[0060] qnrC AF 5'-GGGTTGTACATTTATTGAATC-3'

[0061] qnrC AR 5'-CCTGAATTCTGCATTGCTC-3'

[0062] Step 2 Optimize PCR amplification conditions

[0063] Takara Taq reagents were used to prepare the PCR reaction system, and the annealing temperature was optimized on the TP600 TakaraPCR Thermal cycler dice amplification instrument with gradient heating and cooling functions. The final determination was 94°C for 5 minutes, 94°C for 30 seconds, 50°C for 30 seconds, and 72°C. 30 cycles in 30 seconds, the amplification effect is the best when extending for 5 minutes at 72°C.

[0064] Step 3 Sequence Ana...

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Abstract

The invention belongs to the field of microbiology, and relates to a plasmid of mediateable recipient bacteria with decreased sensibility to quinolones antibacterial drugs, the plasmid-supported mediateable gram negative bacilli to the quinolones drug resistant gene, and preparation method and use of the gene. The invention aims to the productive system of the drug resistance of the quinolones antibacterial drugs to find a new gene capable of mediating bacteria to be drug-resistant, and experiment result shows that the drug resistance of the quinolones antibacterial drugs is caused by the drug resistant gene qnrC. According to the quinolones drug resistant gene, a PCR primer to the gene is designed, and is capable of detecting other bacterial strains having the same gene. The invention can be used for the research of quinolones drug resistant system, further controlling the formation of the drug resistance and the detection of the drug resistant bacterial strains, and possibly used asthe potential target acted by the antibacterial drugs.

Description

technical field [0001] The invention belongs to the field of microbiology, and relates to a plasmid that can mediate the decrease of the sensitivity of recipient bacteria to quinolone antibacterial drugs, the gene carried by the plasmid that can mediate the drug resistance of Gram-negative bacilli to quinolones, and the expression of the gene Preparation method and use. Background technique [0002] Quinolones are widely used in the treatment of urinary tract infections, respiratory infections, abdominal infections, etc., but with the increase of clinical application, bacterial resistance to quinolones is rising rapidly. In China, the drug resistance of Escherichia coli to quinolones has risen rapidly, and the drug resistance rate currently ranks first in the world, much higher than other countries. According to domestic bacterial resistance monitoring data, when quinolones began to be used in China in 1988, almost all Escherichia coli were sensitive to them, and the drug r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
Inventor 王明贵王明华徐晓刚张婴元
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV