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Novel drug resistant gene of Klebsiella pneumoniae

A technology of bacteria and drugs, applied in the field of new drug-resistant genes of Klebsiella pneumoniae

Active Publication Date: 2013-06-05
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports of pneumonia caused by gram-negative bacteria (GNB) other than Klebsiella

Method used

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  • Novel drug resistant gene of Klebsiella pneumoniae
  • Novel drug resistant gene of Klebsiella pneumoniae
  • Novel drug resistant gene of Klebsiella pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Discovery of new resistance gene KPC-15

[0026] -Materials and methods

[0027] 1. Materials

[0028] VITEK2 from French bioMerieux; bacterial identification card; VITEK2GN Test kit; bacterial susceptibility card; AST-GN13 from French bioMerieux; supplementary susceptibility paper from Oxoid Ltd, UK (75μg / 30μg cefoperazone / sulbactam, 30μg minocycline Element); miniBEST plasmid purification Kit from TakaRa company; high-fidelity polymerase, other reagents are commercially available.

[0029] 2. Method

[0030] 2.1 Isolation and identification of Klebsiella pneumoniae KP1241 strain

[0031] The blood culture-positive strains from patients in the cardiothoracic surgery department of Taizhou Municipal Hospital were transferred to blood plates for isolation and culture, and cultured at 35°C in a 5% CO2 incubator for 16-18 hours. Bacterial identification and drug susceptibility analysis system and drug susceptibility plate agar diffusion method (supplement drug s...

Embodiment 2

[0068] Example 2 Bacterial plasmid transduction experiment and drug resistance detection

[0069] 1. Materials and methods

[0070] 1. Materials

[0071] Klebsiella pneumoniae (accession KP1241); wild type E. coli J53.

[0072] 2. Methods and results

[0073] Klebsiella pneumoniae KP1241 and wild-type E.coli J53AzR (sodium azide resistance) were used as donor and recipient bacteria respectively, and 0.5ml of each in the logarithmic growth phase was added to 4ml of fresh LB Incubate overnight in broth. Conjugates were screened on tryptic soy agar (TSA) plates containing sodium azide (300 mg / L) and ciprofloxacin (0.03 mg / L), and incubated at 35°C for 18-24 hours. The screened zygotes were analyzed for their sensitivity to drugs with bioMerieux's VITEK2 bacterial identification and drug susceptibility analysis system and drug susceptibility plate agar diffusion method (supplemented with drug susceptibility discs). The results are shown in Table 2:

[0074] Table 2E.coliJ53Az...

Embodiment 3

[0078] Example 3 Construction of constitutive KPC-15 gene expressing bacteria

[0079] 1. Materials and methods

[0080] 1. Materials

[0081] E.coli JM109; pMD18T vector from TaKaRa company; pBR322 vector; restriction endonucleases Pst I and EcoR I from TaKaRa company; agarose gel recovery kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.; TaKaRa company's T4 DNA ligase.

[0082] 2. Method

[0083] 2.1 Construction of constitutive KPC-15 gene expressing strain E.coli JM109 (pET32a-KPC-15)

[0084] The PCR product in Example 1 was ligated with the pMD18T vector, and the ligated product was added to 100 μl of JM109 competent cells for transformation, cultured on a plate containing IPTG, x-gal, and Amp, and the gene sequence and its gene insertion direction were verified by sequencing. Then the KPC-15 gene was cloned into the pET32a vector to obtain the pET32a-KPC-15 plasmid containing the KPC-15 gene. The recombinant plasmid was transformed into competent E.coli ...

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Abstract

The invention discloses a novel drug resistant gene of Klebsiella pneumonia. The novel drug resistant gene of the Klebsiella pneumonia is obtained by extracting the Klebsiella pneumonia which is drug resistant against imipenem and ertapenem. The nucleotide sequence of the novel drug resistant gene of the Klebsiella pneumonia is SEQ ID NO. 1 and the amino acid sequence is SEQ ID NO. 2. Functions of the novel gene are researched through a plasmid transduction experiment and a prokaryotic expression experiment, and a result shows that the novel gene is drug-resistant against a plurality of drugs. The novel drug resistant gene of the pneumonia Klebsiella has a great significance on reasonable use of antibiotics, reduction of carbapenems drug-resistant bacteria produced during severe infection, and research and development of new drugs for inhibiting drug-resistance performance of bacteria.

Description

technical field [0001] The invention relates to a new drug resistance gene of Klebsiella pneumoniae, in particular to a new drug resistance gene found in drug-resistant Klebsiella pneumoniae isolated from blood culture specimens of patients and The protein encoded by this gene was confirmed to play an important role in bacterial resistance. Background technique [0002] Klebsiella pneumoniae is a rod-shaped gram-negative bacteria, usually covered with a large amount of capsules formed by sticky polysaccharides. Among them, Klebsiella pneumoniae is highly pathogenic to humans and is an important opportunistic pathogen and One of the iatrogenic bacteria. [0003] Klebsiella pneumoniae, also known as pneumoniae or Friedlander bacillus, is the first Gram-negative bacillus recognized to cause pneumonia. The form of the bacteria is short and thick bacilli with a size of 0.5-0.8×(1-2) μm, arranged individually, in pairs or in short chains. The bacteria have no spores, no flagell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/26C12N15/31C12N1/21A61K38/16A61P31/04
Inventor 王冬国杨林军牟永华侯伟伍海建张瑾
Owner ACROBIOSYSTEMS INC
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