Novel integron In1289 containing multiple drug-resistant gene cassettes

An integron, e.colijm109 technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve problems such as life-threatening, hospital distress, and threat to patient health and safety, to prevent outbreaks and reduce selection. effect of stress

Inactive Publication Date: 2016-10-26
王冬国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pathogenic Escherichia coli causes disease outbreaks and epidemics by contaminating drinking water, food, and polluted water bodies. In severe cases, it can be life-threatening; in addition, with the widespread use of broad-spectrum antibiotics in clinical practice, extended-spectrum β-lactam production has appeared in hospitals. The epidemic of Escherichia coli caused a lot of trouble to the hospital and seriously threatened the health and safety of patients

Method used

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  • Novel integron In1289 containing multiple drug-resistant gene cassettes
  • Novel integron In1289 containing multiple drug-resistant gene cassettes
  • Novel integron In1289 containing multiple drug-resistant gene cassettes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Identification of integron In1289

[0028] 1. Isolation and identification of EC7328 strain

[0029] 1.1 Material

[0030] Bacterial susceptibility card: AST-GN13 from bioMerieux, France. AST-GN13 drug sensitivity types are: amikacin, ampicillin, ampicillin / sulbactam, aztreonam, cefazolin, cefepime, cefotetan, ceftazidine, ceftriaxone, ciproxa Star, ertapenem, gentamicin, imipenem, levofloxacin, nitrofurantoin, piperacillin / tazobactam, tobramycin, SMZ.

[0031] Supplement drug sensitive paper (drug sensitive plate agar diffusion test): The paper comes from Oxoid, UK, with compound trimethoprim (30μg), chloramphenicol (30μg) and rifampicin (5μg).

[0032] 1.2 method

[0033] Apparatus identification: Transplant the strains with positive sputum culture from ICU patients of Taizhou Municipal Hospital to blood plate for isolation and culture (with 5% CO at 35℃) 2 Incubate in an incubator for 16-18h), and then put the separated pure bacteria on the VITEK 2 for bacterial ide...

Embodiment 2

[0055] Example 2 Plasmid transduction experiment to study the function of integron In1289

[0056] 1. Method

[0057] (1) The donor bacteria is the EC7328 strain and the recipient bacteria is E.coli J53 Az R (Resistant to sodium azide). The donor bacteria and recipient bacteria were respectively inoculated on LB plates and cultured overnight at 35°C. A single colony was picked and inoculated into 4 mL of LB broth, and cultured in shaking at 220 r / min at 37°C to the logarithmic growth phase. Take 0.5 mL of donor and recipient bacteria in 4 mL of LB broth, and culture them overnight at 37°C. The zygote was screened with tryptic soy agar (TSA) plates, which contained sodium azide (300mg / L) and amifloxacin (0.06mg / L). Incubate at 35°C for 18-24h. The plasmid of the drug-resistant strain was extracted (the steps were the same as in Example 1), and the sequence of the integron In1289 was detected by PCR.

[0058] The primers for amplifying the sequence of the integron In1289 are shown...

Embodiment 3

[0070] Example 3 Gene recombination experiments to study the function of integron In1289

[0071] 1. Method

[0072] (1) Connect the PCR product (nucleotide sequence SEQ ID NO. 1) in Example 2 to the pMD19-T vector, and add the ligation product to 100 μL of E. coli JM109 for transformation, and then transform it with IPTG, x- Cultivate on gal and Amp plates, extract plasmids from IPTG, x-gal, and Amp resistant strains for sequencing (the steps are the same as in Example 1), and detect whether the integrator In1289 is inserted into the genome. Then the integron In1289 sequence was cloned into the pET32a vector, and the insertion site of the integron was at the BamHI restriction site on the pET32a vector, and prepared according to conventional methods to obtain a pET32a recombinant plasmid containing the integron In1289 sequence. The recombinant plasmid was transformed into competent E.coli JM109 cells.

[0073] (2) Test the resistance of successfully constructed clones

[0074] Using...

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Abstract

The invention discloses a novel integron In1289, wherein the sequence of the integron is shown as SEQ ID NO.1. The integron is discovered from a genome of drug-resistant nontyphoidal salmonella EC7328. Therefore, the integron disclosed by the invention, with discovery thereof, has important theoretical and practical meaning for discussing a molecular mechanism of bacterial drug resistance transfer at a gene level on one side, and on the other side, the integron has a certain guidance function for clinical medication; and the integron is conducive to reduce the application of some antibacterial agents in the clinical field, and the integron is capable of reducing selective pressure of horizontal transfer of the drug-resistant gene cassettes and preventing explosive epidemic of drug-resistant bacteria.

Description

Technical field [0001] The invention belongs to the field of molecular biology technology and drug-resistant bacteria monitoring, and relates to a newly discovered integron closely related to the drug resistance of Escherichia coli. Background technique [0002] Escherichia coli, usually called Escherichia coli, is a component of the normal intestinal flora of the human body. Generally, it is a non-pathogenic bacteria in the intestine, but there are also some serotypes of Escherichia coli that can cause diarrhea or extraintestinal with different symptoms. infection. The pathogenic effects are mainly invasiveness, endotoxin and enterotoxin. According to different biological characteristics, pathogenic Escherichia coli is divided into 5 categories: pathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli ( EHEC), enteroadhesive Escherichia coli (EAEC). Pathogenic Escherichia coli cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C12N15/03C12N15/10C12R1/19
CPCC07K14/245C12N15/03C12N15/102C12N15/70C12Q2521/301
Inventor 王冬国周冬生陈佳玉
Owner 王冬国
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