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Detection of Class Ⅰ Integron in Gram-negative Bacteria

Author: GaoJingHua
Tutor: NieDaPing
School: Dalian Medical University
Course: Clinical Laboratory Science
Keywords: gram-negative bacteria class Ⅰintegron
CLC: R446.5
Type: Master's thesis
Year: 2011
Downloads: 5
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Abstract


Objective: To investigate the prevalence of ClassⅠintegrons isolated gram- negative Bacteria clinically(ESBLs-producting Escherichia coli and Klebsiella pneumoniae. Pseudomonas aeruginosa and Acinetobacter baumannii are resistant to multiple drugs .) in Dalian and study the drug resistance mechanism.Methods: We collect non- duplicate163 strains Gram-negative bacilli (including 40 strains of E. coli, 45 strains of Acinetobacter baumannii, 40 strains of Klebsiella pneumoniae, and 38 strains of Pseudomonas aeruginosa). Antibiotics susceptibility was tested by Kirby-Bauer. ESBLs-producting E. coli and Klebsiella pneumoniae were detected by duble paper Synerguy Test. Template DNA is extracted by boiling. The conserved regions and Variable regions of ClassⅠintegrons were amplified by PCR. The purified amplicons of ClassⅠintegrons were sequenced and the variable region were analyzed by RFLP . 0.15KB and 1KB products were purified and sequended. The sequences are searched in the GeneBank and determine the types and arrangements of gene box of the two fragments.Results: The strains of Escherichia coli and Klebsiella pneumoniae were all pruducing ESBLs and Acinetobacter baumannii and Pseudomonas aeruginosa were multiple drug resistance. The rates of ClassⅠintegron Escherichia coli , Acinetobacter baumannii , Klebsiella pneumoniae and Pseudomonas aeruginosa are 70%(28/40), 64.4%(29/45), 45%(18/40) and 55.2%(21/38) respectively in 163 non-duplicate Gram-negative bacilli. 78 isolates have amplified variable region in 96 strains positive ClassⅠintegrons. The 150bp, 1000bp, 1600bp and 2000bp were detected by RFLP in Escherichia coli , Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. 1000bp and 1600bp were 77.4% , 150bp 10.14% and 2000bp 12.82% of all four fragments, 0.15kb fragments contain 5 ’and 3 ’ conserved region but no gene cassette. The 1kb fragments of variable regions did not detected any gene cassette. the reasons are to be identified.Conclusion: ClassⅠintegron widely exists in Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa,which suggesting that integron classⅠplayed an important role in the development and dissemination of multidrud resistance. The ClassⅠintegron positive rate of Klebsiella pneumoniae was lower. 0.15kb fragments do not contain the gene cassette which are the integron 5 ’and 3 ’ conserved region of the structure. The 1kb fragments variable region did not detected any gene cassette. the reasons are to be identified.

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