Dissemination, Drug Resistance and Molecular Biology Properties of the Integrons among Some Clinical Isolates of Pseudomonas Aeruginosa
|School||Nanjing Medical University|
|Keywords||Class 1 integron Gene cassette Pseudomonas aeruginosa Resistance Plate agar dilution method PCR Pulsed-field gel electrophoresis|
Objective: To understand the integration of the sub-part Pseudomonas aeruginosa the distribution resistance in clinical isolates, analysis integron Pseudomonas aeruginosa resistant and multi-drug correlation. Learn how to integrate variable region carrying the resistant genotype, study its molecular characteristics, and to explore the role of integrons in the spread of bacterial resistance and dissemination. Materials and methods: 1. Strains: 1997-2003 Jiangsu Provincial People's Hospital, collected and randomly selected non-repeatability and clinical significance of Pseudomonas aeruginosa strains, a total of 47. Methods: boiling method to extract a total of 47 Pseudomonas aeruginosa DNA templates, each integration based on 1,3 integrin gene conserved region primer design, PCR amplification in order to filter out the respective positive strains; then integron positive strains of Pseudomonas aeruginosa PU21 incubation metastasis bonding test; 47 strains transfer zygote MIC determination using agar dilution method, and the analysis of drug susceptibility. Integron 5 'and 3' conserved region of design the universal variable region primers, PCR amplification of the variable region of class 1 integron positive strains, according to a class. Variable region PCR product of plastic recycling purified, sequence analysis, and the results compared with Genbank sequence to determine the variable region gene cassettes contained. Class 1 integron positive strains corresponding zygote total DNA as template design, according to the class 1 integron variable region sequencing results contained resistant gene cassette primers corresponding to a resistance gene was amplified by PCR cloning and sequencing to confirm results and preliminary test integron in plasmid. Alkaline lysis method to extract integron positive strains corresponding plasmid zygote. By pulsed-field gel electrophoresis (pulsed field gel electrophoresis, PFGE) analysis integron positive strains homology. Results: 47 Pseudomonas aeruginosa detected by the PCR method 29 containing 1 integrase, accounting for 61.7% of all isolates (29/47), Class 3 integrons were not detected. Susceptibility results than the negative class 1 integron positive strains resistant strains was significantly increased; integron positive strains resistant to piperacillin, ceftazidime and amikacin, gentamicin, chloramphenicol, streptomycin the highest drug rate reached 100%. 29 class 1 integron positive strains were transferred bonding success; transfer zygote compared with the wild-type strains were sensitive to piperacillin / tazobactam, imipenem, ciprofloxacin, the MIC 90 fell by 8,128 and 128 times respectively. 29 carried class 1 integron variable region amplification products were approximately 4.3kb. Sequence analysis showed that: the variable region contains four drug resistance gene cassette, to form a new combination of aac (6 ') - Ⅱ-aadA13-cmlA8-oxa-10, has been submitted to Genbank, serial number EU182575. The which aadA13 was first found in P. aeruginosa. While the third sequence of the open reading frame has not been reported, up to 95% homology with known cmlA5 named cmlA8. Both the wild-type strain or zygote were amplified by PCR and the four gene cassettes, sequenced, cloned and sequenced. Regardless of the wild-type strain or zygote were extracted in the same size, greater than 10Kb resistance plasmid. Pulsed-field gel electrophoresis analysis showed that all Class 1 integron-positive strains with a high degree of homology, the presence of clonal propagation and possible relationship. Conclusion: class 1 integron popular hospital Pseudomonas aeruginosa clinical isolates. Class 1 integron-positive strains negative strains more prone to drug resistance to many antibiotics. The combination drug susceptibility phenotype and integration of sub-genotypes carrying resistance genes, integrons and Pseudomonas aeruginosa resistant and multi-drug-related. And compared to the wild-type strain, transfer zygote piperacillin / tazobactam, imipenem, ciprofloxacin sensitive. The same as the class 1 integron variable region gene carrying the resistance gene cassette combinations. Integration sub-situated engagement plasmid. There is the possibility of horizontal or vertical transmission between these Class 1 integron positive strains.