Research Of The Mechanism Of Integron Mediated Drug-resistance In Gram-negative Bacteria

Backgroud:Integrons are genetic fragments that can recognize and capture mobile gene cassettes by site-specific recombination.Gene cassettes which confer resistance to many antibiotics have been characterized within integrons.Although integrons are unable to move,the gene cassettes carried by integron can be mobilized to other integrons by integrase catalizing at attC site.It was confirmed that integrons associate with the bacterial acquired drug-resistance and play an important role in it.Pseudomanos aeruginos(P.A)induce opportunistic infection in onsocomial enviroments with intrisinc and acquired resistance to various antibiotics.Carbapenems are efficient broad sprectrum antibiotics and they are the best treatment option for serious bactrial infection caused by Gram-negative bacteria.However,it comes up a great challenge to clinical antibacterial therapy due to carbapanem resistance of P.A.The aim of our research is to investigate the mechanism associate with carbapanem resistant P.A,and to provide a guidance for the clinical rational antibiotic prescription and treatment.Objective:To identify the categories and gene cassettes arrangements of integrons,and their distributions in the Gram-negative bacteria.Take Pseudomanos aeruginos for example,to investigate the role of metalβ-lactamase,integrons and outer membrane protein for Carbapenem resistance in Gram-negative bacteria.Material and methods:1.Research on integron in Gram- negative bacteria.From September 2006 to June 2007,183 strains of Gram-negative bacteria were isolated from Third Xiangya hospital of Central South University.PCR was used to classify the integron and amplify the variable region by different primers,PCR-RFLP to screen the homology of integron variable region,and the gene cassettes were detected by DNA sequencing.Results:ClassⅠintegron was found in 100 strains of clinical isolates and there is no classⅡorⅢintegron found.Four kinds of integron variable region were identified:blaVIM-2-aacA-Ib-aadA2-qacE; dfrA12-aadA2-qacE-blaCTX-M-2;dhfrXⅡ-orfF-aadA2;and dhfrX-orfF-aadA2-sul1, which are the drug-resistance genes encoded for carbapanem,β-lactamase,aminoglycosides and sulfamide.2.Analysis of carbapenem-resistance mechanism of P. aeruginose.42 strains of P.aeruginose were isolated.EDTA synergy test was used to analyze metalβ-lactamase(MBL);PCR was used to amplify the integron followed by sequencing of the PCR products;expression of mexA was demonstrated by semi-quantity RT-PCR;and expression of the outer membrane protein OprM,OprJ and OprN were detected by Western Blot.In this way,we intended to figure out the main antibiotic-resistance mechanism of P.aeruginose.Results:The rate of metalβ-lactamase and integron were 52.4% and 64.3%respectively.The expression level of the outer membrane protein was high in carbopenems-resistant P.aeruginose,.Conclusions:1.ClassⅠintegron were prevail in Third Xiangya hospital,they harboured variable gene cassettes which encoded resistance to many antibiotics frequently used in nosocomial enviroments.2.The high level production of metalβ-lactamase and the overexpression of the effiux system were regarded as the mechanism leading to carbopenems-resistance in P.aeruginose.There is a direct relation between integrons and aminoglycosides-resistance.