Research Of The Qnr Gene,ISCR1Element And New Dihydrofolate Reductase Gene In Resistance Bacteria From Aquatic Environment

With the improving and progress of the medical level, varieties of antibiotics have been developed gradually and put into use. But along with the use and abuse of antibiotics, causing a large amount of antibiotics residues in the environment, made the bacteria resistance rate to be rising and the emergence of "superbugs" was a wake-up call to us. Now the new drag research and development speed is far behind the pace of drug resistance emergence and spread. Bacteria resistance, especially the multiple drug resistance has become a serious threat to human health and a global problem, also inevitably become the focus of global concern and research content.Quinolone antibacterial daigs, developing from for the treatment of urinary tract infection in early time to now for the treatment of intestinal infection and respiratory infection, have been widely used in clinical, animal husbandry and aquacullure. Bacteria gradually become resistant to them and resistance mechanism is more and more complicated. Quinolone resistance mechanism is mainly divided into chromosome mediated resistance and plasmid mediated resistance, the latter plays an important role in the spreading of antibiotic resistance. In1998, plasmid mediated quinolone resistance mechanism was reported for the first time, namely the qnr gene mediated fluoroquinolone resistance mechanism, qnr genes can spread rapidly in different bacteria, which causes the infection is not easy to control, makes the nosocomial infection popular in a wide range. Previous studies have indicated that the qnrA and qivB usually locate in the downstream of complex class1integron containing ISCR1element, qnrS have not been found to exist in integron by now. As a new gene capture system, ISCR element can move any DNA sequence nearby, providing efficient medium for drug-resistant genes to spread in different species by the horizontal gene transmission. So qnr genes often relates to aminoglycoside. trimethoprim, chloramphenicol and some other antibiotic resistance gene cassettes that located in the variable region of class I integron upstream of ISCR1element. In addition, the qnr genes are usually associated with β-lactamase resistance gene, qnr genes exist in complex integron and integrate with the other varieties of resistance genes, which narrows the space of clinical medicine choose or drug combinations use to treat related bacterial infection and brings us a serious challenge.This study mainly researched on the antibiotics resistance situation in the water environmenl of Jinan area and the distribution of qnr genes to evaluate the effects of antibiotics pollution to the environment and discover new resistance mechanism in the environment. In addition we also studied the gene mobile element ISCR1to find new horizontal gene transfer mechanism and cloned new resistance genes, expressed and purified their proteins to do subsequent analysis. Hope our results could provide some reference for controlling the increasing antibiotic resistance and its transmission, provide certain theoretical basis for medicine choose or drug combinations use to treat related infections, and provide novel targets for drug development.In this experiment we found31qnr gene positive strains and41ISCR1element positive strains in391antibiotic resistance gram-negative bacteria isolated from hospital and living waste water samples in Jinan area. We further classified them into10different genura and analyzed their antimicrobial susceptibility. The detected qnr genes mainly belonged to qnrA1, qnrB2, qnrB4, qnrB6, qnrB9, qnrS1six normal variants and a new variant qnrB26. The other two PMQR genes aac(6’)-Ib-cr, qepA and other resistance genes were also widespread in the qnr gene positive strains. Eight different gene arrays were found in the ISCRI mediated downstream fragment. Three kinds of qnr gene variants were contained in three kinds of the ISCR1mediated downstream structures respectively and three new dihydrofolate reductase(dfr) genes were also located in ISCR1mediated downstream structures. Though cloning, expression and protein purification of the new dfr genes and subsequent analysis we found they all mediated a certain level trimethoprim resistance, and their protein in vitro could promote the normal growth and proliferation of bacteria to some extent in trimethoprim existing situation.