Study On The Drug-resistant Mechanism Of Imipenem-resisitant Pseudomonas Aeruginosa And The Relationship With MBL And Class 1 Integrons

Pseudomonas aeruginosa is an adaptable bacterial saprophyte found in environment. It is also a major gram-negative opportunistic nosocomial pathogen, and the only Pseudomonas species with a great potential to cause severe, life threatening and multidrug-resistant infection. The infections can range from corneal infections to severe and often fatal infactions in patients with cystic fibrosis(CF), severe burns, and leukemia or lymphomas. Carbapenem, such as imipenem and meropenem, have a broad antibacterial spectrum and play a fundamental role in the treatment of infections due to multidrug-resistant Pseudomonas aeruginosa isolates. Imipenem is the most powerful, widely afforded carbapenem to date, so the rate of imipenem-resisitant Pseudomonas aeruginosa is rising. However, Pseudomonas aeruginosa can also develop resistance to carbapenems through mechanisms often associsted with decreased permeability, derepressed expressed of chromosomal AmpCβ-lactamases, and upregulation of the efflux system. In recent years, carbapenem resistance in nosocomial Pseudomonas aeruginosa has oly occasionally been due to production of IMP and VIM metallo-β-lactamases, which hydrolyze mostβ-lactams, including caraapenems, and are encodes by integron-associated gene cassttes. Integron can't move by itself, but can be sited in plasmid or transposon to transmit.This experiment is on the research of the situation of Pseudomonas aeruginosa producing MBL and carrying class I integron in southwest China and the aim of the experiment is to explore new focus of epidemiology research and offer strategy for clinical research.Seventy-two strains of non-repetitive Pseudomonas aeruginosa were isolated, among them sixty-one strains resistant to imipenem and eleven sensitive. The rate of AMK-resisitance was the lowest. The forty-two strains of imipenem-resistant Pseudomonas aeruginosa were subjected to agar dilution methods, and their drug-resistant rate ranked increasingly as followed: AMK, CAZ, CIP, PIP. For the ceftazidime-2-mercaptoethanol -double-disk synery test of the forty-two imipenem-resistant strains, twenty-seven showed positive and there were seven performance types.All the strains were subjected to PCR assays with primers specific for bla_IMP-1 and bla_VIM, then sequencing the positive one to identify the results. The positive strains were followed by PCR analysis with primers specific for class 1 integrons. Then the lba_Int1-positive strains were subjected to PCR analysis of gene cassettes. The rate of carring- bla_VIM in all was 76%, the noticeable two of the bla_VIM-positve strains were sensitive to imipenem. It is highly likely that the gene pool is more extensive and either in a quiescent state or not being detected or both. Yet eleven 11 imipenem-resistant strains gave negative results for MBL detection, perhaps for other drug-resistant meganism. Besides of these a Pseudomonas aeruginosa was found carrying-bla_IMP-1. For sixty-one imipenem-resistant strains, the incidence of the MBL was 87%. All the positive strains for incidence of ceftazidime-2-mercaptoethanol -double-disk synery test were verificated to carry MBL.It is the first time IMP-1 and VIM-2 MBL gene are reported in West China. The sequenced 906bp PCR product of PA 9917 was verificated to be IMP-1 gene by blasting it with PA AY251052.1 in GenBank. For bla_Int1 PCR, PA 9917 was verificated to contain bla_Int1 as blast with PA DQ219465.1 and for bla_InC PCR, 1800bp gene cassette. But there were no MBL gene found in gene cassette. There are dhfrXII, orfF, aadA2 genes for dihydrofolate reductase and putative protein, streptomycin/spectinomycm 3' adenyltransferase in gene cassette of PA 9917. Plasmid has been extracted successfully from PA 9917, but bla_IMP-1 PCR showed negative possibly the plasmid locate in chromosome. PA 895 were verificated to produce VIM-2. The sequence is the same as PA DQ522236.1. But no class 1 integron or plasmid were found in PA 895. It is need further research to locate the MBL in PA895. PA 9917 and PA 895 were both imipenem-resistant and high-resistant for all the drugs except resisitant to CIP.The spread of class 1 integron and gene cassettes among imipenem-resisitant Pseudomonas aeruginosa was studied. The incidence of class 1 integron was almost half of all, and the the incidence of gene cassettes among bla_Int1-positive strains was 69.2%.A new gene cassette located at class 1 integron was found in imipenem-resisitant PA 9576. It showed negative result for ceftazidime-2-mercaptoethanol -double-disk synery test. The homology of gene cassette align with PA AB189979.1 was 98%. The different gene coded dihydrofolate reductase. Six mutant sites were: 101 (D→N),117 (T→S),118 (V→G),120 (V→D),121 (E→K) .Perhaps the metabolism of acidum folicum are affected.The conclusion is that, the incidence of MBL is high among imipenem-resistant Pseudomonas aeruginosa, CAZ-MPE synery test facilite to the detection of MBL, The incidence of class 1 integron was almost half of all, and the the incidence of gene cassettes among lba_Int1-positive strains was 69.2%. The aim of the study is to approach the imipenem-resistant meganism of Pseudomonas aeruginosa, detect the distribution of class 1 integron gene cassettes among them. The Intll is potential target for controlling the spread of MBL, which due to carbapenem-resisitance.