Study On The Molecular Mechanism Of Imipenem-resistance Pseudomonas Aeruginosa

ObjectivePseudomonas aeruginosa(Pa)is a kind of conditional pathogenic bacteia which causes serious nosocomial infection oftenly. According to its intrinsic or acquired drug resistance, the therapy is a tough problem.Imipenem is a widely used antibiotics of cabapemems for anti-infective treatment of Pa infection.Meanwhile, with the wide use of imipenem, this causes cabapemem-resistant strains appear accordingly. Since the multi-drug resistance of Pa strains,it is a big challenge for the clinical anti -infection treatment.In this study, to verify the regularity of interactivity of different mechanism of imipenem resistance, and to provide reference for clinical medication, we analysised the antibiotic resistance feature of imipenem-resistant Pseudomonas aeruginosa (IRPa), that were isolated from hospitalized patients, detected beta lactamase genes which related to the resistance to imipenem and gene cassettes carried by integrons, and investigated the molecular mechanism of imipenem-resistance of Pa.Methods1.Drug sensitive test and three-dimensional test220 strains of Pa were isolated from hospitalized patients between 2003 and 2007. K-B method was used to tested the susceptibility of 10 different antibiotics. IRPa was screened by testing the minimal inhibitory concentration (MIC) of imipemem by using agar diluiion method.The susceptibility of these IRPa to the antibiotics was analysised. Three-dimensional test was used to identify the different kinds of beta lactamases from 220 strains of Pa.2.Carbarpenems hydrolytic enzyme genes and oprD2 gene were detectedamong the selected IRPa strains, PCR method was performed to detect carbapenemase genes which included GES,KPC,SPM,VIM,IMP,GIM gene and the oprD2 gene;Multiplex PCR were used to detect OXA genes and plasmid-mediated AmpC beta lactamase genes; The expression of the chromosomal AmpC beta lactamases and oprD2 genes in IRPa strains were analyzed by Real-time PCR.3.Identification and characterization of integronsIntegrase gene was detected by PCR, and the classification of integrons was performed by using restriction fragment length polymorphism (RFLP).PCR was performed to detect the qacE△1-sull gene,and the gene cassetes which are located at variable region of integrons in the strains were detected to be positive. PCR products of the variable region of integrons were sequenced and analysised to realize whether the drugs resistance gene cassetes carried by integron mediate the imipenem resistance of Pa.Results1. Totally 74 strains of IRPa were selected, the drug resistance ratio to meropemem,third-generation cephalosporin and aztreonam are more than 50%, expecially to cefotaxim, the ratio is 81.08%.The sensitivity rate of Polymyxin B,aminoglycoside antibiotics and ciprofloxacin are higher than other antibiotics. Polymyxin B sensitive rate is 100%, amikacin is 70.27%, gentamicin is 62.16%, and ciprofloxacin is 51.35%.2. 43 isolates producing beta lactamases was detected by three- dimensional test.The isolation rate of beta-lactammase was 19.55% (43/220), the constituent ratio of AmpC beta lactamases, extended spectrum beta lactamases ( ESBLs), metallo-beta lactamases (MBL), super-spectrum beta lactermbse(sSSBLs=ESBLs+AmpC) and an unknown type of beta lactamases were 58.14%(25/43),18.6%(8/43),4.65%(2/43),2.33%(1/43) and 16.28%(7/43),respectively. Among the 43 isolates producing beta lactamases,26 strains are resistant to imipenem. Except 2 isolates producing MBL and 7 strains producing unknown type of beta lactamases, there are 17(65.4%) strains producing AmpC beta lactamases. According to the three-dimensional test detecting the hydrolytic action of AmpC beta lactamases to imipenem, The crude extract AmpC lactamase of 7 strains can hydrolyze IPM feebly.3.PCR and Multiplex PCR was performed to detect the beta lactamase genes which relate to the imipenem resistance. Except the DHA-1 gene identified in I1197 strain, the beta lactamase genes weren't detected in other strains. OprD2 gene wasn't detected in 40 strains which account for 54.05% in total strains.4.The expression of oprD2 genes in IRPa strains were identified by Real-time PCR. Of the 25 isolates with the expression of increased AmpC beta lactamases, the plasmid-mediated AmpC beta lactamase gene DHA-1 was identified in 1 isolate, 12 isolates with diminished expression of oprD2 were IPM resistance, and the others with normal oprD2 expression mainly were IPM sensitiveness;The crude extract AmpC lactamase in 7 isolates can hydrolyze IPM feebly. of the 7 isolates with increased expression of AmpC beta lactamase gene, 5 strains had decreased expression of oprD2 gene.5. Among 74 strains of IRPa, integrase gene and qacE△1-sull gene was detected in 16 strains (22.86%)and the class1 integron was identified in these strains. The variable region of integrons were amplified in 14 strains. 7 types integron with different gene cassettes were identified by sequencing, Aminoglycoside-modifying enzyme genes were detected in all these integrons, and some of them were beta lactamase genes and unknown genes. Four novel cassette arrays was discovered:①aac(6)-II- unknown-CatB3 and②aac(6)-II-catB3-ORFII-ORFIII including the same resistance genes of aac(6)-II and catB3. The aac(6)-II gene codes the resistance to amikacin, netilmicin and tobramycin, and the catB3 gene codes chloranphenicol acetyl -transferase which confer resistance to chloramphenicol. The protein coded by ORFII gene is RNA-directed DNA polymerase. The unknown gene maybe is a novel gene which codes putative transposase;③In blaIMP-9-aacA4- OXA-10 -aadA2-like, IMP-9 gene codes metallo beta-lactamase which can hydrolyze carbapenem; aacA4 gene codes aminoglycoside (6)N-acetyltransferase which confer resistance to aminoglycosides; OXA-10 gene codes low level resistance to cefotaxime,ceftriaxone and aztreonam; aadA2 gene codes resistance to spectinomycin and streptomycin.④In aac(6)-II-blaP1b- aadA2, blaP1b codes carbenicillinase which confer resistance to carbenicillin. The sequences of these gene cassettes were submitted on Genebank,and the accession numbers are FJ917747, FJ817422, FJ655384 and FJ958368, FJ817423, respectively.Conclusions1.The IRPa strains showed multidrug rsesitant and cross resistant to variety of antibiotics. The antibiotics with higher level sensitivity to IRPa should be used to treat IRPa-caused disease, such as ciprofloxacin and aminoglycosideantibiotics. Regarding to the infection caused by multidrug rsesitant Pa strains ,the Polymyxin B would be a good choice of empirical antibiotics treatment.2. Among the Pa strains producing beta lactamase , the detection rate of strains producing AmpC beta lactamase is higest and some of them can hydrolyze IPM feebly. Maybe AmpC beta lactamase is an important factor for the resistance of Pa to third generation cephalosporin and imipenem.3. The main reason of resitance of IRPa to IPM is the mutation of oprD2 or the diminished production of oprD2. No matter the expression of AmpC beta latamase gene in Pa strains is high or low, when the expression of oprD2 gene decreased or disappeard, that will lead to the imipenem resistance of Pa. If just AmpC beta lactamase with normal orpD2 gene expression in Pa strains , most of the strains will be sensitive to imipenem, the rest will obtain the resistance to IPM by producing other beta lactemase or other mechanisms. In general, The mechanism of imipenem resistence in Pa strains is a result of the interplay between diminished production of oprD2 and incresed activity of AmpCβ-lactamase, but the former plays a more important role in these influence.4. Among the IRPa strains with positive integrase gene, only classI integron was detected. In these classI integrons, 7 kinds of combination forms of resistance gene cassetes carried by integron were identifed by squencing. IMP-9 metal beta lactamase gene was found in Class1 integron of 2 Pa strains ,which is an only case related to imipenem resistance. The resistance gene cassetes carried by integrons include resistant gene to aminoglycosides and beta lactam antibiotics. Maybe the different gene cassettes array beared by class I integron are the material foundation of multi-resistance to antibiotics of Pa.Some gene cassetes with unknown function needs to be investgated in the future.