Resistance Mechanisma Of Clinical Imipenem-resistant Pseudomonas Aeruginosa

Purpose:Pseudomonas aeruginosa (PA) is a common opportunistic pathogenas well as one of the main pathogens caused by nosocomial infections.Imipenem is the common and major drug in clinically treat hospitalinfections caused by Pseudomonas aeruginosa. However, due toPseudomonas aeruginosa with congenital and acquired drug resistance,lead to a variety of antibiotics, including carbapenem antibiotic resistancerates rising, it appears more and more imipenem-resistant Pseudomonasaeruginosa (IRPA). The infectious diseases caused by Pseudomonasaeruginosa become more difficult to treatment, which is a major clinicalchallenge. In this study, the resistance mechanisms of imipenem-resistantPseudomonas aeruginosa isolated from our hospital were detected andanalyzed, such as the production of β-lactamase, the expression of active efflux system and the lack of outer membrane protein OprD2, throughthis we can have an overall understanding of imipenem-resistantPseudomonas aeruginosa resistance mechanisms in our hospital, so as toprovide of reference rational use of antibiotics in clinical.Method:1. Detection and analysis of MIC value measurement andmetalloenzymes genes in imipenem-resistant Pseudomonas aeruginosa:Collected imipenem-resistant Pseudomonas aeruginosa isolated fromSeptember2011to June2013in the hospitalized patients of our hospital.Various specimens such as sputum, blood, urine and pus are collected.With a semi-automatic bacterial identification system ATB expressionand the VITEK-2automatic identification and susceptibility analyzersidentification and susceptibility analysis, base on the standard ofAmerican Society of Clinical Laboratory Standards Institute (CLSI) inthe year of2012, using agar dilution method determination thePseudomonas aeruginosa MIC values of imipenem and meropenem. UseK-B disk diffusion method to add metal ion sequestrants paperethylenediaminetetraacetic acid (EDTA) to filter positive phenotype MBLproducing strains of bacterial. DNA extracted by boiling, the detective ofB metal enzyme gene IMP-1, IMP-2, VIM-1, VIM-2, SPM, GIM andothers genes of Pseudomonas aeruginosa in our hospital by PCRamplification. 2. Detection and analysis the active efflux system ofimipenem-resistant Pseudomonas aeruginosa:The above collection of imipenem-resistant Pseudomonasaeruginosa’ DNA were extracted by boiling, the detection of active effluxsystem-related genes such as OprM, mexA, mexB, mexC and mexE wereuse PCR amplification method, in order to understand whether there wereactive efflux system expression and efflux system which is the main typesof bacteria carrying in imipenem-resistant Pseudomonas aeruginosa ofour hospital.3. The detection of imipenem-resistant Pseudomonas aeruginosaouter membrane protein OprD2gene and protein OprD2:Collected Pseudomonas aeruginosa as said in the first part, the DNAof bacterial were extracted by boiling, and amplified by PCR assay outermembrane protein OprD2genes in order to understand whether ourhospital Pseudomonas aeruginosa outer membrane protein gene deletionOprD2situation. A bacterial membrane protein extraction kit extractedwith bacterial membrane proteins, the expression of outer membraneproteins OprD2were detected by SDS-PAGE electrophoresis, theexperiment of sodium salicylate was used to determine the position ofouter membrane proteins OprD2.Results:1. A total of145imipenem-resistant Pseudomonas aeruginosa of clinical isolates were collected in this study. In which putum samplesaccounted for65.5%(95/145); secretion samples accounted for17.2%(25/145) and remaining17.3%came from various other specimens suchas urine, pus, blood, bile and tissue specimens. Distribution from thedepartment, intensive care unit (ICU) accounted for the largestproportion, which is37.9%(55/145); followed by Burns and PlasticSurgery ward, which was17.9%(26/145), respiratory medicine andpediatric accounted for6.9%(10/145), respectively and there wereremaining others specimens from other wards. The imipenem MICvalues of all imipenem-resistant Pseudomonas aeruginosa strains wereequal or greater than8μg/ml, but as to meropenem, besides sevenbacterial’ MIC values equal4μg/ml (sensitive), others’ MIC values wereequal or greater than8μg/ml.2. In the145imipenem-resistant Pseudomonas aeruginosa strains,there are53strains to produce the phenotype MBL positive strainsaccount for36.6%of the total strains. There were43of145strainscarries MBL gene, and there were13VIM-1genes,20VIM-2genes and13SPM genes, others are negative results after gene amplification.2. In145strains of IRPA, a total of16carries detect active effluxgene OprM gene, in the16strains, there are12strains carry mexA andmexB genes, ie12carries mexAB-OprM efflux system, there are fourcarries only OprM genes. Other strains were not detected efflux genes. 3. In145strains of IRPA of this experiment, a total of115did notgive the product, in the detective of SDS-PAGE, there were115strainsdid not appear OprD2protein bands, deletion rate is79.3%, match withOprD2gene deletion. Standard strains of Pseudomonas aeruginosaATCC27853strain and the strains that sensitive to IMP weren’t deletionboth in gene and protein.Conclusion:1. The Pseudomonas aeruginosa distribution of our hospital in eachward, the separation of the highest rates of sputum specimens, accountingfor65.5%; followed by wound secretion, accounting for17.2%.Distribution from the department, intensive care unit (ICU) accounted forthe largest proportion,37.9%; followed by Burns and Plastic Surgeryward,17.9%.2. The lack of outer membrane protein OprD2is the mainmechanism of Pseudomonas aeruginosa in our hospital3. The production of metal β-lactamase in Pseudomonasaeruginosa is another important resistance mechanism, strains carryingthe β-lactamase genes are VIM-1, VIM-2and SPM.4. Exist of efflux system MexAB-OprM of Pseudomonas aeruginosain our hospital is another important resistance mechanism.5. The presence of two or more resistance mechanisms that mediatePseudomonas aeruginosa highly resistant to imipenem.