Mechanisms Of Multidrug Resistance Of Pseudomonas Aeruginosa Isolated From Surgical Intensive Care Unit

Pseudomonas aeruginosa (P. aeruginosa) is a clinically significant conditional pathogen in hospital with intrinsic resistance to various antimicrobial agents. Treatment of P. aeruginosa infections is a therapeutic challenge, especially in Surgical Intensive Care Unit (SICU). So, it is always an important and difficult problem for the world to investigate the mechanisms of drug resistance of P. aeruginosa. P. aeruginosa infections are very common and serious in SICU. Multidrug resistance becomes more and more prevalent. The objective of our study is to explore the multidrug-resistant mechanisms of P. aeruginosa isolated from SICU of Peking University First Hospital. 1. Resistant Phenotypes and β-Lactamases Genotypes of P. aeruginosa 49 consecutive nonrepeated P. aeruginosa strains were isolated from different patients in SICU, Peking University First Hospital from July, 2001 to July, 2004. The Minimal Inhibitory Concentrations (MICs) of various antibiotics were detected with agar dilution method recommended by National Committee For Clinical Laboratory Standards (NCCLS) to study their phenotypes of resistance; theirβ-Lactamases were extracted, types of their enzymes were identified and then their physical and chemical characteristics were studied by three dimentional extract test and Isoelectric Focusing (IEF); primers specific to ESBLs and MBLs were utilized to amplify their genes ofresistance to study their genotypes of resistance.Results:49 isolates of P. aeruginosa from SICU were all resistant to four or more than four antibiotics. The resistant rates of P. aeruginosa to different antibiotics were Cefotaxime 98%; Ciprofloxacin 80%; Levofloxacin 92%; Imipenem84%; Cefoperazone/Sulbactam 6%; Cefepime 6%; Amikacin 4%. The rate of p-Lactamases production was 98%. Among them 75.5% of isolates were found to produce high level of AmpC enzyme continuously. 22.4% strains produced ESBLs. Of 11 strains producing ESBLs, 8 isolates had oxa genes. We did not find shv, ctx-m1, ctx-m3, ctx-m10, veb genes or imp, vim genes in the isolates.2. Active Efflux SystemsTo investigate overexpression of active efflux systems involved in multidrug-resistance of Pseudomonas aeruginosa, expressions of the MexCD-OprJ and MexEF-OprN efflux systems were demonstrated by Western blot with measurement of the levels of outer membrane protein OprJ and OprN. Expressions of the MexAB-OprM and MexXY-OprM efflux systems were demonstrated by semi-quantity RT-PCR and real-time RT-PCR with measurment of the mexA and mexX mRNA expression respectively. The controling genes mexR, mexZ and nfxB, whose product downregulates the expression of the mexAB-oprM, mexXY-oprM and mexCD-oprJ operon respectively, were amplified. DNA fragments were sequenced by automated with ABI PRISM 3700. Results: among 49 multidrug-resistant P. aeruginosa isolated from SICU, 31 strains overexpressed the efflux systems. Among them, 27 strains overexpressed MexAB-OprM; 13 strains overexpressed MexCD-OprJ; 14 strains overexpressed MexEF-OprN and 3 strains overexpressed MexXY-OprM. 17 strains overexpressed two or more than two efflux systems simultaneously. The nucleotide sequences and deduced amino acid sequences analysis revealed that eight strains carried mutations in mexR gene in 10 isolates randomly selected from 27 strains overexpressed the MexAB-OprM. Among them 7 strains had the amino acid substitutions in MexR protein, 1 strain hadterminal code at the position of amino acid 32. The nucleotide sequences of mexR of PA36, PA41 and PA48 were submitted to Genebank with accession number AY899299,Y899300 and AY899301. All 2 strains of MexXY-OprM overexpression carried mutations in mexZ had the amino acid substitution in MexZ protein. Among 5 strains of MexCD-OprJ overexpression, mutations occurred in nfxB in 2 strains and led to the amino acid substitution in NfxB protein. 3 had intact nfxB.3. Outer Membrane Protein OprDTo investigate the mechanisms for Carbapenem resistance of P. aeruginosa in SICU, we examined OprD by Western blot assay. All of the 41 Carbapenem-resistant isolates showed the loss or decreased levels of OprD except PA42. Among them, 23 strains showed loss of OprD, they were all resistant to Imipenem with a MIC of 16～32 μg/Ml Most of them were sensitve or intermediate to Meropenem (22/23), only 1 strain was reisitant to Meromepem. 18 strains had a decreased OprD expression, 17 of them were resistant to Imipenem, 3 were resistant to Meropenem as well. 8 strains showed normal expression of OprD, 7 of them were sensitive to Carbapenems, 1 strain (PA42) was resistant to Meroenem but sensitive to Imipenem. The expression of OprD in Impenem-resistant group was much lower than that in Impenem-susceptible group (p<0.01), but there was no significant difference between Meropenem-resistant group and Meropenem-susceptible group. Not only the Carbapenem, the β-Lactambiotics, the Fluoroquinolones' susceptibilities were also determined by the loss of OprD to some extent. The resistant rate to Ciprofloxacin was statistically different between OprD normal expression group and OprD loss group and the rate for Ofloxacin, Piperacillin, Aztreonam was significantly different, too (P<0.05).Conclusions1. The rate of Multidrug Resistant P. aeruginosa was high in SICU. Continuously high level production of AmpC enzyme was the dominant enzymic mechanism andOXA-type enzyme was the main type of ESBLs in these isolates. Other types of ESBLs and VIM, IMP Metallo-β-Lactamases were not found.2. The rate of overexpression of the active efflux systems was high in multidrug-resistant P. aeruginosa and overexpression of the active efflux systems was one of mechanismsleading to multidrug resistance. The overexpression of MexAB-OprM, MexXY-OprM and MexCD-OprJ was caused by the mutations in mexR, mexZ, nfxB respectively and other mechanisms also existed.3. The Carbapenem resistance of P. aeruginosa clinical strains in SICU was mostly determined by the loss or decrease of OprD. Not only the Carbapenems, the P-Lactambiotics, the Fluoroquinolones' susceptibilities were also determined by the loss or decrease of d OprD to some extent.