2.The mechanism of resistance of Pseudomonas aeruginosa to beta-lactam antibiotics and clinical significance.
Jianxin SONG ; Qiurong RUAN ; Junying QI ; Meiying GAO ; Yiguang WANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2002;22(4):339-342
To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents
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pharmacology
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Bacterial Outer Membrane Proteins
;
metabolism
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Humans
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Microbial Sensitivity Tests
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Permeability
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Pseudomonas aeruginosa
;
drug effects
;
beta-Lactam Resistance
;
genetics
;
beta-Lactamases
;
metabolism
;
beta-Lactams
;
pharmacology
3.The mechanism of resistance of Pseudomonas aeruginosa to beta-lactam antibiotics and clinical significance.
Jianxin, SONG ; Qiurong, RUAN ; Junying, QI ; Meiying, GAO ; Yiguang, WANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2002;22(4):339-42
To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents/*pharmacology
;
Bacterial Outer Membrane Proteins/metabolism
;
Microbial Sensitivity Tests
;
Permeability
;
Pseudomonas aeruginosa/*drug effects
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beta-Lactam Resistance/*genetics
;
beta-Lactamases/metabolism
;
beta-Lactams/*pharmacology
4.Contributions of efflux pumps to high level resistance of Pseudomonas aeruginosa to ciprofloxacin.
Dan-Dan WANG ; Tie-Ying SUN ; Yun-Jian HU
Chinese Medical Journal 2007;120(1):68-70
Anti-Infective Agents
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pharmacology
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Bacterial Outer Membrane Proteins
;
genetics
;
physiology
;
Ciprofloxacin
;
pharmacology
;
DNA Gyrase
;
genetics
;
Drug Resistance, Bacterial
;
Membrane Proteins
;
genetics
;
physiology
;
Membrane Transport Proteins
;
genetics
;
physiology
;
Mutation
;
Pseudomonas aeruginosa
;
drug effects
5.The resistance mechanisms of b-lactam antimicrobials in clinical isolates of Acinetobacter baumannii.
Na Young KWON ; Jae Deok KIM ; Hyun Joo PAI
The Korean Journal of Internal Medicine 2002;17(2):94-99
BACKGROUND: Despite increasing importance of Acinetobacter baumannii in nosocomial infections and rapid development of multi-antimicrobial resistance in this strain, the resistance mechanisms of beta-lactam antimicrobials in A. baumannii were not clearly defined. In order to observe the resistance mechanisms against beta-lactams and carbapenem, we characterized the production of beta-lactamases and outermembrane protein (OMP) profiles for the 44 clinical isolates of A. baumannii. METHODS: The MICs of antimicrobials were determined by agar dilution test. The secondary beta-lactamases were characterized by isoelectric focusing, polymerase chain reactions and nucleotide sequencing, and the production of chromosomal beta-lactamases was quantitated by spectrophotometric method. For two strains with an elevated MIC of carbapenem, outermembrane protein (OMP) profile was analyzed by ultracentrifugation of the sonicated bacteral cells and SDS-PAGE. RESULTS AND CONCLUSION: Twenty two or 4 of 44 strains produced TEM-1-like beta-lactamase or PER-1 extended-spectrum beta-lactamase, respectively. However, when we analyzed the MICs of several beta-lactams with the beta-lactamase production, the resistance level of beta-lactam was mainly determined by the production of chromosomal beta-lactamase, not by the secondary beta-lactamases in the clinical isolates of A. baumannii. In two strains with an elevated MIC of imipenem, a decrease or loss of about 35 kDa and 22 kDa proteins in OMP was observed, which suggested that the change of OMP played a role in carbapenem resistance.
Acinetobacter/*drug effects/isolation & purification/metabolism
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Acinetobacter Infections/drug therapy/microbiology
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Antibiotics, Lactam/*pharmacology
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Bacterial Outer Membrane Proteins/biosynthesis
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Carbapenems/pharmacology
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Cross Infection/drug therapy/microbiology
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Drug Resistance, Bacterial
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Human
;
beta-Lactamases/biosynthesis
6.Immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.
Li-hui XU ; Jie YAN ; Ping RUAN ; Ya-fei MAO
Journal of Zhejiang University. Medical sciences 2005;34(1):9-14
OBJECTIVETo investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.
METHODSNi-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA.
RESULTSUnder the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h.
CONCLUSIONThe expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.
Bacterial Outer Membrane Proteins ; immunology ; pharmacology ; Cells, Cultured ; Endothelial Cells ; cytology ; Epitopes ; Genotype ; Humans ; Inflammation ; etiology ; Interleukin-1 ; biosynthesis ; Leptospira interrogans ; genetics ; immunology ; Lipoproteins ; immunology ; pharmacology ; Recombinant Proteins ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; Umbilical Veins ; cytology
7.Drug screening model acting on out-membrane protein OprM in pseudomonas aeruginosa efflux pump system.
Rui TIAN ; Li-yan YU ; Chun-ling XIAO ; Lian ZUO ; Tian-jue YAO ; Li-xia YANG
Acta Academiae Medicinae Sinicae 2004;26(4):359-363
OBJECTIVETo establish an efflux pump inhibitor screening model with the out-membrane protein OprM in Pseudomonas aeruginosa efflux pump system as the target point.
METHODSEfflux pump out-membrane protein gene oprM was obtained from standard Pseudomonas aeruginosa PA01 strain. Expression of OprM protein was induced in E. coli strain HS151 with T-easy vector as the cloning vector, and pMMB67EH as the expression vector. In order to evaluate the function of OprM protein, we measured intracellular tetracycline concentrations with liquid scintillation counter, measured the diameters of bacteriostatic circles with paper disc, and then established a screening model accordingly.
RESULTSOprM protein was highly expressed. Using Pseudomonas aeruginosa as the main detecting bacteria, we established a drug screening model acting on OprM. A total of 1 600 microbial fermentation samples were screened with this model, among which 56 positive strains were found, with a positive rate of 3.5%.
CONCLUSIONOprM plays an important role in drug efflux. The established model has good specificity and maneuverability.
Anti-Bacterial Agents ; metabolism ; pharmacology ; Bacterial Outer Membrane Proteins ; biosynthesis ; drug effects ; genetics ; Bacterial Proteins ; genetics ; Drug Evaluation, Preclinical ; methods ; Drug Resistance, Microbial ; Drug Resistance, Multiple ; genetics ; Escherichia coli ; genetics ; Humans ; Membrane Transport Proteins ; biosynthesis ; drug effects ; genetics ; Plasmids ; genetics ; Pseudomonas aeruginosa ; drug effects ; genetics
8.Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages.
Ho Ki SHIM ; Jeoung Yeon KIM ; Mi Jeong KIM ; Hee Sun SIM ; Dae Won PARK ; Jang Wook SOHN ; Min Ja KIM
Experimental & Molecular Medicine 2009;41(10):687-694
Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.
Animals
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Antigens, CD/immunology/metabolism
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Bacterial Outer Membrane Proteins/*pharmacology
;
Cells, Cultured
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Female
;
Histocompatibility Antigens Class II/immunology/metabolism
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Host-Pathogen Interactions
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Interleukin-12/biosynthesis
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Interleukin-6/biosynthesis
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Legionella pneumophila/*immunology/metabolism
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Legionnaires' Disease/immunology/metabolism
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Lipoproteins/*pharmacology
;
Macrophage Activation/drug effects/immunology
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Macrophages, Peritoneal/drug effects/immunology/*metabolism
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Mice
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Mice, Inbred BALB C
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Mice, Inbred C3H
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Mice, Inbred C57BL
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Toll-Like Receptor 2/*metabolism
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Tumor Necrosis Factor-alpha/biosynthesis