PURPOSE: Two Korean nationwide studies showed that metallo-beta-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp. MATERIALS AND METHODS: Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes. RESULTS: Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time. CONCLUSION: Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.
Anti-Bacterial Agents/pharmacology ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Imipenem/pharmacology ; Korea ; Polymerase Chain Reaction ; Pseudomonas Infections/*microbiology ; Pseudomonas aeruginosa/drug effects/genetics/*metabolism ; beta-Lactamases/genetics/*metabolism

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.
Alkanes ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fimbriae, Bacterial ; drug effects ; genetics ; metabolism ; Houttuynia ; chemistry ; Pseudomonas aeruginosa ; cytology ; drug effects ; genetics ; pathogenicity ; Sulfites ; pharmacology ; Virulence ; drug effects

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 ; beta-Lactam Resistance/*genetics ; beta-Lactamases/metabolism ; beta-Lactams/*pharmacology

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 ; Humans ; Microbial Sensitivity Tests ; Permeability ; Pseudomonas aeruginosa ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactamases ; metabolism ; beta-Lactams ; pharmacology

BACKGROUNDCarbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa (P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit (SICU) in China.

METHODSThe molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.

RESULTSForty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the low-expressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301.

CONCLUSIONSThere was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.

Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Carbapenems ; pharmacology ; China ; Humans ; Intensive Care Units ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; drug effects ; genetics ; Real-Time Polymerase Chain Reaction ; beta-Lactamases ; genetics ; metabolism

No abstract available.
Anti-Bacterial Agents/pharmacology ; DNA, Bacterial/chemistry/genetics/metabolism ; Drug Resistance, Bacterial ; Integrons/*genetics ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa/drug effects/*enzymology/isolation & purification ; Sequence Analysis, DNA ; beta-Lactamases/*genetics

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

BACKGROUNDAcute lung infection due to Pseudomonas aeruginosa (P. aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P. aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. aeruginosa in vitro. We used flagellin protein from P. aeruginosa, a key initiator of acute P. aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. aeruginosa can cause fibrogenesis/remodeling.

METHODSWe studied the effect of flagellin from P. aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH(2)-terminal kinase (JNK) and extracellular regulated kinase (ERK).

RESULTSExpression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. control groups ((104.3 ± 20.8) vs. (44.6 ± 4.4) pg/ml (P < 0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1 ± 18.8) vs. (104.3 ± 20.8) pg/ml and (48.1 ± 20.8) vs. (104.3 ± 20.8) pg/ml, respectively (P < 0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. the control groups ((554.9 ± 57.7) vs. (51.4 ± 22.9) pg/ml (P < 0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 ± 155.1) vs. (554.9 ± 57.7) pg/ml (P < 0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P < 0.01), 30 minutes (P < 0.01) and 15 minutes (P < 0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.

CONCLUSIONSFlagellin from P.aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells, BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. aeruginosa.

Blotting, Western ; Bronchi ; cytology ; Cell Line ; Cell Survival ; drug effects ; genetics ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; drug effects ; metabolism ; Flagellin ; pharmacology ; Humans ; Interleukin-8 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Pseudomonas aeruginosa ; metabolism ; Signal Transduction ; drug effects ; genetics ; Transforming Growth Factor beta1 ; genetics ; metabolism

BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a threat to hospitalized patients. We evaluated the effects of antimicrobial combinations on XDR P. aeruginosa and A. baumannii isolates. METHODS: P. aeruginosa and A. baumannii isolates, which were resistant to all antibiotics except colistin (CL), were collected from eight hospitals in Korea. Genes encoding metallo-beta-lactamases (MBLs) and OXA carbapenemases were detected by PCR in eight P. aeruginosa and 30 A. baumannii isolates. In vitro synergy of antimicrobial combinations was tested by using the checkerboard method. RESULTS: Minimum inhibitory concentrations of beta-lactams, aminoglycosides, and fluoroquinolones were very high, while that of CL was low for majority of XDR P. aeruginosa and A. baumannii isolates. Antimicrobial combinations including Imipenem (IPM)-CL, ceftazidime (CAZ)-CL, and rifampin (RIF)-CL exerted only additive/indifferent effects on majority of XDR P. aeruginosa isolates. Proportions of XDR A. baumannii isolates that showed synergistic and additive/indifferent inhibition after treatment with antimicrobial combinations used are as follows: IPM-ampicillin-sulbactam (AMS), 17% and 80% isolates, respectively; IPM-rifampin (RIF), 13% and 81% isolates, respectively; IPM-CL, 13% and 87% isolates, respectively; and RIF-COL, 20% and 73% isolates, respectively. Significant proportion (19%) of XDR P. aeruginosa isolates produced MBLs, and majority (82%) of A. baumannii isolates produced either MBLs or OXA-23. CONCLUSIONS: Our results suggest that combinations of IPM-AMS, IPM-RIF, IPM-CL, and RIF-CL are more useful than individual drugs for treating 13-20% of XDR A. baumannii infections.
Acinetobacter baumannii/*drug effects/genetics/isolation & purification ; Aminoglycosides/pharmacology ; Anti-Infective Agents/*pharmacology ; Bacterial Proteins/genetics/metabolism ; Drug Resistance, Multiple, Bacterial/*drug effects ; Drug Synergism ; Fluoroquinolones/pharmacology ; Imipenem/pharmacology ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Pseudomonas aeruginosa/*drug effects/genetics/isolation & purification ; beta-Lactamases/genetics/metabolism

No abstract available.
Anti-Bacterial Agents/*pharmacology ; Azabicyclo Compounds/*pharmacology ; Bacterial Proteins/genetics ; Ceftazidime/*pharmacology ; Cephalosporins/*pharmacology ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Bacterial/*drug effects ; Gram-Negative Bacteria/drug effects/*isolation & purification ; Humans ; Microbial Sensitivity Tests ; Penicillanic Acid/*analogs & derivatives/pharmacology ; Pseudomonas aeruginosa/drug effects/isolation & purification ; Real-Time Polymerase Chain Reaction