BACKGROUND: Resistance to penicillin of Streptococcus pneumoniae in clinical isolates has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for antibiotics and has been encountered with increasing frequency in Korea. In this study, the identification of altered PBPs of S. pneumoniae in clinical isolates by using polymerase chain reaction (PCR) and relationship of PCR with the conventional antibiotic susceptibility test of penicillin were evaluated. METHODS: Thirty isolates of S. pneumoniae from clinical specimens were used. Four sets of PCR primers of penicillin-sensitive and -resistant S. pneumoniae were designed to amplify (i) PBP 2BS: a 360 base pair fragment of the PBP 2B gene, (ii) PBP 2BCA: a 350 base pair fragment of the class A mutations present in PBP 2B gene, (iii) PBP 2BCB: a 295 base pair fragment of the class B mutations present in PBP 2B gene, and (iv) PBP 1AR: a 434 base pair fragment of the PBP 1A gene. In addition, a set of primers that amplify 273 base pair of the autolysin gene (ALY) was applied in combination with the above to identify S. pneumoniae. PCR results were compared with antibiotic susceptibility test (disk diffusion test and penicillin MIC). RESULT: Among 30 clinical isolates tested, 80% of isolates were penicillin resistant. The results of antibiotic susceptibility test were same as those of PCR methods. Among 24 penicillin resistant isolates detected by PCR methods, 5 isolates revealed PBP 2BCB gene, but 19 isolates revealed both of PBP 2BCB and 1AR genes. Five isolates with PBP 2BCB gene showed lower range of penicillin MIC (0.19 ~ 1.0 g/mL) than 19 isolates with PBP 2BCB and 1AR genes (0.75 ~ 4.0 g/mL). CONCLUSION: Detection of altered PBP genes of S. pneumoniae by PCR may be performed for the study of penicillin resistance. This study indicates that more altered PBPs in 1AR genes are related with higher MICs.
Anti-Bacterial Agents ; Base Pairing ; Diffusion ; Korea ; N-Acetylmuramoyl-L-alanine Amidase ; Penicillin Resistance ; Penicillin-Binding Proteins ; Penicillins ; Pneumonia ; Polymerase Chain Reaction* ; Streptococcus pneumoniae* ; Streptococcus*

BACKGROUND: Resistance to beta-lactams in E. coli is mostly via acquisition of plasmid-mediated beta-lactamase gene. Among the plasmid-mediated beta-lactamases, TEM-1 beta-lactamase is by far the most prevalent among ampicillin-resistant E. coli. The prevalence of TEM-1 or TEM-2 ranged from 61% to 98% across the surveys. Klebsiella species generally have class A chromosomal beta-lactamases, which differ greatly from the class C types. Most K. pneumoniae isolates have chromosomally mediated SHV-1 beta-lactamase in most surveys. There has been only one report of prevalence and types of beta-lactamases in E. coli and K. pneumoniae in Korea. We performed this study to determine the prevalence and types of beta-lactamases in E. coli and K. pneumoniae isolated in Korea. METHODS: Ampicillin resistance was determined by disk diffusion test (E. coli) and agar dilution method (K. pneumoniae). Fifty five isolates of E. coli and 92 isolates of K. pneumoniae which were derived from patients in 2 university hospitals in Korea during 1996 were tested by TEM- and SHV-specific PCR. RESULTS: The ampicillin resistance rate in E. coli and K. pneumoniae was 82% and 94.6%, respectively. TEM-type beta-lactamase gene was found in 53% of E. coli isolates. 93.5% of K. pneumoniae isolates was found to have SHV-type beta- lactamase gene. CONCLUSION: In Korea TEM-type beta-lactamase gene was most prevalent in E. coli, but its prevalence rate was relatively low compared with those in other country. For K. pneumoniae, the isolates with SHV type beta-lactamase gene were predominant.
Agar ; Ampicillin Resistance ; beta-Lactamases* ; beta-Lactams ; Diffusion ; Escherichia coli* ; Escherichia* ; Hospitals, University ; Humans ; Klebsiella pneumoniae* ; Klebsiella* ; Korea* ; Penicillinase ; Pneumonia ; Polymerase Chain Reaction ; Prevalence*

BACKGROUND: Accurate detection of organisms producing extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase is very important for treatment of patients. However, unlike the ESBL confirmatory test, there are no guidelines for detection of organisms producing AmpC beta-lactamase. We evaluated a detection method using boronic acid (BA) for ESBL and AmpC beta-lactamase. METHODS: Clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis showing intermediate resistance or resistance to cefoxitin (FOX) or positive for ESBL were tested. A > or =5 mm increase in zone diameter of ceftazidime/clavulanic acid/BA (CAZ/CA/BA) and/or cefotaxime/clavulanic acid/BA (CTX/CA/BA) versus CAZ/BA and/or CTX /BA was considered positive for ESBL. Likewise, a > or =5 mm increase in zone diameter of FOX/BA and/or cefotetan/BA (CTT/BA) versus FOX and/or CTT alone was considered positive for AmpC beta-lactamase. RESULTS: Among 622 clinical isolates, ESBL positive rates by the CLSI ESBL confirmatory test or by the BA method were 18.1% or 18.4% for E. coli, 38.3% or 40.4% for K. pneumoniae, 8.7% or 8.7% for K. oxytoca, and 14.8% or 14.8% for P. mirabilis, respectively. AmpC beta-lactamase positive rates using the BA method were 3.7% for E. coli, 33.3% for K. pneumoniae, 0% for K. oxytoca, and 7.4% for P. mirabilis. The detection rates of coproducing ESBL and AmpC beta-lactamase were 2.4% in E. coli 27.1% in K. pneumoniae, and 3.7% in P. mirabilis. CONCLUSION: The ESBL confirmatory method using BA was found to enhance the detection of ESBLs, even when potentially masked by AmpC beta-lactamase.
Bacterial Proteins ; beta-Lactamases ; Boron ; Cefoxitin ; Escherichia ; Escherichia coli ; Humans ; Klebsiella ; Klebsiella oxytoca ; Klebsiella pneumoniae ; Masks ; Mirabilis ; Penicillinase ; Pneumonia ; Proteus ; Proteus mirabilis

BACKGROUND: The prevalence of extended-spectrum beta-lactamase(ESBL)-producing organisms has been increasing in Korea. We performed a study to characterize various TEM derivatives with clinical isolates of Klebsiella pneumoniae collected from 3 hospitals in Korea. METHODS: Fifty-seven isolates of ESBL-producing K. pneumoniae collected from 3 hospitals were screened by TEM-specific PCR for the carriage of TEM genes. Thirteen strains were found to have TEM-related genes. Eleven blaTEM genes were amplifed and sequenced. The transfer of resistance was tested by conjugation and isoelectric points of beta-lactamases were determined. MICs were measured to obtain a resistance pattern for each indivudual wild- type and transconjugant strain. The hydrolysis rate of TEM-52 was measured spectrophotometrically. RESULTS: Ten strains carried plasmid-mediated CTEM-52 gene, which sequence showed the substitution of 3 amino acids compared to that of TEM-1: 104 glutamic acid --> lysine (GAG --> AAG), 182 methionine --> threonine (ATG --> ACG), and 238 glycine --> serine (GGT --> AGT). MIC showed that TEM-52 mediated a resistance to ceftazidime and aztreonam at a lower level than to cefotaxime. TEM-52 enzyme hydrolyzed cefotaxime efficiently (Vmax, 340) and showed fairly weak activity for ceftazidime (Vmax, 15.1), but very weak activity for aztreonam. The gene of TEM-52 beta-lactamases was mediated by approximately 77-kb plasmids. CONCLUSION: From these results, we conclude that the TEM-52 beta-lactamase is a common TEM- type ESBL in K. pneumoniae in Korea.
Amino Acids ; Aztreonam ; beta-Lactamases* ; Cefotaxime ; Ceftazidime ; Glutamic Acid ; Glycine ; Hydrolysis ; Isoelectric Point ; Klebsiella pneumoniae* ; Klebsiella* ; Korea ; Lysine ; Methionine ; Penicillinase ; Plasmids ; Pneumonia ; Polymerase Chain Reaction ; Prevalence ; Serine ; Threonine

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.
Amidohydrolases ; isolation & purification ; metabolism ; Arthrobacter ; enzymology ; Bacterial Proteins ; isolation & purification ; metabolism ; Chromatography, DEAE-Cellulose ; methods

Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.
Amidohydrolases/*genetics ; Antitubercular Agents/*pharmacology ; Drug Resistance, Bacterial ; *Mutation ; Mycobacterium tuberculosis/*drug effects/genetics ; Pyrazinamide/*pharmacology

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
Amidohydrolases ; chemistry ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; Molecular Weight ; Nocardia ; enzymology ; Phylogeny

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.
Amidohydrolases ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Glycine/analogs & derivatives* ; Hydrogen-Ion Concentration ; Metal-Organic Frameworks ; Temperature

From 1992 to 2001, penicillin resistance of betalactamase-producing Neisseria gonorrhoeae was supervised by Institute of Dermato-Venereology. Results: the rate of penicillin-resistant gonococcal strains was highest in1995 (about 76%), and in 1996 (73.11%), especially in 3 years (1999-2001) this rate decreased significantly: 51.3%, 47.10%, and 35.54%, respectively
Neisseria gonorrhoeae ; Neisseria ; penicillinase

OBJECTIVETo investigate the effect of Angelicae Pubescentis Radix (APR) on the activity of endocannabinoid hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA), and to demonstrate the mechanism of anti-inflammatory effect of APR by in vitro lipopolysaccharide (LPS)-induced inflammation model.

METHODAPR essential oil was extracted by steam distillation, and the chemical components were identified by GC-MS. Enzymatic activity was performed by using recombinant NAAA-overexpressing protein and detected by LC-MS. Lipids were extracted by methonal/chloroform mixure and analyzed by LC-MS. mRNA and protein expression levels of proinflammatory genes were examined by Real time-PCR and ELISA assay kit, respectively. The content of nitro oxide (NO) was detected by Griess reaction.

RESULTTwenty active components were identified from APR essential oil which inhibited NAAA activity in a dose-dependent manner. On the LPS-induced RAW264.7 cells, APR essential oil reversed LPS-suppressed N-palmitoylethanolamide (PEA) contents in a dose-dependent manner and reduced LPS-induced proinflammatory genes, TNF-alpha and IL-6. Moreover, APR essential oil reduced the mRNA expression of iNOS, subsequently reduced the release of NO, a classic inflammatory marker.

CONCLUSIONThe research demonstrated that the effect of APR on inflammation is mediated by the inhibition of NAAA activity, which increase the cellular endobioactor PEA levels and decrease proinflammatory factor. The results suggest that APR can serve as a nature NAAA inhibitor.

Amidohydrolases ; antagonists & inhibitors ; Angelica ; chemistry ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mice ; Oils, Volatile ; analysis ; pharmacology