A rapid dissemination of carbapenem-resistant Acinetobacter spp. represents a significant clinical threat. Production of OXA carbapenemases and metallo-beta- lactamases (MBLs) is the most important mechanism in acquiring carbapenem resistance in Acinetobacter spp. Carbapenem resistance has also ascribed to non- enzymatic mechanisms, including changes in outer membrane proteins, alterations in the affinity or expression of penicillin-binding proteins, and overexpression of efflux pumps. The most important mechanism in A. baumannii isolates from Korea is the production of OXA-23, while that in other species of Acinetobacter is the production of metallo-beta-lactamases.
Acinetobacter ; Korea ; Membrane Proteins ; Oxytocin ; Penicillin-Binding Proteins

No abstract available.
Amino Acid Substitution ; Korea ; Penicillin-Binding Proteins ; Penicillins ; Streptococcus

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.
Catalysis ; Intramolecular Transferases ; genetics ; Mutation ; Penicillin G ; pharmacology ; Penicillin-Binding Proteins ; genetics ; Streptomyces ; enzymology ; genetics

BACKGROUND: The rate of pneumococcal resistance in Korea has surged up to the world's highest level in a short period. To investigate the genetic relatedness and the spread of resistant pneumococci within Korea, and to obtain the basic data about structural changes of penicillin-binding proteins(PBPs), we performed a fingerprinting analysis of PBP 1A, 2X, and 2B genes of multidrug-resistant pneumococci isolated in Korea. METHODS: A total of 22 pneumococcal strains isolated from clinical specimens in 2 university-affiliated hospitals during the period from 1989 to 1996 were tested. PBP 1A, 2X, and 2B genes were amplified from chromosomal DNA by the polymerase chain reaction with specific primers. Amplified products were digested with HinfI or MseI and DdeI and were followed by end-labeling with [alpha-32P] dCTP. Direct comparison of fingerprinting patterns between resistant strains and dendrogram analysis which was based on the UPGMA method were carried out. RESULTS: Fingerprinting analysis of PBP 1A, 2X, and 2B genes digested with HinfI showed that 17 out of 22 strains had almost identical patterns. Dendrogram showed that clusters with greater than 90% similarities existed in 77%, 77%, and 82% of strains with PBP 1A, PBP 2X, PBP 2B, respectively. Fingerprinting patterns with MseI and DdeI were the same as those with HinfI. CONCLUSION: Data from PCR fingerprinting analysis of PBP 1A, 2X, 2B genes of multidrug- resistant pneumococci in this study indicate the genetic relatedness between the resistant strains and suggest the possible spread of pneumococcal resistance within Korea.
Dermatoglyphics* ; DNA ; Korea* ; Penicillin-Binding Proteins* ; Polymerase Chain Reaction* ; Streptococcus pneumoniae* ; Streptococcus*

OBJECTIVETo develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU).

METHODSA recombinant penicillin-binding protein (PBP) 2x* from Streptococcus pneumoniae R6 was expressed in vitro and six β-lactams were conjugated to HRP by four methods. A rapid multi-residue assay for β-lactams was established with PBP2x* and HRP-conjugate.

RESULTSPBP2x* was expressed and purified successfully and the ideal HRP-conjugate was identified. The multi-residue assay was developed. After optimization, penicillin G, ampicillin, amoxicillin, cloxacillin, dicloxacillin, oxacillin, nafcillin, cephalexin, ceftiofur, cefalonium, cefquinome, cefazolin, cefoperazone, cephacetrile, and cephapirin can be detected at levels below MRL in milk with simple pretreatment.

CONCLUSIONThis assay developed can detect all 16 β-lactams demanded by the European Union (EU). The whole procedure takes only 45 min and can detect 42 samples and the standards with duplicate analysis.

Monofunctional biosynthetic peptidoglycan transglycosylase (MBPT) catalyzes the formation of the glycan chain in bacterial cell walls from peptidoglycan subunits: N-acetylglucosamine (NAG) and acetylmuramic acid (NAM). Bifunctional glycosyltransferases such as the penicillin binding protein (PBP) have peptidoglycan glycosyltransferase (PGT) on their C terminal end which links together the peptidoglycan subunits while transpeptidase (TP) on the N terminal end cross-links the peptide moieties on the NAM monosaccharide of the peptide subunits to create the bacterial cell wall. The singular function of MBPT resembles the C terminal end of PBP as it too contains and utilizes a similar PGT domain. In this article we analyzed the infectious and non infectious protein sequences of MBPT from 31 different strains of bacteria using a variety of bioinformatic tools. Motif analysis, dot-plot comparison, and phylogenetic analysis identified a number of significant differences between infectious and non-infectious protein sequences. In this paper we have made an attempt to explain, analyze and discuss these differences from an evolutionary perspective. The results of our sequence analysis may open the door for utilizing MBPT as a new target to fight a variety of infectious bacteria.
Bacteria ; Cell Wall ; Glycosyltransferases ; Muramic Acids ; Penicillin-Binding Proteins ; Penicillins ; Peptidoglycan ; Peptidoglycan Glycosyltransferase ; Sequence Analysis

BACKGROUND: The resistance of Streptococcus pneumoniae to penicillin has been rapidly increasing during the recent three decades. In Korea, we found the incidence of penicillin resistance (PR) to pneumococci was 81% for the clinical isolates in our hospital, and 89% for the colonizing isolates of day-care center around Seoul. Alterations in penicillin-binding protein (PBP) 2B gene have been known to be associated with a resistance to penicillin. We tried to reveal the characteristics of PR of Korean pneumococcal strains through sequencing analysis of PBP 2B gene. METHODS: We determined the nucleotide sequences of 346 bp of a variable region among PBP 2B gene for 12 PR strains and 6 penicillin susceptible (PS) strains isolated in Korea. Phylogenetic tree using PAUP program was made to compare our DNA sequences with those of South African strains. RESULTS: Sequence homology of PS strains was ranged from 99.4% to 100% in Korean PS strain compared to reference strain, R6, except one strain (93.9%). PR strains showed homology of 95.1% to 100% compared to the South African 56762 strain. Phylogenetic analysis based on 346 bp of PBP 2B gene showed that Korean and South African strains formed different monophyletic groups according to the PR/PS patterns. Five specific amino acid changes compared to the PS R6 strain in the position 228, 232, 233, 244, and 261 were noted with a decreased hydrophilicity by Kyte-Doolittle assay. CONCLUSIONS: Our data suggest that the amino acid changes in the PBP 2B are associated with PR in S. pneumoniae, and that a part of Korean PR strains might be originated from a South African PR strain.
Base Sequence ; Colon ; Hydrophobic and Hydrophilic Interactions ; Incidence ; Korea* ; Penicillin Resistance ; Penicillin-Binding Proteins* ; Penicillins ; Pneumonia ; Seoul ; Sequence Homology ; Streptococcus pneumoniae* ; Streptococcus*

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: During the last three decades, the resistance of S. pneumoniae to penicillin has been rapidly increasing in many parts of the world, especially in Korea. To characterize the clinical features and epidemiology of penicillin-resistant S. pneumoniae (PRSP) infections in the community and hospital, as well as to investigate the possible spread of resistant clone, we performed the antimicrobial susceptibility tests, pulsed filed gel electrophoresis (PFGE) and penicillin-binding protein (PBP) profile analysis of PRSP isolates. METHODS: A total 48 PRSP isolates from patients who visited or were admitted to Korea University Guro hospital during the period form July 1998 to June 1999 were studied. Anitimicrobial susceptibility tests for 48 isolates were performed with microbroth dilution method to determine the minimal inhibitory concentration of 11 antibiotics. 39 isolates and 35 isolates were subjected to PFGE and PBP profile analysis, respectively to investigate the genetic relatedness between PRSP isolates. RESULTS: Pneumonia was most common site of infection in the community and the hospital as 50%. There were no significant differences of clinical features and prognosis between community and hospital isolates. But, patients with serious underlying diseases had poor prognosis despite of acquisition site. High level penicillin resistance were observed in 69%, multi-drug resistance were 64.6% of isolates. PFGE showed that 13 of 29 community acquired infection were identical PFGE pattern but not that of 23F Spanish clone. There were various PFGE patterns were observed from community and hospital acquired infection isolates. Some of them were existed in both. PBP profiles showed more diverse, even if in isoaltes of the same PFGE pattern. CONCLUSOIN: In our study, high level penicillin resistance and multi-drug resistance were observed in PRSP clinical isolates. No clinical and prognostic differeces were observed between community and hospital acquired infections. Molecular epidemiology study were suggest the there were various genotypes of PRSP within our society. Some of them were observed in the hospital and community. Therefore, there was an evidence of communication of PRSP clones between the community and hospital.
Anti-Bacterial Agents ; Clone Cells ; Drug Resistance, Multiple ; Electrophoresis ; Epidemiology ; Genotype ; Humans ; Korea ; Molecular Epidemiology ; Penicillin Resistance ; Penicillin-Binding Proteins ; Penicillins ; Pneumonia ; Prognosis ; Streptococcus pneumoniae* ; Streptococcus*

PURPOSE: Low-affinity penicillin-binding protein PBP 2a encoded by mecA is closely related to methicillin resistance in staphylococci, and the expression of PBP 2a is controlled by regulator elements encoded by mecR1 and mecI. Deletion or mutation which occurred in mecI is considered to be associated with constitutive production of PBP 2a. We investigated the distribution of mec regulator genes and the presence of the mutations in mecI among mecA gene-positive methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase negative Staphylococcus (MRCNS) strains. METHODS: A total of 28 MRSA and 26 MRCNS clinical strains were isolated at Chung-Ang University Hospital. The distribution of mec regulator genes and the presence of mutations in mecI were analyzed using polymerase chain reaction and direct sequencing. RESULTS: In 28 MRSA and 26 MRCNS, only mecR1A-positive pattern (type lll) was detected in 53.6% of MRSA and 46.4% of MRCNS. The mecR1 (mecR1A and mecR1B) and mecI-positive pattern (type l) were detected in 42.3% of MRSA and 38.5% of MRCNS. In 19.2% of MRCNS was type lV in which no mec regulator genes were detected. Our results showed that a greater genomic variation existed in MRCNS than a MRSA. Results in direct sequencing of mecI revealed that mecI gene tested in our study did not harbour mutations and deletions. There was no correlation between the level of resistance and the presence or absence of mec regulator genes. CONCLUSION: The induction of methicillin resistance and the variability of phenotypic expression of methicillin resistance suggested that additional factors on the chromosome are involved.
Coagulase ; Genes, Regulator* ; Methicillin Resistance* ; Methicillin* ; Methicillin-Resistant Staphylococcus aureus ; Penicillin-Binding Proteins ; Polymerase Chain Reaction ; Staphylococcus*