OBJECTIVETo observe the effect of amino acid substitution in conserved sequence of penicillin-binding protein (PBP) 1A, 2B, 2X on antimicrobial activity of beta-lactams against Streptococcus pneumoniae (SP).

METHODMinimal inhibitory concentration (MIC) of 6 beta-lactams was determined by the E-test in 59 SP strains. The penicillin-binding protein genes pbp1a, 2b, 2x in every SP strain were amplified by nested-polymerase chain reaction (nPCR), then the PCR products were sequenced using automatic genetic analyzer directly. To analyze the amino acid substitutions, the DNA sequences were converted to protein sequences and aligned by Clustalx software. According to amino acid substitution in conserved sequence of PBP2B, 3 phenotypes were observed, including: PBP2B phenotype I (no amino acid substitution); PBP2B phenotype II (Glutamine 432-->Leucine and/or Threonine 445/451-->Alanine/Serine, Glutamic 481-->Glycine, 1 strain had proline insertion between residues 431/432); PBP2B phenotype III (Alanine 624-->Glycine with the addition of phenotype II). According to amino acid substitution in conserved sequence of PBP1A, 3 phenotypes were observed, including: PBP1A phenotype I (no amino acid substitution); PBP1A phenotype II (Threonine 574-->Asparagine, Serine 575-->Threonine, Glutamine 576-->Glycine, Phenylalanine 577-->Tyrosine, 574TSQF-->NTGY); PBP1A III (Threonine 371-->Alanine/Serine, Proline 432-->Threonine with the addition of 574TSQF-->NTGY). According to amino acid substitution in conserved sequence of PBP2X, 4 phenotypes were observed, including: PBP2X phenotype I (no amino acid substitution); PBP2X phenotype II (Histidine 394-->Leucine or Threonine 338-->Alanine); PBP2X phenotype III (Threonine 338-->Alanine, Isoleucine 371-->Threonine, Arginine 384-->Glycine and Leucine 546-->Valine); PBP2X phenotype IV (Methionine 339-->Phenylalanine, Methionine 400-->Threonine with the addition of PBP2X phenotype III).

RESULTAmong 59 SP strains antibacterial activities distribution (sensitive strains, intermediate strains and resistant strains) of 6 beta-lactams were penicillin (12, 29, 18); amoxicillin(49, 9, 1); cefuroxime (16, 16, 27); ceftriaxone (47, 1, 11); cefotaxime (47, 3, 9); imipenem (49, 10, 0). beta-lactam antibiotics insensitive strains (intermediate + resistant strain) in PBP2B phenotype III, PBP1A phenotype III, PBP2X phenotype III and IV were significantly increased, the MIC(50) of these strains were significantly higher than that of the others.

CONCLUSIONThe amino acid substitutions in or vicinal conserved sequence of PBP of SP increase MIC for beta-lactam antibiotics.

Amino Acid Substitution ; Aminoacyltransferases ; genetics ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; Peptidyl Transferases ; genetics ; Streptococcus pneumoniae ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactams ; pharmacology

OBJECTIVETo assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD).

METHODSAltogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System.

RESULTSBoth mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.

CONCLUSIONSDTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Alzheimer Disease ; enzymology ; Aminoacyltransferases ; metabolism ; Animals ; Base Sequence ; DNA Primers ; Disease Models, Animal ; Mice ; Reverse Transcriptase Polymerase Chain Reaction

UNLABELLEDStreptococcus pneumoniae is a common cause of potentially life-threatening infections such as meningitis, bacteraemia, pneumonia worldwide, for which children of preschool age are at particularly high risk. Since the late 1970s and 1980s, antibiotic resistance among pneumococci has become an emerging problem. Several multidrug-resistant clones have rapidly spread throughout the world.

OBJECTIVE(1) To investigate the prevalence of penicillin and other antibiotics nonsusceptibility among pneumococci. (2) To analyze the correlation of pbp2b amplicon profiles with penicillin resistance. (3) To serotype 31 isolates of penicillin-resistant pneumococci by latex agglutination. (4) To analyze the chromosomal relatedness of serotype 23F and 6 isolates of penicillin-resistant pneumococci by using pulsed-field gel electrophoresis (PFGE) and characterize these isolates in molecular epidemiology.

METHODS(1) Susceptibility was determined by using broth microdilution, E-test, and K-B disk. (2) The correlation of pbp2b amplicon profiles with penicillin resistance was assessed by restriction fragment length polymorphism (RFLP). (3) Serotyping of penicillin-resistant pneumococcal isolates was performed by using latex agglutination. (4) The properties of serotype 23F and 6 isolates of penicillin-resistant pneumococci were assessed by PFGE.

RESULTSS. pneumoniae with increased nonsusceptibility (including intermediate strains and resistant strains) to penicillin G was 9.9% in 1997, 12.6% in 1998, 14.6% in 2000; to cefuroxime 4.2%, 1.5%, 8.2%; to cefotaxime 0.0%, 1.7%, 1.0% respectively. There were no statistically significant differences (P > 0.05). While resistance to erythromycin, trimethoprim-sulfamethoxazole and chloramphenicol increased significantly from 76.8% in 1997 to 87.4% in 2000, from 74.7% to 88.3%, and from 22.6% to 40.8%, respectively (P < 0.05). RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. Of the 31 strains of penicillin-resistant pneumococci (MICs 0.12 - 2.0 micro g/ml) studied, 6 (19.4%) strains (MICs 0.12 - 0.19 micro g/ml) were serotype 23F and 3 (9.7%) strains (MICs 0.5 - 1.5 micro g/ml) were serotype 6. There were nearly identical susceptibility to antibiotics and identical PFGE patterns in the former, and there were different susceptibility to antibiotics and different PFGE patterns in the latter. Three serotype 6 strains had different susceptibility to antibiotics and different PFGE patterns, which suggested that those strains may be scattered.

CONCLUSIONGenerally beta-lactams retained their activity against S. pneumoniae in Beijing. Resistance to erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol increased drastically. RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. In 6 strains of serotype 23 F there were nearly identical susceptibility to antibiotics and identical PFGE patterns, which suggested the probability that there was a spread of serotype 23F isolates with low-level penicillin resistance in local area.

Aminoacyltransferases ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; blood ; genetics ; Carrier Proteins ; blood ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Hexosyltransferases ; blood ; genetics ; Muramoylpentapeptide Carboxypeptidase ; blood ; genetics ; Penicillin-Binding Proteins ; Peptidyl Transferases ; blood ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Streptococcus pneumoniae ; drug effects ; genetics

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.
Aminoacyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Biotin ; Cysteine Endopeptidases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genes, Reporter ; Ligation ; Mutant Proteins ; genetics ; metabolism

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

OBJECTIVETo investigate the antimicrobial resistance and penicillin resistance-associated genes (TEM and pbp2B) of Streptococcus pneumoniae (S. pneumoniae) isolated from sputum specimens of Guangzhou children with respiratory tract infection.

METHODSE-test and Kirby-Bauer methods were applied to detect the antibiotic susceptibility of 44 strains of S. pneumoniae. PCR was used to detect resistance genes pbp2B and TEM, followed by DNA sequence analysis of pbp2B gene. The sequence results were compared to those of penicillin-susceptible S. pneumoniae R6.

RESULTSOf the 44 isolates of S. pneumoniae, only 5 (11.4%) were susceptible to penicillin. All strains were resistant to erythromycin but susceptible to ofloxacin and vancomycin. The resistance rate of the isolates to clindamycin and trimoxazole was more than 90%. The S. pneumoniae isolates showed a high susceptibility to amoxicillin, imipenem and ceftriaxone, with a resistance rate of 0, 2.6% and 3.9%, respectively. The sequence analysis showed that more than 99% nucleotide sequence of pbp2B gene of five penicillin-susceptible isolates was the same as penicillin-susceptible S. pneumoniae R6, without any amino acid replacement. Site mutation was found in the remaining 39 penicillin-nonsusceptible isolates with a nucleotide mutation rate ranging from 13.2% to 23.1% and amino acid replacement rate from 6.5% to 10.9%. The 39 penicillin-nonsusceptible isolates were classified into 4 types according to the mutation site between Ser391 and Thr492 of pbp2B: type I (n=30), type II (n=7), type III (n=1) and type IV (n=1). No TEM gene was detected in all the 44 S. pneumoniae isolates.

CONCLUSIONSThe S.pneumoniae isolates from Guangzhou children with respiratory tract infection are resistant to penicillin and erythromycin. Amoxicillin and the third generation cephalosporin may be recommended for treating S. pneumoniae infection. The mutation of pbp2B gene plays an important role in the development of S. pneumoniae resistance to penicillin.

Aminoacyltransferases ; genetics ; Child, Preschool ; Drug Resistance, Bacterial ; genetics ; Female ; Humans ; Infant ; Male ; Microbial Sensitivity Tests ; Penicillin Resistance ; genetics ; Penicillin-Binding Proteins ; genetics ; Respiratory Tract Infections ; microbiology ; Streptococcus pneumoniae ; drug effects ; genetics ; beta-Lactamases ; genetics

OBJECTIVETo investigate the relation of pbp2B, ermB, ermA/B and mefA genes to penicillin and erythromycin resistance among isolated Streptococcus pneumoniae (Sp) in children.

METHODSTwenty-six strains of Sp were collected from September 2002 to April 2003 at the Children Hospital of Suzhou University. (1) Twenty-six pneumococcal isolates were obtained from respiratory tract secretions of children with respiratory diseases. (2) Susceptibility of the isolates to penicillin, cefuroxime, ceftriaxone, cefotaxime and erythromycin was determined by E-test. (3) The genes pbp2B, ermB, ermA/B and mefA of the isolates were detected with PCR. (4) The PCR product of pbp2B gene was sequenced. (5) DNA sequences of pbp2B of pneumococcal isolates were compared with those of SpR6 [penicillin sensitive (www.ncbi.nlm.gov/nucleotide, NC-003098)].

RESULTSAmong the 26 isolates studied, pbp2B gene mutation was found in 15(58%) isolates, all were point mutation of A, B, C and D genotypes which were seen in 11(73%), 2(13%), 1(7%) and 1(7%), respectively. The numbers of isolates susceptible to penicillin, cefuroxime, ceftriaxone and cefotaxime were 9(82%), 10(91%), 11(100%) and 11(100%), of 11 non-mutation isolates;numbers of isolates resistant to penicillin, cefuroxime, ceftriaxone, and cefotaxime were 13(87%), 11(73%), 1(7%) and 1(7%) out of 15 isolates with mutation.ErmB, ermA/B, mefA and erm/mef genes were positive in 9(35%), 16(62%), 7(27%) and 21(81%)isolates. MIC of erythromycin was 2 to > 256 mg/L among pneumococcal isolates with erm/mef genes.

CONCLUSIONAmong antibiotic resistant pneumococcal isolates in the area, the main basis of penicillin resistance was the mutation of pbp2B genes. Genotype A mutation had the highest rate among the isolates with mutation and manifested as resistance to penicillin and cefuroxime. Expression of either all or any of the ermA, ermB and mef genes led to erythromycin resistance. Antibiotics resistant Sp strains in this area are forming a challenge to efficacy of penicillin and erythromycin.

Aminoacyltransferases ; genetics ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Child ; Drug Resistance, Bacterial ; Erythromycin ; pharmacology ; Humans ; Membrane Proteins ; genetics ; Methyltransferases ; genetics ; Penicillin Resistance ; Penicillin-Binding Proteins ; genetics ; Streptococcus pneumoniae ; drug effects ; genetics

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.
Amino Acid Motifs ; Aminoacyltransferases ; drug effects ; genetics ; Antigens, Bacterial ; drug effects ; Bacterial Adhesion ; drug effects ; Bacterial Proteins ; drug effects ; genetics ; Biofilms ; drug effects ; Cysteine Endopeptidases ; drug effects ; genetics ; Dose-Response Relationship, Drug ; Humans ; Mutation ; genetics ; Nicotine ; administration & dosage ; pharmacology ; Peptidoglycan ; drug effects ; genetics ; Saliva ; physiology ; Streptococcus mutans ; drug effects ; enzymology ; growth & development ; Sucrose ; pharmacology

Bacterial biofilms can be viewed as a specific type of persistent bacterial infection. After initial invasion, microbes can attach to living and non-living surfaces, such as prosthetics and indwelling medical devices, and form a biofilm composed of extracellular polysaccharides, proteins, and other components. In hosts, biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. The clinical aspects of biofilm formation and leading strategies for biofilm inhibitors will be discussed in this mini-review.
Adhesins, Bacterial ; drug effects ; physiology ; Aminoacyltransferases ; antagonists & inhibitors ; genetics ; Animals ; Antimicrobial Cationic Peptides ; genetics ; pharmacology ; Bacterial Infections ; microbiology ; surgery ; Bacterial Proteins ; antagonists & inhibitors ; genetics ; Biofilms ; drug effects ; growth & development ; Chronic Disease ; Cysteine Endopeptidases ; genetics ; Cysteine Proteinase Inhibitors ; pharmacology ; Humans ; Inflammation ; microbiology ; Quorum Sensing ; drug effects ; physiology ; Wound Infection ; microbiology ; surgery

Dermatofibrosarcoma protuberans is a rare, slowly growing, locally invasive spindle-cell tumor characterized by a protuberant cutaneous mass with a pronounced tendency to recur and by a prominent storiform histopathologic pattern'-'. We present a case of dermatofibrosarcoma protuberans with myxoid area on the chest of a 57-year-old man. The histopathological study showed a dermal tumor of uniform spindle-shaped cells with storiform pattern. Immunohistochemically, the tumor was stained positively to anti-CD34 antibody and negatively to anti-factor XIIIa antibody.
Dermatofibrosarcoma* ; Factor XIIIa ; Humans ; Middle Aged ; Thorax