Estudio teórico de compuestos derivados de cefalosporinas con potencial actividad inhibitoria frente a proteína de unión a penicilina (PBP) y resistencia a enzimas betalactamasa de espectro extendido (BLEE) producidas por Escherichia coli / Theoretical study of compounds derived from cephalosporins with inhibitory activity potential front to penicillin binding proteins (PBP) and resistance to Extended spectrum Beta-lactamase (ESBL) produced by Escherichia coli

RESUMEN Se han utilizado herramientas computacionales para proponer moléculas derivadas de cefalosporinas con potencial actividad antibacteriana, frente a cepas de Escherichia Coli, con mayor afinidad como inhibidores de enzimas de unión a penicilinas y que a su vez disminuyan o no tengan afinidad por betalactamasas de espectro extendido. Se diseñaron 20 moléculas con base en la estructura molecular de la cefalosporina, las estructuras fueron optimizadas utilizando la teoría del funcional de la densidad, se calcularon descriptores moleculares de reactividad, de forma paralela se sometieron a acoplamiento molecular con las enzimas antes mencionadas. Las moléculas presentaron valores de energía de unión negativos, doce moléculas mostraron una orientación e interacciones favorables en el sitio activo de la enzima de unión a penicilinas y trece moléculas presentaron menor afinidad que el ligando nativo (cefotaxima) por la betalactamasa. Tres moléculas pueden considerarse como potenciales inhibidores de enzimas de unión a penicilinas resistentes y betalactamasas.

SUMMARY Computational tools have been used to propose molecules derived from cephalosporins with potential antibacterial activity, against strains of Escherichia Coli, with higher affinity as inhibitors of penicillin-binding enzymes and which in turn decrease or do not have affinity for extended-spectrum beta-lactamases. 20 molecules were designed based on the molecular structure of the cephalosporin, the structures were optimized using the density functional theory, molecular descriptors of reactivity were calculated, and in parallel form they were subjected to molecular docking with the enzymes mentioned above. The molecules showed negative binding energy values, 12 molecules showed an orientation and favorable interactions in the active site of the penicillin binding enzyme and thirteen molecules had lower affinity than the native ligand (Cefotaxime) for betalactamase. Three molecules can be considered as potential inhibitors of binding enzymes to resistant penicillins and betalactamases.

Penicillin-binding proteins genotyping of penicillin resistance Streptococcus pneumonia isolated from children in Guangzhou area / 国际检验医学杂志

Objective To understand the molecular epidemiology of penicillin resistance Streptococcus pneumonia (PNSP) isolated from children in Guangzhou area to provide the experimental basis for clinical prevention and control of Streptococcus pneumonia infectious diseases.Methods Specific primers were designed according to Genebank,penicillin binding protein(PBP) genes PBP1A,PBP1B,PBP2A,PBP2B,PBP2X,PBP3 were amplified by PCR.The sequencing analysis was performed.The PCR products were digested by Hinf I,and the restriction fragment length polymorphism (RFLP) was analyzed.Results DNA of PNSP was successfully extracted,the PCR results showed that in 50 strains of PNSP,the positive rates of bacterial strains containing PBP1A,PBP1B,PBP2A,PBP2B,PBP2X and PBP3 were 48.9%,64.4%,71.1%,31.1%,40.0% and 31.1% respectively.The sequencing showed that their homologies with known sequences in GenBank were 99%,98%,100%,97%,95% and 100% respectively.Using RFLP in Hinf I showed that PBP1A,PBP1B,PBP2A and PBP3 only had one kind of genotype,PBP2B and PBP2X had two kinds of genotypes,the positive rates were 71.4%,28.6%,66.7% and 33.3% respectively.Conclusion The gene distribution of PNSP strains among children in Guangzhou is dominated by PBP2A,PBP1B and PBP1A,there are two subtypes in PBP2B,PBP2X when digested by Hinf I,in which the predominant subtype >65%.

Peptidoglycan recycling and bacterial resistance to β-lactams / 中国药学杂志

To introduce peptidoglycan recycling and the β-lactams resistance mechanisms of bacteria, so that some help would be supplied to corresponding scientific workers and university teachers. By searching literatures, combined with our own studies, the bactericidal mechanisms of β-lactams and the resistance mechanisms of bacteria to β-lactams were summarized. The bactericidal activity of β-lactams is resulted from the inhibition of cell wall biosynthesis through combination with penicillin binding proteins such as transpeptidase destruction of peptidoglycan balances between biosynthesis and hydrolysis. The drug resistance of bacteria is resulted from the induction of β-lactamase, expression of out-pumping proteins, increase of outmembrane permeability, and modification of antibiotic target proteins. The proteins related to peptidoglycan recycling, such as transpeptidase and glycosyltransferase, would be potential targets for screening new β-lactams. The proteins related to β-lactams resistance, such as β-lactamase, would be potential targets for screening adjuvant drugs of β-lactams.

Screening and identification of aptamers against the recombinant transpeptidase domain of PBP 2a / 重庆医学

Objective To screen and identify the aptamers of the recombinant transpeptidase domain of PBP2a(penicillin binding protein 2a ,PBP2a) .Methods By using the recombinant transpeptidase domain of PBP2a as the screening target ,oligonucle-otides which were capable of specifically binding to the protein were screened by a random oligonucleotide library through the stem -atic evolution of ligand by exponential enrichment (SELEX )technique .The ssDNA was cloned and sequenced ,and the secondary structure of aptamer clones was predicted with mfold program .Results After 11 cycles of the selection ,the aptamers which were capable of binding to PBP2a with high affinity have been selected .40 clones from the 8 and 10 cycles were selected randomly and se-quenced .The aptamers obtained had no obvious homology according to their sequences by the sequence alignments ,and the 40 aptamers were classified to three groups according to their secondary structures .The aptamer 13 was found to be specific for the target protein with the highest affinity .Conclusion Aptamers for the recombinant transpeptidase domain of PBP2a with high affili-ty and specificity were successfully screened by SELEX ,which lays a foundation for exploring new ways of diagnosis and treatment of MRSA infection .

Cloning,prokaryotic expression,purification and identification of the transpeptidase domain of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus / 国际检验医学杂志

Objective To construct the prokaryontic expression vector of the gene fragment which encodes the transpeptidase domain of penicillin binding protein 2a(PBP2a) of methicillin‐resistant Staphylococcus aureus(MRSA) ,and to express ,purify and i‐dentify the objective protein .Methods Strains of MRSA were isolated and identified from clinical samples ,according to the se‐quence of mecA gene recorded in GenBank ,the primers of mecA fragment which encoded the transpeptidase domain of PBP2a was designed .The gene fragment from MRSA was amplified by using polymerase chain reaction(PCR) and cloned into pET28a(+ ) plasmid .After being identified by enzyme digestion and sequencing ,the recombinant plasmid was transformed into the strain of Escherichia coli BL21(DE3)plysS .The expression of transpeptidase domain of PBP2a was induced by 0 .7 mmol/L IPTG ,the ex‐pressed products were purified by using Ni afinity chromatography ,then were analyzed by using Western blot .Results The recom‐binant expression vector was digested by BamHⅠ and EcoRⅠ ,and the products were at the expected size .The result of sequencing showed two bases undergoing mutation ,while there were no frameshift mutations .The expressed protein was identified by using SDS‐PAGE and Western blot ,a new protein band was visible at the relative molecular mass of 38 × 103 .Conclusion The corre‐sponding prokaryotic expression vector is successfully constructed ,and the transpeptidase domain of PBP2a is successfully ex‐pressed and purified .

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Development of a rapid multi-residue assay for detecting β-lactams using penicillin binding protein 2x* / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a rapid multi-residue assay for detecting 16 demanded by the European Union (EU).</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>PBP2x* 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.</p><p><b>CONCLUSION</b>This 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.</p>

Direct Detection of Methicillin-Resistant Staphylococcus aureus from Blood Cultures Using Three Non-Molecular Methods: PBP2a Latex Agglutination, PBP2a Rapid Immunochromatographic Assay and MRSA-Chromogenic Medium / 대한임상미생물학회지

BACKGROUND: This study compared three non-molecular methods for the detection of methicillin-resistance directly from blood cultures containing Staphylococcus aureus: penicillin-binding protein (PBP) 2a latex agglutination (LA), PBP2a immunochromatographic assay (ICA) and MRSA chromogenic medium (CM). METHODS: Fifty methicillin-resistant S. aureus (MRSA) and 50 methicillin-susceptible S. aureus (MSSA) were seeded into blood-culture bottles. When isolates returned a positive signal, 5 mL of culture was added to serum separator tubes and centrifuged at 1,300 g for 10 min. The pellets were then used as the inoculum for the PBP2a LA, MRSA-CM and PBP2a ICA. The pure colony was used for PBP2a LA test, additionally. RESULTS: The respective sensitivities and specificities were 98 and 100% for PBP2a ICA, and 100 and 100% for MRSA-CM in direct detection of MRSA from positive blood culture. The results of PBP2a LA test using pure colony were entirely compatible with those by mecA gene PCR but the PBP2a LA test using the pellets directly isolated from positive blood culture showed sometimes ambiguous agglutination; its sensitivity and specificity were 78 and 100%, if ambiguous results were scored as negative, and were 90 and 92%, if ambiguous results were scored as positive, respectively. CONCLUSION: For direct detection of MRSA in positive blood culture, MRSA-CM and PBP2a ICA provided excellent results. The PBP2a LA test using pure colony also gave excellent results but the PBP2a LA test by the direct method using pellet of positive blood culture was slightly less sensitive than the other two methods.

PBP1A, CarO and β-lactamase genes detection in a pandrug-resistant Acinetobacter baumannii / 中华临床感染病杂志

Objective To investigate resistant mechanisms of a pandrug-resistant Acinetobacter baumannii (js01) to β-1actams.Methods Strain js01 isolated from sputum sample of an inpatient from Ningbo First Municipal Hospital in December 2011 was confirmed by PCR amplifying and sequencing of gyrA and parC,and aligning with BLASTn.Thirty-three kinds of β-lactamase genes ( 13 kinds of class A,10kinds of class B,2 kinds of class C,8 kinds of class D),linkage detection of insertion sequences and β-lactamase genes,as well as outer membrane porin gene carO were analyzed by PCR.Genes encoding PBPI A were divided into three fragments,PCR amplified and bidirectional sequenced,and ligated to the full-length gene.Results Four kinds of β-lactamase genes were positive in js01:TEM-I,ADC-30, OXA-23 and OXA-66.Linkage detection of insertion sequences and β-lactamase genes showed that ISabal-ADC-30 and ISabal-OXA-23 were positive. When compared with sensitive strain (SDF) of Acinetobacter baumannii,sense mutations were found in carO gene of js01,and identity of amino acid sequence of carO gene between js01 and SDF was 76.0％ (189/249),and differences owed to loss of 3 amino acids.Sense mutations were also found in genes encoding PBP1A of js01,and identity of amino acid sequence of genes encoding PBP1A between js01 and SDF was 99.6％ ( 848/851 ),and differences owed to variations of 3amino acids.However,compared with three-dimensional structure of PBP1 A of SDF,PBP1 A of js01 lost 2helixes.Conclusion In strain js01,mutations of housekeeping genes ( genes encoding PBP1A and CarO),and genes producing β-lactamase mediated by mobile genetic elements,may play a key role in resistance to β-lactams.

Asociación de los genes implicados en la codificaciónde proteina de unión a la penicilina 2a (pbp2a) conla expresión fenotípica de resistencia a la meticilinaen cepas De staphylococcus spp / Association of genes involved in coding binding protein penicillin 2a (PBP2a) with phenotypic expression of methicillin resistance in strains of Staphylococcus spp

Objetivo: Determinar si existe asociación entre genes implicados en la codificación de PBP2a con la expresión fenotípica de resistencia a meticilina en cepas de Staphylococcus spp. Diseño: Descriptivo Transversal. Metodologias: e determinó la resistencia y sensibilidad de 67 aislamientos, mediante pruebas fenotípicas (difusión en disco, concentración inhibitoriamínima CIM, producción de PBP2a y pruebas genotípicas para detectar los genes mecA y sus reguladores mecR1 y mecI por Reacción em Cadena de la Polimerasa (PCR). Resultados: De 9 cepas de S. aureus resistentes por difusión en disco solo 1 fue sensible por CIM. De 7 cepas resistentes por CIM, fueron sensibles por difusión en disco. Por el contrario las 7 cepas de Staphylococcus coagulasa negativo sensibles por difusión en disco fueron resistentespor CIM.En cuanto a la prueba de producción de PBP2a, los resultados fueron discordantes con la prueba de difusión en disco en 20..

Objective: Determining the association between genes involved in the codifi cation of Penicillin Binding Proteins 2A (PBP2A) with the phenotypic expression of methicillin resistance in Staphylococcus spp. Strains Design: Descriptive cross sectional Methodology: The sensitivity of 67 isolates was determined by means of a phenotypic test (disk diffusion, minimum inhibitory concentration CIM, production of PBP2a) and genotype tests to detect the mecA gene and its regulatory mecR1 and mecI by Polymerase Chain Reaction (PCR). Results: From 9 S. aureus resistant strains by disk diffusion 1 was sensitive by CIM, 7 CIM resistant strains were sensitive by disk diffusion. The 7 coagulase negative (CNS) sensitive strains by disk diffusion were resistant by CIM. By production of PBP2a, the results were discordant with the disk diffusion test in 20% and 34%with CIM. The genotype, reveals that, from 60 S.aureus strains 10(17%), and 7 S. coagulase negative strains 4 (57%) carry the mecA gene. From 10 S. aureus mecA positive strains, 5 carry the mecR1 gene and 7 carry the mecI gene. Of the 4 strains of S.coagulase negative mecA positive 2 carry the mecR1 and 2 carry the mecI gene. Conclusion: There is no association between genotype and phenotype in Staphylococcus spp. methicillin resistant strains, since, the resistance is due to many factors that the classical phenotypic test does not include.

Rapid Identification of Staphylococcus aureus and Its Resistance Against Meticillin Using Slidex-staph Monoclonal Antibody / 中华医院感染学杂志

OBJECTIVE To develop a rapid method for simultaneously identifying Staphylococcus aureus(SA) and its resistance against meticillin. METHODS The target strains authenicated to Staphylococcus by traditional methods(appearance,Gram-stain,catalase) first,then using Slidex~staph.plus to authenticate SA;establishing traditional method or coagulase test and ATB-ID32STAPH system for verification.The penicillin-binding protein 2a(PBP2a) was examined by Slidex MRSA Detection,establishing resistance oxacillin sieving test and mecA gene was examined by PCR for verification. RESULTS The 135 strains were positive and 321 strains were negative for Slidex~staph.plus from 456 strains.The 127 S.aureus strains and eight others were confirmed from 135 positive strains finally,11 SA strains and 310 other strains were confirmed from 321 negative strains,there were 92.0% for sensitivity and 97.5% for specificity in this method.The 52 strains PBP2a positively confirmed to meticillin-resistant S.aureus(MRSA) using Slidex MRSA Detection and 57 MRSA strains were confirmed using resistance oxacillin sieving test or PCR from 138 strains.There were 91.2% for sensitivity and 100% for specifity in this method. CONCLUSIONS The duplex Slidex~-staph monoclonal antibody examinated to SA and confirmed to MRSA has higher sensitivity and specificity.

A Study on resistance of non-PBP 2a type to oxacillin of Staphylococcus aureus / 医学研究生学报

Objective:To investigate whether there is another resistance mechanism besides mecA gene in oxacillin-resistant(OR) isolates of Staphylococcus aureus(S.aureus). Methods:There were 130 oxacillin-resistant phenotype isolates of S. aureus. Of these isolates 125 were resistant to more than 3 of 5 non-?-lactams (gentamicin, ciprofloxacin, clindamycin, tetracycline, erythromycin) (OR-R) and 5 susceptible to more than 3 of the 5 non-?-lactams (OR-S) and 14 were oxacillin-susceptible (OS) phenotype isolates of S. aureus and resistant to more than 3 of 5 non-?-lactams (OS-R). All the strains were detected by the two disks diffusion tests with oxacillin (OXA) and oxacillin/clavulanic acid (OXA/CA), by the three-dimensional extract tests of penicillin (PEN) and OXA for penicillinase and oxacillinase, and by PCR tests for mecA. Results:The 130 OR and 14 OS isolates were all oxacillinase-negative with the two disks diffusion tests, all pencillinase-positive and oxacillinase-negative in the three-dimensional extract tests. The mecA gene was detected in 125 OR-R-type and 2 of the 5 OR-S-type isolates, but was not detected in the other 3 OR-S-type nor in any of the 14 OS-R-type isolates by PCR. Conclusion:In a few Staphylococcus aureus strains which are phenotype of oxacillin-resistant and are susceptible to mostly non-?-lactam agents there are both mecA-negative and oxacillinase-negative strains, 2.3% (3/130) of the OXA-resistant strains. The resistance mechanism may be associated with reduced binding capacity of the modified Preexisting PBPs with OXA.

Reverse Effect of Baicalin/Baicalein on Antibiotic Resistance of Methicillin-resistant Staphylococcus Aureus / 中国药房

OBJECTIVE:To study the reverse effect of Baicalin/Baicalein on antibiotic resistance of methicillin-resistant staphylococcus aureus(MRSA) and its mechanism.METHODS:The synergetic antimicrobial effect of oxacillin and Baicalin/Baicalein against MRSA were detected by plating method,and the synergetic antimicrobial effect of oxacillin and Baicalin/Baicalein on growth density inhibition of MRSA was detected by spectrophotometry.The inhibitory effect of Baicalin/Baicalein on PBP2a synthesis was detected by PBP2a latex agglutination assay kit.RESULTS:The combination of Baicalin/Baicalein with oxacillin showed remarkable synergetic antimicrobial effect on clinical isolated MRSA.16 ?g?mL-1 of Baicalein obviously reversed the strong resistance of MRSA to oxacillin.Baicalin/Baicalein showed remarkable inhibitory effect on PBP2a production in MRSA.CONCLUSION:Baicalin/Baicalein can reverse MRSA's strong resistance to oxacillin by inhibiting PBP2a secretion in MRSA.Theoretically,this phenomenon offers a new approach for the treatment of infections caused by MRSA.

Unique alterations in penicillin-binding protein 2B of multidrug-resistant Streptococcus pneumoniae from Korea / 감염

BACKGROUND: Pneumococcal resistance became a global issue during the past decades. Korea is reported to be the hottest spot in the world with regard to the prevalence of penicillin and multidrug resistance. Previous molecular epidemiologic studies strongly suggested that antibiotic-resistant pneumococci from Korea are genetically related. To investigate the molecular characteristics of multidrug-resistant (MDR) pneumococcal isolates in Korea, we performed the DNA sequencing of the gene encoding penicillin-binding protein (PBP) 2B. METHODS: A total of 9 invasive MDR strains which were collected from 1990 to 1995 in various parts of Korea and one internationally epidemic Spanish 23F clone were analyzed. The 1.5 kb transpeptidase-encoding region (TER) of PBP 2B gene was amplified and directly sequenced using ABI PRISM Big Dye Terminator cycle sequencing kit (Perkin Elmer). Sequence data were compared with that of a penicillin-susceptible R6 strain. RESULTS: Alterations in nucleotide sequence (5.4-7.8%) and amino acids (3.0-4.3%) of the PBP 2B gene were relatively uniform among 9 Korean MDR strains. Most alterations in nucleotides (86-94%) and amino acids (86-100%) were noted in the hypervariable region between 408 and 993 bp. All 9 strains possessed 14 common alterations in amino acids, among which Asn-276-->Lys, Arg-285-->Cys and Ser-305-->Phe substitutions were unique to Korean MDR strains. CONCLUSION: Sequence analysis of invasive MDR strains showed that a limited number of amino acid substitutions were noted in the wild-type Korean MDR strains in the transpeptidase domain of the PBP 2B gene. Data strongly suggest the possibility of the spread of a few epidemic clones of resistant pneumococci within Korea, which could partly explain the rapid increase of pneumococcal resistance.

Detection of Penicillin-resistant Streptococcus pneumoniae by Polymerase Chain Reaction / 대한임상병리학회지

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.

PCR Fingerprinting Analysis of Genes Encoding Penicillin-binding Proteins of Multidrug-resistant Streptococcus pneumoniae Isolated from Korea / 감염

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.

Screening Anti-MRSA Traditional Chinese Materia Medica by Targeting on Soluble Penicillin Binding Protein 2a / 中华医院感染学杂志

OBJECTIVE To screen the anti-meticillin-resistant Staphylococcus aureus(MRSA)traditional Chinese materia medica(TCMM)by biosensor technique,targeted on the soluble penicillin binding protein 2a(PBP2a)of clinical MRSA.METHODS The soluble PBP2a with amino acid sequence from 25 to 668 from clinical MRSA were expressed in Escherichia coli by gene recombination technique.Then,the expressed product was identified and its biological function was analyzed.After the PBP2a was immobilized into the carboxymethyl dextran cuvette(CMD),the anti-MRSA TCMM was screened by means of biosensor.RESULTS The soluble protein PBP2a had been successfully expressed,whose relative molecular mass was 74?103.It was confirmed that the soluble PBP2a had transpeptidase activitiy and ?-lactamase activitiy.Subsequently,10 kinds of anti-MRSA TCMM were screened out by biosensor technique.Moreover,Radix Scutellariae,Rhizoma Coptidis and Spica Prunellae had greater anti-MRSA effect than others.CONCLUSIONS Anti-MRSA TCMM has been successfully screened out by biosensor technique,targeted on the soluble PBP2a of clinical MRSA.