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

Anthracosis ; complications ; Bacterial Proteins ; genetics ; Humans ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; genetics ; Penicillin-Binding Proteins ; Respiratory Tract Infections ; complications ; microbiology ; Sputum ; microbiology

OBJECTIVETo investigate the molecular epidemiology of the penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) in Beijing, China.

METHODSThe resistant profile of 63 PNSP strains isolated from children with upper respiratory infection in the outpatient department from 2000 to 2002 was analyzed. The isolates were compared by detecting restriction fragment length polymorphism (RFLP) of the penicillin-binding protein (PBP) genes pbp1a, pbp2b and pbp2x and by applying chromosomal macrorestriction patterns detected by pulsed-field gel electrophoresis (PFGE).

RESULTSSixty-one (96.8%) out of the 63 PNSP strains were multidrug-resistant Streptococcus pneumoniae (Sp). Overall, 16 resistance profiles were found, 14 of which were multidrug resistant profiles. Seven (33.3%), 6 (24.0%) and 8 (47.1%) strains resistant to one of cephalosporins were respectively isolated in 2000, 2001 and 2002, indicating an increasing trend but without any statistical significance (chi(2) = 2.42, P > 0.05). The RFLP results showed 8, 9 and 18 genotypes of pbp1a, pbp2b and pbp2x, respectively. A total of 30 patterns were found according to the three pbps types. And clearly, the most common 5 patterns had main resistant profiles. In the mean time, 35 different PFGE types were elucidated and the 9 PFGE types, with each consisting of more than 2 strains, covered 59% (37/63) of all isolates. One of the 9 PFGE type included 2 strains, both possibly related to each other, but one of them was detected to be the same PFGE pattern with clones prevalent in Asia, Vietnam-19 serogroup, Singapore-19 serogroup, Taiwan-19 serogroup, and the other was the same as that in Korea-19 serogroup.

CONCLUSIONMultidrug resistance is very common among PNSP isolates in Beijing. The spread of a few multidrug resistant clones is an important factor for the prevalence of PNSP. It deserves the concern that the resistant clones spread in Asia have been found in Beijing.

Child, Preschool ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Bacterial ; genetics ; Genotype ; Humans ; Infant ; Molecular Epidemiology ; Penicillin Resistance ; genetics ; Pneumococcal Infections ; epidemiology ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA ; Streptococcus pneumoniae ; classification ; genetics

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

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.
*Bacterial Proteins ; Carrier Proteins/genetics ; Drug Resistance, Microbial ; *Hexosyltransferases ; Muramoylpentapeptide Carboxypeptidase/genetics ; Oxacillin/*pharmacology ; Penicillin-Binding Proteins ; *Peptidyl Transferases ; Staphylococcus aureus/*drug effects/genetics ; Staphylococcus epidermidis/*drug effects/genetics

OBJECTIVETo clone, express and identify the mecA fragment which encoded penicillin binding protein 2a (PBP2a) from methicillin-resistant staphylococcus aureus (MRSA) isolated from patients by gene recombination method.

METHODSAccording to the sequence of mecA gene recorded in GenBank, the primer of mecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [pREP4], and then its expression was induced by 1 mmol/L Isopropy-beta-D-Thiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy.

RESULTSThe recombinant pQE30- mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases, including 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 (pQE30- mecA), with a relative molecular weight of 74 x 10(3), was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification.

CONCLUSIONThe soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.

Base Sequence ; Cloning, Molecular ; Gene Expression ; Humans ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; metabolism ; Peptide Synthases ; genetics ; metabolism ; Plasmids

OBJECTIVETo investigate the relationship between penA/ponA and penicillin resistance of Neisseria gonorrhoeae.

METHODSAgar dilution method was used to determine the minimum inhibitory concentrations (MICs) of the strains. Polymerase chain reaction-single stand conformation polymerphism (PCR-SSCP) and PCR- restriction fragment length polymorphism (RFLP) were used to detect the mutations in ponA and penA genes, which encoding the penicillin binding protein-1 and -2 (PBP1 and PBP2), respectively.

RESULTSAll the 80 N. gonorrhoeae isolates had a D345 insertion detected in penA while 93.7% of N. gonorrhoeae isolates having a point mutation Leu421 --> Pro in ponA. Most of the penicillinase producing N. gonorrhoeae (PPNG) strains possessed the mutations in ponA and penA.

CONCLUSIONOur data suggested that the plasmid and chromosome mediated penicillin-resistance conjugately increased the level of resistance.

Amino Acid Sequence ; Bacterial Proteins ; genetics ; Base Sequence ; DNA Mutational Analysis ; DNA, Bacterial ; Genes, Bacterial ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Mutagenesis, Insertional ; Mutation ; Neisseria gonorrhoeae ; drug effects ; genetics ; Penicillin Resistance ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational

OBJECTIVE: To evaluate the reliability and clinical practicability of cefoxitin disk diffusion test in the detection of methicillin-resistant staphylococcus (MRS) heterogenic drug-resistant strains. METHODS: Three hundred and ten strains of staphylococcus isolated from clinics were detected by the oxacillin disk diffusion test, the cefoxitin disk diffusion test as well as the oxacillin agar dilution test according to the standard operation procedures of NCCLS, and the detection of mecA gene of staphylococcus was used as a criterion. The sensitivities and specitivities of the 4 methods were compared. RESULTS: By the detection of mecA gene, the ratio for MRSA was 57.1%(113/198) and the ratio for MRCNS was 62.5%(70/112). Both the sensitivity and specificity of cefoxitin disk diffusion test in the detection of MRS were 100%, and those in the detection of MRCNS were 98.6% and 100%. CONCLUSION Cefoxitin disk diffusion test is reliable, simple and convenient, and it can be used as a conventional method for the detection of MRS in clinical laboratories.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Cefoxitin ; pharmacology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; genetics ; Microbial Sensitivity Tests ; instrumentation ; methods ; Oxacillin ; pharmacology ; Penicillin-Binding Proteins ; Reproducibility of Results ; Staphylococcus aureus ; drug effects ; genetics ; isolation & purification

OBJECTIVETo investigate the molecule epidemic for 7 genes interrelated penicillin, erythromycin, tetracycline, vancomycin resistance of isolated Streptococcus pneumoniae (SP) in children at Suzhou area.

METHODS(1) Thirty-one pneumococcal isolates were collected from respiratory tract secretions of children with respiratory diseases from Nov 2002 to Apr 2003 at the Children's Hospital of Suzhou University (reference strain ATCC49619). (2) Penicillin susceptibility was determined by E-test, while erythromycin, tetracycline, vancomycin were determined by K-B disk. (3) The detecting of pbp2B, ermA/B, mefA, tetM, vanA, vanB genes by PCR, Sequencing pbp2B genes, Contrasting pbp2B DNA sequences among pneumococcal isolates and SP R6 [penicillin sensitive (www.ncbi.nlm.gov/nucleotide, NC-003098)].

RESULTSOf thirty-one isolates studied, the results were shown as follows; (1) Penicillin sensibility 38.7% (n = 12), penicillin resistance 61.3% (n = 19), pbp2B mutation 64.5% (n = 20); (2) Erythromycin sensibility 9.7% (n = 3), erythromycin resistance 90.3% (n = 28), ermA/B 71% (n = 22), mefA 32.1% (n = 10), ermA/B + mefA 87.1% (n = 27); (3) Tetracycline sensibility 9.7% (n = 3), tetracycline resistance 90.3% (n = 28), tetM 90.3% (n = 28); (4) Vancomycin sensibility 100% (n = 31), vanA, vanB all 0%.

CONCLUSIONAmong pneumococcal isolates at our area, penicillin, erythromycin, tetracycline resistance were high, vancomycin was sensitive. Detecting 7 genes interrelated penicillin, erythromycin, tetracycline, vancomycin resistance expressed genotypies for antibiotic resistances in pneumococcal isolates.

Anti-Bacterial Agents ; pharmacology ; Child ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Multiple, Bacterial ; genetics ; Erythromycin ; pharmacology ; Female ; Humans ; Male ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Penicillin Resistance ; genetics ; Pneumococcal Infections ; epidemiology ; microbiology ; Streptococcus pneumoniae ; drug effects ; genetics ; isolation & purification ; Tetracycline Resistance ; genetics ; Vancomycin ; pharmacology