The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.
Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pneumoniae ; genetics ; metabolism ; Transcription Factors ; biosynthesis ; genetics ; isolation & purification

OBJECTIVEBacterial cultures from respiratory aspirate or sputum have been the conventional diagnostic method for pneumonia, but the results of culture was often affected by early extensive use of antibiotics, sample collection and delivery. The objective of this study was to explore application of the combined detection of culture and polymerase chain reaction (PCR) assay in hospitalized children with pneumonia.

METHODSTotally 187 hospitalized children with pneumonia were enrolled. The age of the patients ranged from 1 month to 10 years, 124 were male, 63 female; 175 of the patients received antibiotics treatment before admission. Deep respiratory aspirate sample from patients was cultured by Streptococcus pneumoniae selective plate, Hemophilus influenzae selective plate and conventional plate. The aspirate samples were also amplified for DNA of 14 bacteria with target enriched multiplex polymerase chain reaction (Tem-PCR) and detected with Luminex xMAP technology platform.

RESULTSThe total positive rate by bacterial culture was 40.1% (75/187), of which 17.1% (24/187) were Hemophilus influenzae b, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 4.8% (9/187) were Staphylococcus aureus, 3.7% (7/187) were Streptococcus pneumoniae, 1.6% (3/187) were Pseudomonas aeruginosa, 1.1% (2/187) were Acinetobacter baumannii, and 1.1% (2/187) were Enterobacter cloacae. The total positive rate by combined detection of culture and Tem-PCR assay were 78.6% (147/187), of which 28.9% (54/187) were Hemophilus influenzae b, 19.3% (36/187) were Streptococcus pneumoniae, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 5.9% (11/187) were Staphylococcus aureus, 5.9% (11/187) were Acinetobacter baumannii, 2.7% (5/187) were Pseudomonas aeruginosa, and 1.1% (2/187) were Enterobacter cloacae.

CONCLUSIONThe Tem-PCR assay may increase the detection rate of Hemophilus influenzae b, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. The Combined detection may increase the positive rate of bacterial pathogens in hospitalized children with pneumonia, and the results might reflect the real patterns of bacterial etiology. The Tem-PCR needs further improvement for diagnosis of Escherichia coli and Klebsiella pneumoniae.

Child ; Child, Preschool ; Colony Count, Microbial ; Female ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Pneumonia, Bacterial ; microbiology ; Polymerase Chain Reaction ; Streptococcus pneumoniae ; genetics ; isolation & purification

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Child ; DNA Primers/chemistry/metabolism ; DNA, Bacterial/chemistry/genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Pneumococcal Infections/microbiology ; Serotyping ; Streptococcus pneumoniae/*classification/genetics/isolation & purification

BACKGROUND: The present analysis focuses on phenotypic and genotypic characterizations of efflux-mediated erythromycin resistance in Streptococcus pneumoniae due to an increase in macrolide resistance in S. pneumoniae worldwide. METHODS: We investigated the prevalence of efflux-mediated erythromycin resistance and its relevant genetic elements from 186 specimens of S. pneumonia isolated from clinical and normal flora from Tehran, Iran. The presence of erythromycin resistance genes was tested by PCR with two sets of primers, specific for erm(B) and mef(A/E), and their genetic elements with tetM, xis, and int genes. Isolates were typed with the BOX PCR method and tested for resistance to six antibiotics. RESULTS: Antibiotic susceptibility tests revealed that 100% and 47% isolates were resistant to tetracycline and erythromycin, respectively. The erythromycin and clindamycin double-disc diffusion test for macrolide-lincosamide-streptograminB (MLSB) resistance phenotype showed 74 (84%) isolates with the constitutive MLSB phenotype and the remaining with the M phenotype. BOX PCR demonstrated the presence of 7 types in pneumococci with the M phenotype. Fourteen (16%) isolates with the M phenotype harbored mef(A/E), tetM, xis, and int genes. CONCLUSIONS: The present results suggest dissemination of polyclonal groups of S. pneumoniae with the M phenotype carrying resistance genes attributed to transposon 2009.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; DNA, Bacterial/metabolism ; Drug Resistance, Multiple, Bacterial/*genetics ; Erythromycin/*pharmacology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Phenotype ; Pneumococcal Infections/microbiology/pathology ; Polymerase Chain Reaction ; Streptococcus pneumoniae/*drug effects/*genetics/isolation & purification ; Tetracycline/pharmacology

BACKGROUNDDespite the prevalence of Streptococcus pneumoniae serotype 19A, the molecular characteristics of this serotype are yet to be fully elucidated. The aim of this study was therefore to determine the homology of the serotype 19A in China.

METHODSPulsed-field gel electrophoresis and multilocus sequence typing were done to these forty-nine serotype 19A isolates to investigate the relationship between the strains prevalent in Beijing and other regions.

RESULTSFrom 1997 to 2006, the percentage of serotype 19A isolates increased. The susceptibility rate to penicillin and amoxicillin decreased and the resistance rate to cefuroxime increased. ST320 was the most prevalent ST, followed by ST3546. There were six new STs identified in our study. The serotype 19A strains were classified into six different pulsed-field gel electrophoresis (PFGE) patterns. ST320, which was associated with two different PFGE patterns (A and D), accounted for 32 isolates, and ST3546, which was associated with two PFGE patterns (B and E), accounted for eight isolates.

CONCLUSIONSFrom 2003 onwards, ST320 was the most common ST and the rate of resistance to cefuroxime increased significantly. Further long-term surveys of Streptococcus pneumoniae serotype 19A are required to monitor ST prevalence and antimicrobial resistance in this important human pathogen.

Child, Preschool ; China ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Infant ; Infant, Newborn ; Molecular Epidemiology ; Pneumococcal Infections ; drug therapy ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects ; genetics ; isolation & purification ; Time Factors

BACKGROUNDErythromycin-resistant Streptococcus pneumoniae isolates that causing invasive pneumococcal diseases (IPD) in Chinese children remain uncharacterized. This study aims to identify the resistance genes associated with erythromycin resistance and to determine the genetic relationships of IPD isolates in Chinese children.

METHODSA total of 171 S. pneumoniae strains were isolated from 11 medical centers in China from 2006 to 2008. All the isolates were characterized via serotyping and antibiotic susceptibility determination. The erythromycin-resistant isolates were further characterized via ermB and mefA gene detection, multi-locus sequence typing analysis, and pulsed-field gel electrophoresis.

RESULTSA total of 164 (95.9%) isolates showed resistance to erythromycin, of which 162 strains with high high-level resistance (MIC ≥ 256 µg/ml). A total of 104 (63.4%) isolates carry the ermB gene alone, whereas 59 (36.0%) harbor both ermB and mefA genes. Of the 59 strains, 54 were of serotypes 19A and 19F and were identified as highly clonal and related to the Taiwan(19F)-14 clone.

CONCLUSIONSThe erythromycin resistance rate in IPD isolates is significantly high and is predominantly mediated by the ermB gene. Isolates that carry both ermB and mefA genes are predominantly of serotypes 19A and 19F.

Adolescent ; Anti-Bacterial Agents ; pharmacology ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Erythromycin ; pharmacology ; Humans ; Infant ; Multilocus Sequence Typing ; Pneumococcal Infections ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects ; genetics ; isolation & purification

The carriage rate and serotype distribution of Streptococcus pneumoniae (S. pneumoniae) in a healthy population in China remains unclear. In this study, we collected the oropharyngeal swabs from 513 individuals in Xinjiang, China. Real-time PCR targeting the lytA gene and 12 serotypes were assessed to identify S. pneumoniae carriage. The total carriage rate of S. pneumoniae was 70.4% (361/513). The most prevalent serotypes were 19B/F, 18B/C, 5, and 6A/B. The highest carriage rate of S. pneumoniae was noted in children aged 6-10 years (88.6%), which merits further attention. The co-colonization rate of two or more S. pneumoniae serotypes was 79.8% (264/331). This study aimed to investigate the baseline pneumococcal carriage rate among healthy individuals in China to improve our understanding of the epidemiology of S. pneumoniae.
Adolescent ; Adult ; Carrier State ; epidemiology ; microbiology ; Child ; Child, Preschool ; China ; epidemiology ; Cross-Sectional Studies ; Female ; Humans ; Infant ; Male ; Middle Aged ; Pneumococcal Infections ; epidemiology ; microbiology ; Prevalence ; Real-Time Polymerase Chain Reaction ; Serogroup ; Streptococcus pneumoniae ; classification ; genetics ; isolation & purification ; Young Adult

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.
Child ; Child, Preschool ; Chronic Disease ; DNA, Bacterial/analysis ; Haemophilus Infections/*diagnosis ; Haemophilus influenzae/genetics/*isolation & purification ; Humans ; Infant ; Moraxella (Branhamella) catarrhalis/genetics/isolation & purification ; Moraxellaceae Infections/diagnosis ; Otitis Media with Effusion/*diagnosis/*microbiology ; Polymerase Chain Reaction ; Research Support, Non-U.S. Gov't ; Streptococcal Infections/diagnosis ; Streptococcus pneumoniae/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

OBJECTIVETo analyze the mechanisms of macrolide resistance in Streptococcus pneumoniae from children in Beijing.

METHODSThe MICs of penicillin and erythromycin were determined by the E-test methods for 200 Streptococcus pneumoniae isolates collected from 2002 to 2003 at Beijing Children's Hospital. MICs of azithrhomycin, clarithromycin, acetylspiramycin and clindamycin for 147 erythromycin-resistant isolates were detected by the agar dilution methods. For phenotyping, macrolide resistance induction tests were used in erythromycin-resistant isolates. PCR was used to determine the presence of the erythromycin-resistant genes.

RESULTSOf 200 Streptococcus pneumoniae isolates, 89.5% were resistant to erythromycin. In 147 erythromycin-resistant isolates, resistance rates were as follows: azithromycin, 100%; clarithromycin, 100%; acetylspiramycin, 95.2%; and clindamycin, 95.9%. The most common macrolide resistance phenotype was the cMIS phenotype (95.9%), 1.4% had the iMLS phenotype and 2.7% the M phenotype. Erythromycin-resistant isolates were characterized for the underlying resistance genotype, with 79.6% having the ermB genotypes, 17.7% having both ermB and mefA, 2.7% having the mefA, and none having neither ermB nor mefA genotypes.

CONCLUSIONSThe rates of carriage of macrolide-resistant Streptococcus pneumoniae by children were high in Beijing during 2002 - 2003. cMLS was the most prevalent phenotype among erythromycin-resistant Streptococcus pneumoniae isolates, and ribosomal modification (ermB gene coded) was the main resistance mechanism against macrolides in Beijing region.

Anti-Bacterial Agents ; pharmacology ; Child ; China ; Clarithromycin ; pharmacology ; Clindamycin ; pharmacology ; Drug Resistance, Multiple, Bacterial ; genetics ; Erythromycin ; pharmacology ; Genotype ; Humans ; Macrolides ; pharmacology ; Microbial Sensitivity Tests ; Penicillins ; pharmacology ; Phenotype ; Spiramycin ; analogs & derivatives ; pharmacology ; Streptococcal Infections ; genetics ; microbiology ; Streptococcus pneumoniae ; drug effects ; genetics ; isolation & purification