Acute Disease ; Bacteria ; isolation & purification ; Child, Preschool ; Haemophilus influenzae ; isolation & purification ; Humans ; Infant ; Infant, Newborn ; Nasopharynx ; microbiology ; Respiratory Tract Infections ; diagnosis ; microbiology ; Streptococcus pneumoniae ; isolation & purification ; Trachea ; microbiology

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: With the emergence of antimicrobial resistance among Streptococcus pneumoniae, a more accurate and automated antimicrobial susceptibility testing method is essential. We evaluated the BD Phoenix Automated Microbiology System (Becton Dickinson Diagnostic Systems, USA) SMIC/ID-2 panel for antimicrobial susceptibility testing of S. pneumoniae. METHODS: A total of 113 clinical strains of S. pneumoniae (88 penicillin susceptible strains, 8 intermediate strains, and 17 resistant strains by 2008 CLSI criteria) were tested. Minimum inhibitory concentrations (MICs) for penicillin, cefotaxime, clindamycin, erythromycin, levofloxacin, trimethoprim/ sulfamethoxazole, tetracycline, and vancomycin were determined by Etest (AB Biodisk, Sweden) and Phoenix System. The results obtained by Phoenix system were compared to those obtained by Etest. RESULTS: The overall essential agreement of MICs (within one dilution of MICs) defined by the Phoenix and Etest was 92.3%. Neither very major errors nor major errors were produced, and minor errors were 6.5%. Minor errors were frequently observed in susceptibility testings for penicillin (22.1%), cefotaxime (12.4%), and trimethoprim/sulfamethoxazole (11.5%). CONCLUSIONS: The Phoenix SMIC/ID-2 panel provided a simple and rapid susceptibility testing for S. pneumoniae, and the results were in a good agreement with those of Etest. The Phoenix system appears to be an effective automated system in clinical microbiology laboratories.
Anti-Bacterial Agents/pharmacology ; Bacterial Typing Techniques/instrumentation/methods ; Drug Resistance, Bacterial ; Microbial Sensitivity Tests/*methods ; Reagent Kits, Diagnostic ; Streptococcus pneumoniae/*drug effects/growth & development/isolation & purification

Bacteria ; drug effects ; isolation & purification ; Drug Resistance ; Escherichia coli ; drug effects ; Female ; Haemophilus influenzae ; drug effects ; Humans ; Klebsiella pneumoniae ; drug effects ; Male ; Microbial Sensitivity Tests ; Respiratory Tract Infections ; microbiology ; Staphylococcus aureus ; drug effects ; Streptococcus pneumoniae ; drug effects

OBJECTIVEThe purpose of this study was to observe the bacterial infections of respiratory tract in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).

METHODS130 patients with AECOPD in outpatient department, emergency room or in wards were studied prospectively. Patients were divided into different groups according to both Anthonisen's classification and their lung function status. Sputum were cultured together with bacteria positive rate and types of AECOPD as well as the damage degree of lung function were analyzed.

RESULTSOf 130 sputum samples, 50 showed positive through culture (38.5%) and 60 strains of pathogens were isolated. Predominant pathogens isolated would include Haemophilus parainfluenzae (20/60), Streptococcus pneumoniae (5/60) and Haemophilus influenzae (10/60). Positive rate of bacterial culture in type 1 AECOPD was 55.0%, higher than those of type 2 (38.3%) and type 3 (18.5%)(P = 0.01) and was increasing with the decrease of lung function of patients with AECOPD (P < 0.02).

CONCLUSIONPositive rate of bacterial culture in patients of type 1 AECOPD was the highest one. Haemophilus parainfluenzae was one of the most important pathogens in AECOPD. There seemed a correlation between positive result of bacterial culture and the severity of COPD.

Adult ; Aged ; Aged, 80 and over ; Bacterial Infections ; epidemiology ; microbiology ; Female ; Haemophilus influenzae ; isolation & purification ; pathogenicity ; Haemophilus parainfluenzae ; isolation & purification ; pathogenicity ; Humans ; Male ; Middle Aged ; Pulmonary Disease, Chronic Obstructive ; epidemiology ; microbiology ; Streptococcus pneumoniae ; isolation & purification ; pathogenicity

OBJECTIVE: To research the prevalences of four kinds of bacteria including Alloiococcus otitidis, Streptococcus pneumonia, Haemophilus influenzae, and Moraxella catarrhalis in children with chronic otitis media with effusion (SOM) of the middle ear effusion, and the reproduction of the nasopharynx, so as to explore their meaning for the children with SOM. METHOD: Alloiococcus otitidis, Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhal were investigated in the samples obtained from middle ear effusion and nasopharyn- geal swabs, using PCR and conventional bacterial culture methods. RESULT: By bacterial culture, the pathogen detection rate from middle ear effusion was 3.6%,while the nasopharynx was 54.0%, the detection rate of Streptococcus pneumonia, Haemophilus influenza, Moraxella catarrhalis was 10.8%, 27.0%, 4.5%, respectively, the drug susceptibility results for 51 samples of bacterial culture positive showed that 39 cases was sensitivite to the β-lactam antibiotic; By PCR, the number of detecting various kinds of bacteria simultaneously in middle ear effusion or in the nasopharynx were 6 and 34. The bacteria prevalences of S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis are 5.4%, 5.4%, 3.6%, and 42.3% in the middle ear effusion, are 25.2%, 27.0%,13.5% and 34.2% in nasopharyngeal, respectively. CONCLUSION (1) PCR method is more sensitively detecting the bacteria than conventional bacterial culture methods. (2) The chronic SOM of children may be a combination of mixed bacterial infection, A. otitidis may be the most common pathogen of children SOM. (3) For children of SOM, if antibiotics are chosen to be used early in the disease, we suggest using the β-lactam antibiotics.
Bacteria ; Bacterial Infections ; complications ; Child ; Haemophilus influenzae ; isolation & purification ; Humans ; Moraxella (Branhamella) catarrhalis ; isolation & purification ; Nasopharynx ; Otitis Media with Effusion ; complications ; microbiology ; Polymerase Chain Reaction ; Prevalence ; Streptococcus pneumoniae ; isolation & purification

No abstract available.
Acinetobacter baumannii/isolation & purification ; Adult ; Aged ; Aged, 80 and over ; Area Under Curve ; C-Reactive Protein/analysis ; Community-Acquired Infections/*diagnosis/microbiology/pathology ; Female ; Humans ; Klebsiella pneumoniae/isolation & purification ; Leukocyte Count ; Male ; Middle Aged ; Neutrophils/*cytology ; Pneumonia/*diagnosis/microbiology/pathology ; ROC Curve ; Respiratory Tract Infections/*diagnosis/microbiology/pathology ; Severity of Illness Index ; Staphylococcus aureus/isolation & purification ; Streptococcus pneumoniae/isolation & purification

No abstract available.
Acute Disease ; Adolescent ; Alkaline Phosphatase/*blood ; Anti-Bacterial Agents/*therapeutic use ; Arthritis, Infectious/*drug therapy/*enzymology/microbiology ; *Bacterial Infections/drug therapy/enzymology/microbiology ; Child ; Child, Preschool ; Haemophilus influenzae type b/isolation & purification ; Humans ; Infant ; Osteomyelitis/*drug therapy/*enzymology/microbiology ; Staphylococcus aureus/isolation & purification ; Streptococcus pneumoniae/isolation & purification ; Streptococcus pyogenes/isolation & purification