Fifty-five strains of Haemophilus influenzae recovered at a children's hospital in Korea from 1992 through 1997, were analyzed for serotype and antibiotic resistance. Antimicrobial susceptibility was tested by broth dilution method. Among the 55 strains, 26 were from normally sterile body fluids, of which 17 were from the immunocompetent children. Spectrum in the immunocompetent included meningitis (47%), bacteremic pneumonia (18%), and bacteremia without focus (35%). Three (12%) of 26 invasive infections were caused by non-type b: one type d and two type f. Nine of 29 non-sterile body fluid isolates belonged to one of encapsulted serotypes: four a, two c, one of each of b, d and e. Thirty two (58%) strains were resistant to ampicillin, and all of which produced beta-lactamase. All of the strains were highly susceptible to amoxicillin/clavulanate, cefixime, cefuroxime, azithromycin and ciprofloxacin, while 1 (2%), 7 (13%), 4 (7%) and 4 (7%) strains were intermediate to cefprozil, cefaclor, loracarbef, and clarithromycin, respectively. The serotype distribution of H. influenzae in Korean children is similar to those in developed countries before the introduction of Hib conjugate vaccine, and ampicillin resistance rate is among the highest published to date.
Antibiotics/pharmacology* ; Child ; Haemophilus Infections/microbiology* ; Haemophilus Infections/drug therapy ; Haemophilus Vaccines ; Haemophilus influenzae/isolation & purification ; Haemophilus influenzae/drug effects* ; Haemophilus influenzae/classification ; Human ; Korea ; Microbial Sensitivity Tests ; Serotyping

OBJECTIVETo investigate the serotypes and antibiotics-resistance patterns of Haemophilus influenzae isolated from children in Hangzhou.

METHODSIsolates were identified with api-NH card. Serotypes were determined with slide agglutination method. The sensitivities of 13 antibiotics against 247 strains of Haemophilus influenzae were determined in vitro with Kirby-Bauer diffusion methods and MICs of ampicillin were determined with E-test. Nitrocefin test was used to detect beta-lactamase.

RESULTSOf the 247 strains isolated from children during the period from August 2001 to July 2002, 153 strains were non-typable, while 94 strains (38.1%) were typable and 90.4% and 1.1% of them belonged to type d and type b, respectively. Higher incidence of typable Haemophilus influenzae was found in male than in female children and the difference was significant (chi(2) = 5.30, P < 0.05), while between upper and lower respiratory tract infected children the difference was not statistically significant (chi(2) = 3.60, P > 0.05). Forty-one isolates (16.6%) were beta-lactamase-positive and 14 strains could not grow on medium in antibiotics sensitivity test. Of all 233 isolates tested successfully, 85.4% were susceptible to ampicillin, and the sensitivity rate to cefaclor, ceftriaxone, cefotaxime, imipenem, rifampin, clarithromycin, and chloramphenicol were as high as 98.7%, 99.6%, 99.6%, 99.6%, 98.7%, 91.0%, and 90.6%, respectively. All strains were sensitive to amoxicillin/clavulanic acid, ampicillin/sulbactan and ofloxacin, while 107 strains (45.9%) were resistant to trimethoprim-sulfamethoxazole, followed by that of tetracycline (14.6%). Resistance to ampicillin and trimethoprim-sulfamethoxazole in typable isolates was statistically significantly higher than in non-typable strains. Twenty-six strains (10.5%) were multi-resistant isolates and the multi-resistance rate in beta-lactamase-positive strains were significantly higher than that in beta-lactamase-negative strains (chi(c)(2) = 146.8, P < 0.001).

CONCLUSIONNon-typable Haemophilus influenzae was the most common type in clinical strains isolated from children in Hangzhou, while type d was the overwhelming type and type b was uncommon in typable isolates. Incidence of typable isolates was higher in male than in female children, and it was apt to intergrow with other species of pathogenic bacteria. The proportion of beta-lactamase-positive strains was not high and ampicillin or other beta-lactam actibiotics were still the treatment of choice for infections with Haemophilus influenzae.

Anti-Bacterial Agents ; pharmacology ; Child ; China ; Drug Resistance, Multiple, Bacterial ; Haemophilus Infections ; microbiology ; Haemophilus influenzae ; classification ; drug effects ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Serotyping

OBJECTIVETo optimize the isolation and purification conditions for Hap(s) protein of non-typeable Haemophilus influenzae.

METHODSHap(s) protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap weak cation exchange columns of CM Sepharose Fast Flow. The condition of the elution was optimized for pH and ionic strength, the absorbance at 280 nm of the elution samples were detected, and the targeted protein band in the collected samples was observed by SDS-PAGE electrophoresis.

RESULTSThe Hitrap ion exchange column was eluted with buffer 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths.

CONCLUSIONThe pH of the buffer only affect the elution of the irrelevant proteins rather than the Hap(s) protein, and elution with the buffer containing 100 mmol/L NaCl can be optimal for eluting the Hap(s) protein.

Bacterial Proteins ; isolation & purification ; Buffers ; Chromatography, Ion Exchange ; methods ; Electrophoresis, Polyacrylamide Gel ; Haemophilus influenzae ; metabolism ; Hydrogen-Ion Concentration ; Osmolar Concentration

OBJECTIVETo study the status of oral pharyngeal carriage and characteristics of Haemophilus influenzae (Hi) in healthy preschool children in Fuzhou.

METHODSSix hundred and three healthy children in two representative kindergartens in Fuzhou were studied as research subjects, and the rates of oral pharyngeal carriage of Hi were studied in four seasons. All Hi strains were serotyped and biotyped.

RESULTSThe oral pharyngeal carriage of Hi in day care nursery healthy children were 36.7% in winter, 18.0% in autumn, 12.4% in summer and 10.9% in spring respectively. Serotype Hib was preponderant in autumn (6.9%). In winter, the carriage rates of NTHi and Hib were 17.1%, 5.4% respectively. The carriage rates of other serotypes were low. Biotype VII and VIII were preponderant in autumn, spring and summer but biotype VII and VIII were decreasing evidently in winter.

CONCLUSIONThere was evident seasonal difference in the rates of oral pharyngeal carriage and type of Hi in healthy preschool children. The carriage rate of Hi strains was high in autumn and winter. Results suggested that while the inoculation of Hib-binding bacterial vaccine was expanded the study on new bacterial vaccine of Hi still needs to be augmented.

Carrier State ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Haemophilus influenzae ; classification ; isolation & purification ; Humans ; Male ; Pharynx ; microbiology ; Seasons ; Serotyping

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

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

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

OBJECTIVETo establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.

METHODSMultiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.

RESULTSThe sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively.

CONCLUSIONThe multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Haemophilus parainfluenzae ; classification ; genetics ; isolation & purification ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Nasopharynx ; microbiology ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity

Child ; China ; Community-Acquired Infections ; microbiology ; Drug Resistance, Bacterial ; genetics ; Haemophilus influenzae type b ; drug effects ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Pneumonia ; microbiology

BACKGROUNDIt is recognized that Haemophilus influenzae isolated from patients with otitis media forms biofilms both in vitro and in vivo, suggesting that biofilm formation in vivo might play an important role in the pathogenesis and chronicity of otitis media, but the effect of antibiotics on biofilm has not been well studied. We investigated the impact of ciprofloxacin and azithromycin on bacterial biofilms formed by Haemophilus influenzae in vitro in this study.

METHODSEleven strains of Haemophilus influenzae were isolated from sputum specimens collected from patients with acute exacerbation of chronic obstructive pulmonary diseases. Formation of bacterial biofilm was examined by crystal violet assay and a scanning electron microscope. Alterations of biofilms were measured under varying concentrations of azithromycin and ciprofloxacin.

RESULTSStriking differences were observed among strains with regard to the ability to form biofilm. Typical membrane-like structure formed by bacterial cells and extracellular matrix was detected. Initial biofilm synthesis was inhibited by azithromycin and ciprofloxacin at concentrations higher than two-fold minimal inhibitory concentration. Disruption of mature biofilms could be achieved at relatively higher concentration, and ciprofloxacin displayed more powerful activity.

CONCLUSIONSHaemophilus influenzae is capable of forming biofilm in vitro. Sufficient dosage might control early formation of biofilms. Ciprofloxacin exerts better effects on breakdown of biofilm than azithromycin at conventional concentration in clinics.

Anti-Bacterial Agents ; pharmacology ; Azithromycin ; pharmacology ; Biofilms ; drug effects ; Ciprofloxacin ; pharmacology ; Haemophilus influenzae ; drug effects ; isolation & purification ; Humans ; Lung Diseases, Obstructive ; microbiology ; Microbial Sensitivity Tests ; Microscopy, Electron, Scanning