[To develop the therapy strategy of lower respiratory tract infection suited to the special situation in China].
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Biomedical subjects
Publications and source records attributed to You-ning Liu.
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OBJECTIVE: To study the resistant phenotype of a clinical strain of Escherichia coli and to explore the effect of its attenuator mutation on AmpC expression. METHODS: A clinical strain of Escherichia coli 20022 (ECO20022) resistant to cefoxitin was isolated clinically. The phenotype was examined by three-dimensional methods, isoelectric focusing (IEF), and microdilution method. The regulator genes of ECO20022 were amplified and sequenced, and the difference between them was analyzed by BLAST method. Then the regulator genes were cloned into pCAT3-basic vector (a promoterless reporter gene vector). Microdilution method was used to detect the minimal inhibitory concentration (MIC) of chloramphenicol and ampicillin to this strain with E. coli ATCC25922 as quality control bacterium. ELISA was used to detect the content of chloramphenicol acetyl transferase (CAT). RESULTS: Compared to the standard E. coli K-12, there were four base substitutions, i.e., 22C-T, 26, 27TA-GT, and 32G-A in the attenuator region of ECO20022. Three-dimensional method showed that this strain was high AmpC-producing. IEF found that it produced three beta-lactamases with the values of PI of 5.4, 8.2, and 9.0 respectively. The beta-lactamase with the PI of 9.0 could be inhibited by cloxacillin but not by clavulanate. The strain was resistant to not only most of third generation cephalosporins, but also to cefepime; however it was still susceptible to carbapenem. The secondary structure of the attenuator RNA of ECO20022 was different from the traditional structure of E. coli K-12. The regulator gene was successfully cloned into pCAT3-basic vector and direct and indirect tests indicated that this regulator gene enhanced the CAT expressing level as much as 10 times that of Escherichia coli K-12. CONCLUSION: AmpC attenuator mutation leads to high AmpC expression in Escherichia coli, resulting in a significant rise of resistance level to beta-lactamase and a great menace to clinical antibiotic therapy.
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OBJECTIVE: To investigate the pathogenic causes of community-acquired pneumonia (CAP) in adult patients in China, the relation of previous antibiotic use and the Pneumonia Patient Outcome Research Team (PORT) classification to microbial etiology, and the prevalence of drug resistance of common CAP bacteria. METHODS: A prospective study was performed on 665 consecutive adult patients with CAP at 12 centers in 7 Chinese cities during one year. The etiology of pneumonia was considered if one of the following criteria was met: (1) valid sputum sample yielding one or more predominant strains; (2) blood cultures yielding a bacterial pathogen; (3) seroconversion, a > or = 4-fold increase or decrease titers of antibodies to Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila. Minimum inhibitory concentration (MIC) of respiratory tract isolates was determined using the agar dilution method. RESULTS: Pathogens were identified in 324/610 patients (53.1%) with valid serum samples and sputum cultures as follows: Mycoplasma pneumoniae (126, 20.7%), Streptococcus pneumoniae (63, 10.3%), Haemophilus influenzae (56, 9.2%), Chlamydia pneumoniae (40, 6.6%), Klebsiella pneumoniae (37, 6.1%), Legionella pneumophila (31, 5.1%), Staphylococcus aureus (23, 3.8%), Escherichia coli (10, 1.6%), Moraxella catarrhalis (8, 1.3%), Pseudomonas aeruginosa (6, 1.0%). Of 195 patients with a bacterial pathogen, an atypical pathogen was identified in 62 (10.2%) cases. The non-susceptibility rate of Streptococcus pneumoniae to penicillin, azithromycin, and moxifloxacin was 20.3%, 75.4% and 4.3% respectively. CONCLUSIONS: Atypical pathogens have important role in CAP, with Mycoplasma pneumoniae being the most common pathogen, and mixed infection of atypical pathogens with bacteria was found in 10.2% of the cases. Streptococcus pneumoniae and Haemophilus influenzae remain the most important bacteria for CAP. More than 75.0% of Streptococcus pneumoniae was resistant to macrolides and 20.3% was resistant to penicillin.
OBJECTIVE: To describe the clinical features of the infection caused by the highly pathogenic avian influenza A (H(5)N(1)). METHODS: A previously healthy 24 year old woman presented to our hospital on November 7, 2005. She was confirmed to be an H(5)N(1) infected case after death. The clinical, radiological and epidemiological data were analyzed. RESULTS: The patient had a history of direct contact with diseased and dead poultry (chicken and duck). The disease course was 10 days from onset of illness to death, and fever preceded dyspnea by 5 days. On admission, the striking characteristics were acute community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), and the major radiographic abnormalities included extensive infiltration bilaterally, focal consolidation and air bronchograms. The radiographic and clinical deterioration was rapid, and the patient died in less than 3 days after hospitalization. The diagnosis of influenza A (H(5)N(1)) was confirmed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR on specimens of the lower respiratory tract, performed by Chinese Center for Disease Control. The postmortem examination showed bronchial hyperemia, extensive consolidation, serous cavity effusions, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF). CONCLUSIONS: Human infection by the highly pathogenic avian influenza A (H(5)N(1)) is a fatal communicable disease. Information of avian influenza A (H(5)N(1)) virus, more attention to the epidemiologic data, and early intervention are critical in reducing the mortality.
OBJECTIVE: To investigate the effects of aerosolized perfluorocarbon (PFC) on gas exchanges, respiratory mechanics and hemodynamics in a swine model of acute respiratory distress syndrome (ARDS). METHODS: ARDS was induced by intratracheal instillation of detergent in 16 piglets, and the animals were then randomly assigned to a PFC treated group (n = 8) and a control group (n = 8). Animals of the control group were gas-ventilated with 100% O2 (3 L/min), while those of the PFC treated group received an additional continuous aerosolized PFC at 7 - 8 ml.kg(-1).h(-1). Blood gases, average artery pressure, heart rate, platform pressure, compliance, expiratory tidal volume and intrinsic positive end-expiratory pressure (PEEPi) were measured per 15 minutes. RESULTS: Detergent instillation resulted in a marked decrease in arterial oxygen pressure (PaO2) within 60 min, from (383 +/- 53) mm Hg (1 mm Hg = 0.133 kPa) to (49 +/- 12) mm Hg in the control group [fraction of inspired oxygen (FiO2) 100%], and from (377 +/- 55) mm Hg to (56 +/- 13) mm Hg in the PFC group (FiO2 100%). After 2 h treatment, PaO2 was increased from (49 +/- 12) mm Hg to (83 +/- 51) mm Hg in the control group, compliance from (1.4 +/- 0.4) ml/cm H2O to (2.8 +/- 1.8) ml/cm H2O, and expiratory tidal volume from (30.8 +/- 5.5) ml to (50.1 +/- 4.1) ml in the control group; PaO2 from (56 +/- 13) mm Hg to (189 +/- 133) mm Hg, compliance from (1.5 +/- 0.4) ml/cm H2O to (4.1 +/- 1.4) ml/cm H2O, and expiratory tidal volume from (30.8 +/- 3.3) ml to (74.5 +/- 16.9) ml in the PFC group (all P < 0.05). There were no significant differences between groups in arterial carbon dioxide pressure (PaCO2), pH values, blood pressure, heart rates, plat pressure and PEEPi during treatment (all P > 0.05). CONCLUSION: It is suggested that aerosolized PFC increases arterial oxygenation, compliance, and expiratory tidal volume in extended detergent-induced ARDS.
OBJECTIVE: To observe and evaluate the pathophysiological indexes of acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats. METHODS: Thirty-three Wistar rats were randomly divided into normal control group and experiment group. Respiratory rate , mortality, arterial blood gases, compliance and wet weight of right lung/body weight ratio, tumor necrosis factor-alpha (TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF) were determined 2, 4 and 6 hours after injection of LPS or normal saline in both groups. RESULTS: In the experiment group,the following changes were found. Arterial partial pressure of oxygen (PaO(2)) was reduced to 69.18 mm Hg (1 mm Hg=0.133 kPa), marked blood stasis, and edema in lung tissues could be grossly seen and pathological examination showed that there was a large number of inflammation cell infiltration and edema in interstitial spaces with disappearance of normal construction of alveolar. There was also dilatation of capillaries with congestion and adherent leukocytes. Furthermore, compliance was decreased to 47% of the normal value, and wet weight of right lung/body weight ratio increased to 137% of the normal value. Blood TNF-alpha level increased markedly in serum and BALF. CONCLUSION: Specific pathological changes and decreased PaO(2) over 30% of the baseline value are the main signs of successful reproduction of ALI model in rats. Compliance and weight of right lung/body weight value can also reflect the status of ALI as helpful indexes.
OBJECTIVE: To investigate the in vivo activities of ceftazidime and cefepime with susceptibility in vitro in rats with experimental pneumonia caused by extended-spectrum beta-lactamase-producing strain of Klebsiella pneumonia. METHODS: With intratracheal instillation method, 3 Klebsiella pneumonia strains isolated from this hospital were used to produce 3 groups of experimental model of pneumonia in rats. All the 3 strains showed resistant to cefotaxime and susceptible to piperacillin-tazobactam in vitro. To ceftazidime and cefepime, strain 1 was both susceptible. Strain 2 was susceptible to ceftazidime and resistant to cefepime. Strain 3 was resistant to ceftazidime and susceptible to cefepime. The three groups of rats were randomly assigned to one of the following five groups: one control group and four treatment groups. The efficacies were evaluated 96 hours later by the survival rate and the viable bacterial counts of the lungs (lg CFU/g). RESULTS: Group 1: piperacillin-tazobactam, ceftazidime and cefepime significantly improved the survival rate (75.0%, 76.9%, 80.0%) and reduced the bacterial counts [(10.8 +/- 2.8), (11.1 +/- 3.2), (11.0 +/- 3.7) lg CFU/g] compared with cefotaxime and the control group [36.0%, 32.0%; (15.7 +/- 5.6), (16.0 +/- 5.5) lg CFU/g; P < 0.05]. Group 2: piperacillin-tazobactam, ceftazidime significantly improved the survival rate (79.2%, 73.1%) and reduced the bacterial counts [(10.7 +/- 2.3), (11.0 +/- 2.7) lg CFU/g] compared with cefotaxime and the control groups [42.3%, 33.3%; (15.5 +/- 5.4), (15.8 +/- 4.6) lg CFU/g; P < 0.05]. Group 3: the survival rate in piperacillin-tazobactam and cefepime groups (80.8%, 75.0%) were significantly higher and the bacterial counts in piperacillin-tazobactam [(10.4 +/- 2.4) lg CFU/g] were significantly lower compared with the cefotaxime and the control groups [37.5%, 34.6%; (14.2 +/- 5.6), (15.3 +/- 4.9) lg CFU/g; P < 0.05]. CONCLUSION: Cefepime and ceftazidime can reduce the mortality and the number of viable bacteria in rat pneumonia caused by some ESBL-producing Klebsiella pneumonia strains susceptible to either of them and their efficacies were similar to piperacillin-tazobactam.
OBJECTIVE: A variety of in vitro experiments indicated that most of the extracellular virulence factors of Pseudomonas aeruginosa (PA) were controlled by the quorum sensing system. The aim of this study was to explore the role of the quorum sensing system in a rat model of PA pulmonary infection. METHODS: Rats were infected intratracheally with PA strain PAO1 or PAO1-JP2 (lasI rhlI double mutant) which was embedded in seaweed alginate bead. The difference of bacteriology and pathology between the two groups was compared. The in vitro activity of elastase was measured by the Congo Red method and the expression of exotoxin A was detected by Western blot. RESULTS: At the 14th and 28th days after challenge, the bacterial colony counts in the PAO1-JP2 group were (9.6 +/- 3.3) lgCFU/g and (4.2 +/- 3.1) lgCFU/g respectively; however, those of the PAO1 group were (11.3 +/- 2.8) lgCFU/g and (9.1 +/- 1.5) lgCFU/g respectively, which showed significant difference (P < 0.05). The lung index of macroscopic pathology (LIMP) and the score of macroscopic pathology of the PAO1-JP2 group were significantly lower than those of the PAO1 group at 7th, 14th and 28th days after challenge. The size of lung abscesses or granuloma in the lung under the microscope and the score of microscopic pathology of the PAO1-JP2 group were also significantly lower at the 7th, 14th and 28th days. The in vitro activity of elastase of the PAO1-JP2 strain was 0.02 +/- 0.00 and that of the PAO1 strain was 0.35 +/- 0.03, which showed significant difference (P < 0.05). The expression of exotoxin A could be detected by Western blot in the PAO1 strain but not in the PAO1-JP2 strain. CONCLUSION: When the quorum sensing system of PA was deficient the expression of some virulence factors, such as elastase and exotoxin A, were decreased, which resulted in milder pulmonary infection.
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OBJECTIVE: To detect the level and dynamic change of severe acute respiratory syndrome (SARS)-coronavirus-specific IgG antibody in conavalescent SARS patients, and to provide information for prevention and vaccine development. METHODS: IgG antibody against coronavirus was detected by ELISA in the blood of 311 convalescent SARS patients for every 2 - 4 weeks. Stata 7.0 statistics software was used to analyse the results. RESULTS: IgG antibody was detected positive on each testing of all the convalescent patients and its peak appeared 35 days after recovery. IgG antibody level showed a 35.8% decrease within one year. CONCLUSION: Data showed that all the SARS convalescent patients had generated high level of specific IgG antibody against coronavirus in the early stage of recovery, but the antibody level declined along with the progress of convalescence, suggesting that the detection of the IgG antibody should go on until it disappeared.
OBJECTIVE: To investigate the changes in adrenomedullin release in bronchial alveolar lavage fluid (BALF) from different lung areas in acute respiratory distress syndrome (ARDS) of produced dogs, by pulmonary and extra-pulmonary causes, with low tidal volume and positive end expiratory pressure (PEEP) treatment under supine and prone position. METHODS: Twenty-four male mongrel dogs were randomly divided into ARDSp (ARDS caused by pulmonary causes) supine group, ARDSp prone group, ARDSexp (ARDS caused by extra-pulmonary causes) supine group, and ARDSexp prone group. A detergent to cause lung injury in ARDSp dogs, and intravenous oleic acid was given in ARDSexp dogs. The results of adrenomedullin in different areas of dog's lung (upper lobe, middle lobe, and lower lobe) and arterial blood gas under lung protective ventilation treatment were measured. RESULTS: After lung injury, the arterial oxygenation index was lowered, and the levels of adrenomedullin in the upper lobe and middle lobe of ARDSp dogs were higher than that of ARDSexp dogs. After receiving low tidal volume and PEEP ventilation, the conditions of all the dogs were gradually getting ameliorated, and prone position ventilation gave better effects on lung injury dogs by inhibiting adrenomedullin release. CONCLUSION: There are statistical differences in adrenomedullin release in different lung areas between ARDSp dogs and ARDSexp dogs, and in both ARDSexp dogs and ARDSp dogs low tidal volume and PEEP treatment under prone position ventilation give better results compared to supine position.
OBJECTIVE: To study the prevalence, phenotype and genotype of the AmpC and ESBLs-producing clinical isolate of Klebsiella pneumoniae. METHODS: The clinical isolates of Klebsiella pneumoniae were examined by standard disk diffusion susceptibility tests, three-dimensional methods, isoelectric focusing (IEF) and microdilution methods. The conjugation experiment, multiplex PCR and DNA sequencing methods were used for further analysis. RESULTS: Four out of a total of 86 isolates tested were shown to be highly AmpC-producing by three-dimensional method. IEF showed that these strains produced a AmpC like beta-lactamase with a PI of 7.8, and DNA sequencing showed that the gene which expressed this AmpC like beta-lactamase was identical to DHA-1, a plasmid mediated cephalosporinase gene. These strains also produced an ESBL like beta-lactamase with a PI of 8.2 and the gene which expressed this beta-lactamase was identical to SHV-12. These strains were resistant not only to most of the third generation cephalosporins, but also to cefepime. However they were still susceptible to carbapenem. CONCLUSIONS: Highly AmpC-producing DHA-1 accompanied with SHV-12 in Klebsiella pneumoniae was reported here for the first time. They result in a significant rise in antibiotic resistance, which is regarded as a great challenge for clinical antibiotic therapy.
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OBJECTIVE: To establish a method to measure mutant prevention concentration (MPC) in vitro, and to measure MPC of fluoroquinolones for staphylococcus aureus. METHODS: The staphylococcus aureus strain ATCC25923 and 20 ciprofloxacin-susceptible clinical isolates were enriched in broth, and the bacterial concentrations were adjusted to 10(10) colony forming units per milliliter. The minimal inhibitory concentration (MIC), MIC for 99% of input cells (MIC99), provisional MPC (MPCpr) and MPC of moxifloxacin, gatifloxacin, pasufloxacin and ciprofloxacin for staphylococcus aureus were determined by agar plates dilution method. RESULTS: The MPC of moxifloxacin, gatifloxacin, pasufloxacin and ciprofloxacin for staphylococcus aureus strain ATCC25923 were 0.18, 0.3, 0.75 and 1.8 microg/ml, and the MPC/MIC99 were 9.0, 7.5, 8.0 and 10.6 respectively. The MPC for 90% of the isolates (MPCpr90) of moxifloxacin, gatifloxacin, pasufloxacin and ciprofloxacin for 20 staphylococcus aureus clinical isolates were 1, 1, 4 and 8 microg/ml, and the MPCpr90/MIC90 were 8, 8, 16 and 16 respectively. CONCLUSION: The capacity of moxifloxacin and gatifloxacin for restricting the selection of staphylococcus aureus resistant mutants were stronger than that of pasufloxacin and ciprofloxacin. Combined with pharmacokinetic parameters, moxifloxacin and gatifloxacin may restrict the selective enrichment of resistant mutants among ciprofloxacin-susceptible staphylococcus aureus clinical isolates, and ciprofloxacin is expected to selectively enrich mutants easily.
OBJECTIVE: To study the resistant phenotype and molecular biology character of plasmid mediated high AmpC-producing clinical isolates of Escherichia coli and to find new AmpC genotype. METHODS: The cefoxitin highly resistant clinical isolates of Escherichia coli were studied by K-B method, three-dimensional method, Isoelectric Focusing (IEF) and the MIC of these strains were examined by micro-dilution method. The conjugation experiment, multiplex PCR and DNA sequencing methods were used in further study. RESULTS: Above 719 strains studied, there are 6 isolates were showed as high AmpC-producing by three-dimensional method and IEF found they could produce a beta-Lactamase which PI was 8.9 and could be inhibited by cloxacillin but not by clavulnate. The strains were resistant to most of third generation cephalosporins, but were susceptible to cefepime, meropenem and imipenem. The experiment also showed that the gene which express this AmpC like beta-Lactamase could be transferable. Multiplex PCR indicated they belong to Citrobacter freundii family. Sequencing of corresponding DNA revealed 99% identities of the deduced amino acid sequence with CMY-2 and CMY-7 respectively. It is a new CMY type cephalosporinase. CONCLUSION: A new CMY type cephalosporinase has been found in clinical strains of Escherichia coli in our hospital. It was resistant to many antibiotics and its resistance could be transferred horizontally.