OBJECTIVE To investigate the clinical distribution and antimicrobial resistance of infections caused by Enterobacter cloacae in our hospital,for guiding the prophylaxis and treatment of these infections in clinical practice. METHODS Antimicrobial susceptibility tests were done by Kirby-Bauer disk diffusions method,the phenotypes of E.cloacae strains harboring AmpC ?-lactamases and extended-spectrum ?-lactamases(ESBLs) were detected by modified three-dimensional extract test. RESULTS A total of 162 strains of E.cloacae were isolated between Jan 2002 and Dec 2004,the percentages of these strains isolated from sputum,urine and wound secretion were 62.3%,11.7% and 9.9%,respectively.73.5% of all strains isolated from intensive care unit(ICU) in every endemic area.AmpC ?-lactamases,AmpC ?-lactamases combined with ESBLs and ESBLs producing strains were detected in 19.1%,11.7% and 14.2% of E.cloacae,respectively.The resistance of AmpC ?-lactamases or ESBLs producer was obviously higher than that of non-AmpC ?-lactamases and ESBLs producer,while the phenomenon of resistance was serious especially in AmpC ?-lactamases combined with ESBLs producer,but all strains were sensitive to imipenem. CONCLUSIONS E.cloacae causes infections of respiratory tract,urinary tract and wound principally in clinic,the most of which occurred in ICU.AmpC ?-lactamases and ESBLs are popular in E.cloacae,the restricted use of ?-lactams is a considerable measure which reduces prevalence of AmpC(?-lactamases) and ESBLs producing strains.The antimicrobial agents for treating infections caused by E.cloacae should be chosen according to the results of the antimicrobial susceptibility tests,imipenem is the first choice for severe infections.

Objective To evaluate the utility of CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14+ monocytes in the blood of tuberculosis (TB) patients .Methods CD4+ TCR tetramers were used to detect tetramer‐positive CD14+ monocytes in the peripheral blood (PBL ) samples of inpatients with advanced pulmonary TB (PTB) by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14+ monocytes and Mycobacterium tuberculosis (MTB) antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of tetramer‐bound CD14+ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4+ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4+ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14+ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14+ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect M TB antigen‐specific CD14+ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .

[Objective]To explore the intracellular antimicrobial activities of erythromycin,ciprofloxacin,levofloxacin,moxifloxacin against Legionella pneumophila.[Methods]The minimum inhibition concentration(MIC)of each antibiotic was evaluated by E-test method and microdilution method respectively.The minimal extracellular concentration inhibiting intracellular multiplication(MIEC)of each antibiotic was evaluated by the MTT colorimetric assay system.[Results]The MIC concentration for each drug by E-test method were:erythromycin,0.047 μg/mL;ciprofloxacin,0.38 μg/mL;levofloxacin,0.125 μg/mL;moxifloxacin,0.125 μg/mL,the MIC concentrations for each drug by microdilution method were:erythromycin,0.125 μg/mL;ciprofloxacin,0.03 μg/mL;levofloxacin,0.016 μg/mL;moxifloxacin,0.016 μg/mL.The MIEC concentration for each drug were:erythromycin,0.25 μg/mL;ciprofloxacin,0.016 μg/mL;levofloxacin,0.016 μg/mL;moxifloxacin,0.004 μg/mL.[Conclusions]Fluoroquinolones have superior activity than erythromycin in U937 cells infected with L.pneumophila.Moxifloxacin is the most potent drug among the four tested antimicrobials.Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics against L,pneumophila and efficient processing of a large number of samples.

Objective To study the homology and clinical distribution of tigecycline-resistant Acinetobacter baumannii (A.baumannii )in a hospital.Methods Multidrug-resistant A.baumannii (MDRAB,n = 88 )from specimens from clinical departments of a hospital in 2013-2014 were collected and detected susceptibility to tigecy-cline;homology of tigecycline-resistant strains were detected by pulsed-field gel electrophoresis (PFGE),clinical characteristics and distribution of infected patients were analyzed.Results 88 patients didn’t use tigecycline before MDRAB were isolated.Of 88 MDRAB strains,4 (4.55%)were resistant to tigecycline,which were No.10,31 , 33,and 87 strains.PFGE results revealed that No.31 ,33,and 87 strains were of the same genotype,and with high homology,which distributed in three different departments;No.31 strain was detected from general intensive care unit (ICU),No.33 strain was detected from emergency ICU,although strains were detected from different depart-ments,patients were transferred before strains were isolated,and were admitted to departments of gastrointestinal surgery and emergency ICU during the same period;No.87 strain was detected from neurosurgical ICU and patient had never been transferred,the detection time was 7-8 months later than No.31 and 33 strains.No.10 strain was isolated from emergency ICU,patient was not transferred.Conclusion Of MDRAB isolated in this hospital,tigecy-cline-resistant strains are low,most strains are homologous,cross infection may be exists in different departments.

OBJECTIVE To study the genetic supports of OXA-23 in Acinetobacter baumannii isolates and investigate the relationship between imipenem resistance acquiring and use of antibiotics.METHODS Consecutive selection of the 24 highly susceptive A.baumannii clinical isolates by imipenem was carried out.Genes of carbapenemases were detected by PCR and the colonial relationship of these isolates was evaluated by ERIC-PCR.Plasmids conjugation experiments and blaOXA-23 hybridization were performed to explore the gene location of blaOXA-23.RESULTS From all 24 susceptible A.baumannii isolates 10 were selected,including 6 multi-colonial blaOXA23 harboring strains.Plasmid conjugation experiments and Southern blotting experiments demonstrated that blaOXA-23 was not associated with integrons.CONCLUSIONS BlaOXA-23 may exist in a certain subset of apparently carbapenem sensitive Acinetobacter strains.When under consecutive selective pressure,bacteria harboring blaOXA-23 become the predominant group and subsequently the antibiotics resistance properties appear.It highlights the reasonable use of this category of antibiotics.

OBJECTIVE To determine the prevalence of gene qnrA in clinical isolates of Enterobacteriaceae,phenotypes of drug resistance and their molecular mechanisms.METHODS Totally 332 isolates of Enterobacteriaceae from Shenzhen City from 2003 to 2004 were screened for the presence of qnrA by PCR.The gyrA,and parC genes the genes,coding ESBLs and AmpC were amplified and sequenced.K-B method,the international standard plate dilute method and E-test were used to detect MIC of the isolates with qnrA.ESBLs were distinguished by the phenotypic confirmatory test.RESULTS The incidence of qnrA in clinical isolates was 3.9%,the highest incidence of 15.6% occurred in Enterobacter cloacae.The clinical isolates were multi-resistant.gyrA And parC mutation associated with quinolone resistance and ESBLs producing was confirmed in most of the isolates with qnrA.qnrA Was embedded in In37 or InX classified to Sul1 type class Ⅰ integrons.CONCLUSIONS Gene qnrA located in plasmids is an important mechanism mediated quinolone resistance in Enterobacteriaceae.The potential horizontal transferability and multiple resistance hereditary background in these isolates challenge the anti-infective therapy.

Objective To investigate the variations of surface protein genes of avian influenza virus (AIV)before and after infecting mouse.Methods Mouse lung tissue was infected with A/Goose/Guangaong/NH/2003(H5N1)and the virus was isolated 12 hours and 9 days after replication in lung tissue of mouse.The isolated strains were amplified in embryonated chicken eggs,anti the virion RNA was transcribed into cDNA by reverse transeriptase.After amplification and purification,dideoxy-mediated chain termination was performed to detect synthetic oligonucleotide primers and DNA sequence was analyzed.Results The homology of nucleotide sequence for HA gene of three isolated strains was 99.6%-99.8%.and that of amino acid sequences was 99.3%-99.6%.The homology of nucleotide sequence for NA gene of three strains was 99.8%-99.9%.all of them were synonymous mutatinns.No variation was found in M gene.Conclusion After replication in mouse lung tissue,no significant mutation was found in the surface protein genes of AIV except some point mutations in HA genes.

Objective To compare the toxicity of outer membrane vesicles ( OMVs) secreted by Acinetobacter baumannii strains with different drug-resistance spectrums.Methods Four Acinetobacter baumannii strains with different drug-resistance spectrums were collected (strain 33, 3237, B29 and 10), and OMVs produced by these strains were extracted and purified.BCA assay was used to determine the protein concentrations, and RAW264.7 cells were incubated with different concentrations of OMVs for 24 h. Cell viability was measured with CCK-8 assay, and gene expression of tumor necrosis factor-alpha ( TNF-α) , interleukin-6 ( IL-6) , interleukin-1 beta ( IL-1β) , keratinocyte-derived chemokine ( KC) and macrophage inflammatory protein 2 (MIP-2) was assessed by quantitative real-time PCR.One-way ANOVA was used for data analysis.Results According to the result of drug susceptibility test, strain 10 was extensively drug-resistant Acinetobacter baumannii ( XDRAB ) strain, strain B29 was multi-drug resistance Acinetobacter baumannii (MDRAB) strain, while strain 33 and 3237 were non-MDRAB strains.After incubated with different concentrations of OMVs for 24 h, cell viability of RAW264.7 declined with the increase of OMVs concentrations.OMVs released from strain10, B29 and 3237 significantly lowered the cell viability at the concentration of 5 μg/mL, while the cytotoxicity of OMVs released from strain 33 was much weaker, and no remarkable decrease in cell viability was observed even at the concentration of 25 μg/mL.OMVs of all strains induced the release of TNF-α, IL-6, IL-1β, KC and MIP-2 in RAW264.7 cells, and the levels of theses cytokines were increased with the concentration of OMVs.Inflammatory response in cells incubated with OMVs from strain 33 was the weakest, while OMVs from strain 10 induced strongest inflammatory response.KC and MIP-2 levels were significantly higher in RAW264.7 cells incubated with OMVs from strain 10 with a concentration of 5 μg/mL than that incubated with OMVs from other strains ( F=19.094 and 19.032,P<0.05 or <0.01).Conclusions OMVs from Acinetobacter baumannii strains with different drug-resistance spectrums are of different toxicity.OMVs from XDRAB and MDRAB strains have higher toxicities and may induce stronger inflammatory response.

ObjectiveTo investigate the specificity of CD4+ Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis(MTB) infections.MethodsThe above TCR tetramer by using biotinylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell( S2 cell) lines was prepared.The PE-labled TCR tetramer was used to costain with S2 cell lines expressing MTB prptide/HLA-DR complexes on the cell membrane,and also was used to detect tetramer-bound CD14+ monocytes and macrophages in the peripheral blood mononuclear cells (PBMC) of pulmonary tuberculosis (PTB) patients and three control groups by flow cytometric analysis.And the FITC-labled tetramer was used to examine tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells by in situ staining.ResultsThe TCR tetramer was well binding with S2 cell lines expressing C14/HLA-DR *1504 on the cell membrane.By flow cytometric analysis,the percentage of tetramer-bound CD14+ monocytes and macrophages in PTB patients group was higher than the other three control groups( P＜0.001 ).By in situ staining,tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells were positive in PTB tissue and negative in control pneumonia tissue.ConclusionThe spcificity of TCR tetramer in monitoring MTB infections by flow cytometric analysis and in situ staining could be seen,which laid a laboratory foundation in the diagnosis and immune mechanism research of TB by using TCR tetramers.

Pythiosis is a rare and refractory infectious disease in human caused by Pythium insidiosum, which mainly occurs in the tropics, subtropics and some temperate regions. There are few reports of pythiosis in China. The mortality of the disease is very high, however, there is uncertainty in its diagnosis and treatment. This article reviews the epidemiological characteristics, clinical manifestations, diagnostic methods, treatment strategies, and prognosis of pythiosis in human, to provide reference for clinical management of pythiosis.