Objective To investigate the changing patterns of Staphylococcus aureus (SA) and its resistance from a paediatric clinic in Beijing Children's Hospital Affiliated to Capital University of Medical Sciences.Methods SA isolated from hospitalized patients in Beijing Children's Hospital Affiliated to Capital University of Medical Sciences from Jan.2003 to Dec.2012 were analyzed.Antimierobial susceptibility was determined by disk diffusion method and Phoenix 100 microbiological system.Results were analyzed according to the 2012 guidelines of Clinical and Laboratory Standard Institute.WHONET 5.6 software was used to analyze the data.Results The clinical isolates of SA from inpatient were 2 843.The positive rates of SA were 10.0％,8.7％,9.0％,11.6％,12.7％,11.7％,10.5％,10.8％,11.9％ and 11.7％ form 2003 to 2012,respectively.The positive rate of Methicillin-resistant Staphylococcus aureus (MRSA) from inpatients was 10.9％.The positive rates of MRSA were 3.1％,5.4％,6.4％,4.1％,6.0％,10.6％,11.2％,12.7％,14.1％ aid 20.1％ form 2003 to 2012,respectively.The resistant rate of Methicillin-susceptible Staphylococcus aueus and MRSA to Penicillin were 91.4％ and 100.0％,Oxacillin were 0 and 100.0％,Cefuroxime were 0.5％ and 87.7％,Ceftriaxone were 0.1％ and 90.3％,Amoxicillin-clavulanic acid were 0.9％ and 86.7％,Erythromycin were 72.4％ and 92.2％,Clindamycin were 50.6％ and 80.3％,Ciprofloxacin were 2.2％ and 30.1％,Trimethoprim-sulfamthhoxazol were 12.2％ and 13.9％.Vancomycin-intermediate and Vancormycin-resistant Staphylococcus aureus were not found.Conclusions The prevalence of MRSA is increasing in paediatric inpatients.And MRSA isolates exhibit multidrug resistance.The sequential surveillance about SA is very important for guiding rational antimicrobial therapy and effective control of infections in paediatric patients.

Objectives To analyze the main pathogen of otitis media and antibiotics resistance of clinical bacterial isolates in pediatric patients. Methods Secretion specimens from 164 cases of otitis media were cultured. Antimicrobial susceptibility was determined. Results were analyzed according to the guidelines of Clinical and Laboratory Standard In-stitute (2012). WHONET 5.6 software was used to analyse the data. Results Pathogens were cultivated in 121 ear secretion specimens from 164 cases of otitis media and the positive rate was 73.8%. There were 9 cases of mixed bacterial infection, accounting for 5.5%. 130 strains of pathogens were isolated from middle ear secretion and the main pathogens were bacteria (83.1%) and fungus (16.9%). Among bacteria, 50 (46.3%) were Streptococcus pneumoniae and 21 (19.4%) Staphylococcus aureus. Among fungus, 17 (77.3%) were Candida spp and 5 (22.7%) Aspergillus spp. By age, the rates of infection caused by Streptococcus pneumoniae in infancy and early childhood were markedly higher than those in preschool and school-age, respectively (P<0.05). The no-susceptibility rate of Streptococcus pneumoniae to penicillin was 76.0%. The prevalence of meticillin-resistant Staphylococcus aureus (MRSA) was 14.3%. Conclusions Proper treatment depends on the secretion culture and drug sensitive test due to various pathogens of otitis media.

100 parasites/?l, 72.73% with

Objective To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase(GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH. Methods The GDH gene was cloned into prokaryotic expression vector pET23(a) to form recombinant expression vector pET23(a)/GDH. pET23(a)/GDH was transformed into E.coli BL21(DE3). Induced by IPTG(isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication. The soluble recombinant GDH was purified by Source-Q and Source-S chromatography. Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product. Results SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein. By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity. Conclusion The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.

Efficient gene delivery and sustained gene expression are required for successful human gene therapy.Although viral vectors are considered the most efficient vehicles for gene transfer,currently available viral vectors have not fully achieved these two requirements.Lentiviral vectors(LVs)can integrate into host chromosomes,allowing long-term gene expression,in addition,these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome,but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments.In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks,a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed.BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA,the packaging plasmid pGAGPOL and the envelope plasmid pVSVG,and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000TM.After 4 days,the culture supernatant of lentiviral vectors was collected,the RNA from the supernatant was examined by the RT-PCR,the protein from the supernatant was examined by Western blot,and the supernatant was used to transfect normal 293T,HepG2 and Vero,which were observed by the immunofluorescence microscopy.The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies)were considered so critical to the output of this system that 3?3?3 factorial design was used to explore the yield optimization of this system.As judged by the results of RT-PCR and the Western blot,lentiviral vectors were found in the culture supernatant;as judged by immunofluorescence with microscopy,293T,HepG2 and Vero which were transfected by the supnantant expressed the report protein-green fluorescent protein(GFP),the results confirmed the valid infectivity of the lentiviral vector produced by the system.Eventually,the best titers of lentiviral vector stocks was up to 1.3?108 tu/ml,which is one order of magnitude higher than the output of classical manufacture system.The new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established.It provides the basis for the future development of industrial application.

Objective To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E. coli TG1 and analyse its immunocompetence. Methods The LDH gene of the P. falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX 4T 1 vector for expression of fusion protein with glutathione S transferase(GST). The recombinant plasmid was transformed into the E. coli TG1. Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected. The immunocompetence of recombinant protein was analysed by ELISA and Western blot. Results The LDH gene of P. falciparum was successfully expressed in the E. coli TG1. The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P. falciparum . The specific humoral responses were induced in mice and the titer of the specific antibody was 1∶16 by two dimensional diffusion assay. Conclusion The LDH gene of P. falciparum has been successfully expressed in the E. coli TG1 and the expressed protein has high antigencity.

Objective To analyze the antibiotic resistance of the carbapenem no-susceptibility Enterobacteriaceae isolated from pediatric patients and the resistant genes of carbapenemase-producing.Methods In all,46 carbapenem no-susceptibility Enterobacteriaceae strains were isolated from patients at Beijing Children's Hospital between January 2008 and December 2010.Agar dilution method recommended by the Clinical and Laboratory Standards Institute was used to examine the minimum inhibitory concentrations (MICs) of 14 antimicrobial agents.Phenotypic testing for carbapenemase-producing was conducted using Hodge test and double-disk synergy test.PCR was used to detect the expression of the carbapenemase-related genes KPC,GES,IMI/NMC-A,SME,IMP,VIM,GIM,SPM,SIM and OXA.WHONET5.6 was used to perform resistance analysis.Results Among 46 carbapenem no-susceptibility Enterobacteriaceae strains,26 (56.5％) were Klebsiella pneumoniae strains,13(28.3％ ) were Enterobacter cloacae and 7( 15.2％ ) were Escherichia coli.The rates of imipenem and meropenem no-susceptibility Klebsiella pneumoniae were 69.2％ and 80.8％,Enterobacter cloacae were 76.9％ and 100％ and Escherichia coli were 85.7％ and 100％,respectively.40(87.0％ ) strains were positive of Hodge test.41 (89.1％) strains were positive of doubledisk synergy test.38 (82.6％) were positive for the IMP genotype.The carbapenemase-related genes were not found in other 8 strains.Conclusion The prevalence of carbapenem no-susceptibility Enterobacteriaceae strains in Klebsiella pneumoniae isolates is relatively high in children.Resistance to imipenem was lower than that to meropenem from Klebsiella pneumoniae,Enterobacter cloacae and Escherichia coli strains.Many carbapenem no-susceptibility Enterobacteriaceae isolated from pediatric patients carry the blaIMP gene.No the KPC gene was found.

Objective To investigate the etiology, epidemiologic features and drug resistance tendency of acute infectious diarrhea among children in Beijing area. Methods Enteric pathogenic bacteria were isolated, cultured and identified for serotype from the stool specimens of children with the initial clinical diagnosis of acute bacterial diarrhea in the intestinal clinic from January to October in 2009 ,and the antibiotic susceptibility of bacteria was tested by disk diffusion method. The cluster A rotavirus antigen was also detected by the qualitative technique of immunochromatographic double-antibody sandwich assay. Results Of the 256 stool specimens, 87 strains of 4 species of pathogenic bacteria were detected with the detectable rate of 34. 0% ,of which 2 strains were detected in one stool sample ,including 40 strains of salmonella (46. 0%) ,23 strains of shigella (26. 4%) ,2 strains of diarrheogenic escherichia coli (2. 3%) and 22 strains of staphylococcus aureus (25.3%). The positively detected patients consisted of 54 males and 32 females with the ratio of 1.69∶1 ,of whom 55 cases (64. 0%) were under 2 years of age. Of the 13 strains of shigella, 13 were sonnei shigella (56. 5%). And of the 22 strains of staphylococcus aureus,20 were detected among the infants under 1 year of age. The rates of crug resistance to certain antibiotics were lower in salmonella than in shigella (ampicillin :65. 0% vs. 95.7% ;compound sulfamethoxazole:20. 0% vs. 78. 3% ;ciprofloxacin:7.5% vs.8. 7% ;ceftriaxone: 15.0% vs. 73.9%). Of the 256 stool specimens ,47 were found positive for cluster A rotavirus,of whom 13 were also positive in stool bacteria culture. Conclusion Salmonella is the major pathogen among children with bacterial diarrhea in Beijing in 2009, and sonnei shigella is the main epidemic strains of shigella diarrhea. lnfants under 2 years of age are the susceptible population of the above two species of bacteria, while staphylococcus aureus mainly infect the infants under 1 years of age. Multi-resistance in shigella is still serious. The incidence of mixed infections of bacteria and rotavirus increases in children with infectious diarrhea.

Objective To study the molecular characteristics of the Staphylococcus aureus isolated from the intensive care units (ICUs) of children's hospital.Methods From January 2016 to December 2016,a total of 39 S.aureus strains were collected and identified from various clinical specimens that were obtained from patients who were confined in the neonatal and pediatric ICUs of Beijing Childreng Hospital.Methicillin-resistant S.aureus (MRSA) and methicillin-susceptible S.aureus (MSSA) were identified using the cefoxitin disc method and the detection of the mecA gene.Multilocus sequence typing (MLST) and spa typing were analyzed using the PCR,and the staphylococcal chromosomal cassette mec (SCCmec) type was analyzed for the MRSA isolates.Twenty-one superantigen genes and the Panton-Valentine leukocidin (pvl) gene were also detected by PCR.Their susceptibility to 12 antibiotics was evaluated using the E-test method.The differences in prevalence of virulence genes and antimicrobial resistance were compared between the MRSA and MSSA isolates by Fisherg exact test.Results All the S.aureus strains were isolated from secretion inside the airway of pneumonia (including severe pneumonia),the blood of patients with bacteremia,and exudate of skin and soft tissue infections.ST59-SCCmecⅣa-t437 (55.6％) and ST398-t571 (28.6％) were the most predominant clones of MRSA and MSSA,respectively.Of the 39 isolates,26 strains (66.7％) had at least one superantigen gene,and seb (38.5％),sek (30.8％),and seq (20.5％) were the most common genes;seb-sek-seq (18.0％) was the main virulence genotype.The pvl geneg positive rate was 25.6％,and no significant difference between MRSA and MSSA was observed (P ＞ 0.05).Notably,79.9％ of the S.aureus isolates were multidrug resistant,and 94.9％,53.8％,and 51.3％ of the isolates were resistant to erythromycin,clindamycin,and chloramphenicol,respectively.The tested isolates were susceptible to trimethoprim/sulfamethoxazole,rifampicin,and vancomycin.Conclusions The S.aureus strains from the ICUs of childreng hospital were isolated from the secretion inside the airway of pneumonia (including severe pneumonia),the blood of patients with bacteremia,and exudate of skin and soft tissue infections.ST59-SCCmecⅣa-t437 (55.6％) and ST398-t571 (28.6％) were the common clones of MRSA and MSSA,respectively.The prevalence of superantigen genes and the multidrug resistant rate were relatively high.

To develop simple, rapid, and efficacious diagnostic methods for malaria is one of the remaining key tasks for malaria control. Previously, we have created a phage-displayed antibody library against Plasmodium falciparum. Six clones of antibody with good reactivity to HRP-II in ELISA were isolated from the library after 3 rounds of enrichment. Soluble ScFvs were produced and the characteristics were determined. The results of Western blot showed that they could bind to HRP-II specifically and had a relative molecular mass(Mr) about 31 000. The work provided a solid fund for diagnostic kit development for malaria.