Objective Exotoxin A ( encoded by gene toxA ) , one of the most toxic protein secreted by pseudomonas aerugi-nosa(P.a.), and PcrV (encoded by gene pcrV), key component to type Ⅲsecretion system of P.a., both matter significantly to the virulence of P.a.The article was to construct a novel DNA vaccine encoding a mutated toxA gene and the pcrV gene of P .a.and i-dentify gene expressions in eukaryotic cells . Methods The genes of toxA and pcrV were amplified by PCR , and the toxA gene was mutated to reduce the toxicity of Exotoxin A .Then gene fragments toxA m and pcrV were inserted into eukaryotic expression plasmid pIRES simultaneously to construct a recombinant DNA vaccine pIRES-toxAm-pcrV.The novel plasmid was transfected into HEK-293 cells by lipofectamine 2000 .The expressions of toxA m and pcrV were detected by Western blot . Results Gel electrophoresis demon-strated the target gene fragments encoding Exotoxin A and PcrV .Western blot exhibited proteins encoded toxA and pcrV expressed by HEK 293 cells. Conclusion The recombinant plasmid pIRES-toxAm-pcrV was successfully constructed .Western blot analysis indi-cated the expressions of toxA m and pcrV in HEK-293 cells.It may be used as a potential candidate of preventive vaccine of Pseudo-monas aeruginosa .

Objective To investigate the genetic features related to drug-resistance of a strain of pan-drug resistant Acinetobacter baumannii. Methods The susceptibilities to 15 antibacterial agents were detected in Acinetobacter baumannii by K-B paper diffusing method. A strain of pan-drug resistant Acinetobacter baumannii was selected. The drug-resistant genes of the selected strain were amplified by polymerase chain reaction (PCR) , including 15 extended-spectrum β-lactamase genes, 3 metal β-lactamase genes, 2 AmpC enzyme genes, 7 aminoglycoside modifying enzyme genes and the genetic markers of integron I and transposons. Results The strain was resistant to 14 antibacterial agents except cefoperazone sodium/ sulbactam. β-lactamases genes ( TEM, OXA-23 and ADC) , aminoglycosides modification enzyme genes [aac(3)- I , aac(6')- I b and ant(3")- I ] , integron I qacEAi-sull and transposon tnpU were positive in PCR amplification. A new subtype of AmpC (chromosome) desoxyribonucleic acid ( DNA) was detected. Conclusion Acinetobacter baumannii is of high resistance to most antibacterial agents, and the mechanisms of the resistance are complex.

OBJECTIVE To analyze the situation of extended spectrum ?-lactamases(ESBLs)and AmpC enzyme produced by nosocomial Escherichia coli isolates in 2005-2007.METHODS ESBLs were detected by double disk synergy test and disk diffusion confirmatory test.AmpC enzyme was detected by the three dimensional assay.Chi square test was used to test the significance.The application of different kinds of antimicrobials before the results of etiology be presented and the resistence rate of the ESBLs both producing and no producing were compared respectively.RESULTS The detectable rate of ESBLs in E.coli isolates of nosocomial and community infection was 55.1% and 21.3% and the detectable rate of AmpC enzyme nosocomial E.coli isolates was 17.4%.All strains were 100% susceptible to meropenem and imipenem but resistant to 15 other antimicrobials in different degree.The sensitivity to Piperacillin/tazobactam,cefoperazone/sulbactam and amikacin were relatively high.CONCLUSIONS The carrying rate of ESBLs from nosocomial E.coli isolates is high and AmpC enzyme and other resistance genes,which lead to multiple drug resistance.Standardized management of antimicrobials application should be strengthened and the consciousness of rational antimicrobials utilization should be raised.

OBJECTIVE To study the current status of the AmpC enzyme and ?-lactamases genes for multi-resistant Acinetobacter baumannii isolates in clinic in Nanjing.METHODS The samples of 20 multi-resistant A.baumannii isolates were collected from Jul 2007 to Oct 2007 from the patients in hospitals of Nanjing.To determine the sensitivity to the 11 antibacterials by using a broth induction method,and two kinds of AmpC enzyme genes and eighteen kinds of ?-lactamases genes of multi-resistant A.baumannii analyzed by polymerase chain reaction(PCR).RESULTS In all of the 20 A.baumannii isolates,the AmpC genes of chromosome type were found in 14 isolates(70.0%),there were the AmpC genes of plasmid type in 1 isolate(5.0%),the TEM genes in 12 isolates(60.0%),the SHV genes in 1 isolate(5.0%),CTX-M-1 genes in 2 isolates(10.0%),OXA-23 genes in 1 isolate(5.0%),and OXA-24 genes in 1 isolate(5.0%),respectively.The OXA-24 genes from No.9 isolate was the type of OXA-72 after DNA sequencing.The amino acid sequence translated by AmpC genes of chromosome type from No.16 and No.17 isolates was the new subtype.CONCLUSIONS The A.baumannii has not only TEM,VEB,GES,IMP,VIM,OXA-10 and OXA-23 genes,but also the type of chromosome and plasmid of AmpC,SHV,CTX-M-1 and OXA-24 genes in Nanjing.It is the first time that activity of AmpC enzyme and the genotype of chromosome and plasmid of AmpC enzyme for matching are tested at the same time.

OBJECTIVE To explore the clinical characteristics of nosocomial infection of lower respiratory tract by coagulase negative staphtylococci(CNS) and their drug resistance to common antibiotics.METHODS The clinical data and drug resistance of CNS of 37 nosocomial infection cases and 68 cases with colonization of lower respiratory tract by CNS were analyzed retrospectively,and the study mainly was focused on the risk factors and basic clinical features of nosocomial infection of lower respiratory tract by CNS.The resistance to common antibiotics of CNS was also summarized.RESULTS The risk factors were determined,which included long duration,age more than 65 years old,chronic obstructive pulmonary disease,the use of antibiotic suchs as carbapenems,the third generation cephalosporin,fluoroquinolones and concurrent infection of fungus.The mortality rate of infection was higher than after colonization.The clinical characteristics of nosocomial infection of lower respiratory tract by CNS included that the temperature of patients was focus on 37.5-38.5 ℃,peripheral blood routine of patients appearsed normal mostly and the lung imaging displayed the bilateral exudation changing near 50% the total.The 105 isolates of CNS were resistant to multi-antibiotics,the drug resistance rate to rifampicin and sodium fusidate was relatively low and resistant strains to vancomycin and teicoplanin were not detected.CONCLUSIONS The toxemia symptoms of nosocomial infection of lower respiratory tract by CNS are mild and the clinical manifestation is atypical.It is more likely to catch nosocomial infection of lower respiratory tract by CNS for the patients who are with low immunity and applied with broad spectrum antibiotic for long-term.

Objective To screen B and T cell antigen epitopes on Acinetobacter baumannii outer membrane protein 33×103-36×103 (OMP33-36).Methods B and T cell epitopes on OMP33-36 of Acinetobacter baumannii were predicted by bioinformatics methods and synthesized.Recombinant expression plasmid pET-30a-OMP33-36 was cloned and used to express OMP33-36 in a prokaryotic expression system.The expressed OMP33-36 was used to immunize BALB/c mice after purification.Serum sample was collected from each mouse in immunization and negative control groups, and then analyzed by indirect ELISA with synthesized peptides to identify B cell epitopes.Splenocytes were separated from every mouse and then cultured with each of the synthesized peptides, respectively.Double sandwich ELISA was performed to detect IFN-γ secretion in the supernatant of cell cultures for screening of T cell epitopes.Results Candidates of B and T cell epitopes were constructed, which were PB1, PB2, PB3, PT1, PT2 and PT3.Results of the indirect ELISA showed that peptides PB1 and PB2 reacted with the serum samples collected from immunized mice and A450 values of the immunization group were significant higher than those of the negative control group.Compared with the negative control group, enhanced secretion of IFN-γ following peptide PT3 stimulation was observed in the immunization group as indicated by the double sandwich ELISA.Conclusion Two B cell epitopes PB1 and PB2, and one T cell epitope PT3 on the OMP33-36 of Acinetobacter baumannii were successfully constructed and screened out.

Objective To investigate the resistance-mechanism of the carbapenems-resistant Klebsiella pneumoniae isolated from clinical.Methods The clinical isolates of carbapenems-resistant Klebsiella pneumoniae from top three comprehensive hospitals of Nanjing area were examined by 40 beta-lactamase,porin-coding genes and linkage of KPC-ISKpn6 using PCR method,the PCR positive results were picked out for sequencing and sequencing BLAST search for comparison analysis.Results Twenty-four strains of carbapenems-resistant Klebsiella pneumoniae were detected,the positive rate of A beta-lactamase TEM-1 and SHV was 100％ (24/24),KPC-2 and LAP-2 was 95.8％ (23/24),45.8％ (11/24) respectively,and C beta-lactamase DHA was 4.2％ (1/24).Meanwhile,the positive detection rates of KPC-ISKpn6 linkage was 95.8％ (23/24),and the mutation rate of porin-coding genes ompK35 and ompK36 were up to 95.8％ (23/24) and 100％ (24/24).Conclusion High incidence of beta-lactamase TEM-1,SHV,KPC-2 and LAP-2 was found in the group of Klebsiella pneumoniae isolates,and carbapenems-resistant of which was primarily due to the high carrying rate of KPC-2 and the high mutation rate of porin-coding genes ompK35 and ompK36.The Insertion sequence ISKpn6 may be involved in the KPC-2 gene mediated-expression.

Objective: To investigate whether Pseudomonas aeruginosa recombinant protein PA3611 can induce the in vitro epithelial-mesenchymal transformation (EMT) of rat bronchial epithelial cells. Methods: A series of processes including DNA synthesis, gene amplification, vector construction, induction of expression and protein purification was used to synthesize the recombinant protein PA3611. CCK-8 kit was used to detect the proliferation of bronchial epithelial cells after stimulation with the protein PA3611. Morphological changes in bronchial epithelial cells were observed. Western blot and qPCR were performed to measure the expression of E-cadherin (E-CAD) and α-smooth muscle actin (α-SMA). T test was used for statistical analysis. Results: DNA sequencing verified the successful preparation of the recombinant protein PA3611. PA3611 inhibited the proliferation (P<0.05) and induced the EMT of bronchial epithelial cells. Moreover, it also inhibited the expression of E-CAD protein, but promoted the expression of α-SMA (P<0.05). Conclusion The recombinant protein PA3611 can induce the EMT of bronchial epithelial cells and the underlying mechanisms are worthy of further investigation.

The aim of this study was to explore the protective effects of geniposide against Influenza A(H1N1)pdm09 virus in vitro and in vivo. In vitro, geniposide was administered as a precaution drug, a direct deactivation drug or a treatment drug at different doses. Peramivir was applied as a positive control. The quantitative colorimetric MTT assay was applied to test both the cytotoxicity of geniposide on Madin-Darby Canine Kidney(MDCK)cells and the cytopathogenic effect(CPE)of geniposide on MDCK cells infected by influenza A(H1N1)virus. The viral inhibitory rate of geniposide on NT0901 was also calculated. In vivo, we presented a mouse model of influenza A(H1N1)pdm09 virus infection. Geniposide(5, 10, or 20 mg/kg)or peramivir(30mg/kg)were used as treatment procedures. Lung index and the survival rate were calculated to evaluate the therapeutic effects of geniposide or peramivir on NT0901-infected mice. Haematoxylin and eosin(H&E)stain was used to access the pathological alterations of lung tissues. The study in vitro demonstrated that the TD50(median toxic dose)of geniposide was higher than 1 040 μmol/L. Besides, the EC50(concentration for 50% of maximal effect)of geniposide administered for precaution, direct deactivation and therapy were 91. 90, 96. 25, 87. 68 μmol/L, respectively. These results suggested that geniposide could block the damage of NT0901 on MDCK cells in a dose-dependent manner. The results in vivo showed that geniposide could significantly alleviate the lung index elevation and inflammatory responses in lung tissues induced by NT0901, reduce the mortality of infected mice and extend their survival time. In conclusion, our investigation indicates that geniposide is highly effective in inhibiting cytopathogenic effect and acute lung injury caused by influenza A(H1N1)pdm09 virus. Geniposide may be a potential therapeutic agent for the suppression of influenza virus.

Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M . tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo .Methods The affinal exchange sites (AES) of the target gene was built ,and then integrated into the phage genomes of M .tuberculosis for harvesting the phagemids .The phagemids was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES .A high titer of the recombinant phages was harvested through amplification in vitro . The M .tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37℃ .Single clone was picked out and gene knock-out was confirmed by PCR . Then C57BL/6J mice were infected with either wild type strain (WT ) or knockout strain (KO ) of M . tuberculosis .Mice mortality ,lung tissue inflammation and colony-forming units (CFU ) counts in vitro were observed 6 to 8weeks post infection with different strains . Paired-samples t test was used for comparison between groups ,chi-square test was used for comparison of rates .Results The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene . The target fragment of Rv2346c was removed successfully and the mice were infected for 6-8 weeks .Themice infected with Rv2346c KO strain had reduced mortality (53％ vs 20％ ,χ2 =6 .1112 ,P<0 .05) ,lung tissue inflammation (1040 ± 89 vs 1960 ± 56 ,t=7 .1016 ,P<0 .05) and CFU count in vitro (15 .0 ± 0 .8 vs 90 .0 ± 1 .5 ,t=23 .0361 , P<0 .05) compared with WT strain 6-8 weeks post infection .Conclusion Rv2346c gene knockout strains of M . tuberculosis mediated by bacteriophageis are successfully constructed ,which establishes the foundation for the future gene function study of Rv2346c .