OBJECTIVE:To construct the homologous recombination plasmid of sigB gene from staphylococcus aureus so as to provide the basis for further study of the formation mechanism of its biofilm.METHODS:The target fragment“upstream sequence of sigB,kana-resistance gene,and downstream sequence of sigB”were amplified by PCR from staphylococcus aureus DNA and pEGFP-N2plasmid,respectively,in which the terminals of3interlinked fragments were complementary,and re-stricted enzyme EcoRⅠand XhoⅠsite existed in the sequence terminal,respectively.The fragments were ligated with fusion PCR in turn.Then target fragment was recombinated into plasmid pBluescriptⅡSK(+)according to the instruction manual of commercial kits.After both recombination of pBluescriptⅡSK(+)and vector plasmid pGEM-7zf(+),restricted enzyme SacⅠand XhoⅠ,the homologous recombination plasmid pGEM-7zf(+)-sigB was finally made via T 4 ligation enzyme.RE-SULTS:The target fragments were amplified by PCR and were confirmed with the agarose gel electrophoresis and sequencing.CONCLUSION:The homologous recombination plasmid of sigB gene from staphylococcus aureus is constructed successfully.

Objective To investigate the correlation of biofilm forming ability with biofilm associated gene of the clinically isolated Staphylococcus aureus for providing basis for the further studies of the mechanisms of biofilm formation.Methods Safranine staining was conducted for the detection of the forming ability of biofilm in 96-well plates.icaAD,icaBC,sar,agr and sigB was amplified by PCR.Results Formation of biofilm could be found macroscopically in 17 out of 27 strains,especially to X387 and X409.icaAD and icaBC were amplified in 22 isolates and sar,agr and sigB in all 27 S.aureus strains.Conclusion ica operon is the key gene for biofilm formation but cooperative action of other genes is needed during the process of biofilm formation.

Objective To study the macrolide-resistance,ability of biofilm formation and icaA-genetypes of 68 stains of clinically isolated Staphylococcus epidermidis(S.epidermidis) so as to explore the efficacy of macrolide to prevent biofilm-associated infections caused by S.epidermidis.Methods Minimum inhibitory concentration(MIC) were detected by agar-plate dilution method,and microtiter-plate assay was used to investigate the ability of biofilm formation,and icaA genetype was identified by PCR.Results High macrolide-resistant rate(88.2%) was showed for clinical isolated S.epidermidis,and biofilms for macrolide-resistant strains were significantly stronger than that of macrolide-sensitive strains,but there was no dependability found between these two for icaA positive rate.Conclusion Frequently-used macrolide such as erythromycin,azithromycin,and clarithromycin may incompetence to prevent biofilm-associated infections caused by S.epidermidis.

Objective To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of antisense RNA. Methods Two parts of HBV genome were reversedly recombined back into overlength HBV genome, which can produce HBV particle, to express antisense RNA complementary to S or S promoter region respectively. HepG 2.2.15 cell lines were transfected with these constructs and the empty vector pMEP4, then positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Results The mean inhibitory rates of HBsAg were (2.74?3.83)%、(66.54?4.45)%(P

OBJECTIVE:To investigate synergistic effect of carbapenems combined with fosfomycin(FOS)on carbapenems-re-sistant Pseudomonas aeruginosa isolates from urinary tract infections in vitro. METHODS:The minimum inhibitory concentration was detected using agar double dilution method. The fractional inhibitory concentration index was determined by checkerboard meth-od. The effect of carbapenems combined with FOS on biofilm of P. aeruginosa isolates was determined using 96 crystal violet stain-ing. RESULTS:12 strains of carbapenem-resistant P. aeruginosa isolates were highly sensitive to FOS and amikacin,and were com-pletely resistant to imipenem and meropenem. The combination of imipenem with FOS could induce a synergistic effect on 4 strains (33.3%);meropenem combined with FOS could induce a synergistic effect on 5 strains(41.7%);no antagonistic effect of carbap-enems combined with FOS appeared. FOS combined with carbapenems could inhibit the biofilm of carbapenems-resistant P. aerugi-nosa(P<0.05 or P<0.01). CONCLUSIONS:The combination of carbapenems with FOS possesses in vitro synergistic antibacteri-al effect on part of carbapenems-resistant P. aeruginosa isolates,the mechanism of which may be associated with inhibiting the bio-film.

Objective To study the biofilm formation ability and related gene distribution of Staphylococcus (S .) aureus iso‐lated from urinary tract infections to provide the theoretical basis for the prevention and treatment of clinical infection .Methods The minimal inhibitory concentration was detected using the agar double dilution method .The bacterial adhesion ability was deter‐mined by flat colony counting method .The biofilm formation ability was analyzed by the 96‐well crystal violet staining method .The biofilm‐associated genes were detected by PCR amplification .Results Eleven clinical strains of S .aureus were high resistant to pen‐icillin and erythromycin ,whereas were all sensitive to vancomycin and nitrofurantoin .All the isolates had a strong ability of adhe‐sion ,but the biofilm formation ability was weak .Among them ,the icaAD and icaBC genes were amplified in 10 S .aureus isolates . Conclusion The adhesion ability and biofilm formation ability of S .aureus isolated from urinary tract infections have the strain differences ,and ica is an important gene of S .aureus biofilm formation .

Objective To analyze the distribution and drug resistance situation of pathogens in the respiratory department during the recent 9 years to provide the basis for rational use of antibacterial drugs in clinic .Methods All pathogens isolated from the respiratory depeartment from January 2003 to December 2011 and the drug susceptibility test results were retrospectively and statistically analyzed .Results A total of 5 714 strains of pathogenic bacteria were isolated ,which mainly distributed in the sputum (90 .1% ) ,excrement (4 .2% ) and urine (3 .6% );among them ,2 943 strains (51 .5% ) were Gram‐negative bacteria ,596 strains (10 .4% ) were Gram‐positive bacteria and 2 175 strains (38 .1% ) were fungi .The top six of isolated bacteria were Candida albi‐cans ,Pseudomonas aeruginosa ,Acinetobacter baumannii ,Klebsiella pneumoniae ,Candida tropicalis and Escherichia coli .The isola‐tion rates of A .baumannii and C .albicans were increased year by year ,while the isolation rate of E .coli was decreased .A .baumannii and P .aeruginosa had a high resistant to all antibacterial drugs ,whereas the resistant rate of A .baumannii was increased year by year and that of P .aeruginosa showed some fluctuation .K .pneumoniae had a high susceptibility to imipenem and meropenem ,and the sensitivity to other antimicrobial agents had a gradually increasing tendency .The sensitive rate of C .albicans to amphotericin B was almost 100% ,and they had a high susceptible to other antifungal agents .Conclusion Drug resistance of the pathogens is com‐mon in the respiratory department .It is of importance to emphasize the pathologic examination ,carry out the surveillance of drug re‐sistance of pathogenic bacteria ,and use the antibacterial drugs rationally in clinical anti‐infective therapy .

Objective:To investigate the changes and the adjusting effects of brain-gut peptides (BGP) on immunologic function in the rats before and after relief of biliary obstruction.Methods:One hundred Wistar rats were randomly divided into five groups,including sham common bile duct ligation group (group A),common bile duct ligation group (group B),sham relief of biliary obstruction + normal saline group (group C) ,relief of biliary obstruction + normal saline group (group D) and relief of biliary obstruction + Sandostatin (group E).At 1d,7d after the operation of each group,the changes of plasma vasoactive intestional peptide (VIP),substance P(SP),serum interleukin-2(IL-2) and serum T-lymphocytic subsets (CD4 +,CD8 +)were determinded by radioimmunoassay and flowcytometry .Results:Plasma VIP and SP were increased in group B compared with group A(P

OBJECTIVE:To investigate the effect of subinhibitory concentration of erythromycin on adhesion of clinical isolated Staphylococcus(S.) epidermidis.METHODS:The subinhibitory concentration of erythromycin was determined based on the susceptibility test,and the representative strain Se.015 was treated with subinhibitory concentration of erythromycin.The optical density value determined by microtiter-plate assay was used to evaluate the effect of erythromycin on the adhesion of the representative strain Se.015,and electron microcopy was employed to observe the adhesion of Se.015 in samples with blank solvent served as control.RESULTS:Compared with control group,erythromycin(4 mg?L-1) group showed significantly higher optical density value(P

Theory of treatment from spleen is being widely applied in in the process of Chinese medical therapies and had already shown its clinical efficacy. The purpose of this paper was to explain this theory in the fields of the theoretical contents, physiology, pathology and clinical practices.