One hundred and forty-five mice were randomized into 4 groups:Group Ⅰ consisted of 25 mice with sham operation,group Ⅰ of 40 mice with hemisplenectomy,group Ⅲ of 55 mice with hemisplenic autotransplantation,and group Ⅳ of 25 mice with splenectomy.Twelve weeks after operation,it was found under optical microscopy,transmission electron microscopy and scanning electron microscopy that the autografted hemispleen was viable.The contents of alpha-naphthol acetylate esterase (ANAE) and acid phosphatase (ACP) in the splenic tissue were measured with microspectrophotometer.ACP level decreased continuously in group Ⅰ (0.462?0.013),group Ⅱ (0.304?0.009),and group Ⅲ(0.180?0.014) (P

Objective To investigate acquired carbapenemases and prevalence of carbapenem- resistant gram-negative bacill.Methods The antimicrobial susceptibility was determined by agar dilution method.Metallo-B-lactamase(MBLs)were screened by EDTA-disk synergy tesL The encoding genes of MBLs were amplified by PCR followed by sequencing.Strain homology Was investigated by pulsed-field gel electronphoresis(PFGE).Results In 141 carbapenem-resistant Pseudomonas aeruginosa(P.aeruginosa), there were three resistant patterns which were imipenem(IMP)-resistant+meropenem(MEM)-resistant (66.7%),IMP-resistant+MEM-sensitive(32.6%),and IMP-resistant+MEM-sensitive(0.7%).AⅡthe carbapenem-resistan Acinetobacter baumannii(A.baumannii),Acinetobacter lwoffi(A.lwoffi),Citrobactor freundii(C.freundii),Klebsielta pneumoniac(K.pneumoniac)and Serratia were resistant to imipenem and meropenem.Four strains of 141 P.aeruginosa were positive by EDTA-disk synergy test,and they produced VIM-2-type Metallo-B-1actamase.Of 34 carbapenem-resistan A.baumannii,30 strains produced OXA-tllrpe.Among them, OXA23, OXA24 and OXA66 accounted for 79.4%,38.2% and 67.6%,respectively.And 22 of 34 strains(64.7%)produced multiple OXA-carbapenemases.All 7 strains A.lwoffi produced OXA-23-type carbapenemases.A11 11 strains C.freundii,5 strains k pneumoniac and 1 strains Serratia produced KPC-2-type carbapencmases.And 6 of 11 strains C.freundii produced new subtype IMP-8.Of 15 PFGE type in 34 strains A.baumannii,14 strains belonged to A-type,7 strains belonged to B-type.Seven A.lwoffi strains distilbuted in difierent PFGE type.Four strains of P.aeruginosa producing VIM-2.type Metallo-8-lactamase did not have the same PFGE type.Eleven C.freundii strains had the same PFGE type.Five k pneumoniae strains had the sanle PFGE type.Conclusions Drug resistance to 12 common antibiotics in carbapenem-resistant gram-negative bacill was higher than non-carbapenem-resistant gram-negative.The former produced many kinds of carbaponemases and the strains prducing carbapenemases were prevalent in the C.freundii, A.baumanii, and k pneumoniae.

Objective To investigate the profile of antimicrobial susceptibility of the Mycoplasma pneumoniae (Mpn)strains isolated from pediatric patients with respiratory tract infection.Methods Antimicrobial susceptibility testing was conducted with a total of 112 Mpn clinical strains by broth microdilution method.Sequence analysis of full 23S rRNA genes was performed for all Mpn strains.Results One hundred and twelve Mpn strains were isolated from January 2009 to March 2011. Of these clinical isolates,98 (87.5%)were resistant to erythromycin and azithromycin.All macrolide-resistant Mpn strains harbored an A2063G or A2064G transition mutation in domain V of 23S rRNA genes.Mpn isolates were still very susceptible to the tetracyclines and fluoroquinolones tested.Conclusions The Mpn strains from pediatric patients are highly resistant to macrolides.The mechanism of macrolide resistance may be associated withthe transition mutation on 23S rRNA gene.

Objective To screen fosfomycin-resistant genes in the clinical isolates of Enterococcus faecium Efm-HS0661 and verify their functions. MethodsAntimicrobial susceptibility and conjugation experiments were carried out to determine if the antimicrobial resistance in clinical strain was transferable.By Solexa high-throughput sequencing,the genes conferring fosfomycin resistance were screened. The function of resistance gene was identified by cloning.ResultsThe clinical isolates of Enterococcus faecium Efm-HS0661 were resistant to glycopeptide antibiotics and fosfomycin, and the fosfomycin resistance was found to be transferred by conjugation. Within the 2414 bp nucleotide sequence obtained by high-throughput sequencing, fosB, a plasmid-mediated fosfomycin resistance gene was found. The fosB gene was 420 bp in length, which shared 99. 8％ amino acid identity with other fosB from Staphylococcus spp. The minimal inhibitory concentration (MIC) of DH5α transformant containing fosB gene against fosfomycin was higher than that of DHSa transformant without fosB gene. ConclusionsThe high-throughput sequencing can be used to screen unknown resistance genes in clinical isolates. The plasmidmediated resistance gene fosB can confer fosfomycin resistance in Enterococcus faecium.

Objective To learn the current in vitro antimicrobial susceptibility of Mycoplasma pneu-moniae in Shanghai and to understand the mechanisms of resistance to macrolides. Methods M. pneumoniae was isolated from pediatric patients with low respiratory tract infections(RTI) using broth and PPLO agar medi-um. PCR amplification and sequence analysis of P1 adhesion gene were performed to identify all M. pneumoniae strains. Susceptibility testing was carried out for macrolides, tetracyclines and fluoroquinolones using broth mi-crodilution method with SP4 broth. PCR amplification and sequence analysis of 23S rRNA genes were performed for all M. pneumoniae strains. P1 gene PCR-RFLP typing was performed to subtype the M. pneumoniae strains. Results One hundred and two M. pneumoniae strains were isolated in Shanghai from Oct 2005 to Dec 2008. All M. pneumoniae isolates were susceptible to the tetracyclines and fluoroquinolones tested. Of 102 clinical isolates, 83(81.4%) was resistant to erytbromycin and all 83 erythromycin-resistant strains had MIC>128 mg/L. An increasing trend of resistance rates were showed: 16.7% (1/6) in 2005, 76.5% (13/17) in 2006, 100.0% (24/24) in 2007 and 81.8% (45/55) in 2008. All macrolide-resistant M. pneumoniae strains harbored an A2063G transition mutation in domain V of 23S rRNA genes. The P1 gene RFLP type 1 is predominant (85.3%, 87/102) in M. pneumoniae clinical isolates. Conclusion The macrolide resistance rate of M. pneu-moniae is very high in Shanghai. The mechanism of macrolide resistance is associated with transition mutation on the 23S rRNA gene.

Objective To develop a method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation. Methods The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23s rRNA gene of Mycoplasma pneumoniae. By using recombinant plasmids containing 23s rRNA gene fragments, 102 Mycoplasma pneumoniae clinical isolates from 2005 to 2008, and 136 nasopharyngeal suction specimens from pediatric patients with low respiratory tract infections in Shanghai Children's Hospital from November to December in 2009 were investigated to determine the specificity and the sensitivity of the CycleavePCR method. PCR amplification and sequence analysis of 23S rRNA genes were performed for all Mycoplasma pneumoniae strains and Mycoplasma pneumoniae positive specimens to confirm the results of the CycleavePCR method. Results Of 102 clinical isolates, 83 was resistant to erythromycin and sequence results show that all macrolide-resistant Mycoplasma pneumoniae strains harbored an A2063G ( 82/83 ) or A2064G ( 1/83 ) transition mutation in 23S rRNA genes. Twelve was Mycoplasma pneumoniae detected positive by CycleavePCR in 136nasopharyngeal suction specimens. The CycleavePCR results were consistent with those of routine PCR and sequencing. There was no signal production from other bacterial species. Sensitivity and specificity were 100%. The detection limit of the CycleavePCR was 10 plasmid copies per reaction. Experiment can be done within 1.5 h. Conclusion A novel method is developed to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene using CycleavePCR.

Objective To investigate the mechanism of the different levels of ciprofloxacin resistance in qnrA-containing transconjugants.Methods E. coli J53AzR as the recipient,4 qnrA-containing transconiugants were constructed by conjugation from 4 qnrA-carrying clinical isolates.MICs of the transconjugants were measured by E test.aac(6')-Ib-cr was detected by PCR,and qnrA mRNA expression level was determined by real-time RT-PCR.The promoter sequences of qnrA were amplified by PCR from qnrA-bearing plasmids and cloned into plasmid pKK232-8,then transformed into HB101.All promoter fragments were sequenced.Resuits The MICs of ciprofloxacin against 4 transconjugants demonstrated a 10-fold difference from 0.094 μg/ml to 1.000 μg/m1.Of 4 qnrA-bearing plasmids in E.coli J53,ciprofloxacin MICs of pHS4 and pHS5 were 0.094 μg/ml and 0.125 μg/ml,respectively;pHS3,which contained the aac(6')-Ib-cr gene as well,MIC was 0.25μg/ml;and pHS5,which had a high expression level of qnrA and the aac(6')-Ib-cr gene,MIC was 1.00μg/ml.The relative expression levels of qnrA mRNA in J53 pHS6 was 32.5,much higher than the other 3 transconjugants(from 1.0 to 2.5).The promoter in plasmid pHS6 was 12-fold stronger than that in the other 3 plasmids.Compared with pHS3,there was 7 bp(GTTAGCA)deletion between the transcription initiation site and the start of qnrA in pHS6.Conclusion Co-existence of qnrA and aac(6')-Ib-cr in a single plasmid and high level of qnrA expression can account for the different levels of ciprofloxacin resistance in transconjugants.

Objective Through the exploration of virtual simulation surgery to find a way to treat lumbar spinal metastases. Methods Based on 64 row spiral CT continuous 2-dimensional images of lumbar segments, normal lumbar vertebral, destruction of disease, abdominal aorta and kidneys were reconstructed by the Mimics software. 3D visualization structure was contemplated by anterior lesions clear, titanium mesh of bone cement support, and posterior pedicle screw fixation. Results The three-dimensional reconstruction distinctly displayed the structures of lumbar and its adjacent organs, and the entire virtual simulation surgery was intuitive. Conclusion The application of virtual simulation surgery ensures more accurate 3D model of lumbar establishment and its adjacent organs, and it provides an objective basis for in-dividualized treatment programs.

OBJECTIVE:To observe the activity of Pyrazinamide gel against Mycob ac terium tuberculosis in vitro and its security in bronchial interventional therap y METHODS:The MIC and MBC of Pyrazinamide and Pyrazinamide gel were measured b y handwork method and instrument method and secuity of Pyrazinamide gel was asse ssed by bronchial interventional therapy in rabbits RESULTS:The MICs of pyrazi namide gel to M tuberculosis H37 RV,M bovis and M phlei were 1mg/L,1mg/L,1 0mg/L,the MBCs of Pyrazinamide gel to M tuberculosis H37RV,M bovis and M ph lei were 10mg/L,10mg/L,40mg/L respectively;the MIC and MBC of Pyrazinamide gel and those of Pyrazinamide had no significant differences;the animal security ex periment was negative CONCLUSION:These results suggest that Pyrazinamide gel a nd Pyrazinamide have the same efficacy against M tuberculosis,because carbomer dose not affect the activity of Pyrazinamide against M tuberculosis;Pyrazinami de gel which contains carbomer is safe in bronchial interventional therapy

Objective To investigate the resistance profile of Klebsiella pneumoniae isolates in Huashan Hospital, Fudan University. Methods The MICs of fluoroquinolones were determined by agar dilution method against 112 clinical strains of K. pneumoniae. Multilocus sequence typing (MLST) were applied to 48 K. pneumoniae strains. The characteristic sequence type (ST) associated with antibiotic resistance was identified by PCR. Results Lower percentage (＜40％) of K. pneumoniae strains were susceptible to fluoroquinolones. Majority (86.2％) of ciprofloxacin non-susceptible K. pneumoniae strains belonged to CC1 (ST11), ST494 or CC4 (ST15 and ST655), indicating the potential of clonal dissemination. ST494 (18.8％) was the second commonest sequence type, next only to ST11. ST494 strains harbored the genes encoding beta-lactamases, oqxAB, qnrD, aac-(6')-lb-cr and armA and had a single point mutation in gyrA. Therefore, ST494 strains were highly resistant to cephalosporins, fluoroquinolones and aminoglycosides and 22％ of the strains were resistant to carbapenems. However, all the ST494 strains were susceptible to tigecycline and tetracycline. Conclusions ST11 and ST494 are the commonest STs of K. pneumoniae conferring multidrug resistance in this hospital. These STs may contribute to the high resistance rates of K. pneumoniae to fluoroquinolones. The susceptibility of ST494 strains to tigecycline and tetracycline allows us to consider the promising potential of such drugs in managing K. pneumoniae infections.