Objective To determine the ESBLs of Klebsiella pneumoniae and ESBL typing by molecular genetic procedures. Methods MIC test and ESBLs confirmation test were taken by agar dilution method in 80 strains of Klebsiella pneumoniae isolated in Beijing area from July 1999 to December 1999. To the 20 ESBLs positive conjugates isoelectric focusing was given. And nucleotide sequencing was analysed to conjugate strain CK23. Results 28 (35%) of the 80 strains of Klebsiella pneumoniae produced ESBLs. In the 28 strains, 20 were successfully done with conjugation of resistant plasmid. Isoelectric focusing results revealed that 13 (65%) of the 20 strains produced an ESBLs protein with a pI of 8.8 with or without additional pI 7.6 and pI 5.4. These ? lactamases were all inhibited by clavulanite acid. The strains with pI=8.8 protein were highly resistant to cefotaxime and ceftriaxone but were susceptible or intermediate to ceftazidime. We picked CK23 strain out from 13 conjugates for gene cloning by the primers designed for bla CTX M and nucleotide sequencing. The results showed that the ESBLs gene in CK23 was CTX M, highly similar to CTX M 3 but had 3 amino acids, which were Glu39Gly, Leu122Pro, Asp278His. Conclusions 28 (35%) of the 80 strains of Klebsiella pneumoniae produced ESBLs. Resistant plasmid successfully conjugated in 20 of the 28 ESBLs producing strains. 13 of the 20 (65%) conjugates pruduced a pI =8.8 ESBLs,which was CTX M ESBLs,with only 3 amino acids different from CTX M 3. This paper indicated that a type of CTX M ESBLs existed in Klebsiella pneumoniae in Beijing, which specifically confers resistance to cefotaxime.

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Objective To investigate the prevalence and awareness rate of adult diabetes mellitus and the risk factors in Liaoning Anshan. Methods A cross-sectional population survey was performed among residents in Liaoning Anshan and 2 500 subjects participated. Subjects whose fasting plasma glucose ≥ 5.6 mmol/L were confirmed by oral glucose tolerance test (OGTT). Results The standardized prevalence of adult diabetes mellitus over the age of 20 in Liaoning Anshan was 8.2%, and the standardized prevalence of pre-diabetes mellitus was 14.8%. The prevalence of diabetes mellitus increased with age (P<0.05). The awareness rate of diabetes mellitus in Liaoning Anshan was 52.3%(125/239), and the awareness rate of pre-diabetes mellitus was 8.7%(35/402). Logistic regression indicated that age, family history of diabetes, overweight or obesity, waist circumference, hypertriacylglycerolemia was the risk factors (P<0.05), while regular exercise was protective factor (P<0.05). Conclusions The current prevalence of diabetes mellitus and pre-diabetes mellitus in Liaoning Anshan is higher, and the awareness rate is lower.

Objective To analyse the necessity of colposcopic directed biopsy to vaginal intraepithelial neoplasia (VAIN) before hysterectomy due to early stage cervical cancer (stage Ⅰ) or high grade cervical intraepithelial neoplasia (CIN). Methods A total of 669 patients who underwent a hysterectomy due to early stage cervical cancer (stage Ⅰ) and CINⅢin Beijing Obstetrics and Gynecology Hospital, Capital Medical University,from January 1, 2009 to December 31, 2013 and followed up, 99 patients with VAIN were enrolled. The clinical data and following up the prognosis were prospectively analyzed retrospectively. Results The occurrence rate of VAIN before and after hysterectomy due to cervical dysplasia was 14.8%(99/669), the occurrence rate and the grade of VAIN showed that significantly increased from CINⅢto cervical cancer stageⅠ(P<0.05);Only 15 patients enrolled had undergone vaginal wall biopsy by colposcopy pre-hysterectomy, including 11 patients who were diagnosed with VAINⅡ-Ⅲand underwent vagina extended resection during the hysterectomy. The 5 year recurrence rate of vaginal stump VAIN after hysterectomy was 12.1%(12/99) and the progression rate was 4.0%(4/99), the recurrent rate was 2.0%(2/99). Conclusions For all the patients who are planning to undergo hysterectomy due to stageⅠcervical cancer and CINⅢ, routine upper side of the vagina wall colposcopic-directed biopsy pre-hysterectomy is strongly recommended. All the patients after hysterectomy due to cervical dysplasia should be followed up regularly within 3 years after hysterectomy.

Objective To investigate the antimicrobial resistance mechanisms and genetic homogeny of Salmonella from community acquired infections in Shenzhen,China.Methods Ninety-three of Salmonella were isolated from 2002 to 2007 at Shenzhen People's Hospital,China.PCR and DNA sequencing were used to investigate the mutation in QRDR of the gyrA,gyrB,parC and parE.Plasmid mediated quinolone resistance genes including qnr and aac(6')-Ib-cr,β-lactamase genes including blaTEM,blaSHV,blaOXA, blaCTX-M, and class 1 integron were detected. All isolates were typed by PFGE. Results S. enterica typhi and S. enterica paratyphi A were susceptible to ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, ceftriaxone and ciprofloxacin, with the susceptible rate of 96%-100%. Fifty-two percent (13/25) of S. enterica typhi and 95% (61/64) of S. enterica paratyphi A were resistant to nalidixic acid. Twenty-four percent (6/25) of nalidixic acid-resistant S. enterica typhi and 94% (60/64) of nalidixic acid-resistant S. enterica paratyphi A showed decreased susceptibility to ciprofloxacin (MIC of 0. 125-1 μg/ml).All nalidixic acid-resistant (susceptible to ciprofloxacin ) Salmonella (NARS) isolates had a single substitution in the QRDR of GyrA, and 91% (68/75) of these isolates carried the substitution Ser83Phe in GyrA. Two mutations in the QRDR of GyrA were detected in both of two ciprnfloxacin-resistant Salmonella,with the additional one mutation in the QRDR of parC. Plasmid mediated quinolone resistance genes including qnr and aac(6')-lb-cr were not detected in any isolate. The blaCTX-M-14 gene was detected in a ceftriaxoneresistant isolate of S. enterica paratyphi A, with ISEcpl located on the upstream of it. Three muhidrugresistant strains of Salmonella all carried one 1 900 bp classⅠ integron gene cassette dhfrⅫ-orfF-aadA2,with the additional one β-lactamase gene of blaTEM-1, or blaOXA-30. Twenty-two distinct PFGE patterns were observed among twenty-five S. enterica typhi. The PFGE patterns of sixty-four S. enterica paratyphi A showed limited genetic diversity (average similarity of 91% ). Ninety investigated inpatients were infected in the community. Six patients infected by S. enterica paratyphi A had a travel history before infection. Conclusions Nalidixic acid-resistant S. enterica typhi and S. enterica paratyphi A are highly prevalent in Shenzhen,China. The mutation in the QRDR of GyrA is the prevalent mechanism responsible for the resistance to nalidixic acid in Slmonella. The great genetic similarity among S. enterica paratyphi A isolates indicates endemic disease from the presence of a single clone over 6-year period.

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Objective To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. Methods After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C.parvum gene (L16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex-100 method was also tested by PCR. Results One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. Conclusion The three kinds of extraction can all be served as templates for PCR detection of C.parvum oocysts, while Chelex-100 method is simpler, quicker and more reliable for DNA extraction of the parasite.

Objective To investigate the protective immunity of the recombinant thioredoxin of Schistosoma japonicum(reSjcTrx)in mice. Methods Thirty 6-week old female C57BL/6 mice were randomly divided into 3 groups with 10 each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control, and infection control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. The mice in adjuvant group was injected three times with Montanide ISA720 and saline only. Mice in infection control group were given no injection. Three weeks after final injection, each mouse was challenged with 30?1 cercariae of S. japonicum (Chinese strain). At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from liver tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before killing. The anti-SjcTrx antibodies in sera were detected with ELISA. Results ELISA showed a high level of specific IgG antibodies in mice immunized with the reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5% respectively, significantly higher than those of the control groups (P﹤0.05). SDS-PAGE and Western blotting revealed that the molecular weight of expressed protein was around Mr 14 000 and could be recognized by sera from rabbit infected with S.japonicum and from mice immunized with reSjcTrx. Conclusion The reSjcTrx induces certain protective immunity against schistosomiasis japonica in mice.

Objective To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. Methods Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheather sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. Results The results suggested that the size of oocysts was (7.4?0.32)?m by(5.4?0.21)?m and the ratio of length and width was 1.37?0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of C.andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C.andersoni clade based on the sequence of SSU rRNA and COWP gene. Conclusion What isolated from naturally infected cow feces has been identified as C. andersoni.