Objective To analyse the encoding gene sequence of extended spectrum ? lactamase produced by Klebsiella pneumoniae E95 strain in Zhejiang Province and identify its ESBLs subtype. Methods The gene of ESBLs produced by E95 strain was amplified by PCR. The purified PCR product was cloned into pGEM Teasy vector and then sequenced by Sanger's dideoxy chain termination composition method. Results The encoded gene of ESBLs was identified as SHV by PCR. Its PCR product had 812 nucleotides. It had the same gene sequence as the gene encoding SHV 12 discovered in Swiss. Conclusions The ESBLs produced by Klebsiella pneumoniae E95 strain isolated from a patient in Zhejiang Province is SHV 12.

Objective:To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss?), serving as a cell-homing approach for bone formation .Methods: In the study , 32 ICR mice were randomly divided into 4 groups,each group including 8 mice.The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: ( 1 ) 1 ∶50 ( volume ratio ) dimethyl sulfoxide ( DMSO ) /phosphate-buffered saline ( PBS ) solution +collagen scaffold ( blank control group ); ( 2 ) 10 -3 mol/L SIM solution +collagen scaffold ( SIM group ); ( 3 ) 200 mg/L mSDF-1solution +collagen scaffold (mSDF-1 group); and (4) 10 -3mol/L SIM +200 mg/L mSDF-1 solution +collagen scaffold ( SIM +mSDF-1 group) .One week after implantation , the mice were trea-ted by injecting the same drug solution mentioned above around the scaffold once a day for two days .The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis , HE staining and immunohistochemical staining .Angiogenesis of each group was checked by calculation of vessels in each tissue section .Results:Six weeks after implantation , the collagen scaffolds were retrieved.The value of gray scale for the SIM +mSDF-1 group[(421 836.5 ±65 425.7) pixels] was significantly higher than that of the blank control group [(153 345.6 ±45 222.2)pixels, P<0.01], the SIM group [(158 119.2 ±100 284.2) pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4) pixels, P <0.05 ]; HE staining analysis revealed that significant bone formation was achieved in the SIM +mSDF-1 group; The immunohistochemical staining showed the existence of os-teopontin and osteocalcin in the SIM +mSDF-1 group; There were more vessels in the SIM +mSDF-1 group[(46 ±8)vessels/mm2] than in the blank control group [(23 ±7) vessels/mm2, P<0.01], and the SIM group[(24 ±6) vessels/mm2 , P<0.01].Conclusion:The novel tissue-engineered bone com-posed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo.It re-presents a novel method of in vivo bone re-generation without seed cell delivery .

Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.

OBJECTIVE To identify the antibiotic resistance,homology and the carbapenemases determinants of imipenem-resistant strains of Acinetobacter baumannii isolated from elderly in the Zhejiang Hospital.METHODS All 142 strains of A.baumannii were isolated from Zhejiang Hospital through Jan 2005 to Jan 2007.K-B method was used to screen imipenem-resistant strains.The MICs of imipenem-resistant strains to 14 antimicrobial agents were determined by agar dilution method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding genes of carbapenamases and the gene environments were investigated by PCR,clone,and sequencing.RESULTS Ninety-seven strains of imipenem-resistant A.baumannii were isolated from 142 strains.All of the strains of carbapenem resistant A.baumannii belonged to 4 epidemic PFGE-clones.Ninety carbapenem resistant strains contained OXA-23-like carbapenemase gene and 91 isolates were positive for OXA-51-like gene. OXA-23-like gene of 86 strains was just on the down-stream of insert sequence ISAba1.OXA-51-like gene of 6 strains had an ISAba1 sequence just on the up-stream.CONCLUSIONS All imipenem-resistant strains of A.baumannii are pan-resistant isolates.Clone dissemination is the most important style of strains spread.No OXA-24-like,OXA-58like,IMP-like,and VIM-like gene are detected.OXA-23-like and OXA-51-like gene are the most popular carbapenemases coding genes of these strains in the Zhejiang Hospital.ISAba1 has close relationship with OXA type carbapenemases genes in Zhejiang Hospital.

Objective To investigate the prevalence of the genotypes of extended spectrum ? lactamses (ESBLs) in klebsiella pneumoniae and eschrichia coli, which were isolated in Hangzhou City from 2000 to 2001. Methods Total of 198 isolates of E. coil and K. pneumoniae were isolated from five hospitals in Hangzhou. ESBLs-producing isolates were detected by inhibitor-potentiated disc diffusion test, Agar dilution method was used to determine the minimum inhibitory concentrations (MIC) of cefotaxime and ceftazidime.ESBLs genes were analysed by isoelectric focusing (IEF) assay and PCR initially,genotypes were determined by sequencing. Results 51 isolates (25.76%) from 198 E. coil and K. pneumoniae strains were obtained. In ESBLs producers, the ESBLs types of SHV, TEM, CTX-M were 17.65%,84.31% and 80.39% respectively. genotypes were: SHV 11,12;TEM 1;CTX M 3and CTX M 3 like, CTX M 13,14. respectively. Conclusions CTX M type were the major genotypes of ESBLs in klebsiella pneumoniae and eschrichia coli isolated in Hangzhou city.

Objective To investigate the distribution of 16S rRNA methylase genes in Klebsiella pneumoniae strains producing Klebsiella pneumoniae ealbapenenase type 2(KPC-2).Methods A total of 37 Klebsiella pneumoniae isolates producing KPC-2 were collected.The minimal inhibitory concentrations (MICs)of these strains to amikacin,gentamyein and netilmicin were determinated by agal dilution method.Six 16S rRNA methylase genes(armA,rmtA,rmtB,rmtC,rmtD and npmA)were detected by PCR.Results The resistant rates to amikacin,gentamycin and netilmicin were 97.3%(MIC50≥1024μg/mL).Among those resistant strains,8 were armr/A positive,25 were rmtB positive,4 were both armA and rmtB positive.and no other 16S rRNA methylase genes were found.The total positive rate of 16S rRNA methylase genes was 78.4%(29/37).Conclusion 16S rRNA methylase genes armA and rmtB ale prevalent in Klebsiella pneumoniae strains producing KPC-2.

Objective To investigate the resistant mechanism of carbapenem-resistant Escherichia coli and its relationship with endogenous infection. Methods Two carbapenem-resistant Escherichia coli strains were isolated from blood and stool of a same patient, respectively. The minimal inhibition concentrations (MIC) of the two isolates against imipenem and meropenem were determined by E-test. The susceptibility against other antimicrobial agents were done by disc diffusion method. Isoelectric focusing electrophoresis (IEF), polymerase chain reaction (PCR) amplification,cloning and sequencing, conjugation, Southern blotting were carried out to analyze the encoding gene of β-lactamases. Homology analysis of the two strains was done by pulsed field gel electrophoresis (PFGE). Results MIC against imipenem and meropenem of the two strains were both≥32 mg/L.Both strains produced KPC-2 (pI 6.7) and SHV-12 (pI 8.2) β-lactamases. blaKPC2gene was located on a 54 kb transferable plasmid. PFGE showed that the two Escherichia coli strains were derived from the same clone. Conclusions The resistance and enzyme digestion map of chromosome DNA of the two Escherichia coli strains are coincident. The Escherichia coli septicemia of this patient is probably an endogenous infection caused by the immigration of Escherichia coli from the gut.

Objective To evaluate the usefulness of DiversiLab system for genotyping of Acinetobacter baumannii.Methods Fifty-eight non-duplicated clinical Acinetobacter baumannii isolated from 15 cities in China in 2005 were typed by rep-PCR-based DiversiLab system.The results were compared with those of PFGE and multilocus sequence typing. Simpson's index of diversity was used to compare the discriminatory power among DiversiLab system, PFGE and MLST.Results Fifty-eight Acinetobacter baumannii isolates were differentiated into 5 clusters and 25 unique types by DiversiLab system. MLST identified 35 distinct sequence types, which fell into one clone complex of CC22 and 35 singletons, while PFGE resolved 5 pulsotypes and 34 unique types.Simpson's diversity indices for DiversiLab system, MLST and PFGE were O.876, O.944 and 0.961, respectively.Conclusions The discriminatory power of DiversiLab system is lower than that of PFGE and MLST.But as a simple, fast and reproducible typing method, it could be used as a first-line typing tool for the analysis of a large number of isolates.

Objective To investigate the effect of resistance nodulation division (RND) efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing (MLST).The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration (MIC) of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP (N-methyl-2-pyrrolidone) for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transcript of adeABC was not found in control group strains A207 and A1731,and the expressions of adeABC,adeFGH and adeIJK were not up-regulated in other isolates.The single nucleotide polymorphism (SNP) was detected in adeR (E220K) and adeS (A130D),respectively.ISAab1 insertion sequence was identified in adeS of adeABC-over expressed isolates.No mutation was found in adeL.Conclusion High expression of adeABC pump may play an important role in tigecycline resistance in carbapenem-resistant Acinetobacter baumannii,mainly due to the insertion of ISAba1 sequence in its regulator gene adeS,but other mechanism of tigecycline resistance may not be excluded.

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.