Objective To explore the establishment of peptide mapping database of Candida albicans ,laying the foundation for rapid diagnosis of Candida albicans infection .Methods 96 Candida albicans were collected clinically ,and its DNA was extracted . Polymerase chain reaction(PCR) was used to amplify the ITS1-5 .8S-ITS2 gene fragments and restriction endonucleases were a-dopted to identify them .Surface enhanced laser desorption ionization-time of flight-mass spectrometry(SELDI-TOF-MS) instrument was applied to detect the Candida albicans peptide mapping ,and Ciphergen ProteinChip software was used to collect data automati-cally .The established peptide mapping database was verified by confirmed Candida .Results According to restriction fragment length polymorphism analysis ,96 strains were confirmed as Candida albicans .15 peptide peaks were captured by SELDI-TOF-MS chips .Five peptide peaks of them with stable expression were screened out ,and the similarity analysis software was used to estab-lish peptide mapping database of Candida albicans .More than 95% of similarity was found between peptide mapping of Candida albicans and established database ,while less than 50% was found between peptide mapping of other Candida species and database . Conclusion The establishment of peptide mapping database of Candida albicans provides a theoretical basis for the rapid diagnosis of Candida albicans infection .

Objective To establish protein fingerprints of common bacteria in clinics and to lay a foundation for rapid identification of bacteria.Methods Strains of Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa and Staphylococcus aureus were detected by surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS).Stable expression protein peaks were screened and the data was input into the self-constructed Fingerwave software for identification of target bacteria by protein fingerprint comparison.Two hundred and fifty-six clinical isolates,including E.coli,K.pneumoniae,P.aeruginosa and S.aureus were detected and the data was compared with constructed database to evaluate its diagnostic value.Results The protein fingerprints including four common bacteia was used to identify the target bacteria with identification rate of 93.1％ (54/58) for E.coli,87.2％ (75/86) for K.pneumoniae,96.2％ (60/63) for P.aeruginosa and 96.2％ (51/53) for S.aureus,respectively.Conclusion Common bacteria can be rapidly identified by using the protein fingerprint comparison,which provides a powerful tool for bacterial identification.

Objective To study the mutations of outer-membrane porin gerte (oprD) in imipenem-resistant Pseudomonas aeruginosa.Methods The PCR was applied to detect the oprD gene from the 34 clinical imipenem-resistant Pseudomonas aeruginosa.DNA sequence was proceeded to analysis the nuclentide sequence of the oprD gene and the deduced amino acid sequence.To analysis the mutation and the function of the oprD domain,those mutations were compaired with the standard Pseudomonas aeruginosa ATCC27853 and 2 clinical imipenem-susceptibility isolates.Results oprD gene mutation was wide and diverse.The rate of the mutation was 92.3% (12/13),mutations were concluded dot mutation,deletion mutation and insert mutation,those result in the amino sequence change and frame shift in L2 and L3 loops of outer membrane protein D,hampering the combine of oprD and imipenem.Some new mutations were found.They were 1 079,1 114,1 196,1 206,1 288,1 300,1 301 bases and 115,127,154,158,185,189 aminos.All above mutations were not deteced in ATCC 27853 and 2 clinical imipenem-susoeptibility isolates.Conclusions The wide and diverse mutations in oprD gene result in amino acid change and/or frame shift L2 and L3 loops,hampering the binding of IMP and oprD.Those may result in resistance to imipenem in Pseudomonas aeruginosa.

Objective To investigate the mechanism of one carbapenems resistant Raoultella planticola( R.planticola) isolate.Methods This is an experimental study.R.planticola was isolated from a patient′s drainage fluid from orthopedic department in November 2010 in Sichuan Provincial People′s Hospital.Minimum inhibitory concentration of R.planticola to 13 antibiotics was determined by using the agar dilution method.Modified Hodge test was used to detect carbapenemase .EDTA synergistic test was performed to research metallo-beta-lactamase.The genes coded the β-lactamase were amplified by polymerase chain reaction ( PCR ) , including class A carbapenemase ( KPC ) , class B carbapenemases (NDM, IMP, VIM, SIM), extended spectrum beta-lactamases[ESBL(CTX, TEM, SHV)], and AmpCβ-lactamases ( FOX, EBC, ACC, DHA, CIT, MOX).Results The susceptibility test showed that R.planticola was resistant to 9 antibiotics.MIC value of meropenem for R.planticola was up to 32 mg/L.R.planticola kept intermediary to imipenem , whereas it was susceptible to cefepime , amikacin and polymyxin B.Modified Hodge test and EDTA synergistic test were positive in R.planticola.Class B carbapenemase (IMP) gene and two extended spectrum β-lactamases(CTX, SHV) genes were positive by PCR.The genes were conformed as IMP-4, CTX-M3 and SHV-12 by sequencing and compared with GenBank.Other resistant genes were negative.Conclusion IMP-4 was identified in R.planticola, the combined produce IMP-4 and ESBLs might be the main mechanism of R.planticola resistant to carbapenems.

Objective To investigate the genetic location of SCCmec-associated psm-mec in Staphylococcus hominis isolated from blood culture,and to lay a foundation for further functional studies of psm-mec in Staphylococcus hominis.Methods 25 strains of Staphylococcus hominis isolated from positive blood culture were collected.mecA and psm-mec gene were amplified by PCR,and the SCCmec types were determined by the results of multiplex PCR assay.For analyzing the genetic location characteristic of psm-mec in SCCmec,three pair special PCR primers were used to measure mecR1/psm-mec,psm-mec/xylR and fudoh respectively.Results There were 21 strains of methicillin-resistant Staphylococcus hominis and 4 strains of methicillin-sensitive Staphylococcus hominis. The positive rate of psm-mec gene in methicillin-resistant Staphylococcus hominis was 47.6%,and no psm-mec gene was found in methicillin-sensitive Staphylococcus hominis.Among psm-mec positive strains,2 strains belonged to SCCmecⅢ,5 strains belonged to SCCmecⅢ-like,and 3 strains belonged to new SCCmec types.All of the 10 psm-mec positive strains were mecR1/psm-mec,psm-mec/xylR and fudoh gene positive.Conclusion SCCmec-associated psm-mec extensively exists in methicillin-resistant Staphylococ-cus hominis isolated from positive blood culture,which distributes mainly in typical SCCmecⅢ,SCCmecⅢ-like and new SCCmec types and locates between mecR1 and xylR gene.

Objective To construct mutant strains of methicillin resistant Staphylococcus epidermi-dis (MRSE) with psm-mec gene deletion and to investigate the function of psm-mec gene.Methods The drug sensitivity test and DNA sequence analysis were performed to screen out the tetracycline and chloram -phenicol sensitive clinical strains of MRSE , whose upstream and downstream sequences of psm-mec gene were identical to those of the Staphylococcus epidermidis reference strain RP62A.The recombinant plasmid pBT2-Δpsm-mec was constructed by using the fusion PCR and a temperature sensitive shuttle plasmid .After being identified , the plasmid was transformed into the Staphylococcus aureus RN4220 strain by electropora-tion, and then transformed into the selected clinical isolates of MRSE .The mutant strains of MRSE with psm-mec deletion were screened out and identified after homologous recombination .The differences in biofilm formation between the mutant and wild-type strains were analyzed for further elucidation the relationships be-tween the psm-mec gene and biofilm formation in MRSE strains .Results Three clinical MRSE isolates for the construction of mutant strains with psm-mec gene deletion were screened out and identified by using drug sensitivity test and sequence alignment analysis .The mutants constructed via homogenous recombination were screened out and identified .Compared with the corresponding wild-type strains, the three mutants with psm-mec gene deletion showed significantly decreased ability of biofilm formation , demonstrating that the psm-mec genes strains induced the biofilm formation of MRSE .Conclusion The Δpsm-mec mutant strains were successfully constructed .The psm-mec gene played an important role in the biofilm formation of Staphy-lococcus epidermdis.

Objective To investigate the SCCmec types of clinically isolated methicillin‐resistant Staphylococcus epidermidis (M RSE) .Methods Eighty‐four strains of clinically isolated Staphylococcus epidermidis identified by the fully automatic microbio‐logical identification system were collected and performed the MRSE identification by PCR for amplifying esp and mecA genes and SCCmec typing .Its distribution characteristics were analyzed .Results Esp gene was amplified in 84 strains and the detection rate of mecA was 76 .19% (64/84) ,in which the MRSE detection rates in blood ,sputum ,urine and wound secretion were 76 .8% , 68 .8% ,100% and 71 .4% respectively .The multiple PCR amplification displayed that among 64 strains of MRSE ,19 strains were SCCmec simple type ,in which 19 strains were SCCmec type Ⅰ and 3 strains were SCCmec type Ⅲ ;42 strains were SCCmec mixed type ,in which 2 strains were SCCmec mixed type Ⅰ and Ⅱ ,14 strains were SCCmec mixed type Ⅰ and Ⅲ ,12 strains were SCCmec mixed type Ⅰ ,Ⅱ and Ⅲ ,5 strains were SCCmec mixed type Ⅱ and Ⅲ ,a strains were and SCCmec mixed type Ⅲ and Ⅳ .Conclu‐sion The SCCmec type in clinically isolated MRSE shows obvious diversity and its majority is SCCmec mixed type .

Objective To establish protein fingerprinting identification model of Pseudomonas aeruginosa (P. aeruginosa) and to lay a foundation for rapid identification of P. aeruginosa by proteinchip golden array. Methods Sixty-four P. aeruginosa and one hundred and ninety-nine control bacteria identified in our laboratory were collected and divided into training and testing group. Surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and proteinchip golden array were used to detect the protein profiling of the bacteria. Data were automatically collected by Ciphergen Proteinchip Software and protein markers of P. aeruginosa were screened by BioMarker Wizard Software. Classification tree model was developed and validated by BioMarker Patterns Software. The model was blindly tested with twenty-nine P. aeruginosa and sixty-four control bacteria. Results Eighty protein peaks were detected between 3000 and 20 000, among which fifty-eight ones showed significantly difference between P. aeruginosa and the control bacteria (P＜0.01). By BioMarker Patterns Software, one protein peak ( M/Z at 14 045.2) was chosen to develop a classification tree model. The results exhibited with sensitivity of 96. 55% and specificity of 100%. Conclusion Proteinchip golden array has the potential for rapid identification of P. aeruginosa.

Objective To investigate the association of nitrotyrosine with coronary heart disease (CHD) in type 2 diabetes mellitus.Methods The nitrotyrosine levels were determined in 109 patients of type 2 diabetes mellitus without CHD (T2DM).One hundred and fifty-two patients of type 2 diabetes mellitus with CHD (T2DM-CHD) and 103 healthy control subjects by ELISA.Results T2DM-CHD patients had significantly increased nitrotyrosine compared with T2DM group and the control group [ ( 78.17±10.68 )nmol/L,(70.50 ± 9.13) nmol/L vs ( 63.23 ± 11.55 ) nmol/L,Ps ＜ 0.01 ].Nitrotyrosine was correlated with total cholesterol,triglyceride,fasting glucose and Gensini Score (r=0.361,P=0.009;r =0.206,P=0.001 ;r=0.347,P=0.026; r=0.466,P ＜ 0.001 ).Multivariable logistic regression showed nitrotyrosine was independently associated with CHD combined with type 2 diabetes mellitus ( OR=1.094,95％ CI:1.053-1.137 ; P ＜ 0.01 ).Conclusion Nitrotyrosine plays an important role in the formation and development of cardiovascular disease in tvoe 2 diabetes.

Objective To investigate the apoptosis of cultured normal human hepatocyte HL-7702 cells induced by glycodeoxycholate(GCDC)and to explore its possible mechanism.Methods HL-7702 cells were incubated with various concentrations(100,150,200 and 250 ?mol/L)of GCDC.The changes of cellular morphology were observed under optical microscope.The apoptosis rate of HL-7702 was determined by Annexin V-FITC/PI double staining.The changes of HL-7702 cell intracelluar \[Ca2+\]i were determined with Fluo-3/AM load technique.The mRNA expression levels of Bcl-2/Bax in HL-7702 cells were analyzed by RT-PCR.Results Typical apoptotic morphological changes were observed after HL-7702 cells had been treated with 150 ?mol/L GCDC for 24 h;HL-7702 cells could be induced to undergo apoptosis in a concentration-dependent manner after 100,150,200,and 250 ?mol/L GCDC treatment for 24 hours.The apoptosis rates were(13.16?2.9)%,(20.3?3.0)%,(25.02?2.1)% and(45.02?3.5)%,which were markedly higher(P