Objective To investigate the linezolid resistance mechanisms and molecular epidemiology of clinical isolates of methicillin-resistant coagulase-negative staphylococci (MRCoNS).Methods Seventeen MRCoNS,including 10 S.capitis,4 S.cohnii,2 S.haemolyticus,and 1 S.sciuri with various levels of linezolid resistance were isolated from intensive care units in our hospital from March to August 2011. Minimal inhibitory concentration (MIC) was determined by E-test method. Pulsed-field gel electrophoresis was performed to analyze the molecular epidemiology.PCRs and DNA sequencing were preformed to investigate the mechanisms of linezolid resistance in MRCoNS.Results Nine S.capitis with linezolid MIC of ＞256 μg/ml were indistinguishable,and another S.capitis with linezolid MIC of 4 μg/ml was closely related.Four S.cohnii with linezolid MIC of ＞256 μg/ml were belonged to the same clonal strain.MIC of linezolid for S.sciuri was 64 μg/ml,and were 4 μg/ml and 6 μg/ml for 2 S.haemolyticus,respectively.A commom G2576T mutation and a novel C2104T mutation were identified in 9 S.capitis with linezolid MIC of ＞256 μg/ml by DNA sequence analysis of domain V of the 23S rRNA gene.cfr gene was deteeted in all staphylococci except a S.sciuri whose 23S rRNA gene contained the G2576T mutation.Conclusion It is the first report of linezolid-resistant clinical isolates of staphylococci in China.Linezolid resistance in MRCoNS is related to the presence of DNA mutation in domain V of the 23S rRNA gene and cfr gene.It's a clonally dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital.

Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.

0.05). Tanreqing intervention treatment had better influence on the pneumodynamics and arterial blood gas than the therapentic influence on that in the control group, the functional improvement rate of lung and extra-pulmonary organs of study group obviously outstripped the rate of control group, and the incidence of VILI and VAP was also obviously decreased in the former group. The mortality in ICU for multiple organ failure (MOF) of Tanreqing intervention group was 16%, which was obviously lower than that (48%) of the control group (P

Objective The feasibility and effectiveness of bilingual teaching combined with CBL (case-based learning) was evaluated in our study. Methods From 2011 to 2012, 69 students from Shanghai Jiaotong University were randomly divided into Bilingual CBL group (36 students) and Chinese CBL group(33 students). A case of salvage of multiple trauma patients was selected as a text-book case and students' acceptance to textbooks and teachers was assessed. Besides, students' self-evaluation, teachers' evaluation of students, students' achievement in procedures and the final theory test scores were evaluated as the outcome of the assessment. Results Students' acceptance of textbook in Bilingual CBL group is lower than that in the Chinese CBL group(P=0.035). Differences in students' evaluation of teacher (P=0.093), students' self-evaluation (P=0.816), and teachers' evaluation of the student(P=0.812) were not statistically significant. Final written examination scores(P=0.100), operat-ing procedures in tracheal intubations (P=0.489), and cardiopulmonary resuscitation (P=0.764) were not statistically different . Only central venous puncture showed a statistical difference ( P=0 . 011 ) . Conclusions Medical bilingual CBL teaching is feasible, without affecting the original teaching of medical knowledge. Bilingual teaching can improve students' English proficiency and enhance their interest in learning.

Objective To evaluate the capability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF MS) for rapid identification of methicillin-resistant Staphylo-coccus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) strains.Methods Twenty-five MRSA and thirty MSSA isolates were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine as the experiment group .Twelve MRSA and twenty-two MSSA clinical strains were isola-ted as the control .All strains were identified by MALDI-TOF MS and the results were further analyzed by ClinProTools 3.0 software.Results Four algorithms including support vector machine (SVM), genetic al-gorithm ( GA) , supervised neural network ( SNN) and quick classifier ( QC) showed similar results in dis-tinguishing MRSA from MSSA isolates .The sensitivity of GA was 100 .0%and the sensitivities of other algo-rithms were all greater than 95.0%.The specificities of GA, SVM and QC were all greater than 90.0%. The areas under the receiver operating characteristic ( ROC) curves of four characteristic peaks at mass-to-charge ratios (m/z) of 3279, 6485, 6555 and 3299 m/z were all greater than 0.9.The virtual gel view showed that the bands generated by MSSA isolates at 3279 , 6485 and 6555 m/z were obviously deeper in color than those generated by MRSA isolates .However , the bands of MSSA isolates at 3299 m/z were appar-ently lighter in color than those of MRSA isolates .83.3%of MRSA and 90.0%of MSSA isolates from the control group were correctly identified by the GA model .Conclusion MALDI-TOF MS could rapidly and accurately identify MRSA from MSSA isolates under the strictly controlled experimental conditions with the advantages of less time-consuming, high sensitivity and high specificity .The accurate identification of MRSA from MSSA isolates could be applied for the prevention and treatment of MRSA infection .

Objective To investigate the related risk factors,clinical features and prognosis of hospital-acquired acute kidney injury (AKI) in intensive care unit (ICU).Methods We retrospectively analyzed 48 patients with both acute kidney injury and multiple organ dysfunction syndrome (MODS),who received renal replacement therapy from October 2006 to February 2011 in our ICU.According to whether the occurrence time of AKI was 48 hours after admission,they were divided into hospital-acquired AKI (HA-AKI) group and community-acquired AKI (CA-AKI) group,with 13 and 35 cases respectively.We compared the differences between these two groups in gender,age,acute physiology and chronic health evaluation Ⅲ (APACHE Ⅲ),primary diseases,days of mechanical ventilation,times of renal replacement therapy,number and indicators of organ failure,prognosis,renal function recovery,length of stay in ICU and hospital.Results The mean age of HA-AKI group is ( 64.5 ± 21.4) years,which is older than that in CA-AKI group ( 50.2 ± 17.5 ) years (P=0.022).The top three primary diseases in CA-AKI group are severe infection(42.8％ ),chronic kidney disease (CKD) concurrency of AKI ( 11.4％ ) and multiple trauma without head injury ( 8.6％ ).However severe infection still occupies the first in HA-AKI group ( 30.8％ ),followed by stroke (23.1％,P=0.024),multiple trauma with head injury( 15.4％,P=0.018 ) and gastrointestinal bleeding( 15.4％ ) ;Patients in HA-AKI group with more than four organ failures account for 84.6％,significantly higher than 65.7％ in CA-AKI group (P=0.000).On the first day,the levels of serum sodium ( P =0.036 ) and bicarbonate ( P=0.001 ) in HA-AKI group are higher than that in CA-AKI group,and the urinary volume is more(P =0.046).In HA-AKI group,the level of urea nitrogen on the seven day increases so progressively that it becomes significantly higher than that on the first day(P=0.015),but in CA-AKI group,there is no significant change in the levels of serum creatinine and urea nitrogen after AKI,while the levels of seruum sodium ( P=0.023 ) and bicarbonate ( P=0.030) increase significantly;APACHE Ⅲ score in HA-AKI group after admission 24 hours is significandy lower than that in CA-AKI group(53.2 ±22.8) point vs (89.1±25.7) point,P=0.000),and the length of stay in ICU and hospital and days of mechanical ventilation in HA-AKI group are significantly longer than that in CA-AKI group,but there are no significant differences in times of RRT therapy,prognosis and recovery of renal function.Conclusion APACHE Ⅲ score after 24 hours of admission does not accurately reflect the prognosis of patients with MODS and HA-AKI.There are great differences in age,primary diseases,organ function changes and other aspects of HA-AKI when compared with CA-AKI.

Objective To explore the effective evaluation methods of case-based learning (CBL) in critical disease teaching.Methods Totally 53 undergraduate students in department of clinical medicine of Shanghai Jiao Tong University,who practiced in our hospital from 2010 to 2012,were divided into the traditional teaching group and CBL teaching group.We used traditional teaching combining with CBL teaching in CBL group.Common cases of critical illness(severe acute pancreatitis and multiple trauma)in intensive care unit (ICU) were selected for CBL cases.The evaluation of theoretical knowledge,mini-clinical evaluation exercise(Mini-CEX)and direct observation of procedural skills (DOPS) were continued after teaching and practice.Results These was no significant difference in the theoretical knowledge examination between the two groups. In Mini-CEX,the students in CBL group were better concerning the medical interview capacity (P=0.000),humanistic care (P=0.013),clinical diagnosis(P=0.035),counseling(P=0.009) and the overall clinical competence (P=0.008) than those in traditional teaching group.The DOPS scores of endotracheal intubation (P=0.016)and central venous catheterization (P=0.001)discussed in CBL teaching were significantly higher in CBL group.Conclusion Traditional theoretical knowledge examination has little significance in the assessment of CBL teaching,but Mini-CEX and DOPS can reflect the advantages of CBL teaching better in the assessment of clinical abilities and skills.

Objective To obtain the near-infrared fluorescence image in vivo and find the relationship between the detecting depth and fluorescence probe concentration. Methods Signals of fluorescence probe in various depths of vivo and tissue phantom with cooled CCD camera were acquired. Results The fluorescence intensity informations with different fluorescence probe concentrations and depths in vivo and liquid phantom were obtained. Conclusion Relationship between fluorescence intensity,location and depth of detecting probe in vivo is found. The linear relation of fluorescence probe concentration and detecting intensity is simulated,which will be used as a reference for the experiment system.

Objective To compare the capability of ertapenem-hydrolyzing in various concentrations in KPC-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry( MALDI-TOF MS).Methods Nineteen KPC-producing Enterobacteriaceae including ten Proteus mirabilis isolates,three Enterobacter aerogenes isolates,two Serratia marcescens isolates,two Citrobacter freundii isolates,one Klebsiella pneumoniae isolate and one Enterobacter cloacae isolate were isolated from 2nd Affiliated Hospital of Zhejiang University; Seven KPC-producing Morganella morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital; Eleven Escherichia coli transconjugants produced the KPC-2 carbapenemase gene were conjugated from seven M.morganii and four P.mirabilis.MALDI-TOF MS was used to detect the ertapenem-hydrolyzing capability in different KPC-producing Enterobacteriaceae.Results When the concentration of ertapenem was 0.1 g/L,ertapenem can be hydrolyzed by KPC carbapenem within 1.5 h and the sensitivity was 100％,all the three peaks produced by ertapenem disappeared; when the concentration of ertapenem raised to 0.3 g/L,the sensitivity fell to 70.3％ (26/37) within 1.5 h,followed with 89.2％ ( 33/37 ) within 2.5 h and 94.6％ (35/37) within 3.5 h; when the concentration of ertapenem raised to 0.5 g/L,the sensitivity was 48.6％ (18/37) within 1.5 h,83.8％ (31/37) within 2.5 h,94.6％ (35/37 ) within 3.5 h.Two independent samples tests indicated that 0.1 g/L of ertapenem group has significant difference with 0.3 g/L and 0.5 g/L group,while there was no significant difference between 0.3 g/L group and 0.5 g/L group; As for the bacteria groups,there are no significant differences between the bacteria groups except the group of P.mirabilis and E.coli.Conclusion MALDI-TOF MS was easy to operate; it can rapidly detect KPC-producing Enterobacteriaceae with high sensitivity and a low false positive rate.0.1 g/L of ertapenem was recommended to detect KPC carbapenem rather than 0.3 g/L and 0.5 g/L; The detection time is short and can be widely applied clinically.

Objective To investigate the molecular types of carbapenem-non-susceptible Esche-richia coli ( E.coli) isolates and their mechanism of carbapenem resistance .Methods Twenty-two carbap-enem-non-susceptible E.coli strains were isolated from 3 hospitals in Hangzhou from 2007 to 2011.The mini-mum inhibitory concentrations ( MICs) of antimicrobials to those isolates were determined by agar dilution method and E-test.The molecular mechanisms of carbapenem resistance of E.coli isolates were analyzed by conjugation experiment,PCR and DNA sequencing.Pulsed-field gel electrophoresis (PFGE),multilocus se-quence typing ( MLST ) , and phylogenetic typing were performed to analyze the molecular epidemiology of those isolates.Results The MICs of imipenem and meropenem to 22 E.coli isolates were ranged from 1 μg/ml to 16 μg/ml,and the MICs of ertapenem were 2 μg/ml to 64 μg/ml.All E.coli isolates produced the KPC-2 carbapenemase and various β-lactamases , and some of them also produced plasmid-mediated AmpC enzymes.Carbapenem resistance was transferred by conjugation and transformation from 22 E.coli iso-lates to E.coli EC600 strains.The E.coli transconjugants or transformants acquired the blaKPC-2 gene and showed similar antibiotic susceptibility patterns in comparison with donor strains .Only a few isolates were in-distinguishable or closely related as indicated by PFGE .Four sequence types including ST131 (9 isolates), ST648 (5 isolates),ST38 (2 isolates) and ST405 (2 isolates) were identified by MLST.Phylogenetic analy-sis indicated that 9 ST131 isolates belonged to phylogenetic group B 2 and the other isolates belonged to group D (11 isolates),group B1 (1 isolate) and group A (1 isolate),respectively.Conclusion The sequence type of prevalent E.coli isolates producing KPC-2 from Hangzhou was ST131,which is an international epi-demic,multidrug-resistant clone,followed by ST648.