Objective To investigate the pathogen distribution and susceptibility profile of fungal isolates from bloodstream infections,and valuate the clinical utility of G test in diagnosis of fungal infections for the purpose to improve antifungal therapy.Methods A retrospective analysis was carried out to analyze the fungal pathogens isolated from bloodstream infections in the First Affiliated Hospital of Nanjing Medical University during the period from January 2013 through December 2015 and their antimicrobial susceptibility.Results A total of 114 fungal strains were isolated from bloodstream infections during the 3-year period,most of which were Candida (99/114,86.8％),especially Candida albicans (30.7％).About 41.2％ (47/114) of the fungal strains were isolated from Department of Thoracic Surgery (10,5 and 4 strains in 2013,2014 and 2015),Hematology (11 strains in 2014),and ICU (7 strains in 2014).Antimicrobial susceptibility testing showed that all the fungal strains (100％) were susceptible to amphotericin B,but 83.5％ susceptible to itraconazole (the lowest).G test was positive before the result of blood culture in 13 of the 54 patients who received G test.Conclusions Candida was the most common fungus in fungal bloodstream infection.Amphotericin B is the most active antifungal agent in vitro.Blood culture combined with serological test can provide clinicians an earlier and reliable diagnosis.

Objective To investigate the effects of betaine on the formation and dispersion of biofilm of Pseudomonas aeruginosa and its drug-resistance.Methods A total of 20 strains of Pseudomonas aeruginosa were obtained from clinical inpatients.The biofilm formation abilities of the Pseudomonas aeruginosa were evaluated by violet staining,and the effects of betaine on the formation and dispersion of biofilm were studied.The minimum inhibitory concentration (MIC) values of Pseudomonas aeruginosa on ciprofloxacin were compared with the controls when biofilm was formed and inhibited.Results Biofilm was formed in all the 20 strains of Pseudomonas aeruginosa in 24 hours with absorbance (A590 nm) (1.90 ± 0.66).Betaine significantly inhibited biofilm formation of Pseudomonasaeruginosa in 24 hours compared with control group(t =4.36,P ＜ 0.01) and the maximum inhibition reached in 48 hours with absorbance(A590 nm) (1.12 ±0.60).The maximum dispersion of betaine on mature biofilm of Pseudomonas aeruginosa reached in 24 hours.The MIC range of ciprofloxacin to the 20 strains of Pseudomonas aeruginosa was 0.03 to 4 μg/mL with 0.25 μg/mL of MIC50 and 2 μg/mL of MIC90.After the biofilm was inhibited by belaine,the MIC of ciprofloxacin to Pseudomonas aeruginosa did not changed.The MIC of ciprofloxacin to biofilm-formed Pseudomonas aeruginosa was more than 16 μg/mL.Conclusion Betaine could effectively inhibit the formation of biofilm and disperse the mature biofilm of Pseudomonas aeruginosa,which may provide more choices for the treatment of clinical infection.The germicidal efficacy of ciprofloxacin has no changed on the biofilm-formed bacteria when inhibition of betaine was involved.

OBJECTIVETo verify the feasibility and safety of the vascular interventional vascular interventional surgical robot system applied to vascular interventional operation.

METHODSFrom March to September 2013, 10 patients had undergone robot-assisted cerebral angiography. There were 6 male and 4 female patients; aged from 19 to 58 years, with an average age of 38.4 years. The operation were carried out by neurosurgeons and vascular interventional robot. After successfully implanted of femoral artery sheath by hand, the catheter was fixed on the robot, under the guidance of navigation image the surgeon manipulate the master part and control the slave part of robot by sending command through network transmission, finally finished the whole cerebral angiography. The operation time was recorded from placing the sheath into femoral artery to finishing cerebrovascular selective angiography, simultaneously the time of staff under exposure of X ray was recorded, and the position difference between the setted targets and the actual position(positioning accuracy).

RESULTSIt took 25-41 minutes to finish the cerebral angiography, the average time was (31 ± 5) minutes, and the robot-assisted angiography went quickly and smoothly without surgical complications. The remote positioning accuracy was (1.03 ± 0.23) mm. The time of staff under exposure of X ray was 0 minute, the entire experimental process was basically implemented mechanization and automation.

CONCLUSIONThis system basically achieves initial medical purposes, such as reducing the radiation, facilitating interventional procedures on the basis of enhancing the image navigation, shorting the operation time, and improve the quality of operation.

Objective:This study aimed to investigate the possible mechanism of 32P colloid induced apoptosis of craniopharyngi-oma (CP) cells in vitro and the relationship between dose effect and time effect. Methods:This study established a primary cell culture of CP limited subculture cell line. Methyl thiazolyl tetrazolium (MTT) assay was performed to plot the cell survival curve after the CP cells were treated with 32P colloid at different concentrations and time. Apoptotic rate was detected by flow cytometry(FCM). Apoptosis related DNA was investigated by TUNEL fluorescent staining. The morphological characteristics of apoptotic cells were determined by Hoechst33342 fluorescence staining. The ultrastructure of apoptotic cells was investigated by transmission electron microscopy (TEM). Results:Hoechst33342 fluorescence staining, TUNEL fluorescence staining, and TEM revealed that 32P colloid induced the apoptosis of CP cells. 32P colloid reduced the survival rate and increased the apoptotic rate of CP cells as concentration (0 MBq/mL to 14.80 MBq/mL) and time (1 d to 14 d) were increased. Conclusion: 32P colloid could effectively inhibit the growth of CP cells and induce apoptosis in vitro. High concentrations and prolonged time could induce a remarkable effect.

Objective To investigate the bacterial resistance in nationwide and understand the distribution of bacterial and resistance trend.MethodsThe 6507 clinical isolates were collected from 19 hospitals in 17 cities.The susceptibility tests were performed using agar dilution method recommended by Clinical and Laboratory Standards Institute (CLSI) in central laboratory.The values of MIC50,MIC90 and MICrange were calculated by SPSS 17.0 and the susceptibilities of isolates to antimicrobial agents were determined by using CLSI (2010) guideline.Of all 6507 isolates,4691 strains were collected from target wards and 1816 were isolated from others wards.ResultsAmong 4691 strains,1156 were Gram-positive (24.6％ ) and 3535 were Gram-negative (75.4％).Based on the minimum inhibitory concentration results,the prevalence of methicillin resistant Stapylococcus aureus and methicillin resistant Stapylococcus epidermidis are 51.6％ ( 325/630 ) and 87.0％ ( 228/262 ) respectively.Staphylococci showing intermediate or full resistance to vancomycin were not observed. Coagulase negative Staphylococci showed 2.5％ (16/642)intermediate rate and 1.6％ ( 10/642 ) full resistance rate to teicoplanin,and showed 0.5％ ( 3/642 )resistance rate to linezolid.Antibiotic resistance rate of Enterococcus faecalis to ampicillin was 17.1％(19/111),while the resistance rate of Enterococcus faecium to ampicillin reached up to 85.0％(164/193).Three Enterococcus faecium were resistant to glycopeptides.The prevalence of penicillin resistance Streptococcus pneumoniae and penicillin intermediate Streptococcus pneumoniae were 41.2％ ( 145/352) and 37.2％ (131/352) respectively based on oral penicillin criterion,while the prevalence were 0.0％ (0/352) and 6.0％(21/352) based on vein to non-meningitis criterion.A vast majority of Enterobacteriaceae maintained high susceptibility to carbapenems,with resistance rate less than 2.0％.In addition,tigecycline,moxalactam,fosfomycin and amikacin displayed desirable antibacterial activity against Enterbacteriaceae,and resistance rates to these drugs were all less than 10.0％.For non-fermenting Gramnegative isolates,resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii to imipenem were 23.1％ ( 139/601 ) and 53.5％ (419/784) respectively.Resistance rate of Acinetobacter baumannii was much higher than that during the period 2007 - 2008.Colistin,tigecycline,minocycline and fosfomycin demonstrated good antibacterial activity against Acinetobacter baumannii in vitro.Conclusions Compared with MOHNARIN 2007 -2008year surveillance results, significant increase in resistance rate of Acinetobacter baumannii was demonstrated.Resistant strains to linezolid and tigecycline were found.Bacterial resistance has been a widespread problem in our country,which requires much more attention.

Objective To investigate antimicrobial resistance among Streptococcus pneumoniae clinically isolated from 14 teaching hospitals located at different areas in China in 2005-2008 and to give logical guidance for clinical empirical therapy.Methods A total of 1 317 non-repetitive S.pneumoniae isolates in 14 teaching hospitals from 2005-2008 were collected and sent to the central lab for reidentification and susceptibility testing, including 271 isolates collected in 2005, 391 isolates collected in 2006, 363 isolates collected in 2007 and 292 isolates collected in 2008. Most of the isolates were from community-acquired respiratory tract infections, which were isolated from outpatient or emergency department patients with respiratory tract infections or those patients with respiratory tract infections within ≤48 hours hospitalization.The districts where the organisms were isolated include North China, Northeast China, South China, Central and Northwest China and East China.The patients included adults, teenagers and children.The minimum inhibitory concentrations (MICs) or inhibitory zone diameter of 17 antimicrobial agents were determined by Etest method, agar dilution method or disk diffusion method.WHONET5.5 software was used to analyze susceptibility rate, intermediate rate, resistance rate, MIC50 and MIC90.Results Linezolid (100%) and fluoroquinolones (95.2%-99.7%) showed excellent activities against S.pneumoniae.Among β-lactams, amoxicillin-clavulanic acid remained high activities (73.8%-92.1%),followed by penicillin, ceftriaxone and cefepime with year-over-year decrease in activities.The activities of three second-generation cephalosporins were low (36.3%-38.4% in 2008).The activities of erythromycin, azithromycin, clindamycin, trimethoprim/sulfamethoxazole and tetracycline against S.pneumoniae were poor and decreased year over year.The incidence of penicillin non-susceptible S.pneumoniae (PNSP) was increasing especially for PISP (from 4.4% in 2005 to 20.2% in 2008).The incidence of PNSP in North China was low (6.0%), while this value were high in central China and East China (30.1% and 38.7%, separately).The incidence of PNSP in adults (15.7%) was obviously lower than that in children(≤5 years:33.0%) and teenagers (6-17 years:38.2%).Conclusions linezolid and fluoroquinolones showed excellent in vitro activity against S.pneumoniae, followed by penicillin and cephalosporins with year-over-year decrease of activity. Clinicians should pay more attention when using those antimicrobial agents with poor activity against S.pneumoniae, which include macrolides, clindamycin, trimethoprim/sulfamethoxazole and tetracycline.

Based on the in vitro culturing system developed for epithelial cells in mammary gland of Xinong Saanen dairy goats using tissue explant culture, high density cultivation, and continuous passaging, the cultured epithelial cells were evaluated by growth curve fitting, karyotype analysis, immunofluorescence staining (keratin, epithelial membrane antigen (EMA), vimentin, beta-casein), oil red staining and RT-PCR of beta-casein gene. The results showed that the growth of epithelial cells with the model number of chromosome of 60 demonstrated a typical 'S' shape curve, the positive gene expression of keratin, EMA, vimentin and beta-casein was detected, the cytoplasmic lipid droplets were observed following the oil red staining, the cultured cells expressed the mRNA of beta-casein. In conclusion, the current in vitro culturing system can obtain the normal mammary gland epithelial cells with the function of secretion.
Animals ; Cell Separation ; methods ; Cells, Cultured ; Epithelial Cells ; cytology ; Female ; Goats ; Mammary Glands, Animal ; cytology ; Primary Cell Culture ; methods

Objective To evaluate the influences of susceptibility interpretation of Escherichia coli,Klebsiella pneumonia and Proteus mirabilis in China mainland according to the old and new ceftazidime,cefotaxime and ceftriaxone breakpoints in CLSI M100-S20 and CLSI M100-S19. Methods First, We analyzed the antibacterial susceptibility results of the three bacteria by agar dilution method in the SEANIR surveillance item, which were collected from 15 national hospitals between the year of 2005 and 2007 and excluded the AmpC enzyme positive isolates according to the PGR-DNA sequencing method and/or the antibacterial susceptibility phenotype. ESBL phenotype was confirmed by the CLSI phenotypic confirmatory test. Antibacterial susceptibility of the total 2733 Escherichia coli, Klebsiella pneumonia, Proteus mirabilis isolates was retrospectively analyzed by WHONET 5. 4 software according to the breakpoints of the CLSI M100-S19 (S19) and CLSI M100-S20 (S20). Second, 207 isolates of Peking Union Medical College Hospital with the results of both agar dilution method and disk diffusion method were performed by recurrent analysis. Then we observed the inter-method agreement through the scatter diagram according to the breakpoints of S19 and S20. Results First, as to the ESBL positive Escherichia coli, Klebsiella pneumonia and Proteus mirabili.s, the resistant rate of cefotaxime increased from 65.2% , 55.5%, 14. 6% under S19 (64 μg/ml) to 99. 7%, 96. 2% , 93. 8% under S20 (4 μg/ml). The susceptibility rates decreased from 6. 0%, 11.5%, 33.3% under S19 (8 μg/ml) to 0%, 0. 2%, 0% under S20 ( 1 μg/ml). Ceftriaxone had the same trend as cefotaxime. Though ceftazidime was more active than cefotaxime and ceftriaxone, as to the ESBL positive Escherichia coli and Klebsiella pneumonia, the resistant rates slightly increased from 30. 3%,43. 2% under S19 (32 μg/ml) to42.0%, 56. 0% under S20 (16 μg/ml). The susceptibility rates slightly decreased from 58. 1%, 44. 1% under S19 (8 μg/ml) to 44. 7%, 28.0% under S20 (4 μg/ml). Second,as to the ESBL negative Escherichia coli, Klebsiella pneumonia and Proteus mirabilis, all the susceptibility rates of ceftazidime, cefotaxime and ceftriaxone were between 99. 2%-100. 0%, the resistant rate were between 0%-0. 4%. Third, the S20 MIC breakpoints had a good correspondence with the ESBL phenotype.Fourth, according to the recurrent analysis of MIC testing and disk dilution method, r value was 0. 67,0. 79, 0. 77 for ceftazidime, cefotaxime and ceftriaxone, respectively, and all P value were under 0. 01. The intermethod rates of S19 and S20 were both acceptable. Conclusions If the cefotaxime and ceftriaxone S20 new breakpoints were used, the concordance of antibacterial susceptibility results and ESBL phenotype would increase greatly. The clinician could select proper antibiotics according to the antibacterial susceptibility results and clinical symptoms. It is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from susceptible to resistant. However, until laboratories implement the new interpretive criteria,ESBL testing should be performed as described in Supplemental Table 2A-S1. The relationship between the new breakpoints of ceftazidime and clinical outcomes need to be further evaluated.

Objective To express the recombinant caspid of genotype 4 hepatitis E virus(HEV) ORF2. Methods HEV recombinant capsid protein D66 was expressed in E. coli, using the ORF2 fragment (aa368-606, obtained from swine bile) of genotype 4 HEV. Results The recombinant capsid proteins D66 self-assemble to be particle with a radius of 13 nm through dimeric form in neutral solution. Coated particles reacted well with sera obtained from patients during acute or recovered phase of HEV infection. Immunofluorescence and immnoblot assay suggested that D66 bound and penetrated HepG2 cell lines, and the process of attachment was blocked by sera collected from patients during acute or recovered phase of HEV infection.Conclusion Recombinant D66 particles simulate the structure at the surface of genotype 4 HEV well and specifically adhere and penetrate the host cells, which lays the foundation for the investigation of the molecular mechanism of genotype 4 HEV infection.

OBJECTIVE To investigate the disinfectant and antibiotic resistance genotypes in 60 strains of Escherichia coli isolated from urine.METHODS Sixty strains of E.coli isolated from inpatients′ urine were collected from Jan 2006 to Oct 2008.Antibiotic susceptibility tests for fifteen antibiotics were performed by Kirby-Bauer method.And three kinds of disinfectant and antibiotic resistance genes(qacE△1,tehA,merA)were analyzed by polymerase chain reaction(PCR) and DNA sequencing.RESULTS More than 70.0% of the sixty strains of E.coli were susceptible to imipenem,piperacillin/tazobactam,cefoxitin,amikacin and gentamicin,and less than 50.0% were susceptible to the other ten antibiotics.There were 42 strains with qacE△1 gene(70.0%),10 strains with merA gene(16.7%) and all strains with tehA gene.The sequence of the first strain was different from those reported in GenBank,so it was a new subtype.CONCLUSIONS There are 70% of E.coli strains isolated from urine samples with qacE△1 gene.And disinfectant resistance may be one of the main factors for hospital infection in the future.