Discussion on the Problems of Vitek 2 Compact Advanced Expert System to Identify Carbapenemase Phenotypes in Isolates of Enterobacteriaceae / 现代检验医学杂志

Objective To explore the accuracy of Vitek 2 compact advanced expert system (AES) in indicating and analyze the carbapenemases-resisting Enterobacteriaceae phenotypes,and further investigate the methods to make up the AES.Methods 28 Enterobacteriaceae strains with Imipenem-Nonsusceptible by Vitek 2 compact,but AES suggested all production of carbapenemases were isolated.And imipenem susceptibility was determined by the disk diffusion method.Modified Hodge test (MHT) and the metallo-β-1actamase was detected by the double disk synergy method.Resistance genes were detected by the PCR amplification.Results ESBLs gene was amplified from all 28 selected strains,16 of which was detected KPC gene,and no strain of metallo-β-1actamases-producing bacteria.With carbapenemase gene detection as the gold standard,the accuracy of AES was 57.1％.Disc diffusion method detection accuracy rate of imipenem was 100％,and for 100％ of MHT accuracy.PCR amplification,MHT and the disk diffusion displayed the same result in detecting carbapenemases,but different with AES (x2 =10.08,P＜0.05).Conclusion The indications of the presence of carbapenemases using AES was not completely correct with a certain false-positive,and it is necessary to take other methods,such as disk diffusion or MHT methods,and improve the reliability of medicine-sensitivity tests.

Clinical research of the effects of miRNA and hypersensitive C-reactive protein on non-targeted vessels in patients with acute coronary syndromes after percutaneous coronary intervention procedure / 中国综合临床

Objective To investigate the effects of miRNA and hypersensitive C?reactive protein ( hs?CRP ) on non?targeted vessels in patients with acute coronary syndromes ( ACS ) after percutaneous coronary intervention( PCI) procedure. Methods The serum samples were collected from 217 cases ACS patients to detect the level of miRNA?14?5P and hs?CRP during admission and follow?up periods.All patients underwentPCI with stent implantation and coronary angiography,and CAG was performed at the time of 12?month follow?up.According to CAG results,the patients were divided into non target lesion progression group(progressiongroup) with 76 cases and non target lesion no progression group(no progression group) with 141 cases.Results The expression level of hs?CRP was significantly higher in progression group than the no progression group((1.65±0.18) mg/L vs.(1.52±0.37) mg/L,t = 3.478,P<0.001).The expression level of miRNA?142?5Pwas higher in progression group than the no progression group(27.12±2.11 vs.34.73±2.67,t = 23.035,P<0.001).Multi?factor regression analysis indicated that high expression levels of hs?CR and miRNA?142?5Pduring admission were the predictors of advance of non?targeted vessels patients(OR = 3.496,95%CI 2.046-5.981,P =0.001;OR =1.208,95%CI 1.073-1.361,P =0.002).Conclusion The serum level of hs?CRP andmiRNA?142?5P can predict non?targeted vessels in patients with ACS after PCI.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid identification of clinical isolates / 医学研究生学报

Objective Laboratory tests of yeast, with their disadvantages of long identification, complicated operation, and low positive rate, cannot meet the needsof rapid diagnosis and timely treatment.This studyinvestigated the feasibility ofapplying matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI-TOF-MS) in rapid identification and clinical isolationof yeast strains. Methods A total of 120 clinical non-repetitive isolates were collected from clinical culture samples and identified by MALDI-TOF-MS and VITEK 2-Compact/API, respectively.The discordant results were resolved by ITS gene sequencing. Results Of the 120 yeast isolates analyzed, 117 (97.5%) were correctly identified at the species level by MALDI-TOF-MS, 1(0.8%) misidenti-fied, and 2 ( 1.7%) unidentified.ITS gene sequencing of the 3 isolates showed the coincidence rate to be 0( 0/3) for MALDI-TOF-MS and Vitek 2-Compact/API. Conclusion MALDI-TOF-MScan be used as a rapid, accurate, and inexpensive tool for the identification of clinical yeast strains.

Clinical application and evaluation on therapeutic effects of mandibular reconstruction with free fibula muscle flaps / 吉林大学学报(医学版)

Objective To investigate the application of vascularized free fibula flaps in reconstruction of mandibular defect,and to evaluate the survival rate and repair effect of the method.Methods 16 patients with mandibular tumor,having a desire to reconstruct mandible,and their systemic state could tolerate the long time operation, were selected to reconstruct mandibular defect by vascularized fibula flaps,of which 12 were male,4 female,aged 24-66 years old,average 45.2 years old.The resection of primary tumor and free fibula flaps were simultaneously conducted,then the fibular flaps were shaped according to the defect location and size of the mandible,and were fixed with reconstructive titanium plate for repair and reconstruction of mandible. The survival of flaps was determined by skin color, texture, and skin temperature of the flaps. The reconstruction effects were evaluated through the patients’surface like photos and X-ray picture of mandible before and after operation.Results All of flaps were survived and no serious complications were found. The complications of the supplied sites were skin tension and wound dehiscence, which were healed by dressing. The mandibular reconstruction effects were good through 2 or more persons’double blind evaluation.Conclusion Free fibula flaps have high survival rate and good results in mandibular reconstruction,and they can meet the needs of various types of mandibular defect repair.

Study on the Mechanism of Quinolone Resistance in Pseudomonas Aeruginosa / 现代检验医学杂志

Objective 30 Pseudomonasaeruginosa mechanism of resistance to quinolones.Methods For the determination of ciprofloxacin MIC by agar dilution method.Used PCR on DNA gyrase and topoisomerase Ⅳ,resistance genes gyrA,gyrB, parC and parE were amplified,and BLAST,to determine whether there was resistance to bits mutation point;using pulsed-field gel electrophoresis (PFGE)of these 30 strains homology analysis.Results The 28 bacterial strains gyrA gene ampli-fied fragment of 137 points were C→T mutation causes T83I;17 strains gyrB gene amplified fragment of 351 G→C lead to G466A;parC gene amplification 21 bacteria fragment 277 point increase with C→U mutation causes S87L change two differ-ent strains parE gene locus C→U mutation A425V and A473V cause change.PFGE results:30 Pseudomonas aeruginosa could be divided into six clones,Aclone 4,B clone 7,C clone 3,D clone 14,and two other single clones.Conclusion The tar-get mutant strains closely related to the epidemic clone type,the same changes in the same pop-type strains of drug targets, and proportional to the level of ciprofloxacin MICs value,the more the number of mutated genes,MICs value higher.GyrA gene most prone to mutation,the mutation was also the first to be discovered,more than any other target of the mutation mutations on binding of drugs and targets that would be the focus of concern.

The relationship between H-type hypertension and unstable angina in the elderly / 中华老年医学杂志

Objective To investigate the relationship between H-type hypertension and unstable angina (UA).Methods Totally 147 elderly inpatients with hypertension and angina in our hospital were selected.Patients were divided into H-type hypertension group [n=72,serum homocysteine (Hcy) level ≥10 μmol/L] and primary hypertension group [n=75,serum homocysteine (Hcy) level ＜10 μmol/L].All patients underwent coronary angiography.Serum Hcy level was measured by enzyme method and compared between groups.Results There were statistical differences in the UA incidence,Gensini's score and high sensitive C-reactive protein (hsCRP) level between the H-type hypertension group and primary hypertension group [(44.4％ (32/72) vs.12.0％ (9/75),(44.2± 21.3) vs.(31.9±18.4),(4.3±2.1) μg/L vs.(2.0±1.9) μg/L,respectively,all P＜0.01].Serum Hcy level in H-type hypertension group was higher during UA attack than during UA remission [(22.2±7.1)μmol/L vs.(13.7±3.7)μmol/L,P＜ 0.01].Serum Hcy level during UA attack was increased in H-type hypertension group than in stable angina group [(22.2±7.1)μmol/L vs.(12.0± 4.2) μmol/L,P ＜ 0.01].Serum levels of Hcy,total cholesterol and low density lipoprotein cholesterol in primary hypertension group were higher in UA patients than in stable angina patients [(8.9±2.2)μmol/L vs.(6.6± 1.2)μmol/L,(6.9±0.7)mmol/L vs.(4.5±0.5)mmol/L,(4.6±0.8)mmol/L vs.(2.7 ± 0.6) mmol/L,respectively,P＜ 0.01 or P＜0.05].Logistic regression analysis showed that H-type hypertension was the independent risk factor for unstable angina in the elderly (OR =5.691,P ＜ 0.01).Conclusions H-type hypertension is closely correlated with unstable angina,which is the independent risk factor for unstable angina in the elderly.Serum Hcy level has significant correlation with coronary atherosclerotic plaque stability and the severity of coronary artery disease.

The relationship of resistance mechanism of two strains of Enterobacteriaceae resistant to carbapenems isolated from a single specimen / 中华微生物学和免疫学杂志

Objective To investigate the relationship of resistance mechanisms of a Klebsiella pneumoniae strain and a Morganella morganii strain resistance to carbapenems isolated from a single specimen. Methods Sensibility of antimicrobial agents was detected by agar dilution method. Specific PCR and DNA sequence analysis were performed to detect resistance genes. Plasmid feature was detected by plasmid conjugation and electrophoresis analysis. Genetic environment around blaKPC was analyzed with sequencing. The changes of outer membrane permeability were analyzed with electrophoresis of outer membrane proteins. Results blaKPC-2 was detected in 2 original isolates strains and their transconjugants. Carbapenem-resistance was successfully transfered by conjugation experiments. blaKPC-2 was located on dissimilar plasmids, but genetic environment around blaKPC-2 was the same sequence. The Morganella morganii isolate showed a loss of 38 ×103 OMPs and an additional 36 ×103 OMPs appearance, while the Klebsiella pneumoniae isolate showed a loss of OMPK36. Conclusion blaKPC-2 was detected in 2 isolates. This gene encoded by two plasmids with different sizes was located on the same composite transposon. The lack of outer membrane proteins could also play an important role causing isolates to exhibite resistance to carbapenems.

Genome shuffling method of Bacillus subtilis / 生物工程学报

Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53 x 10(-4), 1.64 x 10(-4), 4.47 x 10(-3), respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10(-3) and 10(-2).

Two carbapenem-resistant Escherichia coli strains isolated from blood and stool of a same patient / 中华传染病杂志

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.

Study on the molecular mechanism of carbapenem-resistant Escherichia coli / 中华微生物学和免疫学杂志

Objective To investigate the molecular epidemiology and mechanism of carbapenem resistance of Escherichia coli collected from intensive care units(ICUs)of general surgery.Methods Agardilution were carried out to confirmed the drug-susceptibility,pulsed-field gel electrophoresis(PFGE)were performed to analyze the molecular epidcmiology of carbapenem-resistance isolates.Specific PCR,DNA sequencing,conjugation experiments,plasmids extraction,plasmid transformation assays and SDS-PAGE of outer membrane proteins(OMPs)were carried to confirm genotype of carbapenemase and its transmission mechanism.Results PFGE showed the isolates belonged to 10 clonotype,and all the clinical isolates were resistant to β-lactams including imipenem and meropenem,but uncertain to aminoglycosides,specific PCR and DNA sequencing revealed that all isolates encoded carbapenem-hydrolyzing enzyme gene,KPC-2.Plasmid DNA extraction and plasmid transformation assays from some isolates comfirmed that KPC-2 encoded on a 56 kb plasmid.SDS-PAGE analysis confirmed that there are alterations in OMPs of Escherichia coli.Conclusion Escherichia coli isolates with carbapenem resistance are collected from our hospital,production of KPC-2 carbapenemase mainly contributed to reduced susceptibility of carbapenem in Escherichia coli,the alterations in OMPs may as a cofactor in high-level drug-resistance in Escherichia coli.

Detection and analysis of plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in carbapenem-resistant Escherichia coli / 中华检验医学杂志

Objective To study molecular epidemiology and carbapenem-resistance mechanism of four Escherichia coli strains isolated from general surgery wards. Methods Antibiotic susceptibility was carried out by K-B gar diffusion and agar dilution methods. Carbapenemases were screened by three dimensional test and EDTA-Na_2-disk synergy test. Pulsed-field gel electropboresis (PFGE) was performed to analyze molecular epidemiology of isolates. Plasmid was extracted by using an alkalinelysis technique. Conjunction experiment, transformation assay, specific PCR and DNA sequencing were performed to confirm carbapenemase genotype and its transmission mechanism Results Four Escherichia coli isolates were resistant to most antimicrobials including carbapenem. PFGE showed that the four isolates belong to four different clonal strains. Specific PCR and DNA sequence analysis identified that carbapenem resistance in four clinical isolates was mediated by KPC-2 encoded on an approximately 56 000 bp plasmid, and this plasmid did not harbor aminoglycosides and fluorquinolones resistant genes. Conclusion Four Escherichia coli isolates with carbapenem resistance are obtained from our hospital, and KPC-2 plasmid is main cause of carbapenem resistance in these isolates.

A primary survey of the histopathological characteristic in nontuberculous mycobacteria lymphadenitis / 医学研究生学报

Objective:To approach the distinctive histopathological chages of nontuberculous mycobacteria lymphadenitis. Methods: The experimental animal model of nontuberculous mycobacteria lymphadenitis was developed and the histopathological changes were observed under the light microscope.Results:The distinctive histopathological changes of nontuberculous mycobacteria lymphadenitis were identified as following: ①Granulomas were found in lymph nodes which were infected with nontuberculous mycobacteria.The coagulation necrosis was located at the center of the granuloma and it was not caseation.Neutrophils were distributed over the necrosis area.Surrounding the center necrosis area,many epithelioid cells,lymphocytes and mononuclear cells could be found.The periphery of the granuloma was surrounded by the fibrous tissues.Outside the granuloma,Langhans giant cells could be found.②Serpiginous necrosis was found in the lymph nodes which were infected with nontuberculous mycobacteria.The shape of serpiginous necrosis was a long strip.In the center area,the strip of coagulation necrosis and the strip of the space which was remnant after the desquamation of necrosis tissues could be found.Many neutrophils were distributed over the necrosis area.Around the center necrosis area,many epithelioid cells,lymphocytes and mononuclear cells could be found.The fibrous tissues were in the borderline.Conclusion:Serpiginous necrosis was one of the distinctive histopathological change of nontuberculous mycobacteria lymphadenitis.

Progress on AmpC hyperproduction in Escherichia coli / 医学研究生学报

The ampC ?-lactamases gene in Escherichia coli(E.coli) is different from other Gram-negative bacteria.E.coli contains a chromosomal ampC gene which has a weak promoter as well as a transcriptional attenuator.The promoter of the ampC gene in E.coli is part of the preceding frd operon,the attenuator of the ampC gene is a transcription terminator for the frd operon.The ampC regulatory gene,ampR,is absent.Strains carrying the wild-type gene produce a low basal amount of AmpC.Studies on the molecular basis of AmpC overproduction in E.coli have shown that some hyperproducers contain mutation in the promoter region and/or attenautor and/or ampC-coding region of ampC,while others contain more than one copy of ampC.Acquisition of a stronger promoter or insertion of an insertion element containing promoter sequences or regulatory gene ampR has also been proposed as the molecular basis of hyperproduction of AmpC in some E.coli strains.Plasmid-mediated AmpC ?-lactamases have been discovered frequently in E.coli strains.This is another reason for hyperproduction of AmpC ?-lactamases.

A Study on resistance of non-PBP 2a type to oxacillin of Staphylococcus aureus / 医学研究生学报

Objective:To investigate whether there is another resistance mechanism besides mecA gene in oxacillin-resistant(OR) isolates of Staphylococcus aureus(S.aureus). Methods:There were 130 oxacillin-resistant phenotype isolates of S. aureus. Of these isolates 125 were resistant to more than 3 of 5 non-?-lactams (gentamicin, ciprofloxacin, clindamycin, tetracycline, erythromycin) (OR-R) and 5 susceptible to more than 3 of the 5 non-?-lactams (OR-S) and 14 were oxacillin-susceptible (OS) phenotype isolates of S. aureus and resistant to more than 3 of 5 non-?-lactams (OS-R). All the strains were detected by the two disks diffusion tests with oxacillin (OXA) and oxacillin/clavulanic acid (OXA/CA), by the three-dimensional extract tests of penicillin (PEN) and OXA for penicillinase and oxacillinase, and by PCR tests for mecA. Results:The 130 OR and 14 OS isolates were all oxacillinase-negative with the two disks diffusion tests, all pencillinase-positive and oxacillinase-negative in the three-dimensional extract tests. The mecA gene was detected in 125 OR-R-type and 2 of the 5 OR-S-type isolates, but was not detected in the other 3 OR-S-type nor in any of the 14 OS-R-type isolates by PCR. Conclusion:In a few Staphylococcus aureus strains which are phenotype of oxacillin-resistant and are susceptible to mostly non-?-lactam agents there are both mecA-negative and oxacillinase-negative strains, 2.3% (3/130) of the OXA-resistant strains. The resistance mechanism may be associated with reduced binding capacity of the modified Preexisting PBPs with OXA.

Plasmid-mediated resistance to quinolones in Enterobacteriaceae / 医学研究生学报

Although quinolone resistance results mostly from chromosomal mutations in Enterobacteriaceae,it may also be mediated by plasmid-encoded Qnr determinants.Qnr proteins protect DNA from quinolone action and compromise the effect of quinolones,such as nalidixic acid.Qnr proteins including QnrA,QnrB and QnrS,have been identified worldwide with a quite high prevalence among Asian isolates with a frequent association with clavulanic acid-inhibited expanded-spectrum b-lactamases and plasmid-mediated cephalosporinases.The QnrA genes are embedded in complex sul1-type integrons.A close relative and likely progenitor of the QnrA have been found in the water-borne species Shewanella algae.It may help to determine the location of in vivo transfer of the QnrA genes.Further analyses of the role of quinolones,if any,in enhancing this gene transfer may prevent the spreading of the drug resistance and possibly lead to the finding of a novel mechanism of antibiotic resistance.

Detecting antibody against candida albicans mycelial-phase protein using surrogate peptides from phage display library / 中华检验医学杂志

Objective To use phage display for generating surrogate peptides of candida albicans mycelial-phase protein as mimic antigen to develop an enzyme-linked immunosorbent assay (ELISA) assay for detection of candida albicans antibodies.Methods A monoclonal antibody against candida albicans mycelial-phase protein P47, was used in this study for selecting immunoreactive peptides, mimotopes, from a 12 mer phage library.The peptides were characterized immunologically and used for coating antigen in ELISA to detect antibodies against candida albicans in the patients' serum.Results The sensitivity, specificity and replication of the ELISA assay was good enough for clinical use. The antibody was positive in all of 10 samples of patients with confirmed systemic candida albicans infection and the results were same to those obtained with the purified mycelial-phase protein P47; The positive rates in patients receiving immunosuppressive drug cyclosporin A are 33% and the healthy 2% respectively.Conclusion The surrogate peptides from a 12 mer phage library can be used in ELISA for detecting the antibodies of candida albicans P47.

Characteristics of monoclonal antibody against glycophorin A of human erythrocyte / 医学研究生学报

Objectives: To study and prepare monoclonal antibody against glycophorin A of human erythrocyte(GPA McAb).This antibody is a key reagent in preparation of bispecific antibodies for rapid whole-blood immunoassay.　Methods: BALB/c mice received GPA antigen injection. Hybridoma was produced by traditional techniques. Hybridoma was determined with ELISA and indirect agglutination(IA) method.　Results: All 3 GPA McAb reacted with GPA, and they did not autoagglutenate with four types of red cells(type A,B,O,AB).Of the 3 McAb-GPA, two antibodies were IgG1,one IgG2 subtypes. Two IgG1 McAb-GPA can be used in making bispecific antibody in preparation of rapid whole-blood immunoassay.　Conclusions: Immunogenity of GPA was enhanced after coupling with the native serum albumin of the immunized animal, and high titer GPA McAb could be easily obtained. This result is important in making bispecific antibodies in preparation of rapid whole-blood immunoassay.