Objective To investigate the association of AcrAB-tolC efflux pump and marA-soxS-rob regulon with ciprofloxacin-resistance in Shigella flexneri isolates.Methods Forty five strains of Shigella flexneri were isolated in stool samples collected from outpatient diarrhea clinics in Tianjin from 2009 to 2013.The antimicrobial susceptibility of bacteria was analyzed by Kirby-Bauer disk diffusion method.Ten ciprofloxacin-resistant Shigella flexneri isolates and 10 ciprofloxacin-sensitive isolates were randomly selected.The gyrA and parC genes,plasmid mediated quinolone resistant (PMQR) determinants,efflux pump genes (acrA,acrB) and regulation genes (marA,soxS,rob) were screened and sequenced.Minimum inhibition concentrations (MICs) of the strains were determined before and after efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added.The expression of acrA,acrB,marA,soxS and rob genes in ciprofloxacin-resistant and ciprofloxacin-sensitive strains was detected by realtime fluorescence quantitative RCR (RT-PCR),and the differences of expression were evaluated using t test.Results Both gyrA and parC mutations were detected in all ciprofloxacin-resistant strains; qnrS1 was positive in ciprofloxacin-resistant strain CR2 and aac(6')-Ib-cr was positive in ciprofloxacin-resistant strain CR5.Efflux pump genes and regulation genes were not detected in ciprofloxacin-sensitive strains,while soxRS mutation was detected in all ciprofloxacin-resistant strains except CR10.MICs of quinolones in ciprofloxacin-resistant strains decreased to one-fourth or one-eighth when CCCP added,while not changed or only decreased to one-half in ciprofloxacin-sensitive strains.Expressions of acrA,acrB,marA and soxS were significantly higher in ciprofloxacin-resistant strains than those in ciprofloxacin-sensitive strains (P ＜ 0.05 or P ＜ 0.01).Conclusion Efflux pump may involve in the high-level ciprofloxacin resistance in Shigella flexneri through activating transcription of efflux pump genes by marA-soxS-rob regulon,in which soxRS mutation may play an important role.

Objective To observe the clinical efficacy of plasma exchange(PE),PE combined with plasma bilirubin absorp-tion(PE+PBA),and PE combined with double plasma molecule absorption system(PE+DPMAS),to investigate the best treat-ment options for the patients of hepatitis B-associated subacute severe.Methods Totally 140 patients who had hepatitis B-associat-ed were randomly divided into a PE group,a PE+PBA group,and a PE+DPMAS group.The dinical symptoms and blood rontine, electrolytc and the main biochemical indexes were recorded both before and after treatment and compared among the three groups. Results The total effective rate was higher in the PE +DPMAS group than in the PE group and PE+PBA group,which were 70.8%,60.9%,67.4% respectively,but there was no significant difference(P >0.05).compared with before treatment,serum total bilirubin (TBIL),alanine aminotransferase (ALT),aspartic acid amino shift enzyme (AST),valley aminoacyl transfer peptidase (GGT),alkaline phosphatase (ALP),globulin (GLB)and other biochemical indexes decreased significantly after treatment in 3 groups (P <0.001).PA was increased in the PE group and decreased in the PE+PBA and PE+DPMAS group after treatment,the differences were significant(P <0.05).After treatment,PT was shortened in the PE group,the differences were significant(P <0.05),but there were no significant difference between the PE+PBA and PE+DPMAS groups(P >0.05).Serum K+ and Cl- was obviously declined after treatment in the PE group,there was significant difference(P <0.001).Serum Na+ was obviously declined after treatment between the PE + PBA and PE + DPMAS groups (P < 0.001 ),but the difference was no significant in the PE groups.Serum Ca2+ was significantly decreased in the three groups of patients after treatment(P <0.001).WBC,Hb and PLT were significantly statistical difference after treatment(P <0.05).Conclusion The three groups also can improve liver function and the treatment rate for the patients of subacute hepatitis B-associated severe hepatitis.PE+PBA and PE+DPMAS groups can effective-ly reduce two thirds of the overall usage of the plasma.PE+DPMAS groups that the quantity of Cl- ,Ca2+ ,Hb decline much least in the three groups show larger value in the clinical application.

Objective To study the prevalence of food intolerance and to explore its related factors among adult health check-up receivers.Methods A total of 863 adults who took physical examinations in our hospital from April to October 2011 were enrolled in this investigation.Height,body weight and blood pressure were measured,and serum IgG level was measured by enzyme-linked immunosorbent assay (ELISA).Results The total positive rate of food intolerance was 73％,and the leading intolerance items were crab (40.1％ ),egg (29.8％ ),cod fish ( 21.6％ ),milk ( 20.0％ ) and soybean ( 14.4％ ).Females showed significantly higher prevalence of food intolerance than males.Various positive rate of milk or soybean intolerance was found in different age groups.No correlations of serum specific IgG with body mass index and systolic or diastolic blood pressure were observed.In logistic regression analysis,the odds ratio of food intolerance of women was 1.67 ( 95 ％ confidence interval 1.190 to 2.607 ).ConclusionsThe prevalence rate of food intolerance was high.The risk for food intolerance was significantly increased in women.Specific IgG antibody detection may help to early prevent and diagnose food intolerance-related diseases.

OBJECTIVETo explore the surgical method and its clinical effects of minimally invasive osteosynthesis on the treatment of complex ankle fractures.

METHODSFrom January 2007 to December 2011, 53 patients with complex ankle fractures were treated with minimally invasive osteosynthesis. There were 31 males and 22 females, with an average age of 38.2 years old (ranged, 18 to 65). According to the system of Lauge-Hansen, 32 fractures were supination external rotation injury (grade WV), 13 fractures were pronation external rotation (grade III or IV), 5 fractures were pronation abduction (grade III); and 3 fractures can not be classified due to serious comminution fracture of fibula. According to the system of Denis-Weber, there were 4 cases with type A, 34 cases with type B and 15 cases with type C. Seven cases were open fractures. The duration from injuries to operation ranged from 2 hours to 14 days with an average of 5 days. The sequence of reduction and fixation of ankle fractures was firstly posterior malleolus, then medial malleolus and lateral malleolus, and inferior tibiofibular syndesmosis lastly. The fractures of posterior malleolus were reduced and fixed through anterior ankle approaches; the fractures of medial and lateral malleolus were percutaneously fixed with bolts or blade plate or tensile force band; and inferior tibiofibular syndesmosis were firmly fixed if necessary. Baird-Jackson scoring system was used to evaluate clinical effects.

RESULTSForty-eight patients were followed up from 10 to 36 months with an average of 13 months. The fractures got healing with an average time of 12 weeks (ranged, 10 to 18). According to the Baird-Jackson scoring system, the mean score of ankle function was 94.7 +/- 4.2, and 28 cases obtained excellent results, 15 good, 3 fair and 2 poor. One case experienced superficial infections and was cured by changing dressings, 2 cases experienced fixed syndesmosis screw breakage.

CONCLUSIONThe surgical method of minimally invasive osteosynthesis can ensure the anatomical join restoration, protect the blood supply of fracture end, rebuild the function of ankle joint, obtain satisfactory clinical results in treating complex ankle fractures.

Adolescent ; Adult ; Aged ; Ankle Fractures ; Ankle Joint ; physiopathology ; surgery ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods

Objective To investigate the role of AcrAB-TolC efflux pump in fluoroquinolones resistance by Shigella. spp and to explore the significance of AcrAB-TolC efflux pump on mutation of acrR, soxS and marOR as well as on drug re?sistence. Methods Drug resistant bacteria were selected by Kirby-Bauer disk diffusion test. After addition of efflux pump inhibitor carbonylcyanide-m-chlorophenylhydrazone (CCCP), change of minimal inhibitory concentration (MIC)s of nilidixic acid, Levofloxacin, ofloxacin, ciprofloxacin and Norfloxacin were examined. The DNA binding region of acrA, acrB, soxS, acrR and marOR gene in these mutants were amplified by PCR and sequenced. Results Among the 159 clinical isolates of Shigella,11 strains are resistant to fluoroquinolone. After the addition of CCCP, MICs of 2 fluoroquinolone resistant strains decreased; the MICs of 7 fluoroquinolone resistant strains did not change; MICs of 2 fluoroquinolone resistant strains in?creased. The corresponding nucleotides C, A, T, T on the 36th to 39th of marOR gene were missing, showing by sequencing, in fluoroquinolone resistent strains which might be regulated by the efflux pump gene AcrAB-TolC. Conclusion Efflux pump inhibitor could restrain the activity of efflux partially. The mutations of marOR might play an important role in fluoroquino?lone resistent by shigella.

Objective:To analyze the immunogenicity of the extracellular region of Δ42PD1.Methods: Six fragments ofΔ42PD1 extracellular region-encoding sequence were amplified by PCR, and were cloned into pCTCON2 vector, a yeast surface displaying vector.Yeast cells were transfected with Δ42PD1 fragment-carrying plasmids, then yeast cells were spread on SDCAA plates.Single cell clones were selected and cultured in SGCAA media to induce expression of the target genes.Mouse anti-humanΔ42PD1 anti-serum were generated by immunization of BALB/c mice via intramuscular injection ofΔ42PD1-carrying plasmid plus in-situ electroporation.The binding of anti-serum with yeast cells surface-displaying Δ42PD1 fragments were analyzed using flowcytometry.Results:Nucleotide sequences analysis indicated that the amplified six fragments ofΔ42PD1 sequence length were 110 bp,and the isolated sequence ofΔ42PD1 fragments were 100%homology with PD1 gene previously registered in GenBank.Results from flowcytometry showed that among the six fragments of Δ42PD1 displaying on the surface of yeast cells,F3 and F2 profoundly boundΔ42PD1-specific polyclonal antibodies.Conclusion:F3 and F2 ofΔ42PD1 is an immunogenic dominant region,which pave the way for generation of Δ42PD1-specific monoclonal antibody and epitope mapping.

BACKGROUND:Previous therapies for diabetic foot are not ideal with large cost, and moreover, amputation is often required. OBJECTIVE: To perform the Ilizarov bone transport in the treatment of patients with diabetic foot (Wanger grades 3-4), and to observe the limb salvage conditions. METHODS: Eighteen patients with diabetic foot, Wanger grades 3-4, admitted in the Department of Bone and Joint Surgery, First Affiliated Hospital of Guangxi Medical University from December 2013 to June 2015 were enroled in this trial. Al of patients were subjected to Ilizarov bone transport. RESULTS AND CONCLUSION: Al the 18 patients were folowed up for 3 to 20 months, and presented with ulcer healing. Scores on ankle-brachial index and 10-g nylon line test were both increased significantly in the patients after treatment, but the visual analog scale scores were reduced. These findings indicate that the Ilizarov bone transport is an effective method for treating ulcer of diabetic foot at Wanger grades 3-4.

Objective To obtain antibiotic-resistant mutants producing metabolites with antitumor activity from wild-type actinomycete strains without antitumor activity. Methods An actinomycete strain L35-1 was used as an initial strain for obtaining antibiotic-resistant mutants, which is a marine-derived wild-type strain without antitumor activity with an inhibition rate of 2.8% at the 1000 μg/ml of high sample concentration on K562 cells. The antibiotic-resistant mutants both from auto-mutagenesis and chemical mutagen-induced mutagenesis were selected by single colony isolation on antibiotic-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering. The antitumor activity was assayed by the MTT method using K562 cells for the mutants with aqueous acetone extracts of the whole broth of their fermentation.Results A total of 114 neomycin-resistant (ner) and 68 streptomycin-resistant (str) mutants, all from auto-mutagenesis, was obtained on drug-containing plates. Among them, the 7 ner and 3 str mutants appeared to be bioactive with an inhibition rate above 20% at the 100 μg/ml sample concentration on K562 cells. On the other hand, 41 str and 32 ner mutants from DES-induced mutagenesis and 46 ner mutants from NTG-induced mutagenesis were obtained by mutagen-induced mutation coupled with the single colony isolation on antibiotic-containing plates, among which, one str mutant from DES-induced mutagenesis and one ner mutant from NTG-induced mutagenesis were bioactive with an inhibition rate over 20% at the 100 μg/ml sample concentration on K562 cells. Conclusions The present result has revealed that the wild-type actinomycete strains without bioactivity might become a great source initial strains to obtain bioactive mutants by drug-resistant mutation technique.

Objective To evaluate the value of differential diagnosis on congenital biliary atresia(BA) and infantile hepatitis syndrome(IHS) by technetium-99m-diethyl-iminodiacetic acid(99Tcm-EHIDA)hepatobiliary scintigraphy with phenobarbitol sodium.Methods Fifty-eight infants with persistent jaundice were taken phenobarbitol sodium[5 mg/(kg?d)] ,bid ?7 d).Those who had not bowel and gallbladders radioactivity within 24 hours were diagnosed as the diagnostic criterion of BA.Those with bowel and gallbladders radioactivity within 24 hours were diagnosed as the diagnostic criterion of IHS,who then received 99Tcm-EHIDA hepatobiliary scintigraphy with single photon emission computed tomography(SPECT) instrument.The results of all children were analyzed and compared with pathology and clinical follow up results.Results 99Tcm-EHIDA hepatobiliary scintigraphy correctly diagnosed 24 infants with last diagnosis BA and 29 infants with last diagnosis IHS,5 neonates false positive in all 34 IHS patients.The sensitivity in the diagnosis of BA was 100%,the specificity and accuracy were 85.3% and 91.4%,restectively.The sensitivity was 85.3% in the diagnosis of IHS;the specificity and accuracy were 100% and 91.4%,respectively.Conclusions 99Tcm-EHIDA hepatobiliary scintigraphy with phenobarbitol sodium can accurately differentiate BA and HIS at early stage.

Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.