The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.
Carbon-Oxygen Lyases/biosynthesis* ; Diethylnitrosamine/pharmacology ; Escherichia coli/genetics ; Escherichia coli/drug effects ; Human ; Recombinant Fusion Proteins/biosynthesis*

OBJECTIVETo prepare and evaluate novel chlorine dioxide-based disinfectant powder in single-pack that is more convenient for use and transportation.

METHODSOrthogonal experiment was performed to determine the recipe of the disinfectant powder. Stability test, suspension quantitative bactericidal test, simulation field trial, and animal toxicity test were carried out to observe its bactericidal and toxicological effects.

RESULTSThe orthogonal experiment showed that the type of water solution had no effect on the disinfectant powder and the best ratio of sodium chlorite to solid acid was 1:3. Ten grams of the disinfectant powder was fully dissolved in 20 mL water for 2 min, and diluted to 500 mL in water. After 5-10 min, the concentration of chlorine dioxide (ClO2) solution was 266 mg/L to 276 mg/L. After stored at 54 degrees C for 14 d, the average concentration of ClO2 was decreased by 5.03%. Suspension quantitative bactericidal test showed that the average killing logarithm (KL) value for both Staphylococcus aureus and Escherichia coli in 100 mg/L ClO2 solution for 2 min was over 5.00. in simulation field trial, the average descending KL value for Escherichia coli in the solution containing 100 mg/L ClO2 for 5 min was over 3.00. The mouse acute LD50 in the solution 5 times exceeded 5000 mg/kg. The disinfectant powder was not toxic and irritative to rabbit skin and had no mutagenic effect on mouse marrow polychromatic erythrocytes (PCE).

CONCLUSIONThe stability and bactericidal efficacy of solid chlorine dioxide-based disinfectant powder in single-pack are good. The solution containing 100 mg/L ClO2 can kill vegetative forms of bacteria. The concentration of ClO2 on the disinfecting surface of objects is 100 mg/L. The disinfectant powder is not toxic and irritative.

Chlorine Compounds ; pharmacology ; Disinfectants ; pharmacology ; Escherichia coli ; drug effects ; Oxides ; pharmacology ; Staphylococcus aureus ; drug effects

AIMTo investigate the mechanism of the interaction between montmorillonite and bacteria by studying the reactions of different charges of montmorillonites with bacteria.

METHODSBacteriostatic test: one loop of E. coli and Staphylococcus aureus at the concentration of 1 x 10(6).mL-1 was incubated to the plate culture medium containing different concentrations of montmorillonite, and incubated 24 h to observe the growth of bacteria. Bacterial adsorptive test: different amounts of montmorillonite were added into the artificially simulated intestinal solution (containing bacteria 1 x 10(7).mL-1). After the culture, the bacterial colonies were counted.

RESULTSThe results showed that montmorillonite per se showed no bacteriostatic or bactericidal effect, but after exchange with metal ion and functional groups which inhibits bacteria, then it showed these activities. Adsorption was the main way between montmorillonite and bacteria. The special way of fixing bacteria into the "carriage" of montmorillonite gel which carry this structure was its pharmacological basis of curing diarrhea. The adsorption effect was related to layer charge density of the montmorillonites.

CONCLUSIONMontmorillonite showed adsorption ability of bacteria with minus related to its layer charge, but has no bacteriostatic and bactericidal effect.

Adsorption ; Anti-Ulcer Agents ; pharmacology ; Bentonite ; pharmacology ; Escherichia coli ; drug effects ; Staphylococcus aureus ; drug effects

Polymyxin E shows effective treatment of the infection induced by resistant gramnegative bacteria, but its nephrotoxicity severely limits the clinical application of this drug. In this work, methoxypolyethylene glycols 2000 (mPEG2K)-polymyxin E (PME) was synthesized via chemical grafting reaction and had been characterized. The antimicrobial activity and cytotoxicity of mPEG2K-PME in vitro were investigated on Escherichia coli and HK-2 cells, separately. Intra-abdominal infection model was further established in order to study the therapeutic effect and the toxic effect on kidney of mice. The results showed that mPEG2K-PME exhibited significant inhibitory effect on Escherichia coli and had a lower toxicity on HK-2 cells in vitro. At the same time, mPEG2K-PME had a good efficacy in the treatment of Escherichia coli infected mice in vivo. Moreover, nephrotoxicity caused by mPEG2K-PME was significantly reduced compared to free PME. mPEG2K-PME is promising in development of new preparations with high efficiency and low toxicity.
Animals ; Cell Line ; Colistin ; pharmacology ; toxicity ; Escherichia coli ; drug effects ; Escherichia coli Infections ; drug therapy ; Humans ; Kidney ; cytology ; drug effects ; Mice ; Polyethylene Glycols ; chemistry

OBJECTIVETo investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.

METHODSMurine macrophage J774A cells were co-cultured with TcpC producing wild type E. coli strain CFT073 (TcpC(wt)) or tcpc gene-deleted CFT073 mutant (TcpC(mut)) in Transwell system, respectively. Apoptosis of J774A cells co-cultured with TcpC(wt) or TcpC(mut) was analyzed by Annexin/PI double staining. The levels of reactive oxygen species (ROS) in J774A cells were determined by DCFH-DA staining after treatment with TcpC(wt) or TcpC(mut) at 6 h, 12 h,24 h or 36 h. After the ROS was scavenged by N-acetylcysteine (NAC), the changes of J774A cell apoptosis were also examined. The expression of caspase-3 in J774A cells co-cultured with TcpC(wt) or TcpC(mut) in the presence or absence of 0.1 mmol NAC was detected by Western blot.

RESULTSJ774A cells co-cultured with TcpC(wt) for 24 h or 36 h showed significantly increased apoptosis (27.39% ± 4.05% and 28.45% ± 4.55%,respectively) when compared to control group (7.96% ± 1.63% and 10.55% ± 1.44%,P<0.01) or TcpC(mut) group (11.45% ± 2.77% and 19.26%± 2.89%,P<0.01). Levels of ROS in J774A cells treated with TcpC(wt) for 24 h (108.8 ± 9.73) or 36 h (100.3 ± 10.11) were significantly higher than those in control group (56.8 ± 4.11 and 52.8 ± 4.42,P<0.01) or TcpC(mut) (69.7 ± 5.66 and 62.6 ± 4.56, P < 0.01). The pro-apoptotic effects of TcpC(wt) on J774A cells were reversed by 0.1 or 1 mMol NAC treatment. Expression of caspase-3 in J774A cells co-cultured with TcpC(wt) (0.43 ± 0.04) decreased significantly when compared to control group (0.75 ± 0.08,P<0.05) or TcpC(mut) group (0.80 ± 0.12,P<0.05). However,total caspase-3 expression was restored in J774A cells co-cultured with TcpC(wt) in the presence of 0.1 mmol NAC (0.80 ± 0.09).

CONCLUSIONTcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.

Acetylcysteine ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Reactive Oxygen Species ; metabolism ; Virulence Factors ; pharmacology

OBJECTIVE7 variable but nonculturable-state strains of Enterotoxigenic Escherichia coli (ETEC) during the routine bacterial subculture were found in our lab and their morphology and antigen studied. Biological features, antigens and pathogenicity of the revertants were also tested and compared to that of the initial strains in order to detect their variations.

METHODSBiological variations between the variable but nonculturable-state and the revertant of every strain were detected, using the routine gram-staining, reverting the isolates in animal intestinal, reverting their pathogenicity, serological agglutination, biochemical identifications and antibiotic resistance tests.

RESULTSFor the 7 variable but nonculturable-state strains of ETEC,other than the trains that had changed into sphero vegetale cells, there were no other obvious variations found. However, high pathogenicity of these strains still remained.

CONCLUSIONThe presence of variable but nonculturable-state strains suggested that the routine method of bacteria storage should be changed and more attention should be paid to realize the existence of this kind of bacteria during the routine surveillance of the communicable diseases.

Antigens, Bacterial ; Drug Resistance, Bacterial ; Enterotoxigenic Escherichia coli ; drug effects ; immunology ; pathogenicity ; Microbiological Techniques

OBJECTIVETo discuss the possible mechanism of drug resistance transmission between Staphylococcus and Escherichia coli.

METHODSThe chloramphenicol resistance plasmid of Staphylococcus aureus was extracted to transform the sensitive Escherichia coli, and the drug-resistant Escherichia coli were screened by drug sensitivity test.

RESULTSThe drug-resistant Escherichia coli were successfully obtained.

CONCLUSIONStaphylococcus may have a natural shuttle plasmid of drug resistance, which can transform Escherichia coli under specific conditions.

Drug Resistance, Bacterial ; genetics ; Escherichia coli ; drug effects ; genetics ; Plasmids ; Staphylococcus ; genetics ; Transformation, Bacterial

We analyzed the fluoroquinolone resistance mechanism of 28 isolates of ciprofloxacin-resistant E. coli from patients who received ciprofloxacin as a regimen of a selective gut decontamination. Isolates distinctive by infrequent restriction site polymerase chain reaction (IRS-PCR) were subjected to Hinf I restriction fragment length polymorphism analysis, single-stranded conformation polymorphism (SSCP), and nucleotide sequencing of the quinolone resistance determining region (QRDR) in gyrA. Double mutations in QRDR of gyrA (Ser83 Leu and Asp87Asn) were found from most of the strains. Nucleotide sequencing of the marR locus showed that 18 out of 28 (64%) ciprofloxacin-resistant E. coli strains had three types of base change in marR loci: a double-base change at nucleotides 1628 and 1751, or 1629 and 1751: and a single-base change at 1751. However, all the mutated strains showed no tolerance to cyclohexane test, suggesting the mutation in the marR region had no influence on overexpression of the MarA protein. In conclusion, mutation in gyrA was the main mechanism of ciporfloxacin resistance in E. coli from patients with selective gut decontamination. Therefore, mutation in the mar region did not influence the levels of ciprofloxacin resistance in our isolates.
Ciprofloxacin/pharmacology* ; DNA Topoisomerase (ATP-Hydrolysing)/genetics* ; Drug Resistance, Microbial/genetics* ; Drug Resistance, Multiple/genetics* ; Escherichia coli/genetics ; Escherichia coli/drug effects* ; Human ; Mutation/physiology

The worldwide use of antimicrobials in different fields has created enormous pressure for the selection of resistance among opportunistic bacterial pathogen. One hundred four E. coli isolates were collected and identified from swine with diarrhea in Korea during the period of 2002. The isolates showed highly resistant to streptomycin (99. 0%), tetracycline (97. 1%), neomycin (91. 3%)and carbenicillin (84. 6%)in antimicrobial susceptibility test. Moreover, all of the isolates showed multiple antimicrobial resistant to more than 3, and 85%of them were resistant to more than 7 of total 14 antimicrobial agents. In comparison with isolates in 1998, resistance to antimicrobials was more frequent among the isolates in 2002. Presence of class 1 integrons was investigated through amplification of the gene with PCR, and could be classified 8 groups by pattern of 4 different amplicons. Class 1 integrons were observed in 67 strains (64. 2%)of E. coli from swine in Korea. One and 1. 6 kbp of amplicons were revealed to contain aadA1 and aadB-aadA1 gene cassettes respectively. Two kbp of amplicon had three different gene cassettes, dhfrXII-orfF-aadA2, and 3. 0 kbp of amplicon includes aadB-cmlA1 gene cassettes.
Animals ; Anti-Bacterial Agents ; Diarrhea/microbiology/veterinary ; Drug Resistance, Multiple, Bacterial/*genetics ; Escherichia coli/*drug effects/genetics ; Escherichia coli Infections/microbiology/*veterinary ; Integrons/*genetics ; Swine ; Swine Diseases/*microbiology

OBJECTIVETo investigate the epidemiological status of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) and the drug resistance profiles of such organisms.

METHODSA total of 282 clinical isolates of E. coli and 180 of K. pneumoniae were collected from different districts of Zhejiang Province. Inhibitor potentiated broth dilution tests were performed for detecting extended-spectrum beta-lactamases. Etests were performed to detect the drug resistance of these strains against nine commonly used antibiotics.

RESULTSThe prevalence of extended-spectrum beta-lactamases in E. coli and K. pneumoniae was 34.0% and 38.3%, respectively. The average prevalence of extended-spectrum beta-lactamases in E. coli and K. pneumoniae was 35.7%. The resistance prevalence of extended spectrum beta-lactamase producing strains to ceftazidime and cefotaxime was 40% and 26% respectively, so were those to cefepime, cefoxitin, piperacillin-tazobactam, cefoperazone-sulbactam, amikacin and ciprofloxacin. All these strains were sensitive to imipenem.

CONCLUSIONThe results in this study showed that the prevalence of extended-spectrum beta-lactamases was high, while extended-spectrum beta-lactamase producing strains were resistant to most antimicrobial agents except imipenem.

Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; Microbial Sensitivity Tests ; beta-Lactamases ; biosynthesis