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

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*

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

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.
Apoptosis ; drug effects ; Escherichia coli ; genetics ; Humans ; Leptin ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

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 study the genotype distribution of extended-spectrum beta-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E. coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.

METHODSE. coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011. The strains were identified by VITEK-II. The antibiol susceptibility tests were performed with K-B method. beta-lactamases genes were detected multi-PCR, PCR, sequence and blast.

RESULTSA total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection. There were 20 isolates produced TEM-1 type beta-lactamases and 1 isolate produced SHV-1 typebeta-lactamases. 40 clinical isolates were detected to produce CTX-M type ESBLs, there were 20 CTX-M-1 group and 26 CTX-M-9 group, including 6 stains habouring both CTX-M-1 and CTX-M-9 group. Eight CTX-M genotypes were confirmed by sequencing of the PCR products, including CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-28, CTX-M-31, CTX-M-65 and CTX-M-79.

CONCLUSIONCTX-M genotype ESBLs was the most popular extended-spectrum beta-lactamases in E. coli isolated from liver cirrhosis' patients with bloodstream infection. The CTX-M-14 is the dominant epidemic type.

Bacteremia ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Escherichia coli Proteins ; genetics ; Genotype ; Hospitalization ; statistics & numerical data ; Humans ; Liver Cirrhosis ; therapy ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics ; metabolism

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

Expression strain of des-pGlu1-brazzein was constructed and the conditions using lactose as inducer was also optimized. The Influences of three factors which were lactose concentration, induction time and inducing temperature on the growth of strain and on the yield of des-pGlul-Brazzein was analyzed in detail. The result indicated that high lactose concentration inhibit the growth of strains (P < 0.01) but made no difference on expression of target protein between 0.5%-5% (P > 0.05), Biomass would be improved as time passed (P < 0.01), but the yield of target protein didn't increase obviously at 30 degrees C compared with at 37 degrees C. Further result showed that the greater expressed level of des-pGlul-Brazzein, as high as about 20% of total cell protein, could be achieved after the strain had been induced with 0.5% lactose under 28 degrees C - 30 degrees C for 4 h.
Dose-Response Relationship, Drug ; Escherichia coli ; drug effects ; genetics ; Isopropyl Thiogalactoside ; pharmacology ; Lactose ; pharmacology ; Plant Proteins ; genetics ; Plasmids ; genetics ; Temperature ; Time Factors ; Up-Regulation ; drug effects

OBJECTIVETo investigate the effect and mechanism of baicalin against beta-lactamases Escherichia coli (ESBLs E. coli) mediated by toll-like receptor 4 (TLR4) signal transduction pathway.

METHODThe RAW264. 7 cells monolayer pretreated with different concentration of baicalin were inoculated with ESBLs E. coli. The expression of TLR4 mRNA and protein were analyzed by semi-quantitative RT-PCR and Immunofluorescence, respectively. The activity of NF-kappaB was detected by Western blot using total cellular protein. The production of TNF-alpha in supernatant was determined by enzyme linked immunosorbnent assay (ELISA).

RESULTESBLs E. coli significantly up-regulated the expression levels of TLR4 mRNA and protein in a time-dependent manner, induced the activation of NF-KB in RAW264. 7, enhanced the production of TNF-alpha in supernatant. Baicalin down-regulated the expression of TLR4 mRNA and protein, decreased the activation of NF-KB in RAW264. 7 cells and reduced the production of TNF-alpha in supernatant in a dose-dependent manner.

CONCLUSIONBaicailin could inhibit TLR4 signal transduction pathway. The mechanism of baicalin against ESBLs E. coli may be through inhibiting the expression of TLR4 and its signal transduction pathway.

Animals ; Cell Line ; Escherichia coli ; drug effects ; physiology ; Escherichia coli Infections ; drug therapy ; genetics ; immunology ; microbiology ; Flavonoids ; pharmacology ; Gene Expression ; drug effects ; Humans ; Mice ; Toll-Like Receptor 4 ; genetics ; immunology

This study aimed to provide evidence of persistent uropathogenic Escherichia coli (UPEC) in female patients with recurrent urinary tract infection (UTI) after antibiotic therapy. We collected biopsies of the bladder, and clean-catch urine samples from 32 women who had episodes of recurrent UTI and were given antibiotic therapy. Urine samples and biopsies were analyzed by conventional bacteriological techniques. Phylogenetic group and 16 virulence factors (VFs) of UPEC were determined using polymerase chain reaction (PCR). The infection capability of UPEC was confirmed in a mouse model. Immunofluorescence and electron microscopy were used to detect intracellular bacterial communities (IBCs) in the mouse model. The results showed that all urine specimens were detected sterile. E. coli was found in 6 of 32 biopsies (18.75%), and was identified to be UPEC by PCR. Different VFs associated with the formation of IBCs were identified in all six UPEC isolates. Each UPEC isolate was capable of forming IBCs within the bladder epithelial cells of mice. In conclusion, UPEC with distinctive pathological traits and the capability of IBC formation was first found in the bladders of women after antibiotic therapy, suggesting that the IBC pathogenic pathway may occur in humans and it plays an important role in UTI recurrence.
Adult ; Animals ; Anti-Bacterial Agents ; administration & dosage ; Biopsy ; Escherichia coli Infections ; drug therapy ; microbiology ; Female ; Humans ; Mice ; Middle Aged ; Phylogeny ; Urinary Bladder ; drug effects ; microbiology ; pathology ; Uropathogenic Escherichia coli ; drug effects ; genetics ; pathogenicity