OBJECTIVETo investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.

METHODSThe Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.

RESULTSAfter EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).

CONCLUSIONSEPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Apoptosis ; drug effects ; Caco-2 Cells ; Eicosapentaenoic Acid ; pharmacology ; Escherichia coli ; pathogenicity ; Humans ; Intestinal Mucosa ; metabolism ; microbiology ; RNA, Messenger ; analysis ; Tight Junctions ; drug effects ; Tumor Necrosis Factor-alpha ; secretion ; Zonula Occludens-1 Protein ; genetics

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

INTRODUCTIONIncreasing resistance in Escherichia coli and Klebsiella pneumoniae to firstline antibiotics makes therapeutic options for urinary tract infections (UTIs) challenging. This study investigated the in vitro efficacies of 6 antibiotics against multidrug resistant (MDR) uropathogens.

MATERIALS AND METHODSMinimum inhibitory concentrations to ceftibuten, cefpodoxime, fosfomycin, mecillinam, temocillin, and trimethoprim were determined against 155 MDR-isolates of E. coli and K. pneumoniae. The presence of extended-spectrum beta-lactamases (ESBL) and plasmid-borne AmpC enzymes was determined by phenotypic testing with genotyping performed by multiplex polymerase chain reaction.

RESULTSTemocillin demonstrated highest susceptibility rates for both E. coli (95%) and K. pneumoniae (95%) when breakpoints for uncomplicated UTIs were applied; however, temocillin susceptibility was substantially lower when "systemic infection" breakpoints were used. Fosfomycin demonstrated the best in vitro efficacy of the orally available agents, with 78% and 69% of E. coli and K. pneumoniae isolates susceptible, respectively. The next most effective antibiotics were ceftibuten (45%) and mecillinam (32%). ESBL and ampC genes were present in 47 (30%) and 59 (38%) isolates.

CONCLUSIONThis study demonstrated few oral therapeutic options for MDR-uropathogens, with fosfomycin demonstrating the best in vitro activity.

Amdinocillin ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Ceftizoxime ; analogs & derivatives ; pharmacology ; Cephalosporins ; pharmacology ; Drug Resistance, Multiple, Bacterial ; genetics ; Escherichia coli ; drug effects ; genetics ; Escherichia coli Infections ; microbiology ; Fosfomycin ; pharmacology ; Genotype ; Humans ; In Vitro Techniques ; Klebsiella Infections ; microbiology ; Klebsiella pneumoniae ; drug effects ; genetics ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Penicillins ; pharmacology ; Singapore ; Trimethoprim ; pharmacology ; Urinary Tract Infections ; microbiology ; beta-Lactamases ; genetics

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.
Animals ; Anti-Bacterial Agents/pharmacology ; Cattle/microbiology ; Drug Resistance, Multiple, Bacterial ; Electrophoresis, Gel, Pulsed-Field/veterinary ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Female ; Genes, Bacterial/genetics ; Latex Fixation Tests/veterinary ; Microbial Sensitivity Tests/veterinary ; Multilocus Sequence Typing/veterinary ; Prevalence ; Republic of Korea/epidemiology ; Shiga Toxin 1/genetics ; Shiga Toxin 2/genetics ; *Shiga-Toxigenic Escherichia coli/drug effects/genetics

We aimed to observe antimicrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections. Two hundred sixty-eight E. coli strains were obtained from outpatients with various infections at different polyclinics at the 82nd Year of State Hospital in Rize, Turkey. Susceptibility to antimicrobials was tested using a disk diffusion method. The presence of integrons was examined using PCR with specific primers. Positive PCR results were confirmed by sequencing. A broth mating method was used for conjugation assays. Extragenic palindromic-PCR was performed using the oligonucleotide primer BOXA1R. Resistance frequency for ampicillin, trimethoprim/sulfamethoxazole, and tetracycline was determined as 50.6%, 33.5%, and 36.8% respectively. No strains were resistant to amikacin. Seventy isolates were positive for the intI1 gene, of which 49 carried gene cassettes. Eleven isolates were positive for the intI2 gene, eight of which carried gene cassettes. Seven gene cassettes (dfrA1, dfrA5, dfrA7, dfrA17, aadA1, aadA5, and sat2) were predominantly harbored in integrons. We detected conjugative plasmids harboring integrons in two E. coli strains. Four strain clusters were yielded by BOX-PCR fingerprints showing that they were clonally related. No apparent relationship occurred among class 1 and 2 integron-carrying strains. We conclude that integrons are widespread in genetically variable E. coli strains and will continue to mediate dissemination of resistance genes in the community.
Anti-Bacterial Agents/*pharmacology ; Community-Acquired Infections/*microbiology ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Escherichia coli/*drug effects/isolation & purification ; Escherichia coli Proteins/*genetics ; Humans ; Integrases/genetics ; Polymerase Chain Reaction ; Turkey

The tissue-specific and MeJA-induced transcriptional levels of BcUGT3 and BcUGT6 in Bupleurum chinense were analyzed in the present study. The transcriptional levels of BcUGT3 in root, leaf, flower and fruit were similar and they all were higher than those in stem. The transcriptional level of BcUGT6 was the highest in leaf and the lowest in flower among in all tested tissues. With non-treated adventitious roots as control, BcUGT6's transcriptional levels were elevated to nearly 2 folds for 2 h, 8 h, 24 h, 2 d and 4 d in MeJA-treated adventitious roots of B. chinense. It showed that the transcriptional level of BcUGT6 was slightly affected by MeJA. While, BcUGT3's transcriptional levels were gradually elevated, and till 4 d after MeJA treatment, the expression level was about 7 folds than that of non-treated control. Using pET-28a (+), the expressions of two genes was investigated. Induced by IPTG, the target proteins were expressed in E. coli and then purified. All the results obtained in the present study will be helpful for follow-up bio-function analysis of BcUGT3 and BcUGT6.
Acetates ; pharmacology ; Bupleurum ; cytology ; enzymology ; genetics ; Cell Membrane ; metabolism ; Cyclopentanes ; pharmacology ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation, Plant ; drug effects ; Hexosyltransferases ; chemistry ; genetics ; isolation & purification ; metabolism ; Intracellular Space ; metabolism ; Oxylipins ; pharmacology ; Protein Sorting Signals ; Protein Structure, Secondary ; Protein Transport ; Sequence Analysis ; Transcription, Genetic ; drug effects

OBJECTIVETo construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system.

METHODThe 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI.

RESULTPlant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%.

CONCLUSIONGenetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

Anti-Bacterial Agents ; pharmacology ; Biomarkers ; Cinnamates ; pharmacology ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mannose-6-Phosphate Isomerase ; genetics ; metabolism ; Plants, Genetically Modified ; drug effects ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; metabolism ; Transformation, Genetic

We investigated the occurrence and genetic basis of AmpC beta-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum beta-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 microg/mL, respectively. The MIC values for carbapenem were > or =2 microg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics ; DNA, Bacterial/genetics ; Enterobacteriaceae Infections/*epidemiology/*microbiology ; Escherichia coli/drug effects/enzymology/isolation & purification ; Hospitals, University/statistics & numerical data ; Humans ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification/*physiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Proteus mirabilis/drug effects/enzymology/isolation & purification ; Republic of Korea/epidemiology ; beta-Lactamases/*genetics

Succinic acid production by fermentation from biomass, especially the lignocellulosic hydrolysate, is an alternative to chemical synthesis. Many studies report the inhibition of cell growth and succinic acid production from lignocellulosic hydrolysate, hardly is known about the actual kinetic and mechanism of the inhibition of individual factors. In this study, we studied inhibition effects of furfurals and 5-hydroxymethylfurfural (5-HMF) on cell growth and succinic acid production of engineered E. coli. Cell growth and succinic acid titer were severely inhibited by furfural and HMF with both concentrations higher than 0.8 g/L. Cell growth was totally inhibited when the concentration of furfural was above 6.4 g/L, or the concentration of HMF was above 12.8 g/L. At the concentration of maximum toleration, which was 3.2 g/L, furfural decreased the cell mass by 77.8% and the succinic acid titer by 36.1%. HMF decreased the cell mass by 13.6% and the succinic acid titer by 18.3%. Activity measurements of key enzymes revealed that phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarate reductase all were inhibited by furfural and HMF. This study gave a quantitative view to the succinic acid production under the inhibition of lignocellulose degradation products and will help overcome the difficulties of the lignocellulosic hydrolysate fermentation.
Escherichia coli ; drug effects ; genetics ; metabolism ; Fermentation ; Furaldehyde ; analogs & derivatives ; pharmacology ; Industrial Microbiology ; methods ; Lignin ; metabolism ; Metabolic Engineering ; methods ; Succinic Acid ; metabolism

We report a case of CTX-M-55-type extended-spectrum beta-lactamase (ESBL)-producing Shigella sonnei infection in a 27-year-old Korean woman who had traveled to China. The patient was admitted to the hospital due to abdominal pain, watery diarrhea, and fever (39.3degrees C). S. sonnei was isolated from her stool specimens, and the pathogen was found to be resistant to cefotaxime due to CTX-M-55-type ESBL. Insertion sequence (IS)Ecp1 was found upstream of the blaCTX-M-55 gene. The blaCTX-M-55 gene was transferred from the S. sonnei isolate to an Escherichia coli J53 recipient by conjugation. Pulsed-field gel electrophoresis and Southern blotting revealed that the blaCTX-M-55 gene was located on a plasmid of approximately 130 kb.
Adult ; Anti-Bacterial Agents/pharmacology ; Asian Continental Ancestry Group ; Cefotaxime/pharmacology ; China ; Drug Resistance, Bacterial/drug effects ; Dysentery, Bacillary/diagnosis/*microbiology ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli/metabolism ; Feces/microbiology ; Female ; Humans ; Plasmids/chemistry/genetics ; Republic of Korea ; Shigella sonnei/enzymology/*isolation & purification ; Travel ; beta-Lactamases/genetics/*metabolism