HSP2 recombinant in BL21 (DE3) pLysS strain was expressed at high level, but had varied in variable conditions from 22 - 370C. After simple steps of processing, HSP2 in soluble form could be purified by ion-exchange chromatography through CM-sepharose on FPLC system
Escherichia coli ; proteins ; isolation & purification

Animals ; Cattle ; Chickens ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; isolation & purification ; pathogenicity ; Sheep ; Swine

Child ; China ; Diarrhea ; diagnosis ; microbiology ; Escherichia coli Infections ; diagnosis ; Humans ; Shiga-Toxigenic Escherichia coli ; isolation & purification

OBJECTIVETo understand the distribution of virulence gene and the epidemiological characteristics of diarrheagenic Escherichia(E.) coli (DEC) from diarrheal patients in Beijing.

METHODSStool specimens from diarrheal patients were cultured which were collected from the hospitals under sentinel surveillance program, during 2012-2013. DNA was examined by real-time PCR.

RESULTS253 out of 6 370 specimens were positive for DEC detection with the rate as 4.0%. A total number of 262 DEC strains were isolated. Two different pathotypes of DEC strains with mixed infection, were isolated from 9 specimens. Different pathotypes would show the following profiles: 42.8% for enteropathogenic E. coli (EPEC) including 42.0% atypical and 0.8% typical; 38.9% for enterotoxigenic E. coli (ETEC) including 24.8% st positive, 9.9% lt positive and 4.2% st and lt both positive;15.3% for enteroaggregative E. coli(EAEC);2.7% for enteroinvasive E. coli (EIEC); one strain STEC with serotype O26:K60. ETEC had obvious characteristics on age. All kinds of DEC were isolated throughout the year with seasonal fluctuation.

CONCLUSIONDEC isolates from diarrheal patients in Beijing were dominated by EPEC and ETEC, with atypical ones accounted for the majority of EPEC. One specimen was found under mixed infection. Pathotypes DEC were found to have different age and seasonal distributions.

China ; epidemiology ; Diarrhea ; microbiology ; Enteropathogenic Escherichia coli ; genetics ; isolation & purification ; Enterotoxigenic Escherichia coli ; genetics ; isolation & purification ; Epidemics ; Escherichia coli ; genetics ; isolation & purification ; Escherichia coli Infections ; epidemiology ; microbiology ; Genotype ; Humans ; Virulence

OBJECTIVETraditional detection approaches for non-O157 STEC are both time and labour consuming in diseases surveillance. Virulence genes detection based on multiplex PCR could not only improve the detection efficiency but also increase the accuracy.

METHODSSix virulence genes of non-O157:H7 (stx1, stx2, eae, hly, etpD, katP6) were detected by two groups of trebling PCRs. The multiplex PCRs were optimized by melting curve analysis in SYBR Green I real-time PCR. Testing result of multiplex PCR was consistent with serological testing.

RESULTSThe sensitivity limits of the multiplex PCR for stx1, stx2, eaeP, etpD, katP, and hly were 10 ng/ml, 120 ng/ml, 110 ng/ml,165 ng/ml, 85 ng/ml, and 15 ng/ml, respectively, which is similar with that of single PCR. When the multiplex PCR was applied in 120 adults and 90 children diarrhea samples detection, 13 cases were detected for non-O157 positive.

CONCLUSIONThe method we established can be used for non-O157 STEC virulence genes detection and screening with high efficiency and accuracy.

Escherichia coli Infections ; diagnosis ; microbiology ; Escherichia coli Proteins ; genetics ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Shiga-Toxigenic Escherichia coli ; genetics ; isolation & purification ; Virulence Factors ; genetics

OBJECTIVETo establish a database and to understand the molecular epidemiological features of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from different animal reservoirs and patients.

METHODSPulsed-field gel electrophoresis (PFGE) was performed according to the PulseNet protocol with minor modifications. A dendrogram was constructed using the BioNumerics.

RESULTSUnder the PulseNet protocol, 62 PFGE patterns were obtained from 76 non-O157 STEC isolates and then divided into A to M groups. Isolates from different sources were widely distributed in different groups, but were predominant seen in certain groups.

CONCLUSIONThe non-O157 STEC isolates in China were highly polymorphic. PulseNet protocol seemed to be suitable for the typing of Chinese non-O157 STEC isolates.

Animals ; China ; epidemiology ; DNA, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; genetics ; isolation & purification ; Feces ; microbiology ; Genotype ; Humans ; Shiga-Toxigenic Escherichia coli

Escherichia coli ; classification ; genetics ; isolation & purification ; Escherichia coli Infections ; urine ; Humans ; Urinary Tract Infections ; microbiology ; urine ; Urine ; microbiology

China ; epidemiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; isolation & purification ; metabolism ; Humans ; Shiga Toxin 1 ; biosynthesis ; isolation & purification ; Shiga Toxin 2 ; biosynthesis ; isolation & purification ; Sorbitol

OBJECTIVETo evaluate the detective efficacy of Chromogenic Coliform and Escherichia Coli Agar (CCEA).

METHODSA new chromogenic medium CCEA prepared by Huankai laboratory was used to compare with a classical medium of violet red bile agar (VRBA), and other two Chromogenic media Agar I and Agar II by detecting separately 11 reference strains, thirteen sterile samples with Coliform or E.coli and other four samples, and the accordant rates of detection were observed.

RESULTSCCEA had the good selectivity. To seven kinds of quality strains in the resultant analysis, CCEA with VRBA and Agar I had not shown salience difference (P > 0.05), and CCEA with Agar II had significant difference (P < 0.05). CCEA showed more advantages than the Agar II. To thirteen sterile samples with Coliform or E.coli in resultant analysis, CCEA with Agar I and Agar II had shown no significant difference (P > 0.05), while CCEA with VRBA had significant difference (P < 0.05). CCEA might be more advantageous than the VRBA. In analysis of the four actual samples of Coliform, CCEA with VRBA, Agar I and Agar II showed no significant difference (P > 0.05). The accordant rates were 90%, 71.88%, 86.25% and 81.25% respectively, showing CCEA > Agar I > Agar II > VRBA. To two actual samples of E.coli in the resultant analysis, the CCEA with Agar I and Agar II had not shown significant difference (P > 0.05). The accordant rates were 100% respectively.

CONCLUSIONSThe CCEA might be more advantageous than the VRBA, having the same efficacy as with Agar I and Agar II.

Bacteriological Techniques ; Culture Media ; Enterobacteriaceae ; isolation & purification ; Escherichia coli ; isolation & purification

Annexin A5 ; biosynthesis ; isolation & purification ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Recombinant Proteins ; biosynthesis ; isolation & purification