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Objective: To investigate the association between phage-mediated shiga toxin and molecular distribution of CRISPR in Escherichia ( E. ) coli O26∶H11 or NM. Methods: A total of 135 E. coli O26 ∶ H11 or NM strains were collected from NCBI database. Software CRT and CRISPR Finder were used to extract CRISPR and Excel was used to assign the spacer of unique number and type CRISPR. And the relationship between CRISPR and stx phage was analyzed. Results: All the 135 E. coli O26 ∶ H11 or NM strains had the CRISPR. For CRISPR1, CRISPR2.1, CRISPR2.2 and CRISPR3-4, 19, 22, 1 and 1 subtypes were found, respectively. According to the four CRISPR sites, the strains could be divided into 40 subtypes. Stx-phage was only observed in the group C of CRISPR. Compared with E. coli of stx-phage negative, E. coli with stx-phage harbored more spacers. Conclusions: CRISPR loci was extensively existed in E. coli O26∶H11 or NM, and many subtypes were found in these strains. The presence of stx-phage was related to the molecular distribution of CRISPR in E. coli O26∶H11 or NM. CRISPR might be a valuable biomarker to identify strains with high virulent potential.

Objective: To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study. Methods: Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes ( tetA , tetB , tetC , tetD ) and beta-lactam resistance genes ( blaSHV -1, blaCTX - M , blaTEM ). Results: Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes ( tetA , tetB and tetC ) were detected among the 16 strains. Conclusion: The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.

Population genomics, an interdiscipline of genomics and population genetics, is booming in recent years with the rapid growth number of deciphered genomes and revolutionizes the understanding of bacterial population diversity and evolution dynamics. It also largely improves the prevention and control of infectious disease through providing more accurate genotyping and source-tracing results and more comprehensive characteristics of emerging pathogens. In this review, taking one of the best characterized bacteria, Escherichia coli, as model, we reviewed the phylogenetic relationship across its five major populations (designated A, B1, B2, D and E); and summarized researches on molecular mutation rate, selection signals, and patterns of adaptive evolution. We also described the application of population genomics in responding against large-scale outbreaks of E. coli O157:H7 and E. coli O104:H4. These results indicated that, although being a novel discipline, population genomics has played an important role in deciphering bacterial population structures, exploring evolutionary patterns and combating emerging infectious diseases.

Objective: To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation., Methods: A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain., Results and Conclusion: We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.

Objective: To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics., Methods: The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining., Results and Conclusion: We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

Objective: To express and identify enterohemorrhagic Escherichia coli (EHEC) O157:H7 Shiga toxin 1 A subunit (Stx1A)., Methods: Stx1A encoded gene was amplified from EHEC O157:H7 genome by PCR, confirmed by sequencing and cloned into vector pET-22b(+). The recombinant plasmid pET-22b(+)-Stx1A was transformed into E.coli BL21(DE3) which was induced by IPTG to express the target protein. After purified by AKTA(TM);-His affinity chromatography, the recombinant protein was identified by mass spectrometry. With the recombinant protein, BALB/c mice were immunized to develop the anti-sera and evaluate its specific reaction with the natural Stx1A by Western blotting., Results: The Stx1A gene with a size of 945 bp was amplified and cloned into prokaryotic expression vector pET22b(+) to form pET-22b(+)-Stx1A. The recombinant protein was effectively expressed in E.coli BL21(DE3) and purified by 6×His-based affinity chromatography. The mass spectrometry analysis showed that the target protein was Stx1A. Western blotting demonstrated that its immunized sera could react specifically with the natural Stx1A., Conclusion: The EHEC O157:H7 Stx1A gene was successfully cloned and expressed, which laid a solid foundation for the following researches.

Objective: To 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., Methods: Pulsed-field gel electrophoresis (PFGE) was performed according to the PulseNet protocol with minor modifications. A dendrogram was constructed using the BioNumerics., Results: Under 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., Conclusion: The non-O157 STEC isolates in China were highly polymorphic. PulseNet protocol seemed to be suitable for the typing of Chinese non-O157 STEC isolates.

Aim: To prepare translocated intimin receptor-cytoskeleton coupling protein (TccP) of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and its polyclonal antibody., Methods: TccP was amplified from the genome of EHEC O157:H7 Sakai strain by PCR and used to construct the recombinant prokaryotic expression vector pET28a-TccP. The recombinant vector was transformed into E.coli BL21( DE3) to express the protein in the bacteria under the induction of isopropy-D-thiogalactoside (IPTG). After purification, the protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by ELISA and Western blotting for its sensitivity and specificity. The rabbit anti-TccP polyclonal antibody was then applied in the study on the localization of TccP within the host cells adhered by EHEC O157:H7., Results: The sequence of TccP cDNA we amplified was the same as reported by GenBank. The recombinant prokaryotic expression vector pET28a-TccP was constructed successfully. Western blotting revealed that M(r); of the target protein expressed in E.coli BL21(DE3) was 37 000 and the rabbit anti-TccP polyclonal antibody had a specific reaction with the target protein, which demonstrated that the recombinant protein and its polyclonal antibody were prepared successfully. Immunofluorescence detection using rabbit anti-TccP polyclonal antibody showed that TccP aggregated in the cell membrane of the host cell adhered by EHEC O157:H7., Conclusion: We successfully prepared the recombinant vector pET28a-TccP and the anti-TccP polyclonal antibody and applied the antibody to confirm the localization of TccP in EHEC O157:H7 adhesion host cells.

Objective: To develop an assay for the simultaneous detection of 7 common foodborne pathogens with xMAP liquid chip in a single-tube reaction., Methods: Seven specific primers and probes were designed and synthesized based on the target gene sequences from GenBank. Target bacterial sequences were amplified by asymmetric PCR. The biotinylated products were hybridized to seven probe beads in a multiplex reaction and analyzed by using streptavidin conjugated to a fluorescent reporter molecule. The developed liquid chip xMAP assay was used to test 140 strains of bacteria and then 56 food samples., Results: No cross-reaction and false signals were observed. The detection limit was 1 - 100 pg and 10(5) - 10(6) cfu/ml. The results tested by xMAP were in accordance with the traditional culture method., Conclusion: The processing of xMAP liquid chip assay, including DNA preparation and sample detection, could be finished within 3.5 hours and could be applied to the classification and identification of foodborne pathogens in the food safety monitoring.

Objective: To investigate the presence of tellurite resistance (ter) gene cluster and its resistance in Escherichia coli 0157 strains collected by national foodborn disease surveillance system from 2005 to 2007., Methods: The ter gene cluster was tested by PCR. Tellurite resistance was evaluated by plate dilution method., Results: Of the investigated 89 E. coli strains,the presence of ter gene cluster was found in 51 strains. They were separately from 41 of 42 E. coli O157: H7 strains,all of 6 strains of O157 : NM (non-mobile flicH7 positive) and only 3 of 41 O157: NM (flicH7 negative)/O157: hund strains. The 51 strains with ter gene cluster were resistance to the high level of potassium tellurite. The MIC was from 64-512 microg/ml. In contrast, the MIC of most of 38 strains without ter gene cluster was from 2-16 microg/ml. Only one strain was 128 microg/ml. 28 of 29 stx-positive E. coli isolates contained the ter gene cluster and had the high levels of tellurite resistance., Conclusion: E. coli 0157 from food had the diversity levels of tellurite resistance. The presence of the ter gene cluster had significant associated with the high levels of tellurite resistance.

Objective: To identify suspected Escherichia coli O157 strains collected by national foodborne disease surveillance system from 2005 to 2007, as well as to investigate their virulence gene' s distribution in different kinds of food., Methods: All suspected strains were primarily tested by biochemical test strips API20E, and typed by agglutination with O/H serum, the virulence genes were detected by PCR., Results: (1) A total of 154 strains were identitied as Escherichia coli by API20E. (2) A total of 89 strains from 154 suspected strains were identified as Escherichia coli O157 by serotype test and PCR of special gene, of them 42 were O157:H7 (47%) and the rest were O157: NM and O157: hund (3) Virulence genes test by PCR: all of 42 O157: H7 strains and 6 O157: NM strains carried eaeA and hlyA: 28 sorbitol-non fermenting O157: H7 strains carried stx with a total of 29 positive ones. (4) The strains carrying stx gene of E. coli O157: H7 were identified in different groups of food, such as mutton, beef, pork, chicken and salad., Conclusion: E. coli O157 strains with different virulence genes were isolated from parts of the surveyed food. From now on, research should be carried out to enhance the surveillance of E. coli O157 not only in food but also from environment in order to provide more scientific data for establishing effective measures of protection.

Objective: To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories., Methods: Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method., Results: The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection., Conclusion: LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.

Objective: To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157 : H7 (EHEC O157 : H7) Shigela toxin 2B subunit (Stx2B) and vibrio cholera toxin B subunit (CTB) as well as to detect the immunogenicity and GM1-binding ability of fusion protein., Methods: To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified, then to transform constructed plasmid into E. coli BL21 (DE3) induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDS-PAGE and Western-blot., Results: The amplified ctb-stx2b fragments appeared to be 750 bp and gene sequence was identical to designed sequence. The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about M(r) 20 x 10(3) and the expressed protein could react to CTB monoclone anti-body. The fusion protein CTB-Stx2B could bind GM1., Conclusion: CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability. This study provided information on further EHEC O157 : H7 vaccine research.

Objective: To understand the epidemiological characteristics of enterohaemorrhagic Escherichia coli (EHEC) O157 and to determine the degree of its genetic relations., Methods: Polymerase chain reaction (PCR) techniques and chromosomal DNA digested by restriction enzyme Xba I according to PulseNet directions by pulsed field gel electrophoresis (PFGE) method were applied to 300 E. coli O157 strains isolated from patients and animal sources from 1988 to 2005 from Henan, Jiangsu and Anhui provinces., Results: Very high prevalence of stx2 gene in EHEC O157:H7 strains isolated from some provinces of China was found and variation existed in some strains. We got 161 PFGE patterns from 300 strains. The stx2-producing strains could be clearly separated from stx2 variation-producing strains., Conclusion: The variability of restriction enzyme-digestion patterns of O157 genomes suggested that the presence of some genomic diversity among the strains did exist.

Objective: To understand the variation of Shiga toxin (stx) genes of Escherichia coli O157:H7 strains isolated in China., Methods: Polymerase chain reaction (PCR) was used to identity the types of stx genes and the nucleotide sequences of the amplified stex variants genes were determined. Compare to the cytotoxicity of Stx,variants were tested by HeLa cell assay., Results: We found novel stx2 genes in 3 of 289 strains of Shiga toxin-producing E. coli O157:H7 isolated from 1999 to 2002 in China. The novel stx2 genes were inserted by a 1.3-kb insertion sequence (IS) and the nucleotide sequences of IS showed 100% homology with that of IS1203 variant (IS1203v). The IS1203v inserted in the stx2 genes of three E. coli O157:H7 strains at different sites and the direction of the open reading frames (ORFs) of IS1203v of each strain was different. In addition to the above mentioned findings, the nucleotide sequences of three stx2 genes were completely identical and the type of the three Stx2 was Stx2 prototype. Compare to the cytotoxicity of Stx2 prototype, the novel Stx2 was found to be obviously lower., Conclusion: E. coli O157:H7 strains harboring stx2::IS1203v genes were isolated in China. Consequently, the results of HeLa cell assay showed that the insertion of IS1203v could lead to low cytotoxicity of Stx2.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen. One of the important virulence traits of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property encoded by a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE contains ler (LEE-encoded regulator) gene. The product of ler is a central up-regulator of many virulence genes of the LEE. Another important virulence factor of EHEC O157: H7 is Shiga toxin (Stx), encoded by a prophage integrated into the chromosome of O157:H7. In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157: H7 was constructed by use of suicide vector pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. A ler/stx deletion mutant of EHEC O157:H7 was constructed, termed as O157:H7(deltaler/deltastx). The cultural supernatant of O157 ler/stx deletion mutant was inoculated in vero cell culture, and the result indicating that O157 ler/stx deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the O157 ler/stx deletion mutant and the virulent strain O157:H7 EDL933, respectively. Mice were observed daily for clinical signs and weight changes. After inoculation of the deletion mutant, test group of mice (inoculated with O157:H7(deltaler/deltastx)) gained weight normally and experienced no clinical signs. In contrast, control group of mice (inoculated with O157: H7) exhibited weight loss and all died in four days. In another experiment, pregnant mice were orally vaccinated by O157:H7(deltaler/ deltastx) twice at interval of 14 days. Subsequently, the suckling mice were orally challenged with O157:H7 EDL933 at 7 days of age. The results showed that 78.34% of the sucking mice born by vaccinated mice were survival and 12.73% of the sucking mice born by non-vaccinated mice were survival. This study demonstrated that O157 ler/stx deletion mutant lost the toxigenicity to vero cell and to be safety to mice. Oral immunization can induce specific immune responses, and this mutant strain could be used as an attenuated vaccine candidate against EHEC O157.

Objective: To study the change of Escherichia coli O157:H7 resistance to disinfectants in continuous disinfection and the relationship between the resistance and pO157, chromosome DNA., Methods: Using 4 disinfectants in a separate manner to disinfect 5 Escherichia coli O157:H7 50 generations continuously, we made a before-after comparison of their resistibility and analyzed the change of the structure of pO157 and chromosome DNA., Results: After the 50-generation-continuous disinfection, the bacteria resistance to sodium dichloroisocyanurate, iodophor and quaternary ammonium increased, but the resistance to chlorhexidiniacetas did not any change; the maps Cal I and Rsr II cutting pO157 revealed some changed after disinfection by sodium dichloroisocyanurate and quaternary ammonium, but the maps showed no change after disinfection by iodophor and chlorhexidiniacetas; the chromosome DNA PFGE maps change considerably after 50 generations of disinfection by sodium dichloroisocyanurate, iodophor and quaternary ammonium, but the chromosome DNA PFGE maps were similar after 50 generation of disinfection by chlorhexidiniacetas., Conclusion: The Escherichia coli O157:H7 resistance to disinfectants will increase after the 50-generation-continuous disinfection by sodium dichloroisocyanurate, iodophor and quaternary ammonium. There may be genes both in chromosome DNA and in pO157 which resist sodium dichloroisocyanurate and quaternary ammonium, the reason for increase of resistance may be related with the change of chromosome DNA and pO157. There may be genes in chromosome DNA which resist iodophor, the reason for increase of resistance to iodophor may be related with the change of chromosome DNA, but not related with pO157.

Objective: To study epidemiological characteristics of Escherichia coli O157:H7 isolated in Sichuan., Methods: Multiplex PCR was used to analyze the slt1, slt2, eaeA, and hly gene. Plasmid profile and PFGE was utilized to investigate the homology among the strains. Drug and disinfectant resistance patterns of 67 stains were determined., Results: The overall virulence gene prevalence was 62.7% (42/67); and the main virulence gene type was slt1 + slt2 + eaeA + hly. Of the 67 Escherichia coli O157: H7 strains, 5 plasmid profile and 6 PFGE types were acquired. 64.2% (43/67) Escherichia coli O157:H7 strains resisted to 7 antibiotics, namely, Ampicilin, Chloramphnicol, Tetracyline, Gentamicin, Cephlothin, SMZ, TMP-SMZ, and there were 23, 35, 34 strains resisted to Ampicilin, Tetracyline and SMZ respectively. The resistance types were different in Escherichia coli O157: H7 strains isolated from humans, animals, food, and environment. 80.6% (54/67) Escherichia coli 0157:H7 strains resisted to alcohol and quaternary ammonium, but all the stains were sensitive to chlorhexidiniacetas., Conclusion: The virulence gene prevalence rate was familiar of 67 Escherichia coli O157: H7 Strains isolated from Sichuan, but virulence gene type was not the same in different sources. Bacterial drug resistance was high. Bacterial resistance to common disinfectants was so high that the strains could be killed by big dosage disinfectants. There were some familiarity with drug resistance, plasmid profile and PFGE types of 67 Escherichia coli 0157:H7 strains isolated from different sources, which indicated homology of the strains.

Objective: To type and group the Shiga-toxin producing Escherichia coli O157:H7 strains isolated recent years in China to understand the epidemiological features caused by the pathogen., Methods: Pulsed field gel electrophoresis of large restriction fragments of bacterial chromosomal DNA was used., Results: The 51 isolates of E. coli O157:H7 collected in recent years in China could be divided into 8 Pulsed Field Gel Electrophoresis (PFGE) types based on the size and number of restriction fragments and patterns, that were digested by XbaI. Strains isolated from Ningxia province showed only two types- PFGE1 and PFGE2. Strains isolated in Xuzhou of Jiangsu province had 6 PFGE types. Isolates identified between 1986 - 1988 belonged to PFGE7. Strains isolated from patients in 1999 - 2000 were PFGE5 and PFGE3. Strains isolated from stool samples of domestic animal, food and vegetable were PFGE3 - 6, of which the predominant type was PFGE5. All of the 5 strains isolated from patients with diarrhea and hemolytic uremic syndrome (HUS) belonged to PFGE type 5, which was the dominant type of the isolates from stool samples of domestic animal and samples of food and vegetable contaminated., Conclusion: Data suggested that the cluster patients with diarrhea and HUS might have been related to the pathogens from domestic animas and contaminated food or vegetables. The distribution of PFGE types also varied in different provinces of China.

Objective: To detect and characterize the virulence genes in E. coli O157:H7 isolated from various reservoir in six areas of Jiangsu province., Method: The virulence genes of Shiga-like toxin (SLT(1) and SLT(2)), intimin (eaeA) and hemolysin (hlyA) were chosen as the target genes and amplified in multiplex PCR assays., Results: Of the eighty-five E. coli O157:H7 strains, the overall virulence gene prevalence was found to be 56.5% (48/85). The prevalence rates virulence genes of isolates from various areas were different from 0% up to 90.5%. It seemed to exist a relationship between the virulence gene prevalence and the level of incidence. In the areas where rates of incidence were divided into high, low, sporadic or zero, the prevalence rates were 85.7% (36/42), 52.6% (10/19) and 8.3% (2/24), respectively. The prevalence rates of isolates were also different from various reservoirs, decreasing by sheep, cattle, pig and poultry. One isolate from a rabbit was positive for SLT(2), eaeA and hly genes. Of forty-eight isolates carrying virulence genes, 38 (79.2%) had SLT(2), eaeA and hly genes, taking the dominate virulence gene pattern, 8 (16.6%) had all of the four virulence genes 2 (4.2%) had both SLT(2) and hly genes respectively. In addition, SLT(1) gene showed a lower prevalence, which was different from some findings abroad., Conclusion: Since virulence gene pattern of E. coli O157:H7 is an important molecular epidemiological marker, it can provide an useful information for epidemiologic studies, and helpful to the design of prevention and control strategies. For virulence gene detection, multiplex PCR seems to be a simple, rapid, specific and sensitive method.