Background: aaP, aggR, and astA have been found to play important roles in diarrheal pathogenecity of EAEC. They may be exist in other diarrheagenic E.coli (DEC). Objectives: To determine the distribution of aaP, aggR, and astA in ETEC, EPEC, EIEC and non-diarrheagenic E.coli. Subjects and method: 75 strains of ETEC, EPEC, EIEC and 100 non-DEC have been screen by PCR with primers specific toaaP, aggR, and astA. Results: aaP, aggR, and astA have been seen in DEC with the prevence from 7 to 72,7%. The highest prevence was in EIEC, 72,7% for aap; 45,5% in EIEC for aggR; and 50% in ETEC for astA. 14% of non-DEC harbored aggR and more than 30% harbored aap and astA. Conclusion: This finding has contributed to understanding the distribution of aap, aggR and astA in ETEC, EPEC, EIEC and non-DEC as well.
Enterotoxigenic Escherichia coli ; Enteropathogenic Escherichia coli ; Escherichia coli ;

BACKGROUND: We investigated the prevalence of various pathogens (13 enteric bacteria and 5 viruses) which cause diarrhea using multiplex PCR of stool specimens and compared two multiplex PCR methods for detecting diarrheagenic Escherichia coli. METHODS: A total of 405 stool specimens submitted between November 2010 to February 2011 for routine culture of enteric pathogens were included and screened for five viruses (astrovirus, Group A rotavirus, enteric adenovirus, norovirus G1/G2) and eight bacteria (Salmonella spp., Shigella spp., Campylobacter spp., Vibrio spp., C. difficile Toxin B, C. perfringens, Y. enterolytica, Aeromonas spp.) using the Seeplex(R) Diarrhea ACE detection kit (Seegene). In addition, virulence-associated genes of enteropathogenic E. coli, (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, (EIEC), enterotoxigenic E. coli (ETEC), and enteroaggressive E. coli (EAEC) were detected using 16-plex PCR and a commercial diarrheagenic E. coli detection (DEC) PCR kit (SSI Diagnostica). RESULTS: Overall, 138 (34.1%) of 405 samples was positive for pathogen. The positive rate for virus was 18.5%. norovirus G2, Group A rotavirus, enteric adenovirus, astrovirus and norovirus G1 were detected in 40, 23, 8, 3 and 1 samples, respectively. The positive rate for bacteria was 24.4% (99/405). C. difficile toxin B was the most frequently detected, followed by C. perfringens, EPEC, and EAEC. The agreements of the two multiplex PCR methods for detecting EPEC and EHEC were 99.3% and 100%, respectively. CONCLUSION: The detection rate was high (34.1%) including various diarrheagenic E. coli (6.2%) and C. perfringens (5.2%). Multiplex PCR is thus useful for detecting various pathogens.
Adenoviridae ; Aeromonas ; Bacteria ; Campylobacter ; Diarrhea ; Enterobacteriaceae ; Enterohemorrhagic Escherichia coli ; Enteropathogenic Escherichia coli ; Enterotoxigenic Escherichia coli ; Escherichia ; Escherichia coli ; Multiplex Polymerase Chain Reaction ; Norovirus ; Polymerase Chain Reaction ; Prevalence ; Rotavirus ; Shigella ; Vibrio ; Viruses

Verotoxin-producing Escherichia coli O157 is a primary cause of severe and bloody diarrhea. Campylobacter spp. are one of the commonly reported bacterial cause of gastrointestinal infections throughout the world. Only a few cases involving both E. coli O157 and Campylobacter species have been reported. The authors simultaneously isolated verotoxin-producing E. coli O157 and Campylobacter species from the stool of a 3 year-old male with bloody diarrhea, fever and abdominal pain.
Abdominal Pain ; Campylobacter* ; Child, Preschool ; Diarrhea ; Escherichia coli O157* ; Escherichia coli* ; Escherichia* ; Fever ; Gastroenteritis* ; Humans ; Male ; Shiga Toxins ; Shiga-Toxigenic Escherichia coli

BACKGROUND: The Polymerase Chain Reaction (PCR) for the Shiga toxin has been widely used for diagnosis of Shiga toxin-producing Escherichia coli (STEC) infection including Escherichia coli O157 (O157) instead of using a culture. However, bacteriological isolation must be followed for final diagnosis. Our study was aimed to compare the detection limit between the culture after the acid treatment and the PCR after enrichment. METHODS: The standard strain of O157 was cultured, diluted and mixed with the stool of normal adult in order to make a final concentration of the 10(1)-10(5) colony forming unit (CFU)/g of stool. Each concentration of samples was enriched in a trypticase soy broth for 6 hours at 42degrees C and treated with acid to suppress normal flora. Then it was streaked on cefixime-tellurite-sorbitol MacConkey (CT-SMAC) agar evenly and cultured for 18 hours at 37degrees C. The same concentrations of bacterial suspension in the stool were enriched in a Luria-Bertani (LB) broth overnight at 37degrees C. The centrifuged pellets from 1 mL of each concentration of the samples were boiled and DNA was extracted using the resin method and PCR was performed to amplify stx2. RESULTS: The detection limit for the culture after acid treatment was 10(3) CFU/g of the stool and that for PCR after enrichment was 101 CFU/g of the stool. CONCLUSIONS: Culture after acid treatment for O157 would be an effective method for isolation of O157 from a patient's stool. However, this method is less sensitive than the PCR after enrichment as far as detection limit is concerned. A combination of both methods would be an effective method for detecting O157 from patient stools.
Adult ; Agar ; Diagnosis ; DNA ; Escherichia coli O157* ; Escherichia coli* ; Escherichia* ; Humans ; Limit of Detection ; Polymerase Chain Reaction* ; Shiga Toxin ; Shiga-Toxigenic Escherichia coli ; Stem Cells

Attaching and effacing Escherichia coli (AEEC) cause enteric infections in humans and animals. Attaching indicates the intimate attachment of bacteria to the enterocyte, and effacing relates to the localized effacement of brush border microvilli. Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) infections are characterized by the formation of attaching and effacing (AE) lesion on the intestinal epithelial cells. Therefore, they are often grouped together as AEEC. Development of multiplex PCR allowed us to type five of the most important genes implicated in the formation of the AE lesion. A total of 60 AEEC strains isolated from diarrheal patients were investigated by multiplex PCR for the presence of the insertion site of locus of enterocyte effacement (LEE) and LEE-related (eae, tir, espA, espB, and espD) genes. Associating the results of LEE genes typing in the AEEC strains, three different pathotypes are determined: eae(gamma)-tir(gamma)-espA(gamma)-espB(gamma)-espD(gamma) (O157:H7), eae(beta)-tir(beta)-espA(beta)-espB(beta)-espD(beta) (O26:H11), and eae(alpha)-tir(alpha)-espA(alpha)-espB(alpha)-espD(alpha) (O55:H6). These results indicate that AEEC are a heterogenous groups of organisms.
Animals ; Bacteria ; Enterocytes* ; Enterohemorrhagic Escherichia coli* ; Enteropathogenic Escherichia coli* ; Epithelial Cells ; Escherichia coli ; Humans ; Microvilli ; Multiplex Polymerase Chain Reaction

Escherichia coli O157 is an important serotype of enterohemorrhagic E. coli that causes hemorrhagic colitis worldwide. Outbreaks of E. coli O157 have been assocoated with contaminated food like meat, raw milk, and water, but recently vegetables and fruits have accounted for a growing number of recognized outbreaks. We isolated verotoxin producing E. coli O157 from the stool of a 3 year-old female with bloody diarrhea and abdominal pain. The child had been eating salad with vegetables and fruits frequently.
Abdominal Pain ; Child ; Colitis ; Diarrhea ; Disease Outbreaks ; Eating ; Enterohemorrhagic Escherichia coli ; Escherichia ; Escherichia coli ; Escherichia coli O157 ; Female ; Fruit ; Humans ; Meat ; Milk ; Shiga Toxins ; Vegetables

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

Aims: Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. Methodology and results: A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces. Conclusion, significance and impact of study The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).
Escherichia coli O157 ; Crassostrea

OBJECTIVE: To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7. METHODS: The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains. RESULTS: At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion. CONCLUSIONS We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.
Escherichia coli O157 ; Escherichia coli Proteins ; Polyphosphates

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