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 construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation.

METHODSA 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 CONCLUSIONWe 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.

Carrier Proteins ; genetics ; DNA Primers ; Escherichia coli O157 ; genetics ; Escherichia coli Proteins ; genetics ; Gene Deletion ; Plasmids ; Polymerase Chain Reaction

OBJECTIVETo construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.

METHODSThe 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 CONCLUSIONWe 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.

DNA Primers ; Escherichia coli O157 ; genetics ; Escherichia coli Proteins ; genetics ; Gene Deletion ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction

OBJECTIVETo investigate the possible roles of adenosine and the cytokines TNF-alpha and IL-10 in the pathogenesis of acute bacterial prostatitis (ABP) in rats.

METHODSForty-eight male Wistar rats were randomly divided into groups A (ABP), B (ABP + theophylline intervention), C (sham) and D (blank control). ABP models were established by injecting Escherichia coli 0157 into the prostate, and those in group B were treated by intraperitoneal injection of theophylline immediately after modeling. At 4 and 14 days, the prostate tissues of the rats were collected for detection of the expressions of TNF-alpha and IL-10 by immunohistochemistry and the concentration of adenosine by high-performance liquid chromatography.

RESULTSAt 4 and 14 days, the concentrations of adenosine were significantly higher in group A ([48.38 +/- 17.27] and [26.54 +/- 11.22] microg/g) than in C ([0.45 +/- 0.25] and [0.46 +/- 0.29] microg/g) and D ([0.41 +/- 0.23] and [0.43 +/- 0.27] microg/g) (P < 0.05), and so were the expressions of TNF-alpha in A (0.23 +/- 0.08 and 0.21 +/- 0.03) than in C (0.07 +/- 0.03 and 0.07 +/- 0.01) and D (0.07 +/- 0.06 and 0.07 +/- 0.06) (P < 0.05), and those of IL-10 in A (0.13 +/- 0.03 and 0.25 +/- 0.01) than in C (0.07 +/- 0.03 and 0.07 +/- 0.03) and D (0.07 +/- 0.01 and 0.07 +/- 0.02) (P < 0.05). Compared with group A, the rats in group B showed significant increases at 4 and 14 days in the severity of inflammation, concentration of adenosine ([86.64 +/- 32.87] and [51.17 +/- 22.96] microg/g, P < 0.05) and expression of TNF-alpha (0.37 +/- 0.08 and 0.32 +/- 0.06, P < 0.05), but exhibited no remarkable difference in the expression of IL-10 (0.12 +/- 0.06 and 0.15 +/- 0.06, P > 0.05).

CONCLUSIONAdenosine may affect the progression of inflammation by regulating the expressions of the cytokines TNF-alpha and IL-10 in ABP rats through the adenosine receptor signaling pathway.

Adenosine ; physiology ; Animals ; Escherichia coli O157 ; Interleukin-10 ; metabolism ; Male ; Prostate ; drug effects ; metabolism ; Prostatitis ; metabolism ; microbiology ; Random Allocation ; Rats ; Rats, Wistar ; Theophylline ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

In the event of an infectious disease outbreak in cattle, carcasses must be disposed of in a rapid and contained manner. This brief communication details injection of a barbiturate to euthanize cattle inoculated with Escherichia coli O157:H7 followed by carcass composting in a manner that prevents the spread of infectious agents.
Animals ; Cattle ; Cattle Diseases/*microbiology ; Disease Outbreaks/*veterinary ; Escherichia coli Infections/microbiology/*veterinary ; *Escherichia coli O157 ; Euthanasia, Animal/*methods ; Hypnotics and Sedatives/administration & dosage/pharmacology ; Male ; Pentobarbital/administration & dosage/*pharmacology ; Soil

OBJECTIVESThis study is to monitor the survival of E. coli O157:H7 in the aquatic microcosm from Han River and explore the feasibility of fluorescence staining, heterotrophic plate count and ELISA in detection of viable but nonculturable E. coli O157:H7.

METHODSE. coli O157:H7 were added into aquatic microcosm from Han River and cultured with low temperature and oligo-nutrition. Then the survival of E. coli O157:H7 were real-time monitored by acridine orange direct count (AODC), direct viable count (DVC)-CTC staining, DVC-nalidixic acid (NA) staining, heterotrophic plate count (HPC) and ELISA.

RESULTSE. coli O157:H7 can be converted to a viable but nonculturable state in the aquatic microcosm from Han River 58 days after cultured at 4°C with oligo-nutrition. The amount of viable E. coli O157:H7 was measured as 1.2 × 10(5) CFU/ml by DVC-CTC and 9.0 × 10(4) CFU/ml by DVC-NA, whereas the amount of culturable bacterial determined by HPC is 0. The amounts of bacteria determined by ELISA are basically stable within 58 days around 10(6) CFU/ml.

CONCLUSIONE. coli O157:H7 can be converted into a viable but nonculturable state in Han River water at 4°C with oligo-nutrition, and ELISA combined with fluorescence staining and heterotrophic plate count can be used in quantitative detection of the viable but nonculturable E. coli O157:H7.

Colony Count, Microbial ; Culture Media ; Escherichia coli O157 ; isolation & purification ; physiology ; Microbial Viability ; Rivers ; microbiology ; Water Microbiology

OBJECTIVETo establish a rapid method of loop-mediated isothermal amplification (LAMP) for detecting enterohemorrhagic Escherichia coli (EHEC) O157:H7.

METHODSSix primers that specifically recognized the rfbE gene of EHEC O157:H7 were designed. Under the optimized reaction conditions, LAMP and PCR were evaluated for the sensitivity and specificity in the detection of 39 laboratory samples of EHEC O157:H7 strains, and their detection results of contaminated fresh pork samples were compared.

RESULTSLAMP assay correctly identified all the 7 EHEC O157:H7 strains and showed negative results for all the 32 non-EHEC O157:H7 strains. The detection limit of LAMP was much lower than that of rfbE-PCR (10 vs 100 cfu/ml). In the detection of the contaminated pork samples, both LAMP and PCR yielded results consistent with those by the conventional detection method.

CONCLUSIONThe rfbE-based LAMP assay can serve as a rapid, sensitive, specific and low-cost means for detecting EHEC O157:H7 strain.

DNA, Bacterial ; Escherichia coli O157 ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.
Animals ; Argentina/epidemiology ; Brazil/epidemiology ; Cattle ; Cattle Diseases/epidemiology/*microbiology ; Enterohemorrhagic Escherichia coli/genetics/*isolation & purification/pathogenicity ; Escherichia coli O157/*genetics/*isolation & purification/pathogenicity ; Food Microbiology ; Gene Expression Regulation, Bacterial/physiology ; Genetic Markers ; Humans ; Polymerase Chain Reaction/veterinary ; Shiga Toxin 1/genetics/metabolism ; Shiga Toxin 2/genetics/metabolism ; Virulence

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.
Abattoirs ; Animals ; Cattle ; Escherichia coli O157/*genetics/isolation & purification ; *Immunomagnetic Separation ; Meat/microbiology ; *Polymerase Chain Reaction ; Rectum/microbiology ; Shiga Toxin 1/*genetics ; Shiga Toxin 2/*genetics ; Turkey