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

Diagnosis of thrombotic microangiopathy (TMA) is challenging due to its close association with other forms of microangiopathic hemolytic anemia, such as malignant hypertension and disseminated intravascular coagulation, and because other manifestations including cytopenia and acute kidney injury are manifestations of other medical comorbidities. Further challenges for accurate diagnosis include distinguishing between primary and secondary TMA, as well as between hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). TTP is typically differentiated from HUS by the presence of more severe thrombocytopenia, along with a higher frequency of altered mental status with relatively preserved renal function. However, the clinical course can vary among patients, requiring polymerase chain reaction testing of patient stools for enterohemorrhagic Escherichia coli and a disintegrin and metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS13) assay. To reduce the mortality rate, prompt initiation of plasmapheresis is important in cases where TPP cannot be excluded. Future advances enabling more rapid testing for ADAMTS13 levels will reduce the need for unnecessary plasmapheresis, so that treatment strategy can be more optimized.
Acute Kidney Injury ; Anemia, Hemolytic ; Comorbidity ; Diagnosis ; Diagnosis, Differential ; Disseminated Intravascular Coagulation ; Enterohemorrhagic Escherichia coli ; Hemolytic-Uremic Syndrome ; Humans ; Hypertension, Malignant ; Mortality ; Plasma Exchange ; Plasmapheresis ; Polymerase Chain Reaction ; Purpura, Thrombotic Thrombocytopenic ; Thrombocytopenia ; Thrombospondins ; Thrombotic Microangiopathies

BACKGROUND/AIMS: To compare the epidemiological aspects of enterohemorrhagic Escherichia coli (EHEC) between Korea and Japan by analyzing the current state of EHEC infection outbreaks and related risk factors. METHODS: We investigated the epidemiological aspects of EHEC infection cases between Korea and Japan from 2006 to 2010. The following factors were analyzed: national prevalence rate (PR), regional prevalence rate, epidemic aspects (i.e., Cases related to gender), male to female morbidity ratio, age, and seasonal distribution. RESULTS: In total, there were 254 cases of EHEC with an average PR of 0.11 per 100,000 populations in Korea from 2006 to 2010. During the same period in Japan, there were 20,883 cases of EHEC with an average PR of 3.26 per 100,000 populations. The PR in Japan was significantly higher than that in Korea (p < 0.01). In both countries, more females than males had EHEC infections, with the highest incidence of infections (> 50%) observed for individuals younger than 9 years. EHEC is an emerging zoonosis and may be caused by consumption of raw or undercooked meat products from ruminants. CONCLUSIONS: This study provides a quantitative analysis of the epidemiological aspects and risk factors of EHEC infections in Korea and Japan and will provide insight on effective future strategies to reduce these infections.
Disease Outbreaks ; Enterohemorrhagic Escherichia coli* ; Epidemiologic Studies ; Female ; Humans ; Incidence ; Japan* ; Korea* ; Male ; Meat Products ; Prevalence ; Risk Factors ; Ruminants ; Seasons

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

PURPOSE: This study was performed to evaluate the compliance with, and adequacy of, the Korean national guidelines which had been recommended until 2011 for isolation of patients with group 1 nationally notifiable infectious diseases (NNIDs), namely cholera, typhoid fever, paratyphoid fever, shigellosis, and enterohemorrhagic Escherichia coli (EHEC) infection. MATERIALS AND METHODS: We evaluated the clinical and microbiological characteristics of confirmed cases of group 1 NNIDs and compliance with the guidelines in 20 Korean hospitals nationwide in 2000-2010. We also compared the Korean guidelines with international guidelines. RESULTS: Among 528 confirmed cases (8 cases of cholera, 232 of typhoid fever, 81 of paratyphoid fever, 175 of shigellosis, and 32 EHEC infections), strict compliance with the Korean guideline was achieved in only 2.6% to 50.0%, depending on the disease. While the Korean guidelines recommend isolation of all patients with group 1 NNIDs, international guidelines recommend selective patient isolation and screening for fecal shedding, depending on the type of disease and patient status. CONCLUSION: Compliance with the previous national guidelines for group 1 NNIDs in Korea was generally very low. Further studies are needed to evaluate whether compliance was improved after implementation of the new guideline in 2012.
Cholera ; Communicable Disease Control ; Communicable Diseases* ; Compliance* ; Dysentery, Bacillary ; Enterohemorrhagic Escherichia coli ; Guideline Adherence ; Humans ; Korea* ; Mass Screening ; Methods ; Paratyphoid Fever ; Patient Isolation ; Typhoid Fever

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

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in childhood and is primarily diagnosed in up to 4.5% of children who undergo chronic renal replacement therapy. Escherichia coli serotype O157:H7 is the predominant bacterial strain identified in patients with HUS; more than 100 types of Shiga toxin-producing enterohemorrhagic E. coli (EHEC) subtypes have also been isolated. The typical HUS manifestations are microangiopathic hemolytic anemia, thrombocytopenia, and renal insufficiency. In typical HUS cases, more serious EHEC manifestations include severe hemorrhagic colitis, bowel necrosis and perforation, rectal prolapse, peritonitis, and intussusceptions. Colonic perforation, which has an incidence of 1%-2%, can be a fatal complication. In this study, we report a typical Shiga toxin-associated HUS case complicated by small intestinal perforation with refractory peritonitis that was possibly because of ischemic enteritis. Although the degree of renal damage is the main concern in HUS, extrarenal complications should also be considered in severe cases, as presented in our case.
Acute Kidney Injury ; Anemia, Hemolytic ; Child* ; Colitis ; Colon ; Enteritis ; Enterohemorrhagic Escherichia coli ; Escherichia coli ; Hemolytic-Uremic Syndrome* ; Humans ; Incidence ; Intestinal Perforation* ; Intussusception ; Necrosis ; Peritonitis ; Rectal Prolapse ; Renal Insufficiency ; Renal Replacement Therapy ; Shiga Toxin ; Thrombocytopenia

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

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