Low-level release of Shiga-like toxin (verocytotoxin) and endotoxin from enterohemorrhagic Escherichia coli treated with imipenem

Shiga-like toxin (SLT) and endotoxin may participate in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) infection. Levels of release of SLT and endotoxin from EHEC treated in vitro with antibiotics were estimated. There were differential levels of release of SLT and endotoxin from EHEC treated with different antibiotics. Treatment of EHEC strains, namely, E. coli O157, O111, and O26, with imipenem induced much lower levels of release of SLT and endotoxin than treatment with ceftazidime.     

Abundance in sewage of bacteriophages that infect Escherichia coli O157:H7 and that carry the Shiga toxin 2 gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Acute and chronic diarrhoea and abdominal colic associated with enteroaggregative Escherichia coli in young children living in western Europe

BACKGROUND: Enteroaggregative Escherichia coli (EAggEC or EAEC) can spread and cause disease in developing countries, but it is not presently known whether it spreads disease in industrialised countries. Therefore, we did a prospective study to assess the incidence and the clinical manifestations of infections due to EAEC in children in Germany. METHODS: 798 children with diarrhoea, admitted to hospital within a defined geographical area during a 24-month period, were included in the trial. EAEC were cultured from stool specimens, screened by PCR, and identified by colony hybridisation from DNA sequences found on the virulence plasmid. The findings were confirmed by aggregative adherence to HEp-2 cells. Stool samples from 580 children admitted to hospital without diarrhoea were also studied as controls. FINDINGS: EAEC were found in the stools of 16 (2%) of 798 children with diarrhoea, but in none of 580 children without diarrhoea. Only four of the EAEC-infected children had travelled to developing countries. Most EAEC infections were acquired in the summer months. Infection with EAEC was associated with acute, watery diarrhoea in 12 children, and with chronic diarrhoea of up to 5 months' duration in four. Five children had abdominal colic that lasted for 2-4 weeks as their main symptom. The incidence of EAEC infection was 7.7 patients admitted to hospital per 100,000 children in the general population aged younger than 16 years. INTERPRETATION: EAEC infection is associated with acute, watery diarrhoea and may be acquired in industrialised countries. Chronic diarrhoea or abdominal colic of unknown aetiology in young children may also be caused by EAEC infection.     

Bordetella pertussis adenylate cyclase toxin: proCyaA and CyaC proteins synthesised separately in Escherichia coli produce active toxin in vitro

We analyzed the role of Fyn tyrosine kinase in cell cycle progression of B lymphocyte progenitor (pro B cell). Whereas there were no substantial defects in the intramarrow B cell genesis in the fyn(-) mouse, and long-term proliferation of fyn(-) pro B cells was maintained in vitro under a serum containing culture condition, the cell cycle was arrested at G2/M upon serum deprivation. Morphological analyses demonstrated that the cytokinesis of fyn(-) pro B cells was retarded in the presence of serum and that the entry of fyn(-) pro B cells into late telophase was completely blocked under the serum-free condition. In contrast, the earlier phases of mitosis of fyn(-) pro B cells proceeded normally without FCS. This failure to initiate late telophase resulted in the accumulation of elliptical binucleated cells that might be the outcome of the nuclear division without cytokinesis. Consistent with this defect in the progression of cytokinesis, Fyn was localized in the midspace of dividing pro B cells at anaphase. These results suggested that Fyn localizes at the midspace of dividing pro B cells and regulates the progression of cytokinesis.     

Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157:H7 in rural Wisconsin

The epidemiology and clinical aspects of Escherichia coli O157:H7 (E. coli O157:H7) infections in rural Wisconsin have rarely been reported. In the last six years, 66 cases of E. coli O157:H7 infection were encountered at our institution. Bloody diarrhea was the universal presentation and all cases represented apparent sporadic infection as institutional or community-wide outbreaks were not found in our study. The mean age was 31 (range 7 months to 86 years), 25% less than 10 years old and 60% were female. Most cases were seen in summer and early autumn (88%). Two patients (3%) developed hemolytic-uremic syndrome. Case-fatality rate in this study was 1.5%. Antibiotic treatment and hospitalization did not change the course and outcome of the infection. Routine screening of E. coli O157:H7 during winter time (December and January) may not be necessary in our rural area. The understanding gained from our study might foster better infection control.     

Distribution of serogroups of Vibrio cholerae non-O1 non-O139 with specific reference to their ability to produce cholera toxin, and addition of novel serogroups

A total of 1898 strains of Vibrio cholerae non-O1 non-O139, which had been collected worldwide for the past 3 year period of 1994-1996, were serogrouped. The strains were also examined for presence of cholera toxin (CT) gene (ctx) and NAG-ST gene, and strains which carried to ctx were further analyzed for their ability to produce CT. In addition, attempts were made to establish novel serogroups for those serologically untypable strains. Of those examined, 1,774 strains of V. cholerae non-O1 non-O139 was classified into 128 known serogroups while 50 strains were found to belong to R type, and the rest of the 74 strains could not be serotyped. Distribution of the serogroups did not seem to correspond to either the strains geographic distribution or sources of isolation. Of those serologically untypable strains, 38 novel serogroups (O156-O193) were established and added to our reference of V. cholerae antigenic schema. It was also found that antisera raised against many V. cholerae strains included R antibodies. This indicates that any V. cholerae antisera for diagnostic purpose should be absorbed with the reference R strains, CA385, before use. There were luminescence producing strains among those sucrose and VP reaction negative strains. Subsequent DNA/DNA homology analysis revealed that they were identified as V. cholerae. This points to a possibility that strains tentatively identified as Vibrio mimicus by conventional biochemical tests may have included luminescent strains of V. cholerae. It is thus highly recommended that strains in question should be tested for the luminescence production in order to differentiate V. cholerae from V. mimicus. Of those 1989 strains examined, 37 strains (ca. 2%) were found to produce CT. Interestingly, CT producing strains were prevalent in serogroup O141; 10 strains out of 16 strains (63%) were positive for CT. The evidence calls for a caution to possible occurrence of cholera-like diarrhea caused by V. cholerae O141 in the future.     

Application of multiplex PCR for detection of non-O157 verocytotoxin-producing Escherichia coli in bloody stools: identification of serogroups O26 and O111

Primers were designed to amplify sequences of verocytotoxin genes and eaeA genes of Escherichia coli O26:H11, O111:H8, and O157:H7 in a multiplex PCR assay. This assay successfully detected E. coli O26:H11 in bloody stool specimens in which other enteric pathogens were not detected by culture-based methods. Rapid assays to detect non-O157:H7 verocytotoxin-producing E. coli is important to improve methods for the etiologic diagnosis of hemorrhagic colitis.     

Influence of incubation conditions on survival and acid tolerance response of Escherichia coli O157:H7 and non-O157:H7 isolates exposed to acetic acid

OBJECTIVE: Recently a few cases of long QT syndrome were reported during treatment with cisapride. In most of these cases, risk factors for cardiac arrhythmias or pharmacologic interactions might have been involved, and the role of cisapride remained unclear. Macrolides such as clarithromycin potentially interact with the metabolic elimination of cisapride and have overlapping indication areas. We therefore studied whether combined treatment with clarithromycin and cisapride leads to pharmacokinetic changes and increased QT intervals. METHODS: The study was an open, randomized, 2-way crossover study with washout periods of 1 week. Twelve healthy volunteers were recruited. Treatments were cisapride (10 mg 4 times a day) for 10 days with concomitant clarithromycin (500 mg twice a day) from days 6 through 10, or clarithromycin (500 mg twice a day) for 10 days combined with cisapride (10 mg 4 times a day) from days 6 through 10. Frequent ECG recordings were performed for 24 hours before drug treatment (baseline). After 5 days of monotherapy and combination therapy, frequent ECG recordings and assessments of plasma drug levels were performed for 24 hours. RESULTS: Clarithromycin alone was associated with a minimal increase in QTc intervals. Monotherapy with 10 mg cisapride 4 times a day led to a concentration-dependent QTc elevation, amounting to 6 ms during steady state. Combination of cisapride and clarithromycin caused an average QTc increase of 25 ms above pretreatment values and 3-fold increases in cisapride concentrations. CONCLUSIONS: QTc elevations after cisapride or clarithromycin alone remained within the normal range of diurnal variation. Coadministration of cisapride and clarithromycin produced a substantial QT prolongation. The data support the recently purported interaction between cisapride and clarithromycin and thus the filed contraindication to combine these drugs.     

The maintenance of Plasmid-containing organisms in populations of Escherichia coli

Populations of Escherichia coli containing a small non-conjugative plasmid were grown in carbon-limited continuous culture. For all plasmids tested the presence of the plasmid lowered the growth rate of the host bacterium, and the proportion of plasmid-containing organisms in the total population declined initially. However, periodically, adaptive changes occurred in plasmid-containing organisms which increased their growth rate. This resulted in oscillations in the proportion of plasmid-containing organisms, and the delayed loss of the plasmid from the population.     

An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA

1. We describe the first application of microdialysis to monitor the pharmacokinetics of a drug in the blood of man. 2. The aims of the study were to ascertain patient acceptability and tolerability of a new microdialysis probe and to assess its accuracy in determining the pharmacokinetics of levodopa and its principal plasma metabolite 3-O-methyldopa (3-OMD). 3. Eight patients with parkinsonism on chronic levodopa therapy were investigated. 4. After an overnight fast, a flexible microdialysis probe, perfused with isotonic saline, was inserted into a forearm vein and a blood sampling cannula was inserted in a forearm vein of the other arm. After ingestion of a levodopa preparation (Madopar Dispersible), dialysate was collected over 5 or 10 min periods and blood samples were taken every 15 or 30 min for 2-6 h. 5. Dialysate drug profiles were similar to those of plasma, and levodopa and 3-OMD concentrations exhibited significant (P < 0.001) correlation with those observed in the corresponding plasma samples. 6. The mean (+/- s.d.) blood dialysate concentrations for levodopa and 3-OMD were 36.1 +/- 9.2% and 43.4 +/- 8.4% respectively of the plasma content. 7. The tolerability of the probe was excellent, and all eight patients found it preferable to conventional blood sampling. 8. Microdialysis of blood is less invasive than frequent intermittent direct blood sampling, and can readily be used to continuously monitor levodopa pharmacokinetics. In a clinical setting, a combination of drug monitoring by this technique together with clinical evaluation of motor function can be used to optimize levodopa treatment in patients with Parkinson's disease.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

Enterohemorrhagic Escherichia coli O157 outbreak in Obihiro-city--biological and molecular characteristics among O157 isolates]

The outbreak of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in Obihiro City, Japan, occurred in late October 1996. The infection affected a total of 169 kindergarten pupils and school staff members in a private kindergarten. Twenty-one children (12.4%) progressed into hemolytic uremic syndrome (HUS). Moreover, the person-to-person infections in 9 families and the duration of excretion of EHEC in 13 patients were observed. The contaminated food was identified as the potato-salad served at lunch. Analysis of biological characteristics, the ability of toxin production, and the DNA analysis by PCR-based fingerprinting, the RAPD tests, among all clinical isolates, clarified a homologous origin of contamination.     

Isolation and characterization of sorbitol-fermenting Shiga toxin (Verocytotoxin)-producing Escherichia coli O157:H- strains in the Czech Republic

Two sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H- strains were isolated from patients with hemolytic-uremic syndrome in the Czech Republic in 1995. Their phenotypic and genotypic characteristics and genomic DNA fingerprints were identical or closely related to those of SF STEC O157:H- strains isolated in Germany in 1988 to 1997. This indicates that the Czech isolates belong to the SF STEC O157 clone which is widespread in Germany. It is the first finding of the clone outside Germany.     

Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.     

Present status and advances in detecting Shiga toxin-producing Escherichia coli O157

Currently, detection of Shiga toxin-producing Escherichia coli(STEC) in stool samples is based on the isolation method in most clinical laboratories. The procedures are as follows: i) isolation with selective agar plates, ii) biological test with differential media, iii) serological test of anti-O antisera, iv) detection of toxin or toxin gene. These procedures take 4 days, therefore more rapid method is required. In the near future, a rapid detection method that detects STEC directly from stool samples will be introduced. Polymerase-chain reaction (PCR), enzyme-linked immuno-sorbent assay (ELISA), detection of serum anti-O157 antibodies are now available in clinical laboratories. Result of PCR for detection Shiga toxin gene and serum anti-O157 antibodies are described. Fifteen stool and serum samples from patients suspected of STEC infection were examined. With the culture and PCR method, 2 patients were positive by both methods and the results were confirmed in both cases. Six patients were positive by the antibodies detection method. From these results, the PCR method using stool samples was useful as a rapid detection method in clinical laboratories. Detection of serum antibodies has been simplified and is not an expensive method. Therefore, the method is useful for clinical diagnosis of STEC infection, especially, for diagnosing HUS or after antimicrobial agents have been administered to patients.     

Rapid detection of Shiga-like toxin gene in feces with rapid-cycle polymerase chain reaction

A rapid detection for Shiga-like toxin in feces was developed with the nucleic acid extraction method by silicondioxide-guanidine thiothianate and rapid-cycle polymerase chain reaction by RapidCycler (model 1002; Idaho Technology, RC-PCR here after). Twenty-two fecal samples that were collected from patients with diarrhoea caused by E. coli O157:H7 and frozen for 6 months were examined directly by RC-PCR, conventional PCR assay using by ThermalCycler 9600-R (Roche, TC-PCR here after) and by the culture method using tellurite-cefixime sorbitol MacConkey (direct method). These examinations were done also after being injected into TCV-TSB and incutated at 35 degrees C overnight (indirect method). The sensitivity of RC-PCR and TC-PCR using a diluted suspension of broth enriched at 35 degrees C overnight were 4.1 pg and 410 fg, respectively. Positive results in the direct method were obtained in 7 for RC-PCR, 10 for TC-PCR and 5 for culture. Positive results on indirect assay were obtained in 9 for RC-PCR, 9 for TC-PCR and 7 for culture. It was demonstrated that the RC-PCR assay was able to detect Shiga-like toxin gene in feces in less than 90 minutes after being received at the laboratory.     

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC

OBJECTIVES: The purpose of this study was to survey the efficiency of visible light curing units in dental practices across Australia. METHODS: Survey forms were distributed to representatives of 3M Health Care to complete when visiting dentists in their working areas. The information collected included the type and age of the unit, curing times used, history of maintenance, replacement of components, and the light intensity reading. RESULTS: Of the 214 light curing units surveyed, approximately 27% recorded a light intensity of 200 mW cm-2 or less, a level regarded as inadequate to cure a 2-mm thick increment of composite resin. An additional 26% registered an output of between 201 and 399 mW m-2. This level would be considered acceptable with additional curing time; however, 44% of practitioners were curing for 20 s or less. A negative correlation was found between the age of the unit and the intensity recorded. Nearly 50% of respondents had never checked the light output of their unit. CONCLUSIONS: The results indicate that just over one-half of the light curing units surveyed were not functioning satisfactorily. An obvious reduction in intensity was noted with the older units. There is a substantial lack of awareness among dentists of the need for maintenance and regular checking of the light intensity of these units.     

Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.     

Recovery of thermally-stressed Escherichia coli O157:H7 by media supplemented with pyruvate

Unheated and heat-stressed (57 degrees C, 50 min and 60 min) cells of Escherichia coli O157:H7, were enumerated using three media supplemented with 1% sodium pyruvate (NaPyr): plate count agar (PCA), tryptic soy agar (TSA) and phenol red sorbitol agar (PhRSA) using the spread plate method. The medium recovering the greatest numbers of severely heated E. coli O157:H7 was PCA with 1% NaPyr. Recovery of heat stressed E. coli O157:H7 on this medium was significantly higher (P < 0.05) than the two other media with pyruvate: 16.3% (50 min heating) and 0.55% (60 min heating) of the total population was recovered with TSA + 1% NaPyr when compared to those numbers found on PCA + 1% NaPyr. The ability of PhRSA + 1% NaPyr to recover heat-stressed E. coli O157:H7 was similar to that of TSA + 1% NaPyr. Using PhRSA + 1% NaPyr media. 12.9% (50 min heating) and 0.61% (60 min heating) of the total population were recovered when compared with the cells enumerated on PCA + 1% NaPyr. Recovery of the heat-stressed cells using the spread plate method was greater than using pour plate method. Recovery was significantly higher (P < 0.05) on the spread plates for highly stressed E. coli O157:H7(1.2 log) heated for 60 min than on the pour plates. Overall, the populations on the TSA spread and pour plates were low compared with the same heat-stressed cells recovered on media containing pyruvate. The     

Immunomagnetic separation with mediated flow injection analysis amperometric detection of viable Escherichia coli O157

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.