Escherichia coli STb toxin binding to sulfatide and its inhibition by carragenan

Escherichia coli heat-STb is an important cause of diarrhea in piglets. STb was shown to interact specifically with sulfatide (3'-sulfogalactosyl-ceramide) present on the surface of epithelial cells of piglet jejunum. Basic data are lacking on STb binding to sulfatide in solution and more precisely on the possible inhibition of this interaction. Using surface plasmon resonance technology, we compare binding of STb to sulfatide and other glycoshingolipids previously shown, with a multiplate-binding assay, to also interact to various degrees with the enterotoxin. In addition, inhibition of STb-sulfatide binding was studied using free galactose, galactose-sulfate residues and a polymer of sulfated galactans known as carragenan. We determined a dissociation constant of 2.4±0.61 nM for the STb-sulfatide interaction. These data indicated that STb was binding to sulfatide with greater affinity than previously determined using radiolabeled toxin. Much lower affinities were observed for lactoceramide and glucoceramide. The binding of STb to sulfatide was clearly inhibited by λ-carragenan but not by galactose, 4-SO₄-galactose or 6-SO₄-galactose. Inhibition of STb binding to its receptor was achieved using λ-carragenan at picomolar concentrations. Then, using IPEC-J2 cells in culture and flow cytometry, we showed that λ-carragenan was able to inhibit the permeabilization process associated with STb.     

Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.     

A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies

Abstract STb is a heat-stable enterotoxin elaborated by enterotoxigenic Escherichia coli strains associated with weaning piglets and is responsible for diarrhoea in those animals. The maltose binding protein (MBP) of E. coli was used as a carrier for STb, a poorly immunogenic molecule. Constructions were produced where the gene coding for mature STb toxin (MBP-STb) and a fragment of the gene spanning the major epitopic region of STb (AA8–AA30)(MBP-STb₂) were fused to malE gene coding for MBP. The fusion proteins accumulated in the periplasm and were detected with a polyclonal antibody raised against the purified toxin. MBP-STb induced secretion in the biological model whereas MBP-STb₂ was non-toxic. Immunization of rabbits evoked an antibody response to STb for these two fusion proteins. However, only MBP-STb elicited antibodies that effectively neutralized the toxicity of pure STb toxin as determined in the rat loop assay.     

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I , three for cdt-II , four for cdt-III , three for cdt-IV and four samples were positive for cdt-V , as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I , one cdt-II , three cdt-III, three cdt-IV and four with cdt-V . Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA , cnf1 , eaeA , cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens . Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.     

Pathogenesis of enteroaggregative Escherichia coli infection

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in 'typical EAEC strains'. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis.     

Nitric oxide-mediated apoptosis in rat macrophages subjected to Shiga toxin 2 from Escherichia coli

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.     

Detection of Iss and Bor on the surface of Escherichia coli

AIMS: To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. METHODS AND RESULTS: Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. CONCLUSIONS: These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.     

60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains

Aims:  To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens.
Methods and Results:  Bacteriophages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The morphological characterization of the bacteriophages was made by transmission electronic microscopy (TEM) observations and the genetic comparison between bacteriophages DNA was performed by restriction fragment length polymorphism (RFLP) patterns. Results showed that 70·5% of the tested E. coli strains were sensitive to a combination of three of the five isolated phages, that seemed to be virulent and taxonomically belong to the Caudovirales order. Two of them look like 16–19, T4-like phages ( Myoviridae ) and the third is a T1-like phage and belongs to Syphoviridae family. All of them are genetically different.
Conclusions:  It was possible to obtain a combination of three different lytic bacteriophages with broad lytic spectra against the most prevalent O-serotypes of APEC.
Significance and Impact of the Study:  Data reported in this study, presents an in vitro well studied phage product to be used as antimicrobial agent to treat colibacillosis in poultry industry.     

Amino acid sequence of heat-labile enterotoxin from chicken enterotoxigenic Escherichia coli is identical to that of human strain H 10407

Abstract The DNA sequence of heat-labile enterotoxin from the chicken enterotoxigenic Escherichia coli 21d strain was determined by direct dideoxy sequencing of polymerase chain reaction (PCR)-amplified DNA and was compared with those of heat-labile enterotoxins from porcine and human enterotoxigenic E. coli strains EWD 299 and H 10407. The structural genes of the A and B subunits of chicken heat-labile enterotoxin were identical to those of human heat-labile enterotoxin from the human H 10407 strain. Moreover, 67 base pairs of the upstream and 60 base pairs of the downstream region of the chicken heat-labile enterotoxin gene were also identical to those of the human heat-labile enterotoxin from strain H 10407. However, the patterns of plasmids from the 21d and H 10407 strains were different. The 21d strain had no band corresponding to the 42-MDa plasmid of the H10407 strain encoding the heat-labile enterotoxin gene but it had a smaller plasmid. These data suggest that although the DNA sequence of chicken heat-labile enterotoxin is identical to that of human heat-labile enterotoxin, the plasmid encoding the chicken heat-labile enterotoxin gene in the chicken might be different from that encoding the human heat-labile enterotoxin gene in the H10407 strain.     

牦牛肠毒素型大肠杆菌的分离鉴定和某些生物学特性的研究

从西藏地区腹泻死亡牦牛中分离出 1株肠毒素型大肠杆菌并对其某些生物学特性进行了研究。结果表明 ,该菌在形态特征、培养特性和生化特性方面与大肠杆菌基本一致。血清学试验表明 ,该菌株O抗原属O14 8,K88,K99,987P单因子血清均不能凝集本菌 ;该菌不产生溶血素 ;对绵羊、豚鼠、马、鸡的红细胞表现强凝集 ,而K88,K99,987P抗血清均不能抑制其对绵羊、豚鼠、马、鸡红细胞的凝集 ;该菌株在营养肉汤中经 37℃ ,4 8h培养表达菌毛 ;肌肉接种兔、腹腔接种小白鼠均具有高致病性 ;乳鼠胃内投服试验和兔回肠结扎试验证明 ,该菌能产生热稳定肠毒素和热敏性肠毒素 ;分离菌株对恩诺沙星、环丙沙星、阿莫西林、羧苄青霉素等高度敏感 ,而对链霉素、四环素、土霉素等表现耐药性。     

Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of food and water-borne E. coli-mediated human diarrhoea worldwide. The incidence in developing countries is estimated at 650 million cases per year, resulting in 800 000 deaths, primarily in children under the age of five. ETEC is also the most common cause of diarrhoea among travellers, including the military, from industrialized nations to less developed countries. In addition, ETEC is a major pathogen of animals, being responsible for scours in cattle and neonatal and postweaning diarrhoea in pigs and resulting in significant financial losses. Studies on the pathogenesis of ETEC infections have concentrated on the plasmid-encoded heat-stable and heat-labile enterotoxins and on the plasmid-encoded antigenically variable colonization factors. Relatively little work has been carried out on chromosomally encoded virulence factors. Here, we review the known virulence factors of ETEC and highlight the future for combating this major disease.     

Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.