Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I,Shiga-like toxin II,andEscherichia coli O157:H7 lipopolysaccharide in human serum

Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Prevalence of enterohemorrhagic Escherichia coli in raw and treated municipal sewage.

Municipal sewage was screened for DNA encoding Shiga-like Toxin (SLT) II, a key protein involved in the virulence of enterohemorrhagic Escherichia coli. PCR analysis of sewage concentrates showed that DNA encoding SLT II was present in a single sample of untreated sewage and absent in all other samples tested (n = 6). Thermotolerant E. coli cultured from the sewage (n = 1,520) also tested negative for SLT II by colony hybridization.     

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.     

Extraction of lithium from spodumene by bioleaching

A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-O157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.     

Exploiting retrograde transport of Shiga-like toxin 1 for the delivery of exogenous antigens into the MHC class I presentation pathway.

Shiga-like toxin 1 (SLT) from Escherichia coli O157:H7 enters mammalian cells by endocytosis from the cell surface to the endoplasmic reticulum before translocating into the cytosol. Here, SLT was engineered at its N- or C-terminus to carry a peptide derived from influenza virus Matrix protein for delivery to major histocompatibility complex (MHC) class I molecules. We show that SLT N-Ma was capable of sensitising cells for lysis by appropriate cytotoxic T-lymphocytes whilst no killing of SLT-resistant cells was observed. Our results demonstrate that peptide was liberated intracellularly and that retrograde transport of a disarmed cytotoxic protein can intersect the MHC class 1 presentation pathway.     

Growth of Escherichia coli in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay

Abstract Subinhibitory concentrations of trimethoprim-sulfamethoxazole increased the total yield of Shiga-like toxin (SLT), produced by Shigella dysenteria 1 and by enterophathogenic and enterohemorrhagic strains of Escherichia coli . Stimulation of SLT synthesis by trimethoprim-sulfamethoxazole was demonstrated by an increase in cytotoxic activity for HeLa cells and the diameter of the zone formed around bacterial colonies probed with monoclonal antibodies to SLT. Thus, supplementation of culture media with trimetroprimsulfamethoxazol will facilitate SLT purification and detection of SLT-producing bacteria.     

Molecular cloning and nucleotide sequence of another variant of the Escherichia coli Shiga-like toxin II family

Escherichia coli strain H.I.8 (O128:B12) produces low levels of a Shiga-like toxin (SLT) which we have called SLTIIva because of its close relationship with SLTIIv. The Vero cell cytotoxicity of SLTIIva is neutralized by antisera against SLTII and SLTIIv but not by antisera against SLTI. These data indicate that the SLT of strain H.I.8 is a member of the SLTII family. Since SLTIIva shares with SLTIIv the property of having low cytotoxicity to HeLa cells compared with Vero cells, it is appropriate to consider both toxins as variants of SLTII. SLTIIva differs from SLTIIv in that it is more heat-stable. Further, SLTIIv-producing strains of E. coli have only been isolated from pigs while the SLTIIva-producing E. coli strain examined in this study was isolated from a human infant with diarrhoea. The genes for this SLT were cloned from a cosmid library of total cellular DNA by screening recombinants for Vero cell toxicity and with a DNA probe derived from SLTIIv structural genes. Nucleotide sequence analysis was performed on a 2.0 kb AvaII-HincII fragment which encodes the toxin gene. The nucleotide sequence data confirm the close relationship between SLTIIva and SLTIIv: they have 98% nucleotide sequence homology in the B subunit gene and 70.6% homology in the A subunit gene. Comparison of DNA sequences indicated that SLTIIva was most closely related to SLTIIv, closely related to SLTII and less closely related to SLTI.     

Effects of human intravenous immune globulin on diarrhea caused by Shiga-like toxin I and Shiga-like toxin II in infant rabbits.

Shiga toxin and the related Shiga-like toxins (SLT), produced by Escherichia coli, can cause hemorrhagic colitis and hemolytic uremic syndrome (HUS). Human intravenous immune globulin (HIVIg) blocks the cytotoxicity of some SLTs in vitro. To examine the ability of HIVIg to modify disease caused by Shiga-like toxin I or Shiga-like toxin II (SLT-I or SLT-II), we injected 3-day-old rabbits intraperitoneally with SLT-containing cell-free supernatants from Escherichia coli O157: H7. A subset of rabbits was treated with subcutaneous HIVIg. All rabbits given 10(4) CD50 of SLT-I developed severe diarrhea, and 5 died. When HIVIg 500 mg/kg was given in addition to SLT-I, only 6 of 18 rabbits (33.3%) developed diarrhea (P < 0.0001), and 1 died. HIVIg 500 mg/kg or 1,000 mg/kg protected against diarrhea when given one hour prior to toxin. HIVIg 1,000 mg/kg was protective when administered one hour after toxin, but not at 6 or 24 hr. Seventeen of 18 rabbits given 10(6) CD50 of SLT-II developed severe diarrhea, and 4 died. In contrast to SLT-I-associated disease, HIVIg had no effect on diarrhea in rabbits given SLT-II. We conclude that HIVIg protects infant rabbits from diarrhea and death caused by intraperitoneally administered SLT-I, but does not affect the course of SLT-II-associated illness.     

Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity

Bschehchia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids. The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues. By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments. Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol. In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur. However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells. We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alterNatlve accessible sites to compensate for loss of target sites within the loop.     

A mutant Shiga-like toxin IIe bound to its receptor Gb(3): structure of a group II Shiga-like toxin with altered binding specificity

BACKGROUND: Shiga-like toxins (SLTs) are produced by the pathogenic strains of Escherichia coli that cause hemorrhagic colitis and hemolytic uremic syndrome. These diseases in humans are generally associated with group II family members (SLT-II and SLT-IIc), whereas SLT-IIe (pig edema toxin) is central to edema disease of swine. The pentameric B-subunit component of the majority of family members binds to the cell-surface glycolipid globotriaosyl ceramide (Gb(3)), but globotetraosyl ceramide (Gb(4)) is the preferred receptor for SLT-IIe. A double-mutant of the SLT-IIe B subunit that reverses two sequence differences from SLT-II (GT3; Gln65-->Glu, Lys67-->Gln, SLT-I numbering) has been shown to bind more strongly to Gb(3) than to Gb(4). RESULTS: To understand the molecular basis of receptor binding and specificity, we have determined the structure of the GT3 mutant B pentamer, both in complex with a Gb(3) analogue (2.0 A resolution; R = 0.155, R(free) = 0.194) and in its native form (2.35 A resolution; R = 0.187, R(free) = 0.232). CONCLUSIONS: These are the first structures of a member of the medically important group II Shiga-like toxins to be reported. The structures confirm the previous observation of multiple binding sites on each SLT monomer, although binding site 3 is not occupied in the GT3 structure. Analysis of the binding properties of mutants suggests that site 3 is a secondary Gb(4)-binding site. The two mutated residues are located appropriately to interact with the extra betaGalNAc residue on Gb(4). Differences in the binding sites provide a molecular basis for understanding the tissue specificities and pathogenic mechanisms of members of the SLT family.     

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Diarrheal and Environmental Isolates of Aeromonas spp. Produce a Toxin Similar to Shiga-Like Toxin 1

Diarrheal and environmental isolates of 39 strains of Aeromonas spp. were studied for detection of virulence factors. Although these 39 strains did not produce either heat-labile or heat-stable enterotoxins, culture filtrates of 31 strains produced cytopathic effects on Vero cells. Among these, culture filtrates of three strains of Aeromonas hydrophila and one strain of Aeromonas caviae could be neutralized by Escherichia coli O157:H7 Shiga-like toxin 1 antiserum. A single band of plasmid DNA of 2.14 kbp was isolated from these strains of Aeromonas spp. and E. coli O157:H7, which could be amplified by the polymerase chain reaction (PCR), employing oligonucleotide primers from the Shiga-like toxin 1 (SLT1) gene of E. coli O157:H7. E. coli HB 101 cells when transformed with the same plasmid showed cytopathic effects on Vero cells, which indicates that the SLT 1 homolog gene(s) of Aeromonas spp. is plasmid encoded. These results suggest that Aeromonas spp. may also produce Shiga-like toxin 1, or at least a cytotoxin with some homology with the Shiga-like toxin 1 of E. coli O157:H7.     

Genes for tRNA(Arg) located in the upstream region of the Shiga toxin II operon in enterohemorrhagic Escherichia coli O157:H7.

We found two genes for tRNA(Arg) in the region upstream of genes for Shiga-like toxin type II (SLT-II) in Escherichia coli O157:H7. The two encoded forms of tRNA(Arg) recognize rare codons in E. coli K12 but these rare codons occur in the toxin genes at high frequency.     

Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.     

Role of bacterial toxins in pathogenesis of hemolytic-uremic syndrome, caused by enterohemorrhagic Escherichia

The pathogenesis of the hemolytico-uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) has been studied previously rather completely. HUS is characterized by the signs of microangiopathic hemolytic anemia, thrombocytopenia with renal lesions and manifestations of transient disturbances in the functions of the central nervous system. The adherence of EHEC to enterocytes was found to occur in the terminal section of the ileum and the large intestine. This process is realized with involvement intimin, EHEC outer membrane protein. Shiga-like toxins (SLT) produced by EHEC are the leading factor of their pathogenicity. The mechanism of the toxin translocation through enterocytes is not yet clear, still there is no doubt that SLT penetrates into the systemic blood stream. This is indicated by the results of histopathological studies it possible to find the toxin traces on the membranes of endothelial cells of blood vessels. The study reveals that the cells of the vascular epithelium are highly sensitive to SLT. These cells carry receptors Gb3, also known as CD77, on their membranes. Enterohemolysin, serine protease, causing disturbances in the barrier function of the intestine, can be regarded in the pathogenesis of hemorrhagic colitis which develops as the result of the damaging action of EHEC and the above-mentioned toxins. This leads to the increased level of blood systemic bacterial lipopolysaccharides, which may play, in combination with the action of SLT, an important role in the development of multi-organ pathology in HUS patients.     

Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.

A rapid and sensitive method for detection of Shiga-like toxin (SLT)-producing Escherichia coli (SLT-EC) with the polymerase chain reaction (PCR) is described. Two pairs of oligonucleotide primers homologous to SLTI and SLTII genes, respectively, were used in multiplex PCR assays. The first pair generated a ca. 600-bp PCR product with DNA from all SLTI-producing E. coli tested but not from E. coli strains that produce SLTII or variants of SLTII. The second pair generated a ca. 800-bp PCR product with DNA from E. coli strains that produce SLTII or variants of SLTII but not from SLTI-producing E. coli. When used in combination, the SLTI and SLTII oligonucleotide primers amplified DNA from all of the SLT-EC tested. No PCR products were obtained with SLT primers with DNA from 28 E. coli strains that do not produce SLT or 44 strains of 28 other bacterial species. When ground beef samples were inoculated with SLT-EC strains 319 (O157:H7; SLTI and SLTII), H30 (O26:H11; SLTI), and B2F1/3 (O91:H21; SLTII variants VT2ha and VT2hb) and cultured in modified Trypticase soy broth for 6 h at 42 degrees C, an initial sample inoculum of as few as 1 CFU of these SLT-EC strains per g could be detected in PCR assays with DNA extracted from the broth cultures.