In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.

An outbreak of edema disease (ED) was monitored in 80 piglets after weaning over a period of 4 weeks. The shedding of Shiga-like toxin-II(e)1) producing Escherichia coli strains, the serum bactericidal activity (SBA) against SLTEC-II(e), and the antibody response against SLT-II(e) were investigated. The antibody response was monitored by utilizing a glutathion-S-transferase (GST) + SLT-II(e) B/SUB fusion protein (FRANKE et al., in press) for immunoblot assays.E. coli-strain GO15III (0141:K85ac) was diagnosed as SLT-II(e)-producing E. coli by polymerase chain reaction, DNA hybridization and cytotoxicity assays. Maximum excretion of GO 15III appeared between days 8 and 15 after weaning. On day 1 after weaning no piglet shed GO15III, while the number increased on day 8 to 53 (66.2%) and on day 15 to 59 (73.8%) of the piglets. 4 week after weaning, GO15III was only isolated from 23 (28.8%) of the piglets. In parallel, serum bactericidal activity against GO15III increased significantly in the sera of 73 (91.2%) piglets, reaching a stable maximum from day 15 on. During the first two weeks after weaning, no piglet yielded detectable SLT-II(e)-IgG. However, the number of SLT-II(e)-IgG positive piglets increased steadily from day 15. On day 15, 5(6.2%) piglets were positive in SLT-II(e)immunoblot analysis and 29 days after weaning the number increased to 31 (38.8%).These data represent the first serological monitoring of a natural outbreak of edema disease in piglets after weaning by using a recombinant fusion protein (GST+SLT-(e) B/SUB). The recombinant protein proved to be a useful diagnostical tool for monitoring the specific antibody status of piglets.

An outbreak of edema disease (ED) was monitored in 80 piglets after weaning over a period of 4 weeks. The shedding of Shiga-like toxin-IIe) producing Escherichia coli strains, the serum bactericidal activity (SBA) against SLTEC-IIe, and the antibody response against SLT-IIe were investigated. The antibody response was monitored by utilizing a glutathione-S-transferase (GST) + SLT-IIe B/SUB fusion protein (FRANKE et al., in press) for immunoblot assays. E. coli-strain GO15III (0141:K85ac) was diagnosed as SLT-IIe-producing E. coli by polymerase chain reaction, DNA hybridization and cytotoxicity assays. Maximum excretion of GO15III appeared between days 8 and 15 after weaning. On day 1 after weaning no piglet shed GO15III, while the number increased on day 8 to 53 (66.2%) and on day 15 to 59 (73.8%) of the piglets. 4 week after weaning, GO15III was only isolated from 23 (28.8%) of the piglets. In parallel, serum bactericidal activity against GO15III increased significantly in the sera of 73 (91.2%) piglets, reaching a stable maximum from day 15 on. During the first two weeks after weaning, no piglet yielded detectable SLT-IIe-IgG. However, the number of SLT-IIe-IgG positive piglets increased steadily from day 15. On day 15, 5 (6.2%) piglets were positive in SLT-IIe immunoblot analysis and 29 days after weaning the number increased to 31 (38.8%). These data represent the first serological monitoring of a natural outbreak of edema disease in piglets after weaning by using a recombinant fusion protein (GST+SLT-IIe B/SUB). The recombinant protein proved to be a useful diagnostical tool for monitoring the specific antibody status of piglets.

In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe. A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region. Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene. Pure cultures of E. coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test. Of 50 E. coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR. All 43 of these strains reacted with the EAggEC probe. Six EAggEC strains gave negative results by both molecular techniques. In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E. coli demonstrated a positive PCR result. The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens. Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory.

We constructed and purified recombinant B-subunits of the SLT-IIv as well as tested their usefulness in an immunoblot assay. The slt-IIvB gene amplified by PCR was ligated into the fusion vector pGEX-2T, and expressed in E. coli K 12 laboratory strains. Deletion of the signal sequence was necessary for optimal expression. High quantities of the fusion protein could be purified by affinity chromatography and subsequently used as antigen for immunoblot analysis with serum samples from diseased pigs and healthy controls. IgG antibodies against SLT-IIv were detected in the sera of 11 of 52 (21.15%) healthy pigs. By contrast, only in 1 of 28 (3.57%) serum samples of pigs with edema disease caused by SLT-IIv-producing E. coli we could demonstrate SLT-IIv-specific antibodies. During an outbreak of edema disease, sera from 10 pigs were taken at 4, 20, and 40 days after disease onset to investigate the immune response elicited by SLT-IIv. Immunoblot analysis with the recombinant SLT-IIv fusion protein revealed that the number of IgG-positive serum samples increased within this period of 40 days from one on day 4, to seven on day 20, to ten on day 40; the number of IgM-positive samples also increased from one after 4 days to eight after 20 days. Forty days after disease onset, IgM reactivity was no longer detectable. Since all animals seroconverted in the follow-up sera, the antigenicity of SLT-IIv during infection of pigs seems to differ from that of SLT-II in human hemolytic uremic syndrome where only a minority of patients are known to mount an immune response. The recombinant SLT-IIvB described here may be a possible candidate for vaccination trials.

Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans. In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors. The slt genes of five E. coli O101 strains were cloned and sequenced. For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs. The A subunit revealed more than 99% homology to that of SLT-IIe. DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates. None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E. coli hemolysin). The absence of virulence factors typical of SLT-I- and SLT-II-producing E. Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E. coli, suggests a new pathogenic mechanism for E. coli O101 infection of humans. For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases.

Journal Article Acute renal failure following high intravenous doses of naftidrofuryloxalate Get access A. Heidland, A. Heidland Dept. of Internal Medicine, University of WürzbergJosef-Schneider Str. 2, 97080 Würzberg, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar G. Schindler, G. Schindler Dept. of Radiology, University of WürzbergJosef-Schneider Str. 2, 97080 Würzberg, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar S. Franke S. Franke Dept. of Surgery, University of WürzbergJosef-Schneider Str. 2, 97080 Würzberg, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Nephrology Dialysis Transplantation, Volume 10, Issue 12, December 1995, Page 2371, https://doi.org/10.1093/ndt/10.12.2371a Published: 01 December 1995

The pheno- and genotypes of Shiga-like toxins (SLTs) in non-O157 Escherichia coli strains from patients with haemolytic uraemic syndrome were determined. The clinical isolates investigated were from Italy and Germany and belonged to serotypes O22:H8, O26:H-, O26:H11, O91:H-, O111:H- and O128:H-; one isolate was non-typable. SLT genotypes were analysed by complete nucleotide sequence analysis of the B-subunit genes. The results showed that 14 strains possessed slt-I alone, two contained slt-II alone and five isolates harboured both slt-I and slt-II genes. In only two strains were slt-II-related genes found, together with either slt-I or slt-II. These findings indicate that variants of SLT-II are rarely found in non-O157 E. coli isolates from patients with haemolytic uraemic syndrome. Polymerase chain reaction (PCR) with Taq cycle sequencing was found to be a suitable method for classification of slt genotypes.

In order to conduct molecular typing of Eikenella corrodens strains by macrorestriction fingerprinting, we evaluated different restriction enzymes for digestion of genomic DNA and determined the optimal parameters for separating E. corrodens DNA by pulsed-field gel electrophoresis. Ten E. corrodens strains isolated from oral and extraoral infection sites in different individuals were analyzed. The rare-cutting restriction endonucleases DraI, SmaI and XbaI usually used for pulsed-field gel electrophoresis analyses were not suitable for digestion of E. corrodens genomic DNA because they either did not digest the DNA or produced bands of similar molecular weights that could not be separated. Accordingly, among additional enzymes including BamHI, BglII, EcoRI and Hind III, we found BamHI and BglII to be the most suitable rare-cutting enzymes for pulsed-field gel electrophoresis analysis. They cleaved the genomes of all the above strains into 15-20 fragment bands that were clearly separated by the following pulsed-field gel electrophoresis conditions: 140 V with a running time of 40 h, pulse times of 5 to 50 s with linear ramping and an electrical field angle of 120 degrees. These conditions enabled us to distinguish 8 individual pulsed-field gel electrophoresis patterns from the 10 strains analyzed. However, only 4 identical outer membrane protein profiles were differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data obtained in this analysis showed clonal divergence among members of the E. corrodens species, at the same time revealing this pulsed-field gel electrophoresis as being a highly attractive procedure for epidemiological investigation of this organism, including its acquisition and transmission.

In this study 98 Escherichia coli strains which belonged to traditional enteropathogenic (EPEC) serotypes and 82 enterohemorrhagic E. coli (EHEC) strains were screened by polymerase chain reaction (PCR) for the presence of E. coli -attaching and -effacing (eae) genes. These strains were also hybridized with the enteropathogenic adherence factor (EAF) probe and examined in the fluorescence actin staining (FAS) test. The results obtained from the individual strains demonstrated that all 26 class I EPEC with localized adherence to HEp-2 cells carried EAF and eae genes. In contrast, of 72 EPEC strains with no or diffuse adherence only 1 strain was EAF positive and 6 strains had eae. Of 82 EHEC strains a total of 75 carried eae sequences. Of considerable interest, 15 of 21 E. coli strains that lost their slt genes during subcultivation were found to be eae positive. As controls a total of 53 enterotoxigenic and enteroinvasive E. coli, and 125 E. coli strains from the normal flora were investigated and all displayed negative results in the eae-PCR. From the 201 strains comprising classical EPEC serotypes, EHEC and E. coli with lost slt genes, a total of 126 displayed a positive FAS test and 122 reacted in the eae-PCR. Only 4 strains were FAS test positive but eae-PCR negative. Our data indicate that E. coli strains possessing the eae genes are heterogenous with respect to their virulence determinants. Loss of virulence plasmids and phage-encoded slt genes either in the host or during storage may contribute to this heterogeneity. The eae-PCR detected all class I EPEC and 91.5% of the EHEC. For diagnostic purposes we, therefore, recommend the combination of eae- and slt-specific gene probes which allowed a 100% detection of the class I EPEC and EHEC strains investigated here.

The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pK18. The nucleotide sequence of this fragment was determined, and a set of primers was designed to amplify a 397-bp region associated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known genes. However, between bases 701 and 787 of the fragment, an 82.8% homology between the EAF and the insertion sequence IS630 of Shigella sonnei exists. The results of PCR with primers of the EAF sequence demonstrated that all of the 151 EAF probe-positive EPEC strains with localized adherence to HEp-2 cells yielded positive EAF PCR results. In contrast, none of the 277 EAF probe-negative strains reacted to the EAF PCR. In addition, the PCR assay was successfully used to generate vector-free digoxigenin-labeled EAF fragments that gave valid results in colony blot hybridization assays. The EAF PCR appears to be a specific and efficient method for the detection of EPEC strains carrying the EAF plasmids.