Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii

A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E. coli O157 O-antigen. A strain of Citrobacter freunndii showing similar cross-reaction with E. coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure [ 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-]. This O-antigen differs from that of the E. coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E. coli O157 antisera. The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.     

Bacterial penetration of bladder epithelium through lipid rafts

Type 1 fimbriated Escherichia coli represents the most common human uropathogen, owing much of its virulence to invasion of the uroepithelium, which is highly impermeable due to the preponderance of uroplakins and highly ordered lipid components. We sought to elucidate the molecular basis for E. coli invasion of the bladder epithelium by employing human 5637 bladder epithelial cells, and we found the following: (i) intracellular E. coli associated with caveolae and lipid raft components; (ii) RNA(i) reduction of caveolin-1 expression inhibited bacterial invasion; (iii) a signaling molecule required for E. coli invasion was located in lipid rafts and physically associated with caveolin-1; (iv) bacterial invasion was inhibited by lipid raft disrupting/usurping agents. In the mouse bladder, the E. coli type 1 fimbrial receptor, uroplakin Ia, was located in lipid rafts, and lipid raft disruptors inhibited E. coli invasion. Cumulatively, E. coli uroepithelial invasion occurs through lipid rafts, which, paradoxically, contribute to bladder impermeability.     

Antigenic properties of genetic recombinants of serologically typed E. coli]

The conjugation between the typed strains of E. coli belonging to various serological groups and conjugation between typed and untyped E. coli strains were studied. Genetic determinant controlling the synthesis of the O100 antigen proved to be closely linked with histidine locus. Among recombinants obtained in crossing the typed E. coli strains there were such belonging to the serological type different from the serological types of donor and recipient cells.     

Characteristics of microbial cultures in bacteriuria and various data on the immunological reaction in children with pyelonephritis]

A study was made of 268 cultures isolated from the urine of 263 children suffering from pyelonephritis. Of the total number of different cultures E. coli constituted 79.3 percent; the percentage of the rest varied from 5.2 to 0.4. Examination of 87 urinocultures of E. coli isolated from sick children with the specific immune response showed that the majority of bacterial signs (urease activity, capacity to produce alpha-hemolysin to utilize saccharose and raffinose, to synthesize colicine) failed to correlate with their pyelopathogenicity. The reference to individual serological groups also failed to serve as a sufficient foundation for the separation of these microbes into individual nephropathogenic or pyelopathogenic groups. In experiments with 3H-glucose labeled bacteria there was revealed a marked adhesive capacity in 94 percent of E. coli strains towards the epithelial cells of the RH strain. A positive radioactive label failed to correlate with the presence in E. coli of common pili and with the bacterial agglutinability with the sera K88, K99, and KH-III. The latter pointed to the presence of a factor of unknown nature in the nephropathogenic E. coli strains imparting adhesive properties to bacteria.     

Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis.

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.     

Serological Relatedness of Bacterial Deoxyribonucleic Acid Polymerases

A number of bacterial species have been surveyed for serological activities with antiserum to Escherichia coli B deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7.). The degree of serological cross-reaction is taken as a measure of relatedness of both the enzyme molecules from various species and the bacterial species themselves. Extracts were assayed by complement fixation only after treatment with deoxyribonuclease, since DNA bound to DNA polymerase alters the serological activity of the enzyme. Antiserum to E. coli DNA polymerase I did not react with either purified E. coli DNA polymerase II or the phage T4-induced DNA polymerase.     

The mosaic of proteins synthesized by Salmonella typhimurium

A profusion of proteins with heterologous serological specificities was synthesized by S. typhimurium grown on artificial media; accordingly, sera prepared in rabbits with these proteins displayed an abundance of antibodies reacting, in agar gel, against numerous heterologous proteins. the absorption of the sera with different Enterobacterial proteins proved that the S. typhimurium proteins are a mixture of specific proteins, and common E. coli and Salmonellae determinants; in addition, a group of strongly cross-precipitating proteins common to S. typhimurium and S. choleraesuis and to S. typhimurium and S. kentucky were identified that were not present in the proteins common to S. enteritidis, S. typhi and E. coli, or in the S. paratyphi A proteins used absorptions. The specific proteins of S. typhimurium were synthesized on artificial media in, apparently, smaller amounts than the common proteins; their role in the protection of mice against infection with their natural pathogen was, however, proof of their specificity and contrasted with the ineffectiveness, in protecting the mice, of the common proteins.     

Cloning and expression in Escherichia coli K-12 of the genes for major outer membrane protein OmpA from Shigella dysenteriae, Enterobacter aerogenes, and Serratia marcescens.

The outer membranes of many gram-negative bacteria contain a major heat-modifiable protein which shows serological cross-reactivity with the OmpA protein of Escherichia coli K-12. Using the cloned gene for the E. coli K12 protein as a DNA-DNA hybridization probe, we were able to identify the corresponding genes from Shigella dysenteriae. Enterobacter aerogenes, and Serratia marcescens. These were cloned in a phage lambda vector, and their expression in E. coli K-12 was studied. All three OmpA proteins were fully produced and correctly exported to the outer membrane. In several cases, complete or partial restoration of known function of the E. coli K-12 protein was observed.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Kidney bean lectin-induced Escherichia coli overgrowth in the small intestine is blocked by GNA, a mannose-specific lectin

The reversible and dose-dependent hyperplastic growth of the small intestine and accelerated epithelial cell turnover caused by feeding rats with diets containing kidney bean lectin (PHA) increased the proportion of immature cells on the villi whose membrane and/or cytoplasm contained mainly simple, polymannosylated glycans. These new α-linked mannosyl terminals, particularly of the damaged epithelium, facilitated the preferential adherence of opportunistic Escherichia coli with mannose-sensitive Type 1 fimbriae, and other coliforms, to the glycocalyx. Accordingly, the growth of the gut was accompanied by a reversible and PHA dose-dependent overgrowth with E.coli. As expected from their common carbohydrate specificity, the inclusion in the diet of the mannose-specific agglutinin from snowdrop ( Galanthus nivalis ) bulbs (GNA) significantly reduced the extent of E.coli overgrowth, but abolished neither the growth nor the damage caused by PHA to the small intestine. Thus, GNA and perhaps other mannose-specific lectins, especially when used in a preventive mode, can be used to specifically block the proliferation of Type 1 E.coli in the small intestine.     

Phage conversion of the antigens in Escherichiae and Shigellae. 2. Conversion of the somatic antigen in Shigella flexneri with the moderate E. coli 0129 phage]

The authors studied the converting activity of the moderate EF5 phage isolated from the lysogenic E. coli 0129 strain. It was shown that this phage converted the O-antigen with the detection of the type antigen V in the strains of Sh. flexneri of the serological type la and y-variant. The converted cultures contained the type antigen V and were identical by the antigenic properties to one another and the Sh. flexneri of the serological type 5 and E. coli 0129. A conclusion was drawn that phages converting the antigens of Sh. flexneri could be encountered in escherichia and could modify the antigens in Sh. flexneri and escherichia possessing the antigenic factor 3,4.     

Determination of colonization factor antigens CFA in diagnosis of enterotoxigenic strains of Escherichia coli]

This study was aimed at establishment whether preliminary determination of colonization factor antigens CFA may be useful in selection of potentially pathogenic strains of Escherichia coli with serological types belonging to ETEC and 750 isolates of E. coli from children with symptoms of diarrhoea. Enterotoxigenicity of strains was evaluated by suckling mice test and culture of Y1 cell tissue. Colonization factor antigens CFA were evaluated on the basis of slide agglutination and agar gel immunodiffusion with application diagnostic sera prepared for this study. Ability of enterotoxin production was found in 25% strains of E. coli with serological types belonging to ETEC. In 90% these strains were isolated from cases of epidemic diarrhoea. ETEC strains were found in 11% of hospitalized children and in 5% who were treated outside of hospital because of diarrhoea. MRHA adhesins occurred on 80% of ETEC strains were all diagnosed as CFA/I. CFA/II were not found and in only three strains non-fimbrial CFA/IV was present. Preliminary determination of CFA during selection of ETEC strains presents as a very sensitive method (97%) and is also highly specific (99%). Application of this method will result in significant increase of affectivity of biological tests directed toward determination of E. coli enterotoxigenicity.     

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.     

Escherichia coli STb toxin and colibacillosis: knowing is half the battle.

Expression of both adherence and enterotoxin expression are required for enterotoxigenic Escherichia coli (ETEC) strains to cause colibacillosis. ETEC strains are responsible for diarrhea in humans and animals by production of various enterotoxins. For many years, the role of the heat-stable E. coli enterotoxin STb as a diarrhea-causing toxin in animals, and in particular in swine, has been controversial. In fact, although the presence of STb-positive E. coli strains and diarrhea in animals is frequently observed, the difficulty of reproducing the pathology in an animal model was interpreted as a lack of toxicity. Recently, new light was shed on the activity of STb in intestinal ligated loops and in pigs orally inoculated with STb-positive E. coli strains. This minireview revisits the effects of STb on the intestinal epithelium and enlightens the significance of STb in swine colibacillosis. The interaction of STb toxin with other E. coli enterotoxins and dual ETEC/enteropathogenic E. coli or ETEC/attaching effacing E. coli infections are also discussed.     

临沂市肉鸡致病性大肠埃希菌的分离鉴定及耐药性研究

目的查清临沂市肉鸡大肠埃希菌的优势血清型及耐药情况,为研制疫苗和科学防治本病提供依据。方法对病料进行细菌分离培养,应用形态学检查、生化试验、致病性试验和血清学试验对大肠埃希菌及其血清型进行鉴定,通过药敏试验研究其耐药性。结果从365份病料中,分离出大肠埃希菌311株,分离率为85.2%。优势血清型为O78、O1、O11、O18、O15和O2;对链霉素、氨苄西林、利福平、庆大霉素、恩诺沙星、环丙沙星和左氧氟沙星等药物表现出很高的耐药性,耐药率分别为98.0%、87.3%、86.0%、77.3%、73.4%、70.0%和64.6%,而对大观霉素、头孢噻呋、多黏菌素、氟苯尼考和痢菌净高敏。结论临沂市肉鸡大肠埃希菌的优势血清型有O78、O1、O11、O18、O15和O2,分离菌株对大部分抗菌药物产生耐药性。因此,治疗鸡大肠埃希菌病应通过药敏试验,合理地选择药物。     

Escherichia coli O157:H7 induces attaching-effacing lesions in large intestinal mucosal explants from adult cattle

Attaching-effacing (A/E) lesions following natural and experimental infection with Escherichia coli O157:H7 have been seen in neonatal and 3-4-month-old weanling but not older cattle. To test the hypothesis that the adult bovine large intestinal epithelium is resistant to the development of A/E lesions, colonic and rectal mucosal tissue explants from 18-month-old steers were inoculated with E. coli O157:H7 and examined. Epithelial cells of inoculated explants developed A/E lesions at the bacterial attachment sites, providing evidence that the large intestinal mucosal epithelium may be a site of infection that contributes to carriage of E. coli O157:H7 in adult cattle.     

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.     

The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli

Patients with haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC) produce serum antibodies to the lipopolysaccharides (LPS) of Escherichia coli O157 and certain other E. coli serogroups. Patients may also make salivary antibodies to the LPS of E. coli O157. Serological tests based on these antibodies can be used to provide evidence of infection in the absence of culturable VTEC or the toxins they produce. Serum antibodies to LPS persist for several months following onset of disease, enabling both current and retrospective serological testing. The LPS of E. coli O157 shares epitopes with strains of Brucella abortus, Yersinia enterocolitica O9, Vibrio cholerae O1 Inaba, group N Salmonella and certain strains of Citrobacter freundii and E. hermanni. Serological tests for serum antibodies to E. coli O157 should be evaluated in the light of these cross-reactions. Serological tests to supply evidence of infection with E. coli O157 have been shown to provide a valuable adjunct to bacteriological procedures for detecting culturable VTEC and VT. The use of well characterized LPS antigens in association with the techniques of ELISA and immunoblotting provide valuable procedures for detecting evidence of infection with E. coli O157 and possibly other VTEC.     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

Effect of culture conditions on Escherichia coli O157:H7-mediated attaching-effacing lesions in a bovine large intestinal mucosal explant model

The effects of culture conditions on the extent of Escherichia coli O157:H7 attaching-effacing (A/E) adherence in an adult bovine large intestinal mucosal explant model were assessed by three different morphometric methods. Measurement of the percent of tissue sections with A/E adherence and the number of foci of A/E adherence mm(-1) of surface epithelium was more sensitive than measurement of the percent of surface epithelium with A/E adherent bacteria for detection of treatment effects. Culture of bacterial inoculum in tryptic soy broth, incubation of explants in 5% CO(2), and rocking of explants on a platform rocker at 18 cycles min(-1) provided optimal conditions for A/E adherence. In future studies, the model may be used for preliminary testing of intervention strategies aimed at reduction of E. coli O157:H7 intestinal colonization of cattle.