Mechanisms involved in protection provided by immunization against core lipopolysaccharides of Escherichia coli J5 from lethal Haemophilus pleuropneumoniae infections in swine.

In an investigation of the potential protective effects of immunity against common lipopolysaccharide core antigens of gram-negative bacteria during a severe gram-negative infection in the natural host, we induced Haemophilus pleuropneumoniae infections in weanling pigs immunized with a vaccine of an Rc mutant of Escherichia coli (strain J5). To help define the mechanism involved in J5-mediated protection, we compared the clinical, hematologic, bacteriologic, and serologic responses following an H. pleuropneumoniae infection in J5-immunized pigs with those following an H. pleuropneumoniae infection in nonimmunized control animals. As a result of an intranasal inoculation, all of the control animals and the J5-immunized animals were infected with H. pleuropneumoniae. However, while 80% (4 of 5) of the nonimmunized pigs died within 24 h as a result of the infection, no deaths occurred in the J5-immunized animals. In the immunized group, J5 titers dropped during the acute stages of the infection and rebounded to well above the prechallenge levels during convalescence. The J5 titer also increased in the single surviving control animal. These findings suggest that antibodies against common subsurface components of gram-negative bacterial cell walls correlate with protection from an otherwise lethal challenge of H. pleuropneumoniae but do not prevent infection. Important growth-phase-dependent antigenic changes have been recognized to occur during the growth of H. pleuropneumoniae in cultures (R. Nielson, Nord. Veterinaermed. 28:337-348, 1976). In a study of these changes and during an inquiry into the mechanism of J5 antibody-mediated protection, measured quantities of H. pleuropneumoniae were removed from a broth culture at hourly intervals and used to absorb hyperimmune equine J5 antiserum. Significantly greater amounts of J5-specific antibodies were absorbed during the log phase of bacterial growth than during the early or late phase. The availability of epitopes recognized by J5 antibodies appears to be closely related to the rate of bacterial multiplication. The results of these experiments suggest a mechanism of protection provided by increased immunity to E. coli J5 during gram-negative infections.     

Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.     

Comparative analysis of Haemophilus influenzae type b and Escherichia coli J5 lipopolysaccharides

Haemophilus influenzae type b (HIB) and Escherichia coli J5 (J5) lipopolysaccharides (LPS) were examined to explore the basis of previously observed cross-protection. HIB-LPS and J5-LPS contained ketodeoxyoctonate, glucose, glucoheptose and glucosamine as common carbohydrate moieties, and laurate, myristate, beta-hydroxymyristate and palmitate as common fatty acids, although in different ratios. J5-LPS was five times more lethal than HIB-LPS for chick embryos. Weak serological cross-reactivity was observed by haemagglutination and two-dimensional immunoelectrophoresis. No significant cross-reactivity was demonstrated by enzyme-linked immunosorbent or toxicity-neutralisation assays. The cross-reactivity observed between HIB-LPS and J5-LPS was probably due to common components in the core glycolipid.     

Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes.

The effects of the lipopolysaccharide (LPS) of Escherichia coli J5 and 0111B4 on the function of human polymorphonuclear leukocytes (PMN) were tested. E. coli J5 is a UDP-galactose-4-epimerase-deficient mutant of E. coli 0111B4, and its LPS, therefore, contains mainly lipid A, as it lacks the polysaccharide side chains. PMN which had been incubated with J5 LPS showed decreased phagocytic, chemotactic, and metabolic activities as compared with control PMN. In contrast, incubation of PMN with 0111B4 LPS had no effect or even an enhancing effect on PMN function. When lipid A and the polysaccharide fraction were isolated from 0111B4 LPS, it was shown that lipid A had the same deleterious effect on PMN function as did J5 LPS and that the LPS fraction had no effect. When PMN were incubated with J5 LPS or lipid A, it could be shown that these structures were able to induce PMN to generate superoxide and chemiluminescence. 0111B4 LPS and the polysaccharide component were able to generate a metabolic burst by the PMN to a lesser extent. The induced defects in PMN function by J5 LPS could be prevented when polymyxin B or an oxygen-radical scavenger was present. We hypothesize that the lipid A portion of LPS is toxic for PMN due to the induction of toxic oxygen species by the PMN. These toxic oxygen species destroy the phagocytic, chemotactic, and metabolic activities of the PMN.     

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.     

Distribution of serotypes and virulence-associated genes in pathogenic Escherichia coli isolated from ducks

The objective of the present study was to investigate the serotypes and virulence-associated genes of avian pathogenic Escherichia coli (APEC) isolated from duck colibacillosis cases. Two hundred and fifty-four APEC isolates from duck colibacillosis cases were serotyped and amplified for 12 known virulence-associated genes and the betA gene (encoding choline dehydrogenase) by polymerase chain reaction assays. One hundred and forty-three E. coli isolates from cloacal swabs of healthy ducks were also amplified for the same genes. A total of 53 O-serogroups were found in 254 APEC isolates, among which O93, O78 and O92 were predominant serogroups. Polymerase chain reaction results showed that Shiga-toxin-producing E. coli distributed in only 2.4% of ducks compared with 49.2% of the APEC isolates harbouring the irp2 gene, and 44.9% the fyuA gene, respectively. The ibeA gene was only present in 27 APEC isolates and was not found in healthy ducks. The rfaH gene was detected in 20.5% of APEC isolates, whereas 5.6% was found in healthy ducks. A total 79.5% of APEC isolates harboured the betA gene, which was significantly higher than in healthy ducks (16.1%), suggesting that betA may be associated with virulence.     

Antibody responses to Escherichia coli O157 and other lipopolysaccharides in healthy children and adults

In Mexico, diarrheal disease due to different serotypes of Escherichia coli is highly prevalent, with only sporadic isolation of O157 non-H7 strains. This could be due to exposure to the O157 or related E. coli lipopolysaccharide (LPS), such as O7 or O116, at an early age. By using enzyme-linked immunosorbent assay (ELISA) and Western blotting, the present study analyzed 605 serum samples from Mexican adults and infants without clinical symptoms of disease for the presence of antibodies to these three E. coli LPSs. The bactericidal activities of homologous and heterologous rabbit and human serum samples against O7, O116, and O157 E. coli LPSs were also determined. By using a cutoff point of 0.7, it was found by the ELISAs that 28 of 562 (5%) of the serum samples from adolescents and adults and 2 of 43 (5%) of the serum samples from infants less than 1 year of age reacted with the O157 LPS. By using cutoff points between 0.4 and 0.699, the proportion of serum samples from both age groups that reacted with the O157 LPS increased to 20%. Western blotting analysis of selected serum samples that showed an intermediate response against the O157 LPS by the ELISAs showed that 61 of 88 (69%) reacted with the same LPS. A similar result was observed for maternal milk samples. The bactericidal activities of rabbit serum samples against the O7, O116, and O157 LPSs showed that they were positive for both homologous and heterologous antigens. Similar results were observed with the human serum samples. O157 non-H7 strains were identified in only 10% of the E. coli strains isolated from 263 Mexican children with and without diarrhea over the past 15 years. This absence of O157:H7 strains in Mexico may be associated with the presence of antibodies against O157 or related E. coli LPSs.     

Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin

Type 1 fimbriae with mannose-specific lectins are widely distributed among members of the family Enterobacteriaceae and confer the ability to attach to a range of host cells, including colonic epithelial cells. The mucosal surfaces are protected by secretory immunoglobulin A (IgA), which agglutinates microorganisms and prevents their attachment to host epithelial cells. This action has been attributed to a specificity of the antigen-combining site of mucosal immunoglobulins for bacterial and viral surface components. Here, we report a novel mechanism for the antibacterial effect of secretory IgA. Secretory IgA and IgA myeloma proteins, especially those of the IgA2 subclass, were shown to possess carbohydrate receptors for the mannose-specific lectin of type 1-fimbriated Escherichia coli. The presence of the high-mannose oligosaccharide chain Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc correlated with binding activity. The interaction between bacterial mannose-specific lectins and IgA receptor oligosaccharide resulted in agglutination of the bacteria and in inhibition of bacterial attachment to colonic epithelial cells. Thus, this interaction could form the basis for a broad antibacterial function of secretory IgA against enterobacteria regardless of the specificity of antibody molecules.     

Monosaccharide composition of lipopolysaccharides from Campylobacter jejuni and Campylobacter coli

The monosaccharide composition of the LPS from 5 Campylobacter jejuni strains and 7 Campylobacter coli strains has been studied. All LPS's contained KDO, heptose, glucosamine, glucose, and (with one exception) galactose. All C. jejuni and 3 C. coli LPS's contained greater than 1% galactosamine. 3-Amino-3.6-dideoxyglucose was present in all but one C. coli LPS and in only one C. jejuni LPS.     

Distribution of saa gene variants in verocytotoxigenic Escherichia coli isolated from cattle and food

The pathogenesis of verocytotoxigenic Escherichia coli (VTEC) infection in humans is multifactorial, given that verocytotoxins are the principal virulence factor. Most strains causing serious diseases possess the eae gene that encodes the adhesin intimin, but its presence is not essential for virulence as some cases are caused by eae-negative strains. An autoagglutinating adhesin designated Saa was found in some eae-negative strains. This protein varies in size as a consequence of variation in the number of copies of a 37-aa repeat unit in the C-terminal region. Based on these findings, we designed PCR primers to amplify the region coding for these differences to detect saa gene variants present in VTEC strains isolated in Argentina from cattle and meat. The gene saa was detected in 36 (31.6%) eae-negative strains and 5 variants were found. Strains isolated from cattle possessed 4 saa variants, whereas 2 variants were present in isolates from meat. Saa variant 1 predominated (18 strains) and was distributed in strains isolated both from cattle and from meat. Our study revealed the existence of two novel saa variants, termed 4 and 5, which have a higher number of 111-bp repeats than saa genes previously studied.     

Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.     

Distribution and evolution of virulence factors in septicemic Escherichia coli

Bacterial septicemia is an emerging clinical problem which is increasing in significance due to the rapid spread of antibiotic resistance. In order to combat this problem, it is essential to identify the critical virulence factors of these septicemic strains and study their functions and role in pathogenesis. Here, we survey some of the virulence factors which are essential for sepsis and are potential candidates for development of new drugs or vaccines.     

Effects of Escherichia coli and E. coli lipopolysaccharides on the function of human ureteral epithelial cells cultured in serum-free medium.

Escherichia coli is the microorganism most commonly isolated from human urinary tract infections. Earlier studies by others have shown that bacterial attachment and production of toxins (e.g., lipopolysaccharides [LPS]) enhance recruitment of leukocytes to the infection site and mucosal inflammation. The mechanisms by which these changes occur have not been completely defined. In the present study, epithelial cell cultures isolated from the human ureter (UT cells) (A. Elgavish, J. J. Wille, F. Rahemtulla, and L. Debro, Am. J. Physiol. 261:C916-C926, 1991; J. J. Wille, J. Park, and A. Elgavish, J. Cell. Physiol. 150:52-58, 1992) served as a model system with which to explore the mechanisms of action of Escherichia coli and E. coli LPS in UT cells. E. coli adhered to UT cells and inhibited carrier-mediated sulfate uptake to half of that in untreated UT cells, suggesting that the intracellular pool of sulfate available for sulfation may be lower in infected cells and may lead to the production of undersulfated glycoconjugates. Incubation of UT cells with E. coli LPS inhibited carrier-mediated sulfate uptake to an extent similar to that caused by whole E. coli, indicating that the effect of E. coli on sulfate uptake may be mediated by LPS. LPS caused an increase in Na+ content in rapidly proliferating UT cells but not in quiescent cells. We postulated that this change in the intracellular ionic environment or changes coupled to it (e.g., pH or Ca2+ levels) may serve as a transducing signal. This possibility was supported by the fact that LPS stimulated clustering of ICAM-1 on the cell surface of rapidly proliferating but not quiescent UT cells. This study suggests that, in vivo, LPS stimulation of ICAM-1 clustering on the surface of the urothelium may allow more effective binding of leukocytes. This may be the mechanism underlying earlier findings in vivo indicating a role for LPS in the recruitment of leukocytes to the urinary tract as a host defense mechanism following urinary tract infection.     

Physicochemical surface properties of Escherichia coli strains isolated from different types of urinary tract infections.

Escherichia coli strains isolated from three groups of patients with urinary tract infections, such as acute pyelonephritis, acute cystitis, and asymptomatic bacteriuria, were analyzed with respect to their physicochemical surface properties by means of polymer two-phase partitioning in dextran-polyethylene glycol systems and hydrophobic interaction chromatography on Octyl-Sepharose. Strains causing acute pyelonephritis constituted a homogenous group which, depending on the growth conditions, demonstrated smooth-type lipopolysaccharide, elevated negative charge, and liability to hydrophobic interaction, whereas strains isolated from acute cystitis and asymptomatic bacteriuria showed a more heterogenous pattern.     

培养肝细胞,肝窦壁内皮细胞及贮脂细胞对大肠杆菌脂多糖的摄入作用

目的和方法：应用免疫荧光及激光共聚焦扫描技术，观察原代培养大鼠肝细胞、肝脏贮脂细胞及窦壁内皮细胞对不同分子结构大肠杆菌脂多糖的摄取作用。结果：无血清条件下，肝细胞、贮脂细胞、窦壁内皮细胞加入Ｓ型脂多糖（Ｓ－ＬＰＳ）及Ｒｄ型脂多糖（Ｒｄ－ＬＰＳ）培养５ｍｉｎ后，胞浆内均出现强烈抗脂多糖荧光显色；胞浆抗脂多糖荧光强度显著高于空白对照水平；且Ｒｄ－ＬＰＳ培育后肝细胞、窦壁内皮细胞胞浆内荧光强度增高幅度显著高于Ｓ－ＬＰＳ培养后的增高幅度。抗脂多糖显色局限于胞浆，胞核内未见明显着色，但经与Ｒ型多糖培养后，肝细胞核内亦出现强烈抗脂多糖荧光显色。结论：分离培养的大鼠肝细胞、贮脂细胞及肝窦壁内皮细胞对细菌脂多糖具有摄入作用，且脂多糖与这些细胞的相互作用依脂多糖分子结构不同而异     

Distribution and transferability of plasmids in trimethoprim resistant urinary Escherichia coli

Urinary isolates of Escherichia coli that were resistant to trimethoprim were collected in Glasgow Royal Infirmary during 1979 and 1980. Eighty-eight were resistant to trimethoprim 1024 micrograms/ml and 80 (92%) were also resistant to sulphamethoxazole 1024 micrograms/ml; 73% were multiresistant. Plasmids were detected in 98% of strains and 60% carried two or more. Half of the isolates transferred trimethoprim resistance to E. coli K12 and 70% of these cotransferred resistance to sulphonamide although these markers were often not linked. Trimethoprim resistance was carried on 12 different plasmids, four of which also conferred sulphonamide resistance. All except two carried streptomycin resistance which suggests that Tn7 was probably present. The results are discussed in relation to current prescribing policy.     

Distribution and genetic location of Tn7 in trimethoprim-resistant Escherichia coli

A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III. Of the isolates, 57% carried Tn7. Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon. The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70%. This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss. The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance.