Identification of Salmonella typhi genes expressed within macrophages by selective capture of transcribed sequences (SCOTS).

Salmonella enterica serovar Typhi (S. typhi) is a human-restricted pathogen which causes typhoid fever. Relatively little is known about S. typhi host interaction as animal models of this disease are severely limited by the lack of virulence of S. typhi in other hosts. The virulence of other Salmonella serovars in animal models is dependent on the abilities of these bacteria to survive within host macrophages. We have used selective capture of transcribed sequences (SCOTS) to identify S. typhi genes expressed during growth in human macrophages. This positive cDNA selection technique identified 28 distinct clones representing previously identified as well as novel, uncharacterized and hypothetical gene sequences that are expressed intracellularly. Transcripts for the Vi capsular antigen and genes whose products are involved in stress responses and nutrient acquisition were obtained from intracellular bacteria using SCOTS. Most of these clones are present in the S. typhimurium genome and are also expressed in murine macrophages. Nineteen of these gene sequences were disrupted insertionally in S. typhi, and most of the resulting mutants exhibited a lower level of survival within macrophages compared with the wild-type parent strain. Mutant strains, transformed with a copy of a wild-type gene, exhibited a macrophage survival level similar to that of the parental strain.

Effects of global regulatory proteins and environmental conditions on fimbrial gene expression of F165(1) and F165(2) produced by Escherichia coli causing septicaemia in pigs.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae. F165(1) fimbriae belong to the P fimbrial family and F165(2) fimbriae belong to the S fimbrial family. Regulatory regions of foo (F165(1)) and fot (F165(2)) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised. Expression of F165(1) and F165(2) fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters. Differential expression of fimbrial genes was observed. Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression. foo and fot expression was optimal at 37 degrees C and under aerobic conditions. Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium. This could reflect an in vivo differential expression.

Lack of a role of cytotoxic necrotizing factor 1 toxin from Escherichia coli in bacterial pathogenicity and host cytokine response in infected germfree piglets.

Some Escherichia coli strains isolated from intestinal or extraintestinal infections in pigs produce cytotoxic necrotizing factor 1 (CNF1). In order to analyze the role of CNF1 in the pathogenesis of porcine colibacillosis, newborn colostrum-deprived germfree piglets were orally inoculated with a wild-type CNF1-producing strain (M623) or with an isogenic cnf1 mutant (M623DeltaCNF1). The two isogenic strains induced a high mortality with similar lung and serosal inflammatory lesions, indicating that both strains were pathogenic in these piglets. Bacterial counts in various organs of inoculated piglets revealed an intestinal predisposition of M623 and M623DeltaCNF1 strains for the cecum and colon. Extraintestinal organs (lungs, liver, spleen, and kidney) were also colonized by both strains. Similar colonization of intestinal and extraintestinal tissues in animals inoculated with either strain was observed, except in the ileum, where M623 showed a higher colonization than M623DeltaCNF1. Intestinal (ileum and colon), extraintestinal (lung and kidney), and immune (mesenteric lymph nodes and spleen) tissues were sampled at 1 day postinoculation and analyzed for cytokine expression by a reverse transcriptase PCR technique. Inoculation with E. coli M623 induced an enhanced expression of inflammatory cytokines (interleukin-1alpha [IL-1alpha], tumor necrosis factor alpha, and IL-12p40) in the intestinal organs compared to uninoculated piglets or piglets inoculated with nonpathogenic intestinal E. coli 862B, which is also able to colonize the intestinal tract. There was little difference in cytokine transcript levels in the intestinal and extraintestinal organs in piglets inoculated with E. coli strains M623 or M623DeltaCNF1, except in the ileum, where IL-1alpha and IL-8 mRNA levels correlated with bacterial colonization. Expression of regulatory cytokines (gamma interferon and IL-4) was weak in immune tissues from piglets inoculated with M623 or M623DeltaCNF1. Taken together, our data indicate that the CNF1-producing strain, M623, is pathogenic and induces inflammatory cytokine expression in germfree, colostrum-deprived piglets. Nevertheless, in this model, the CNF1 toxin does not appear to be a major factor for pathogenicity or cytokine response, as demonstrated by the use of an isogenic cnf1 mutant.

Phase variation of F165(1) (Prs-like) fimbriae from Escherichia coli causing septicaemia in animals.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and synthesize fimbriae involved in virulence, designated as F165(1). F165(1) fimbriae belong to the P fimbrial family and are encoded by the foo gene cluster. The foo regulatory region of strain 5131 possesses characteristics similar to that of members of the P regulatory family, including papI and papB homologues, and two GATC sites separated by 102 bp, targets of differential Dam methylation. In wild-type strains, the synthesis of F165(1) is repressed by leucine and the fimbriae undergo phase variation. Immunofluorescence staining showed that phase variation of F165(1) results in a majority of cells (98%) in the ON phase, in contrast with phase variation of other members of this regulatory family, for which the majority of the cells are in the OFF state. Using a translational fusion in strain 5131 between phoA and fooA, encoding for the major structural subunit of F165(1), it was shown that leucine inhibits the OFF to ON switch and modulates the basal transcription of the foo operon.

Optimisation of a reagentless laccase electrode for the detection of the inhibitor azide.

Here we report on the optimisation of a reagentless enzyme sensor for the detection of azide based on the mediated reduction of O2 by a laccase enzyme co-immobilised in a redox hydrogel on electrode surfaces. The sensor response is shown to be influenced by the enzyme loading, the electrolyte pH and ionic strength. The response of the sensor is stable, decreasing by only 25% over a sixteen-hour period. Reproducible inhibition curves for the determination of azide levels from cyclic voltammetric scans can be obtained by normalisation of the sensor response. The resulting enzyme inhibition biosensor can detect levels of azide as low as 2.5 microM under these conditions. Constant potential amperometric detection at the laccase enzyme electrode in a flow injection set-up yields a peak current for inhibition of the mediated reduction of O2. Reproducible peak currents and areas (8.0 and 6.3% RSD, respectively, for n = 11) are obtained for repeated injections of 100 microM azide. Reproducible response curves can be obtained by injection of a 25 mM azide sample and assuming that the peak height and peak area obtained represent 100% inhibition of the enzyme.

Development of an assay for urinary free cortisol determination on the Technicon Immuno 1 system.

OBJECTIVES: To develop and evaluate a method using an ethyl acetate extraction procedure for the determination of urinary free cortisol on the Technicon Immuno 1 system from Bayer Corporation. DESIGN AND METHODS: We tested the assay precision, linearity, and correlation with the Urinary Kallestad Quanticoat Cortisol radioimmunoassay. We also studied the efficiency of the extraction procedure, performed a cross-reactivity study with different cortisol metabolites, and determined the reference values. RESULTS: The assay shows within-run CVs varying from 1.6 to 5.3% and between-day CVs from 2.7 to 6.1% for urinary free cortisol concentrations from 58 to 1097 nmol/L. The assay demonstrates an excellent linearity and a very good correlation with the Kallestad Quanticoat Cortisol assay (slope = 0.94, y-intercept = 29 nmol/L, Sy/x = 54 nmol/L, r = 0.996). The reference values were estimated at 42-281 nmol/d. The extraction procedure shows an average recovery of 99.0% and minimal interference with the cortisol metabolites tested with the exception of cortisone. CONCLUSIONS: The evaluation shows that the developed assay has the analytical characteristics required for its utilization in a clinical laboratory.

A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase.

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.

Mutations in the f165(1)A and f165(1)E fimbrial genes and regulation of their expression in an Escherichia coli strain causing septicemia in pigs.

Transposon (TnphoA) mutagenesis was used to study the expression of F165(1) fimbriae, related to Prs fimbriae, in the pathogenic Escherichia coli strain 5131 (O115:K "V165":F165). This strain causes septicemia in swine and also expresses F165(2) fimbriae, related to F1C. Adhesin-defective mutants from the wild-type pathogenic strain were produced and TnphoA insertions were localized either in the f165(1)A gene, which encodes the major fimbrial subunit or in the f165(1)E, gene, which encodes a minor fimbrial subunit. TnphoA gene fusions were used to measure expression of F165(1) fimbrial genes. Similar pattern of regulation of expression was observed in both f165(1)A and f165(1)E genes. Optimal expression of F165(1) fimbriae was obtained on solid minimal medium. Production of F165(1) fimbriae was negatively regulated by addition of glucose, leucine or alanine to the media, by growth at 18 degrees C, and by pH above or below 7.0.

Reagentless mediated laccase electrode for the detection of enzyme modulators.

We have investigated aerobic mediation of electron transfer to a laccase enzyme by the solution redox couples [Os(bpy)(2)Cl(2)](+/0) and [Os(bpy)(2)(MeIm)Cl](2+/+), where bpy is 2,2-bipyridine and MeIm is N-methylimidazole. The factors that influence the homogeneous mediation reaction are investigated and discussed. Investigation of ionic strength, pH, and temperature effects on the kinetics of intermolecular electron transfer elucidates the governing factors in the mediator-enzyme reactions. Coimmobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the reagentless addressing of enzyme activity, consuming only oxygen present in solution. Thus, these immobilized enzyme biosensors can be utilized for the detection of modulators of laccase activity, such as the inhibitor sodium azide. The enzyme inhibition biosensor can detect levels of azide as low as 2.5 × 10(-6) mol dm(-3) in solution.

Performance evaluation of automated immunoassays on the Technicon Immuno 1 system.

We performed an immunoassay evaluation for various analytes on a fully automated random-access analyzer, the Technicon Immuno 1 system from Bayer Corp. This system involves latex agglutination, magnetic separation sandwich, and magnetic separation competitive immunoassay configurations. The evaluated analytes were thyrotropin (TSH), triiodothyronine, thyroxine, free thyroxine, follitropin, lutropin, prolactin, beta subunit of human chorionic gonadotropin, cortisol, ferritin, alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen. We tested the assay precision, linearity, and correlation with comparison methods for these analytes. The functional sensitivity of the TSH assay and the sample-to-sample carryover were also studied. Excellent results were obtained for within-run and between-day precision studies, with most assays showing within-run CVs <4% and between-day CVs <6%. The linearity for all assays was acceptable and the correlation between Immuno 1 assays and comparison methods showed satisfactory results. The functional sensitivity of the TSH assay was estimated at 0.04 mU/L. No sample-to-sample carryover was detected.

Identification of a mutation in the pst-phoU operon that reduces pathogenicity of an Escherichia coli strain causing septicemia in pigs.

We used transposon (TnphoA) mutagenesis to study the role of virulence factors of pathogenic Escherichia coli strains associated with septicemia in calves and piglets. We have produced an avirulent and serum-sensitive mutant of wild-type pathogenic strain 5131 O115:K"V165":F165 and have localized and identified the TnphoA insertion in the pstC gene of the pst-phoU operon. This operon encodes the PstSCAB transporter and PhoU protein that negatively regulate the phosphate (Pho) regulon. This mutation is pleiotropic and could have an effect on pathogenicity and on the production of the surface polysaccharides of strain 5131. The mutant demonstrated restored repressibility of alkaline phosphatase and regained the capacity to resist serum and to survive systemically for at least 5 days in experimentally inoculated pigs when complemented with plasmid pAN92, bearing the pst-phoU operon.

Expression and detection of pap-, sfa-, and afa-encoded fimbrial adhesin systems among uropathogenic Escherichia coli.

Thirty-five Escherichia coli isolates from young children and women with pyelonephritis, cystitis, or asymptomatic bacteriuria were characterized genotypically and phenotypically. The isolates were examined genotypically by using DNA probes specific for the hemolysis gene and for the pap, sfa, and afa adhesin systems. Genes for the adhesin systems were also detected by polymerase chain reaction, using multigene amplification. The isolates were serotyped, tested for hemolysin production, and classified for their adhesion specificity by hemagglutination and by binding specificity assays. Twenty-seven of the 35 isolates were pap positive. Results showed that pap-positive isolates expressing class I or class II G adhesins were more frequent in cases of pyelonephritis than in cases of cystitis and asymptomatic bacteriuria. Expression of the class III G adhesins was more frequent in isolates from cystitis and asymptomatic bacteriuria than in isolates from pyelonephritis. Multiple adhesin systems and hemolysin were more frequently found in isolates from cases of pyelonephritis than in isolates from cases of cystitis and asymptomatic bacteriuria. There was perfect correlation between the results obtained by polymerase chain reaction and and those obtained by hybridization.

Pathogenicity of an Escherichia coli O115:K"V165" mutant negative for F165(1) fimbriae in septicemia of gnotobiotic pigs.

To evaluate the role of the F165(1) fimbrial system in the pathogenesis of septicemia, 2-day-old germfree pigs were inoculated intragastrically with Escherichia coli O115:K"V165":F165 wild-type strain 5131, its F165(1)-negative TnphoA mutant M48, or E. coli O115:K(-):F165(-) wild-type strain 862B. Pigs were sacrificed at different times (3, 6, 12, 24, 48, and 96 h) postinfection (p.i.). Pigs inoculated with strain 5131 developed clinical signs (anorexia, lameness, reluctance to move, or lack of motor coordination) and were moribund within 48 h p.i., and, at necropsy, infecting bacteria were isolated in various extraintestinal organs. Strain 5131 was isolated as early as 6 h p.i. from the blood of inoculated pigs. Pigs inoculated with mutant M48 developed only mild clinical signs at 96 h p.i. Mutant M48 colonized extraintestinal organs of pigs but to a lesser extent than the parent strain did. In contrast to the parent strain, this mutant was not isolated in the blood of inoculated pigs. Pigs inoculated with strain 862B remained normal during the experiment. All of the strains colonized the mucus layer of the intestine, but no histological changes of intestinal mucosa were observed by either light or electron microscopy. The parent strain, but not the mutant M48, expressed F165(1) in vivo. In a competitive study in which the parent strain and its afimbrial mutant were inoculated simultaneously, clinical signs of septicemia developed 24 h after inoculation, and only the parent strain 5131 was isolated from the blood of inoculated pigs. Our results suggest that the F165(1) fimbrial system of E. coli O115:K"V165" strains may play an important role in the ability of the bacteria to survive in the blood and spread systemically through the porcine host.

Molecular cloning of a determinant coding for fimbrial antigen F165(1), a Prs-like fimbrial antigen from porcine septicaemic Escherichia coli.

The genetic determinant coding for F165(1) fimbriae was cloned from the chromosome of the porcine Escherichia coli wild-type strain 4787 (O115:K-:H51:F165). The fimbrial determinant was further subcloned into the BamHI site of pACYC184 and a restriction map was established. On Southern hybridization, identity between the chromosomally encoded prs-like determinant of strain 4787 and its cloned counterparts was demonstrated. The cloned F165(1) fimbriae and those of the wild-type strain possessed a major protein subunit of molecular mass 18.5 kDa. Strains expressing F165(1) fimbriae were detected using an F165-specific polyclonal antiserum and caused mannose-resistant haemagglutination and agglutination of Forssman latex beads. Antiserum against the cloned F165(1) fimbriae recognized a 18.5 kDa band in the parent strain 4787.

Occurrence of pap-, sfa-, and afa-related sequences among F165-positive Escherichia coli from diseased animals.

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.