Biomedical subjects

J Harel

Publications and source records attributed to J Harel.

At least 55 records · Page 3Linked to original sources

PubMed1995

Alterations in penicillin-binding proteins in strains of Streptococcus suis possessing moderate and high levels of resistance to penicillin.

We examined the penicillin-binding proteins (PBPs) of certain field strains of Streptococcus suis, as well as those from laboratory variants having different degrees of resistance to penicillin. Results indicated that (i) S. suis possesses three distinct groups of PBPs, arbitrarily named here PBP 1, PBP 2, and PBP 3, with approximate molecular weights of 97, 82, and 45 kDa respectively; (ii) PBP profiles of field strains of S. suis having different MICs (< or = 0.03) to 16.0 micrograms/ml) were not uniform (PBP 2 being difficult to detect in strains whose MICs exceeded 0.10 micrograms/ml, and PBP 3 which exhibited shifts in molecular weight of approximately 5 kDa); (iii) laboratory variant PBPs 1 and 2 showed decreased affinity for penicillin as compared to the parent strain in antibiotic competition experiments, even though the PBP profiles of both were similar. We suggest that PBP modifications (altered molecular weight and/or decreased affinity for penicillin) are involved in the mechanism of resistance to penicillin by S. suis.

Bacterial Proteins↗

PubMed1995

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs.

We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA (E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA+ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA- or eaeA+ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli. In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and effacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli.

Adhesins, Bacterial↗

PubMed1995

DNA probe and polymerase chain reaction procedure for the specific detection of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae, a Gram-negative anaerobic spirochete, is the causative agent of swine dysentery, a mucohaemorrhagic diarrheal disease in which lesions are confined to the large intestine of pigs. A DNA probe and polymerase chain reaction (PCR) amplification procedures which are specific, rapid , and sensitive for the detection of S.hyodysenteriae have been developed. Clone pF12 from a plasmid library of S.hyodysenteriae B204 genomic DNA was identified as a clone specific for S.hyodysenteriae but not for S.innocens by differential hybridization screening with S.hyodysenteriae and S.innocens genomic DNA probes. A DNA probe consisting of a 1.3 kb restriction fragment from pF12 was found to be highly specific for S. hyodysenteriae and detected 10(5) bacterial cells. A PCR procedure using primers derived from this fragment yielded a single product which was specifically generated for S.hyodysenteriae template DNA and not for other control cells DNA. PCR provided increased sensitivity with the direct detection of as few as 10 S.hyrodysenteriae cells. The PCR procedure could detect S.hyodysenteriae cells in seeded faecal matter. Moreover the PCR assay was able to detect most S. hyodysenteriae field isolates of serotypes 8 and 9. These tools have diagnostic application in veterinary microbiology.

Animals↗

PubMed1995

Cloning of determinants encoding F165(2) fimbriae from porcine septicaemic Escherichia coli confirms their identity as F1C fimbriae.

Cloning of the f165(2) operon that encodes F1C-like fimbriae in Escherichia coli indicates that this operon is a member of the S/Foc family. The genetic determinant coding for the F165(2) fimbriae was cloned from the chromosome of the porcine E. coli wild-type strain 4787 (O115:K-1:H51:F165). The cloned F165(2) and the wild-type operon expressed a major fimbrial protein subunit of molecular mass 17.2 kDa that was detected by anti-F165 and anti-F1C polyclonal sera. The sequences of the f165(2)A and f165(2)FGH genes are reported. Major subunit gene f165(2)A encodes a mature protein of 156 amino acids. Minor subunit genes F, G and H encode mature proteins of 148, 145 and 276 amino acids, respectively. The amino acid sequences of the four proteins share similarities with those of the known S and F1C fimbrial antigens that are produced by extraintestinal E. coli which is associated with sepsis, urinary tract infections and newborn meningitis. The F165(2)A protein was identical to the major subunit protein of F1C, with a difference only at the first position. It was also similar, to a lesser extent, to the major subunit proteins of SfaI and SfaII fimbriae. F165(2)F was identical to FocF and SfaG/SfaIIS, and F165(2)H was more closely related to FocG than to SfaIS/SfaIIS, and F165(2)H was more closely related to FocH than to SfaIH/SfaIIH.

Amino Acid Sequence↗

PubMed1995

Production of Escherichia coli STb enterotoxin is subject to catabolite repression.

Enterotoxigenic Escherichia coli are known to secrete several types of toxins including STb, a heat-stable enterotoxin. STb enterotoxin production was studied in wild-type E. coli strains. Using a quantitative STb-specific inhibition ELISA, the amount of toxin present in the culture supernatant fractions of various E. coli strains was determined. Variation in the production of STb toxin was observed for the wild-type strains. For E. coli strain 82-4247 grown in trypticase soy broth, the toxin was produced after 4 h of growth and was maximal after about 57 h of growth. The amount of toxin in the culture supernatant fraction increased concomitantly with bacterial growth. Using the rat loop assay, the biological activity of STb was retained even after the logarithmic phase of growth when STb production levelled off (i.e. from 24 to 74 h). STb production by E. coli strain 82-4247 varied with the culture medium used. In particular, addition of 1.0% (w/v) compared to 0.1% glucose to Davis minimal medium decreased STb production, whereas addition of 1.0% (w/v) glycerol did not affect STb production. Addition of exogenous cAMP reversed the repressive effect of glucose. Using mutant strains, STb production was shown to be subject to catabolite repression.

Adenylyl Cyclases↗

PubMed1995

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.

Animals↗

PubMed1995

Interaction with pig ileal explants of Escherichia coli O45 isolates from swine with postweaning diarrhea.

We have previously observed that Escherichia coli O45 isolates from swine postweaning diarrhea (PWD) induced attaching-effacing (A/E) lesions in experimentally inoculated gnotobiotic piglets. In the present work, ileal explant culture has been used as an in vitro model for the study of the development of A/E lesions due to these isolates. The characteristic intimate bacterial attachment and microvilli effacement with cupping and pedestal formation, identical to that observed in gnotobiotic piglets, was demonstrated in pig ileal explants inoculated with O45 E. coli isolates. The initial attachment of bacteria to the enterocytes was observed from 2 to 4 h postinoculation (PI) and full development of A/E lesions was observed within 8 h PI. In this model, we observed that 22 of 25 eaeA-positive O45 isolates induced A/E lesions. However, A/E lesions were not observed for any of 7 eaeA-negative O45 isolates. Thus, we describe a useful in vitro model for the study of A/E capacity of porcine E. coli. Use of this model has enabled us to demonstrate the relatedness of the eaeA gene to A/E capability among porcine O45 E. coli from PWD.

Animals↗

PubMed1994

Analysis of genes coding for the major and minor fimbrial subunits of the Prs-like fimbriae F165(1) of porcine septicemic Escherichia coli strain 4787.

Septicemic Escherichia coli 4787 of porcine origin produces a Prs-like fimbrial antigen, F165(1) which agglutinates sheep erythrocytes in a similar manner to Prs fimbriae, but unlike the latter, also agglutinates pig erythrocytes. In a previous study, we reported the cloning of the f165(1) operon and showed that it encodes a Prs-like adhesin. Here, we report the sequence of the f165(1)A, and f165(1)EFG genes. f165(1)A encodes a protein of 161 amino acids preceded by a signal peptide of 21 amino acids. A size of 19.3 kDa was calculated for the processed F165(1)A protein. The E, F, and G open reading frames potentially give rise to mature proteins of 149, 148 and 313 amino acids respectively. The F165(1)A protein showed significant similarity with the major subunit protein of P-fimbriae of F11 serotype, differing only in four positions. F165(1)E and F165(1)G were found to be closely related to the PrsE and PrsG proteins of Prs-fimbriae variants, whereas F165(1)F was found to be almost completely identical to the F protein of various P and Prs fimbriae. Our results indicated that the f165(1) is a mosaic operon consisting of sequences related to both the pap operon and the prs operon.

Amino Acid Sequence↗

PubMed1994

Neighboring plasmid sequences can affect Mini-Mu DNA transposition in the absence of expression of the bacteriophage Mu semi-essential early region.

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.

Bacteriophage mu↗

PubMed1994

Virulence properties and attaching-effacing activity of Escherichia coli O45 from swine postweaning diarrhea.

Escherichia coli O45 isolates associated with swine postweaning diarrhea in Québec were characterized with respect to virulence determinants genetically and investigated for their attaching and effacing (A/E) activities by experimental inoculation of gnotobiotic piglets and by the HEp-2 cell adherence assay. All of 32 isolates tested were negative for enterotoxigenic and verotoxigenic E. coli virulence determinants, heat-labile enterotoxin (LT), heat-stable enterotoxins (STap, STb), verotoxins (VT1, VT2), and F4 (K88), F5 (K99), F6 (987P), and F41, except one STb-positive and two F4-positive isolates. A total of 25 isolates hybridized with an EaeA probe, and 11 hybridized with an enteropathogenic E. coli adherence factor (EAF) probe. None of 32 isolates hybridized with a bundle-forming pilus (BFP) probe. The EAF, EaeA, and BFP factors have been associated with human enteropathogenic E. coli strains. A total of 10 of 12 eaeA-positive porcine O45 isolates induced A/E lesions characterized by intimate adherence of bacteria to the intestinal epithelial cell membrane with effacement of the microvilli, similar to those of human attaching-effacing E. coli. However, A/E lesions were not observed in the piglets inoculated with any one of three eaeA-negative O45 isolates. All E. coli O45 isolates were non-adherent to HEp-2 cells. Thus, we have demonstrated the production of typical A/E lesions by nonenterotoxigenic E. coli O45 isolates from swine postweaning diarrhea. The results indicate the significance of the eaeA gene in A/E activities of these isolates and suggest that EAF and BFP are not involved in O45 E. coli infection of weaning piglets.

Adhesins, Bacterial↗

PubMed1994

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.

Adhesins, Escherichia coli↗

PubMed1994

Genomic relatedness among reference strains of different Streptococcus suis serotypes.

Hybridization studies using genomic DNA and a rDNA probe revealed genetic relatedness among reference strains of different Streptococcus suis serotypes. Although most serotype 22 isolates are biochemically atypical, the reference strain of capsular type 22 is genetically related to other S. suis serotypes, but not to Streptococcus pneumoniae. Using DNA digested with BamHI and BglII for ribotyping, some S. suis reference strains had common patterns, but this analysis mainly revealed variations in patterns of S. suis strains of different serotypes.

Blotting, Southern↗

PubMed1994

Characterization of Serpulina hyodysenteriae isolates of serotypes 8 and 9 from Quebec by restriction endonuclease fingerprinting and ribotyping.

This study was undertaken to assess the discriminatory value of restriction endonuclease fingerprinting (REF) analysis and ribotyping of 21 Serpulina hyodysenteriae isolates of serotypes 8 and 9. For REF analysis, DNAs were digested with the BglII restriction enzyme and the resultant fragments were separated by polyacrylamide gel electrophoresis. For ribotyping, hybridization of BglII genomic fragments with a probe of rrnB operon using an Escherichia coli rDNA probe was performed on all isolates. Although many isolates shared a common pattern by BglII REF and BglII ribotyping analysis, differences among some S. hyodysenteriae isolates were observed. REF and ribotyping using BglII restriction enzyme, were not specific for serotypes. The predominance of an REF and a ribotype pattern among S. hyodysenteriae isolates from Quebec suggested that epidemiologically important S. hyodysenteriae types occur in different swine herds.

Bacterial Typing Techniques↗

PubMed1994

Attaching and effacing and enterotoxigenic Escherichia coli associated with enteric colibacillosis in the dog.

The objective of this study was to describe observations from cases of enteric colibacillosis in the dog. Thirteen cases of canine enteric colibacillosis were diagnosed from routine necropsy submissions to our diagnostic laboratory from 1980 to 1992. In all cases there was a clinical history of gastrointestinal disease associated with histological and bacteriological evidence of either attaching and effacing Escherichia coli (AEEC) or enterotoxigenic Escherichia coli (ETEC) infection. Of these 13 cases of enteric colibacillosis, 12 were associated with AEEC and one with ETEC. Eight of the 12 AEEC isolates were available for study. They were of various serogroups, non-hemolytic, and negative for the genes coding for fimbrial antigens F4, F5, F6, F41 and F165; enterotoxins STap, STb and LT; and verotoxins VT1 and VT2. These eight isolates were EAE-positive (E. coli attaching and effacing) by colony hybridization; six of these were also EAF-positive (EPEC adherence factor), and six were BFP-positive (bundle-forming pilus). The ETEC isolate was negative for the EAE, EAF and BFP determinants and for the fimbrial antigens tested but was positive for the STap and STb genes. Most of the dogs affected with enteric colibacillosis originated from kennels and pet shops and were aged between 1.5 and 3 months. Coinfection with other enteric pathogens was identified in eight of these 13 cases. This study showed that Escherichia coli should be considered of causal significance when investigating diarrheal disease in dogs, particularly in puppies.

Animals↗

PubMed1993

High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli.

Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible Ptac promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb. This genetic construction provides a readily obtainable source of biologically active STb toxin.

ATP-Binding Cassette Transporters↗

PubMed1993

Virulence factors associated with F165-positive Escherichia coli strains isolated from piglets and calves.

In this study, 91 F165-positive Escherichia coli isolated from calves and piglets with diarrhea or septicemia were characterized with respect to receptor binding specificity, presence of the aerobactin system, production of colicin V, resistance to the bactericidal effects of serum. Although most F165-positive isolates shared similar DNA sequences with pap operon sequences, less than half of these isolates demonstrated MRHA to P antigen of human red blood cells and Forssman antigen of sheep red blood cells recognized by P and F (or Prs) adhesins respectively. Certain F165-positive isolates sharing similar DNA sequences with both pap and sfa operon sequences demonstrated mannose-resistant hemagglutination of sheep erythrocytes, as observed in human uropathogenic E. coli possessing the prs operon. Most isolates caused mannose-resistant, neuraminidase-resistant hemagglutination of human, equine, feline, and bovine erythrocytes. Thus, F165-positive isolates express one or more adhesins with different receptor binding specificities. An association was observed between the various receptor binding specificities and serogroup. Most F165-positive isolates possessed the aerobactin system and were resistant to the bactericidal effects of serum, but only 38.5% isolates produced colicin V.

Adhesins, Escherichia coli↗

PubMed1993

Fusion of the genes encoding Escherichia coli heat-stable enterotoxin b (STb) and the maltose-binding protein to obtain mature STb enterotoxin.

The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.

ATP-Binding Cassette Transporters↗

PubMed1993

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.

Animals↗