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Biomedical subjects

B W Wren

Publications and source records attributed to B W Wren.

At least 73 records · Page 4Linked to original sources
PubMed1993

Characterisation of Clostridium difficile strains by polymerase chain reaction with toxin A- and B-specific primers.

A total of 218 Clostridium difficile strains was examined for production of toxin A by ELISA, production of toxin B by a cytotoxin assay and the presence of toxin A and B gene-associated sequences by the polymerase chain reaction (PCR). After saturation amplification with toxin B-specific primers, the characteristic amplification product (591 bp) was detected in all 184 toxigenic strains examined. PCR with toxin A-specific primers gave positive results with all but one of the toxigenic strains. By contrast, PCR with toxin A- and toxin B-specific primers yielded negative results with all 34 non-toxigenic strains tested. This suggests that PCR detection of either the toxin A or B gene is a good indication of toxin production. PCR did not require DNA extraction or hybridisation and was convenient, sensitive and rapid. Toxigenic C. difficile could be detected in mixed cultures, suggesting a role for PCR in the identification of toxigenic C. difficile in primary culture.

Bacterial Proteins↗
PubMed1992

Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.

Amino Acid Sequence↗
PubMed1992

Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.

Animals↗
PubMed1991

A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences.

Analysis of the derived amino acid sequences of toxins A and B from Clostridium difficile has identified an extraordinarily large number of repeat amino acid units in the C-terminal regions of the proteins. Nearly one third of each of the proteins consist of repeating units which appear, at least in the case of toxin A, to be responsible for carbohydrate binding. Similar repeat units are also found in the C-terminal region of four glucosyltransferases from Streptococcus mutans and Streptococcus downei, and in four lytic enzymes from Streptococcus pneumoniae and its bacteriophages (HB-3, Cp-1 and Cp-9). In each case the repeats constitute the ligand-binding portion of the respective enzymes. A glucan-binding protein from S. mutans, which lacks enzymatic activity, has similar repeats spanning almost the entire molecule. This family of ligand-binding proteins appears to be of modular design, with one module consisting of a repetitive ligand-binding domain located in the C-terminal region and the other module(s) providing enzymatic functions.

Amino Acid Sequence↗
PubMed1991

Antigenic cross-reactivity and functional inhibition by antibodies to Clostridium difficile toxin A, Streptococcus mutans glucan-binding protein, and a synthetic peptide.

A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits. Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide. A polyclonal antibody against the glucan-binding protein, which cross-reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. We recently identified toxin A and the glucan-binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules. This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction.

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

Amino Acid Sequence↗
PubMed1990

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

Bacterial Toxins↗
PubMed1989

Hybridization analysis of three chloramphenicol resistance determinants from Clostridium perfringens and Clostridium difficile.

The chloramphenicol resistance determinant from a nonconjugative strain of Clostridium perfringens was cloned and shown to be expressed in Escherichia coli. Subcloning and deletion analysis localized the resistance gene, catQ, to within a 1.25-kilobase (kb) partial Sau3A fragment. The catQ gene contained internal HindII, HaeIII, and DraI restriction sites and was distinct from the catP gene, which was originally cloned (L. J. Abraham, A. J. Wales, and J. I. Rood Plasmid 14:37-46, 1985) from the conjugative C. perfringens R plasmid, pIP401. Hybridization studies were carried out with a 0.35-kb DraI-P fragment of pJIR260 as an internal catQ-specific probe and a 0.38-kb EcoRV-HinfI fragment of pJIR62 as an internal catP-specific gene probe. The results showed that the catP and catQ genes were not similar and that neither probe hybridized with cat genes from other bacterial genera. However, the catP gene was similar to the cloned catD gene from Clostridium difficile. Comparative studies with both catP and catD probes showed that these genes had significant restriction identity. We therefore suggest that these genes were derived from a common source.

Blotting, Southern↗
PubMed1989

Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.

Antigen-Antibody Reactions↗
PubMed1988

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile.

Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.

Antigens, Bacterial↗
PubMed1988

Molecular cloning and genetic analysis of a chloramphenicol acetyltransferase determinant from Clostridium difficile.

A gene bank from a clinical isolate of Clostridium difficile expressing high chloramphenicol acetyltransferase activity was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the plasmid vector pUC13. Among 1,020 clones tested, 11 were resistant to chloramphenicol; 1 of these, with an insert size of 1.9 kilobases (pPPM9), was studied further. The clone pPPM9 was mapped using a variety of restriction enzymes, and a 0.27-kilobase EcoRV-TaqI restriction fragment was shown to be within the chloramphenicol resistance (Cmr) gene by using transposon (Tn1000) mutagenesis. The 0.27-kilobase fragment and the 1.9-kilobase insert were radiolabeled and used as DNA probes in hybridization studies. Southern blot analysis with the gene probes against chromosomal DNA from Cmr strains of C. difficile obtained from five distinct geographical locations revealed that at least two copies of the same chloramphenicol acetyltransferase gene were present for each strain. Hybridization of the gene probes against Cmr strains of Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella edwardsii, Escherichia coli, and to four other clostridial species revealed no homology even under conditions of low stringency.

Blotting, Southern↗