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Biomedical subjects

D E Taylor

Publications and source records attributed to D E Taylor.

At least 145 records · Page 8Linked to original sources
PubMed1990

Genetic analysis of transfer and incompatibility functions within the IncHI plasmid R27.

This study was undertaken to establish a transfer complementation system for IncH plasmids and to locate regions of incompatibility within the HI1 plasmid, R27. Two regions of R27 were found to contribute to incompatibility as determined by incompatibility testing with fragments of R27 cloned in cosmid vectors. One of these regions hybridized with the IncHI1 rep probe (Couturier et al., Microbiol. Rev. 52, 375-395, 1988). Complementation analysis was carried out using transfer-deficient mutants of R27 in combination with pHH1508a. Cosmid vectors, which contained cloned restriction fragments of R27, were able to complement selected R27 Tra- mutants, enabling the transfer-deficient plasmid to transfer at near-normal frequencies. Complementation of R27 Tra- plasmids by pHH1508a at both 26 and 37 degrees C was shown to occur, but was host-dependent in its degree. These results suggest that the transfer mechanisms of IncHI and IncHII plasmids are related.

Cloning, Molecular↗
PubMed1990

Use of non-radioactive DNA probes for detection of Campylobacter jejuni and Campylobacter coli in stool specimens.

DNA probes specific for C. jejuni (pDT1720 containing a 1475 base pair fragment) and for C. jejuni and C. coli (pDT1719 containing a 1845 base pair fragment) were isolated from a bacteriophage lambda gt11 genomic library of C. jejuni, using antiserum prepared against a 46 kDa major outer membrane protein of C. jejuni. The two probe-fragments had different restriction maps and were only moderately related by DNA hybridization analysis. A non-radioactive labelling kit which consisted of alkaline phosphatase conjugated anti-digoxigenin antiserum and 5-bromo-4-chloro-3-indoyl phosphate with nitroblue tetrazolium as the colour substrate, which gives a purple colour for positive hybridization, was used to test 140 stool specimens, 70 of which were culture positive and 70 of which wer culture negative for Campylobacter spp. The pDT1720 fragment (C. jejuni probe) could detect a minimum of 1 x 10(5) C. jejuni cells on filters, whereas the pDT1719 fragment (C. coli probe) was 100-fold less sensitive. The C. jejuni probe demonstrated a sensitivity of 93% with culture positive stool samples, however, 15% of culture negative samples were also recorded as positive using this non-radioactive DNA probe.

Blotting, Southern↗
PubMed1990

Simultaneous resistance to two or more broad-spectrum anthelmintics by gastrointestinal nematode parasites of sheep and goats.

The detection, by means of faecal egg count reduction tests and larval cultures, of two cases of multiple anthelmintic resistance in goats and two in sheep, is reported. The former cases appeared to involve resistance to all three broad-spectrum drench groups (benzimidazole, levamisole, ivermectin) by mainly Ostertagia spp., the latter resistance to two of them (benzimidazole, levamisole) by Trichostrongylus spp.

Journal Article↗
PubMed1990

Molecular studies on the mechanism of tetracycline resistance mediated by Tet(O).

The mechanism of resistance to tetracycline in Escherichia coli mediated by the Campylobacter jejuni-derived resistance determinant Tet(O) was investigated. The cloned Tet(O) protein had no detectable effect on the intracellular accumulation of tetracycline. The presence of Tet(O) markedly diminished the inhibitory effect of tetracycline on protein synthesis both in vivo and in vitro. Ribosomes prepared from tetracycline-resistant and susceptible E. coli cells bound almost identical amounts of radiolabeled tetracycline. Thus, a reduction in the binding of the antibiotic to its target site on the ribosome is not the primary mechanism of resistance. Poly(U)-directed polyphenylalanine synthesis revealed that an S-100 fraction prepared from tetracycline-resistant cells made the ribosomes prepared from susceptible cells considerably more resistant to the inhibitory action of tetracycline. The N-terminal portion (1 to 150 residues) of Tet(O) is highly homologous to the GTP-binding domain of elongation factor Tu and to elongation factor G, indicating that the Tet(O) protein has the potential to bind GTP. These data suggest that the Tet(O) protein could function either as a tetracycline-resistant analog of this elongation factor(s) or by modifying the target sites on the ribosomes in a catalytic fashion.

Amino Acid Sequence↗
PubMed1990

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.

Campylobacter↗
PubMed1990

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.

Base Sequence↗
PubMed1989

Nomenclature for tetracycline resistance determinants.

Tetracycline resistance determinants are widespread and distinguishable genetically and biochemically. The nomenclature for this increasing number of determinants has been varied and inconsistent. This communication suggests ways of naming these determinants and their genes and gene products consistent with current genetic terminology.

Bacteria↗
PubMed1989

Comparison of tellurite resistance determinants from the IncP alpha plasmid RP4Ter and the IncHII plasmid pHH1508a.

The tellurite resistance (Ter) determinants of the IncHII plasmid pHH1508a and the broad host range IncP alpha plasmid RP4Ter were cloned into pUC8, creating plasmids pDT1364 and pDT1558, respectively. The Ter region of pDT1364 was localized to a 1.25-kilobase region by using Tn1000 insertion mutagenesis. Insertions of Tn1000 into pDT1558 which resulted in tellurite sensitivity spanned 1.75 kilobases of DNA. No similarity between the restriction maps of these two plasmids was observed, and no homology could be detected by DNA-DNA hybridization. Expression in an in vitro transcription-translation system showed that pDT1364 encoded two polypeptides with molecular masses of 23 and 12 kilodaltons (kDa) which were not expressed by pUC8. Some of the Tn1000 insertion mutants did not express the 23-kDa protein. pDT1558 encoded a 40-kDa polypeptide which was not expressed by pUC8. Both Ter determinants were expressed constitutively. Our findings suggest that the mechanisms of Ter encoded by these two plasmids are different.

Bacterial Proteins↗
PubMed1989

Isolation and characterization of catalase-negative and catalase-weak strains of Campylobacter species, including "Campylobacter upsaliensis," from humans with gastroenteritis.

During 1987 and 1988, nine strains of catalase-negative or -weak Campylobacter species were isolated in Alberta, Canada. DNA hybridization studies demonstrated that seven strains were "Campylobacter upsaliensis," one strain was highly homologous with Campylobacter jejuni DNA, and one strain was a campylobacter unrelated to the other two species. All "C. upsaliensis" strains were hippurate negative, and six of seven were susceptible to cephalothin. The unusual variant of C. jejuni was hippurate positive and cephalothin resistant, whereas the unclassified strain was hippurate negative and resistant to intermediate levels of cephalothin. All patients from whom "C. upsaliensis" was isolated had diarrhea. Five of the patients were children two years old or younger, and two were adults. In this study, all catalase-negative and -weak strains were isolated from stool specimens by using a charcoal-based selective medium containing 32 micrograms of cefaperazone per ml and which was described by Hutchinson and Bolton (D. N. Hutchinson and F. J. Bolton, J. Clin. Pathol. 37:956-957, 1984).

Adult↗
PubMed1989

Biophysics of impact injury to the chest and abdomen.

The physical mechanism of blunt impact injuries to thoracic and abdominal viscera is often conveniently described simply in terms of "crush"--this is an over-simplification. Any impact to the torso does result in the rapid displacement of the body wall which may lacerate and contuse underlying viscera, but this simple explanation does not account for pathology at sites some distance from the contact point and does not adequately describe the dependence of the severity and location of injury upon the rate of energy transfer. Quite minor displacements of the body wall may produce serious injury if the body wall velocity is high. The motion of the body wall generates waves that propagate within the body and transfer energy to internal sites. The nature and properties of these waves are discussed in simple terms and the role of waves in the production of the characteristic injuries resulting from impact to the torso is presented.

Abdominal Injuries↗
PubMed1989

Mapping of transfer and H pilus coding regions of the IncHII plasmid pHH1508a.

The IncHII plasmid pHH1508a (208 kilobases) encodes resistance to potassium tellurite, trimethoprim, and streptomycin. Conjugative pili encoded by pHH1508a were isolated, purified, and used for preparation of anti-H pilus antiserum. Immuno-gold labelling experiments using H pilus specific antiserum showed that antigenic determinants were located along the entire length of the H pilus. Immuno-gold labelling and lysis studies using pilH alpha, a bacteriophage specific for H pili, were used to investigate transfer-deficient mutants of pHH1508a obtained by Tn5 mutagenesis and an in vitro constructed derivative of 96 kilobases, pDT1178, which also conferred resistance to potassium tellurite, trimethoprim, and streptomycin. The transfer-deficient mutants did not specify H pili, whereas pDT1178, which transferred at low frequency (1 x 10(-4) transconjugants per recipient), specified a small number of H pili. A naturally occurring plasmid, pMG110, was found to encode the production of H pili, but was completely transfer deficient (less than 1 x 10(-7) transconjugants per recipient). This study suggests that genes required for H pilus production and assembly as well as low level transfer are located separately within the 96-kilobase fragment of pDT1178 and that other genes, located outside this region, are essential for the regulation and full expression of conjugative transfer.

Conjugation, Genetic↗
PubMed1989

Emergency room use and primary care case management: evidence from four Medicaid demonstration programs.

Claims-based utilization data collected in four Medicaid demonstration programs are used to examine the impact of primary care case management on patterns of reliance on the emergency room as a source of services. The experience of stratified random samples of AFDC (Aid to Families with Dependent Children) adults and children in the demonstration programs is compared with that of equivalent samples from comparison groups in traditional Medicaid programs. Data were analyzed in multivariate models controlling for person characteristics in a pre/post, demonstration/comparison design. Results indicated large reductions in the proportion of persons with at least one emergency room visit ranging from 27 to 37 per cent for children and 30 to 45 per cent for adults. Use levels for persons with at least one ER visit are less substantially affected. The findings suggest that gatekeeping designs can alter patterns of enrollee use with respect to the emergency room and some discussion of these implications is presented.

Adult↗
PubMed1988

Nucleotide sequence analysis and expression of a tetracycline-resistance gene from Campylobacter jejuni.

Resistance to tetracycline in the microaerophilic Gram-negative bacterium Campylobacter jejuni is plasmid-mediated. A 6.9-kb HindIII DNA fragment containing the tetracycline-resistance (TcR) gene (designated tetO) from the C. jejuni conjugative plasmid pUA466 was cloned into pUC8, and the resultant plasmid pUOA1 was used to transform Escherichia coli to Tc resistance. The tetO gene was localized at a 2.0-kb region comprising 0.2-kb and 1.8-kb HincII fragments, and the nucleotide sequences were determined. The protein coding region of tetO contained a 1911-bp open reading frame which corresponded to a 72.3-kDa protein. Upstream from the start codon were hexanucleotides that resembled the canonical sequences found at the -10 region, -35 region and the ribosome-binding site of the prokaryotic promoter. The tetO gene product was expressed utilizing an E. coli-derived in vitro transcription/translation system. The polypeptide had an apparent Mr of 68,000. Comparison of the amino acid sequences of TetO to those of TetM (derived from the Gram-positive Streptococcus pneumoniae) revealed 76% homology. Hydrophilicity plot analyses of TetO and TetM proteins provided almost identical profiles. These results clearly support our earlier [Taylor et al., J. Bacteriol. 169 (1987) 2984-2989] suggestion that TcR determinants found in Gram-positive bacteria and in C. jejuni may have a common ancestry.

Amino Acid Sequence↗
PubMed1988

Structure and location of tellurium deposited in Escherichia coli cells harbouring tellurite resistance plasmids.

The plasmids RP4Ter and pHH1508a, which belong to the P and HII incompatibility groups, respectively, confer resistance to potassium tellurite (K2TeO3) on Escherichia coli. The genes for tellurite resistance were cloned from each plasmid onto the vector pUC8 to create pDT1366 and pD1364, respectively. Unstained, unfixed bacteria carrying these plasmids contained black intracellular deposits when grown on media containing tellurite. Thin sections of these bacteria fixed with glutaraldehyde were prepared and examined by electron microscopy. The black deposits were located inside the cell and were frequently associated with the inner membrane of the bacterium. Bacteria containing pDT1366 or pDT1364, and therefore a higher gene dosage of the Ter determinant, contained more black deposits, but had a decreased resistance, as measured by the minimum inhibitory concentration using the agar dilution method. Using the technique of electron spectroscopic imaging, the black intracellular deposits were shown to contain predominantly reduced metallic tellurium, and significant amounts of oxygen or carbon, thereby confirming earlier results using X-ray diffraction analysis of whole cells.

Cell Membrane↗