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Biomedical subjects

D E Taylor

Publications and source records attributed to D E Taylor.

At least 127 records · Page 7Linked to original sources
PubMed1992

Plasmid-mediated resistance to tellurite: expressed and cryptic.

The ability of some bacteria to grow in the presence of high concentrations of tellurium compounds has been recognized for almost 100 years. Since then, interest in this phenomenon has generated a slow but steady trickle of literature. In the past few years, the use of modern techniques in molecular biology has led to a dramatic increase in our understanding of the genetics of several bacterial determinants for resistance to tellurium compounds. These determinants are frequently found to be encoded by plasmids which carry multiple antibiotic resistance determinants. Our understanding of the biochemistry of these systems remains limited. In this article, the history of the study of bacterial resistance to tellurium compounds is briefly reviewed. This is followed by an analysis of the recent developments in the study of plasmid-mediated resistance determinants. Finally, preliminary investigations on the possible mechanisms of bacterial resistance to tellurium compounds are presented.

Bacteria↗
PubMed1992

Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.

Campylobacter fetus↗
PubMed1992

Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.

Base Sequence↗
PubMed1992

Genome maps of Campylobacter jejuni and Campylobacter coli.

Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.

Bacterial Proteins↗
PubMed1992

The arsenical ATPase efflux pump mediates tellurite resistance.

The ars operon of the resistance plasmid R773 was found to produce moderate levels of resistance to tellurite. A MIC of 64 micrograms of TeO3(2-) per ml was found for Escherichia coli cells harboring plasmids which contained all three of the structural genes (arsA, arsB, and arsC) of the anion-translocating ATPase. MICs specified by plasmids carrying only one or two structural elements or the cloning vector alone were 2 to 4 micrograms/ml. The rate of TeO3(2-) uptake was found to be on the order of 55% less for cultures containing the resistance plasmids.

Adenosine Triphosphatases↗
PubMed1992

Pulsed-field gel electrophoresis for epidemiologic studies of Campylobacter hyointestinalis isolates.

Campylobacter hyointestinalis was isolated from five members of the same family who had previously consumed raw milk. Pulsed-field gel electrophoresis of genomic DNAs from the five strains, after digestion with restriction endonuclease SalI, revealed that three strains had identical genome patterns and therefore appeared to be related, whereas the other two had completely different genome patterns and appeared to be unrelated. We report here for the first time the isolation of C. hyointestinalis from family members who had consumed raw milk. Our study also demonstrates the usefulness of pulsed-field gel electrophoresis for epidemiologic studies of this unusual campylobacter.

Bacteriological Techniques↗
PubMed1992

Genetics of Campylobacter and Helicobacter.

This article reviews the current state of genetic analysis of Campylobacter and Helicobacter. Chromosomal genes cloned from Campylobacter and Helicobacter species are listed along with the method used to identify the cloned gene. Campylobacter plasmid genes that have been cloned and expressed in Escherichia coli and that specify resistance to tetracycline, kanamycin, or chloramphenicol are presented. This review also examines our current knowledge of genetic exchange in Campylobacter, including conjugative plasmid transfer, natural transformation, electrotransformation, and bacteriophage transduction. In Helicobacter, natural transformation has been described and both plasmids and bacteriophages have been observed. Plasmid cloning vectors have been constructed for Campylobacter. Available vectors are discussed and restriction maps of some useful vectors that we have constructed are included. The genome sizes of C. jejuni and C. coli are approximately 1.7 megabases (Mb), whereas the genome size of H. pylori ranges from 1.60 to 1.73 Mb. The positions of various genes on the C. jejuni and C. coli genome maps have been determined using both homologous and heterologous DNA probes. Genomic maps of these organisms are presented.

Caulobacter↗
PubMed1992

Use of disposable skin staplers for bowel anastomosis to reduce laparotomy time in war.

Laparotomy for penetrating wounds to the abdomen is demanding in terms of time, skill and resources. Any modification of existing techniques that will lessen these demands will be of benefit at surgery in forward areas in war. A previous paper (1) compared a method of small bowel anastomosis using disposable skin staplers with a two-layer handsewn anastomosis using fresh pig ileum. The stapled method was both significantly faster to perform and mechanically superior. In two further studies on a total of 12 pigs the stapled method has been compared with a single-layer and a double-layer handsewn small bowel anastomosis. All 12 pigs recovered from laparotomy without complication. Construction times were recorded. The quality of healing of the anastomoses was assessed by clinical observation, post-mortem and histological examination, and tensiometry. The skill required to perform the stapled anastomoses was estimated subjectively. The stapled technique was consistently faster than the handsewn methods. A Kolmogorov two-sample test showed an improvement in times when comparing the stapled method with the single layer, with a significance of P = 0.05. An improvement was also seen when the stapled method was compared with the double layer, with a significance of P = 0.01. There was no statistically significant difference between the construction times for the two handsewn methods. There was no evidence of anastomotic failure or haematoma at post-mortem and no adverse reaction to the metal staples. No features were identified on histology to indicate impaired healing with the stapled method. Tensiometry of the anastomoses showed that the stapled anastomosis is as mechanically reliable as the handsewn anastomosis. The level of skill required to perform the stapled anastomosisis judged to be within the capabilities of relatively junior surgeons once the technique has been mastered in an anastomosis workshop.

Anastomosis, Surgical↗
PubMed1991

Nucleotide sequence and overexpression of the tellurite-resistance determinant from the IncHII plasmid pHH1508a.

The transcription and translation of the tellurite-resistance (TeR) genes of the HII incompatibility group plasmid, pHH1508a, were studied. The nucleotide (nt) sequence of the TeR region was determined and two possible open reading frames, tehA and tehB, were identified. The direction of transcription and translation of these genes was confirmed through the preparation of lacZ and phoA (encoding alkaline phosphatase) fusions. The transcription start point was identified in the sequence using RNA primer extension. The tehA gene codes for a 36-kDa polypeptide which is highly hydrophobic. The TehA protein appears to be located in the inner membrane of the bacterial cell since tehA fusions with both phoA and lacZ were obtained and expressed. The tehB gene codes for a 23-kDa polypeptide which appears to be relatively hydrophilic and is probably located in the cytoplasm. Both proteins were overproduced using a T7 RNA polymerase/promoter system. No nt or amino acid sequence homology could be found between this TeR determinant and the TeR genes from the IncHI-2 plasmid, pMER610, and the IncP alpha plasmid, RK2.

Amino Acid Sequence↗
PubMed1991

Sizing and mapping of the genome of Campylobacter coli strain UA417R using pulsed-field gel electrophoresis.

Agarose-immobilized chromosomal DNA from the nalidixic-acid-resistant Campylobacter coli strain UA417 and its streptomycin-resistant (StrR) derivative, UA417R, were digested with the restriction enzymes SalI (GTCGAC) and SmaI (CCCGGG). The sizes of the resulting fragments were determined using pulsed-field gel electrophoresis. The two genomes showed similar restriction patterns of seven and 13 fragments for the two respective enzymes and the total genome size was determined to be approx. 1.7 Mb. Analysis of partial digestion fragments, as well as Southern-blot hybridization, were used to construct a physical map of the C. coli UA417R genome. Natural transformation studies using DNA fragments extracted from UA417R, as well as the erythromycin-resistant (EryR) C. coli strain UA585, were used to locate the StrR and EryR resistance markers on the genomic map.

Blotting, Southern↗
PubMed1991

Bacteriophages for incompatibility group H plasmids: morphological and growth characteristics.

Two independently isolated temperature-sensitive bacteriophage that are specific for enterobacterial hosts harboring HI and HII plasmids were characterized to determine if any identifiable differences existed between them. The traits examined included adsorption pattern of phage to H pili, bacteriophage size, sensitivity to chloroform, RNA strandedness, reaction with F-specific antiphage serum, virion protein pattern, temperature range of lytic ability, and plaque morphology. No differences between the phages were observed for any of the features analyzed. Ecological questions on the origin and maintenance of temperature-sensitive phages are discussed.

Bacteriophages↗
PubMed1991

Pulsed-field gel electrophoresis of Campylobacter jejuni and Campylobacter coli genomic DNA and its epidemiologic application.

Genomic DNA from 12 different Campylobacter jejuni and 10 Campylobacter coli isolates was digested with SmaI and analyzed by pulsed-field gel electrophoresis (PFGE) and 16S rRNA hybridization studies. Although the two Campylobacter species displayed species-specific restriction and hybridization patterns, significant intraspecies differences were observed. Combined PFGE and hybridization pattern analysis failed to provide any more epidemiologic information than was obtained from PFGE restriction profiles alone. Therefore, results from these studies indicate that PFGE analysis of SmaI-restricted genomic DNA provides a reliable means of differentiating C. jejuni from C. coli and may represent a more practical approach to epidemiologic studies than combining conventional DNA restriction digestion pattern with RNA hybridization procedures.

Animals↗
PubMed1991

Characterization of erythromycin resistance in Campylobacter jejuni and Campylobacter coli.

The mechanism of resistance to erythromycin, the drug of choice in the treatment of campylobacter gastroenteritis, was investigated. Erythromycin resistance (MICs, greater than 1,024 micrograms/ml) in three clinical isolates of Campylobacter jejuni and one C. coli isolate was determined to be constitutive and chromosomally mediated. In vivo protein synthesis in erythromycin-susceptible C. jejuni and C. coli strains was completely inhibited by low levels of erythromycin (5 micrograms/ml), whereas a high concentration of the antibiotic (100 micrograms/ml) had no effect on protein synthesis in erythromycin-resistant strains. Biological assays showed that extracellular degradation of erythromycin was not responsible for erythromycin resistance in strains of Campylobacter species. The rates and amounts of uptake of [14C]erythromycin by resistant and susceptible campylobacter cells were determined to be similar. Binding assays with purified campylobacter 70S ribosomes as well as 50S ribosomal subunits showed that those from erythromycin-resistant strans bound much less [14C]erythromycin than did those from susceptible strains. Genomic DNA from C. coli UA585 was used to transform erythromycin resistance to C. coli UA417. The erythromycin resistance marker was associated with a 240-kb SmaI fragment of the C. coli UA585 genome. Our results rule out erythromycin inactivation or efflux and are not consistent with the production of an RNA methylase, although they are consistent with a mutational mechanism of resistance due to a change in a ribosomal protein gene. This study constitutes a detailed biochemical and genetic characterization of erythromycin resistance in Campylobacter species.

Bacterial Proteins↗
PubMed1991

A DNA sequence upstream of the tet(O) gene is required for full expression of tetracycline resistance.

The DNA sequences upstream of the tet(O) and tet(M) open reading frames (ORFs) (ca. 300 bp) were found to share a higher degree of homology than those of the tet(O) and tet(M) ORFs themselves. A transcription initiation site for tet(O) was located by primer extension analysis. Campylobacter coli was found to use a promoter sequence different from that used by Escherichia coli. The sequence upstream of tet(O) was shown to be required in cis for high-level resistance to tetracycline.

Base Sequence↗
PubMed1991

Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter.

The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control. Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu. These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA. The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified. The first ORF codes for KilA, a 28-kDa hydrophilic protein. The second ORF, telA, codes for a hydrophilic protein of 42 kDa. The third ORF, telB, codes for a hydrophobic protein of 32 kDa. This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA. All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system. The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system. A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter.

Alkaline Phosphatase↗
PubMed1990

Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction.

A chloramphenicol-resistance determinant (CmR), originally cloned from Campylobacter coli plasmid pNR9589 in Japan, was isolated and the nucleotide sequence determined, which contained an open reading frame of 621 bp. The gene product was identified as Cm acetyltransferase (CAT), which had a putative amino acid sequence that showed 43% to 57% identity with other CAT proteins of both Gram+ and Gram- origin. Although expression of the cat gene was constitutive in both C. coli and Escherichia coli, results of primer extension experiments indicated that transcription was initiated at different sites in these two species. A kanamycin-resistance determinant, identified as the aphA-3 gene, was located downstream from the cat gene. The codon usage of the cat gene is very different from that used in E. coli, however, the CAT polypeptide was synthesized in large amounts in E. coli maxicells. Therefore, the codon usage bias is not one of the obstacles which affects Campylobacter spp. gene expression in E. coli. New Campylobacter cloning vectors were constructed in this study.

Amino Acid Sequence↗