Biomedical subjects

G Dougan

Publications and source records attributed to G Dougan.

At least 253 records · Page 14Linked to original sources

PubMed1985

Impact of recombinant DNA technology on studies of bacterial pathogenicity.

Gene manipulation techniques along with other molecular approaches have aided studies on bacterial pathogenicity in a number of ways. Potential virulence determinants have been isolated from a wide variety of pathogenic bacteria and some of these have been characterized in order to determine their genetic organization and identify specific gene products. Proteins such as toxins, adhesins, extracellular enzymes and surface antigens have been analysed. Once a potential virulence determinant has been characterized opportunity exists for further manipulations to be undertaken. For example, natural toxoid producing strains have been constructed including organisms expressing high levels of E. coli heat-labile toxin B-subunit and cholera B-subunits. Gene fragments can be used as hybridization probes to screen for silent or related virulence genes. Mutated genes can be re-introduced into parental pathogenic bacteria, following manipulations in E. coli, to construct specific mutants. Examples of some of these approaches will be discussed.

Bacteria↗

PubMed1984

Simultaneous outbreaks of infection due to Serratia marcescens in a general hospital.

Seventy isolates of Serratia marcescens were obtained from 30 patients in different units of one hospital between April 1982 and February 1983. No common source was found. Not all isolates were multi-resistant and nearly all that were, fell into two main groups, A and B. These groups were defined by phage typing and cephalosporin sensitivity, all apart from one Group B isolate were multi-resistant, whereas Group A isolates contained multi-resistant and sensitive strains. Plasmid screening, resistance transfer studies and plasmid elimination experiments demonstrated that the multi-resistant phenotype was due to a 120 Mdal transmissible plasmid. Resistance to cephalosporins was chromosomally encoded.

Anti-Bacterial Agents↗

PubMed1984

The impact of new technologies on vaccine development.

The ability to move genetic determinants between species using in vitro gene-manipulation techniques has opened up new approaches to vaccine development. This has rapidly grown into an exciting area of research in both academic and industrial laboratories. There are numerous scientific challenges which require multidisciplinary teams to solve problems in creating new immunogens. This has challenged our existing knowledge about protein structure and conformation, microbial pathogenicity and the immune system. Recombinant-DNA techniques are invaluable as tools of analysis and antigen production. The surface of micro-organisms can also be minutely explored with the use of synthetic peptides and monoclonal antibodies. Nevertheless, these new technologies do not allow us to circumvent the need for detailed understanding of pathogens and the disease process. What is apparent from the work carried out so far is that there are few easy answers to vaccine development and it is not realistic to expect rapid solutions to these problems. As there are many potential targets for constructing novel vaccines for both human and animal diseases, it is helpful to establish some priorities. There is a tendency to look at the existing effective vaccines and simply direct research at producing them more economically or with enhanced safety and stability. The advantage of this approach is that considerable background work will have already been carried out establishing the basis for the application of recombinant DNA techniques. However, this can also lead to conflicts (often within the same institute or company) between the new and old technologies. This could be to the detriment of the new technologies which are still only partly developed and may not be good enough yet to compete with existing vaccines in cost or efficacy. The more ambitious, and eventually more rewarding, approach is to attempt to develop new vaccines where none had existed before. There is a vast untapped market, especially in the parasitic diseases, but the scientific problems may be considerable and much more background work is likely to be necessary. Indeed, most of the work in this area is more accurately referred to as basic research rather than vaccine development as totally new, effective vaccines are still some way off. Having directed research towards a specific organism or disease there are still many options available as to the scientific strategy to adopt. As discussed in this review it may be possible to consider subunits, synthetic antigens and live (attenuated or heterologous) organisms as possible vaccines.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvants, Immunologic↗

PubMed1984

Genetically engineered vaccines: problems and promises.

Determinants of important protective antigens from a variety of viral, bacterial and parasite pathogens have been successfully cloned and expressed in novel hosts using recombinant DNA techniques. The use of these cloned determinants in the development of new vaccines, including immunologically defined subunit vaccines, genetically defined live attenuated vaccines, and synthetic peptide vaccines is described.

Animals↗

PubMed1983

Gentamicin and methicillin resistant Staphylococcus aureus in Dublin hospitals: molecular studies.

A large number of Staphylococcus aureus strains resistant to gentamicin, methicillin and other antibiotics, isolated in several Dublin hospitals during a 4-year period, were screened for plasmid DNA. Isolates assigned to four principal phage groups showed uniform plasmid profiles. A plasmid of mol. wt 21.0 x 10(6) encoding penicillin resistance was present in all isolates screened. A tetracycline resistance plasmid of mol. wt 24.0 x 10(6) was present in 40% of isolates of phage types 90 and 5/47/54/84/85 whereas a plasmid of mol. wt 3.0 x 10(6) was responsible for tetracycline resistance in isolates of phage types 77 and 85. Aminoglycoside, methicillin and erythromycin resistances were apparently not encoded on plasmids. Gentamicin resistance was transduced at low frequency between several strains of S. aureus and the resistance phenotype was due, in part at least, to the production of an inactivating enzyme.

Genes, Bacterial↗

PubMed1983

Cloning, expression, and mapping of the Staphylococcus aureus alpha-hemolysin determinant in Escherichia coli K-12.

A fragment of Staphylococcus aureus DNA encoding the alpha-hemolysin determinant was cloned from strain Wood 46 by inserting Sau3A-generated genomic DNA fragments between the BamHI sites of the lambda replacement vector L47.1. Phages expressing alpha-hemolysin were detected by overlaying plaques formed from several thousand independent recombinant phage with erythrocytes and looking for zones of hemolysis. One phage expressing alpha-hemolysin was purified and named lambda w alpha 3. This was subsequently shown to contain a 10.2-kilobase pair insert of S. aureus DNA. A 7.6-kilobase pair HindIII fragment encoding the alpha-hemolysin was subcloned from lambda w alpha 3 into the plasmid vector pACYC184 to form the hybrid plasmid pDU1148. Escherichia coli K-12 cells harboring pDU1148 synthesized a low level of alpha-hemolysin which remained associated with the cells and was not secreted into culture supernatants. When the same strain was stabbed onto blood agar plates, no zones of hemolysis were detected after overnight growth at 37 degrees C but hemolysis developed if the plates were left at room temperature for 48 h. By introducing specific deletions or Tn5 insertions into plasmid pDU1148, the alpha-hemolysin gene was mapped to a region within a 3.3-kilobase pair EcoRI-HindIII fragment which was subcloned onto the vector plasmid pBR322. A specific enzyme-linked immunosorbent assay with peroxidase-labeled rabbit anti-alpha-hemolysin antibodies was used to measure the levels of alpha-hemolysin antigen expressed in E. coli K-12 cells harboring pDU1148 or a variety of pDU1148::Tn5 and pDU1148 deletion mutants.

Chromosome Mapping↗

PubMed1983

Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus.

A DNA sequence encoding Staphylococcus aureus alpha-hemolysin, which had been previously cloned and mapped in Escherichia coli K-12, was introduced into Bacillus subtilis BD170 and several strains of S. aureus by using plasmid vectors, some of which could replicate in all three organisms. The determinant was cloned on a 3.3-kilobase pair DNA fragment into B. subtilis by using the vector plasmid pXZ105 to form the hybrid plasmid pXZ111. B. subtilis cells harboring pXZ111 produced large zones of alpha-hemolysis after 18 h of growth at 37 degrees C on rabbit blood agar plates, and alpha-hemolysin activity was detected in supernatants prepared from growing cultures of this strain. The alpha-hemolysin was apparently secreted across the B. subtilis cell envelope. Polypeptides of molecular weights 34,000 and 33,000 were precipitated with anti-alpha-hemolysin serum from lysates prepared from BD170 cells harboring pXZ111. A hybrid replicon which could replicate in both E. coli and S. aureus was constructed in E. coli by ligating a HindIII fragment encoding the replication functions and chloramphenicol resistance genes of S. aureus plasmid pCW59 to the pBR322 alpha-hemolysin hybrid plasmid pDU1150. The DNA of this plasmid, pDU1212, was prepared in E. coli and used to transform protoplasts prepared from a non-alpha-hemolytic, nonrestricting strain of S. aureus RN4220. Some of the transformants contained plasmids which had suffered extensive deletions. Some plasmids, however, were transformed intact into RN4220. Such plasmids were subsequently maintained in a stable manner. pDU1212 DNA was prepared from RN4220 and transformed into alpha-hemolytic S. aureus 8325-4 and two mutant derivatives defective in alpha-hemolysin synthesis. All three strains expressed alpha-hemolysin when harboring pDU1212.

Bacillus subtilis↗

PubMed1983

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili.

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.

Antigens, Bacterial↗

PubMed1983

Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.

A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.

Cloning, Molecular↗

PubMed1983

Expression of a cloned K88ac adhesion antigen determinant: identification of a new adhesion cistron and role of a vector-encoded promoter.

The determinant for the K88ac adherence antigen of porcine enterotoxigenic Escherichia coli has been cloned previously onto the vector plasmid pBR322 to form the K88ac-pBR322 hybrid plasmid pMK005 (M. Kehoe et al., Nature [London] 291:122-126). Further studies on the expression of the K88ac antigen from pMK005 are presented in this paper. Expression was found to be dependent mainly on the P1 promoter of the pBR322 vector. The natural K88ac promoter was apparently not cloned from the original parental K88ac plasmid. The P1 promoter was deleted and replaced by a DNA sequence encoding the promoter-operator region of the E. coli tryptophan (Trp) operon. Cells harboring the Trp-pMK005 hybrid plasmid expressed high levels of K88ac antigen when the Trp promoter was repressed. If the promoter was derepressed either by growing the cells in low concentrations of tryptophan or in the presence of indole acrylic acid, growth of the cells harboring the Trp-pMK005 hybrid plasmid was inhibited. A quantitative assay was used to measure the levels of K88ac antigen expressed by cells harboring different pMK005::Tn5 plasmids. All cells were found to express a reduced level of K88ac antigen, providing evidence that a single transcription unit, initiating at promoter P1 of pBR322, may be involved in the expression of the K88ac antigen. By constructing specific deletion and insertion mutants of pMK005, a fifth adhesion cistron, tentatively named adhE, was identified and mapped at the proximal end of the K88ac determinant. Although the cistron is required for high-level expression of K88ac surface-associated fimbriae, as yet no gene product has been assigned to adhE.

Antigens, Bacterial↗

PubMed1983

Studies on the expression and organization of the K88ac adherence antigen.

The genetic determinant for the K88ac adhesion antigen has been cloned on a 6.5 kilobasepair DNA fragment into the multiple copy plasmid pBR322. The resulting hybrid plasmid named pMK005 was used to study the organization and expression of polypeptides involved in K88ac antigen assembly. Five cistrons named adh A, B, C, D and E were mapped on the cloned DNA and maximal expression of K88ac antigen was found to be dependent on a pBR322 encoded promoter. Four polypeptides of molecular weights 70,000 daltons, 25,000 daltons, 17,000 daltons, and 23,500 daltons (K88ac fimbrial subunit) were identified as the products of the adh A, B, C and D cistrons respectively. The subcellular location of each of these polypeptides was determined by fractioning minicells. pMK005 encodes sufficient information to promote adhesion of a wild-type E. coli strain 09:K13:H19 to the porcine small intestine in vivo.

Animals↗

PubMed1982

Properties of Escherichia coli grown in vivo using a chamber implant system.

Strains of Escherichia coli were examined for their ability to grow in diffusion chambers implanted into the peritoneal cavities of mice and rabbits. In rabbit chambers E. coli K12 strains grew poorly, whereas isogenic strains harbouring ColV plasmids and wild-type isolates from extra-intestinal infections grew well. The difference was much less marked with strains grown in mouse chambers. Differences in sensitivity to the bactericidal action of human or rabbit serum were found in some cases between organisms of the same strain grown in vivo or in vitro. Host immunoglobulins were bound to the surface of all in vivo bacteria. Comparison of the polypeptide composition of bacterial cell envelope preparations on SDS-PAGE gels revealed differences between in vivo and in vitro grown E. coli growing in vivo may possess properties significantly different from the same organisms growing on laboratory medium.

Animals↗

PubMed1981

Genetic analysis of K88-mediated adhesion of enterotoxigenic Escherichia coli.

Four cistrons (adh) involved in the expression of the K88 adhesion system have been identified and mapped. Three of these (adh A, adh B and adh C) are located in a single operon (I) whereas the fourth (adh D) is expressed from a separate promoter (operon II). The polypeptides encoded by these cistrons have been identified and their role in the formation and regulation of K88 fimbriae (pili) is discussed.

Chromosome Mapping↗

PubMed1981

Plasmids in epidermolytic strains of Staphylococcus aureus.

Thirty-four epidermolytic toxin-producing strains of Staphylococcus aureus obtained from a variety of sources were screened for the presence of plasmid DNA. All serotype ii toxin producers harboured a large 42 kilobase pairs (kb) plasmid. Elimination of these plasmids resulted in the simultaneous loss of a bacteriocin determinant (Bac+) and type ii toxin production (Toxii+). Some strains producing serotype i toxin (Toxi+) contained similar 42 kb plasmids. Elimination of these plasmids resulted in the loss of only bacteriocin production. Strains producing both toxin serotypes readily lost Toxii+ and Bac+, which were carried on the same plasmid, but Toxi+ could not be eliminated. Thus Toxi+ was probably chromosomally determined, while Toxii+ was a plasmid-encoded marker. In some strains cadmium resistance was also linked to the 42 kb plasmid. The 42 kb plasmids from seven strains with different phenotypes were analysed with restriction endonucleases EcoRI and HindIII. The plasmids shared 19 of 22 HindIII fragments indicating that they are closely related to each other.

Bacterial Toxins↗

PubMed1981

Identification and cloning of the genetic determinant that encodes for the K88ac adherence antigen.

Strains of Escherichia coli capable of causing diarrhea in young pigs are often able to proliferate in the upper small intestine of the infected animal due to the presence of a specific surface antigen, K88. The genetic determinants for K88 antigen production and the ability to utilize the trisaccharide raffinose (Raf) are carried on a 50-megadalton plasmid. Recombinant deoxyribonucleic acid techniques were used to insert an 8.2-megadalton HindIII fragment carrying the K88ac gene(s) from the K88/Raf plasmid pPS100 into the vector pBR322. At lease six polypeptides encoded by this fragment were expressed in minicells. These polypeptides ranged in size from 18,000 to 70,000 daltons. The K88ac antigenic subunit, which has an apparent molecular weight of 23,500, was identified by immunoprecipitation with staphylococcal protein A as the coprecipitant.

Antigens, Bacterial↗

PubMed1981

Antibiotic resistance in Providencia stuartii isolated in hospitals.

A total of 238 isolates of Providencia stuartii obtained from infected patients in six Dublin hospitals were grouped by using serological and bacteriocin typing methods and tested for sensitivity to a number of antimicrobial agents. Most isolates were resistant to several of these agents. Resistance to tetracycline, resistance to penicillin, resistance to polymyxin, and probably resistance to nitrofurantoin was intrinsic. Plasmid screening coupled with resistance transfer studies showed that both chromosome-encoded and plasmid-coded resistance mechanisms were clinically important. Ampicillin resistance was both chromosomally and plasmid encoded, whereas resistance to kanamycin and resistance to carbenicillin were exclusively plasmid encoded. Gentamicin resistance was more common than kanamycin resistance, and although gentamicin-resistant strains contained aminoglycoside acetyltransferase activity, no association could be demonstrated with plasmid deoxyribonucleic acid in the strains tested. Unlike minimal inhibitory concentrations for kanamycin, minimal inhibitory concentrations for gentamicin varied over a wide range. P. stuartii isolated obtained from several different countries were tested for comparison. As a group, these strains were less resistant, but they did exhibit similar resistance properties.

Anti-Bacterial Agents↗

PubMed1980

Temperature-sensitive mutants of equine arteritis virus.

Seventeen temperature-sensitive mutants of equine arteritis virus, a nonarthropod-borne togavirus, have been isolated. 5-Fluorouracil, o-methylhydroxylamine and ethyl methanesulphonate were used as mutagens. The mutants were characterized by their ability to synthesize virus RNA and virus proteins at the permissive (35 degrees C) and restrictive temperature (40 degrees C) using autoradiography of cells labelled with 3H-uridine in the presence of actinomycin D and immunofluorescence respectively. Among the mutants, four were unable to synthesize virus RNA and virus proteins at 40 degrees C (RNA-/protein-). The other mutants were RNA-/protein+ (3); RNA +/-/protein- (2); RNA+/protein+ (6) and RNA+/protein- (1).

Equartevirus↗

PubMed1979

Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3.

Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements. Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides. Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis. A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site. Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J. Bacteriol. 136: 742--756, 1978; Heffron et al., Proc. Natl. Acad. Sci U.S.A. 72:3632--3627, 1975). No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.

Bacterial Proteins↗