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M Wilks

Publications and source records attributed to M Wilks.

At least 19 recordsLinked to original sources
PubMed2001

Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG1363 and its use as a vaccine delivery system against H. pylori infection in mice.

The use of Lactococcus lactis as an antigen delivery vehicle for mucosal immunisation has been proposed. To determine whether L. lactis could effectively deliver Helicobacter pylori antigens to the immune system, a recombinant L. lactis expressing H. pylori urease subunit B (UreB) was constructed. Constitutive expression of UreB by a pTREX1 vector resulted in the intracellular accumulation of UreB to approximately 6.25% of soluble cellular protein. Five different oral regimens were used to vaccinate C57BL/6 mice and the immune response measured. One regimen, which consisted of four weekly doses of 10(10) bacteria, followed after an interval of approximately 4 weeks by three successive daily doses, was able to elicit a systemic antibody response to UreB in the mice, although subsequently, a similar regimen produced a significant antibody response in only one out of six mice. The other three regimes, in which mice were vaccinated with two or three sets of three consecutive daily doses of recombinant bacteria over 30 days, failed to elicit significant anti-UreB serum antibody responses. In three regimens, the immunised mice were then challenged by H. pylori strain SS1 and no protective effect was observed. These findings suggest that any adjuvant effects of L. lactis are unlikely to be sufficient to produce an effective immune response and to protect against H. pylori challenge, when used to deliver a weak immunogen, such as UreB.

Animals↗
PubMed2001

A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane.

Nitric oxide (NO) is a mediator of essential biological processes, including vasodilatation, anti-microbial activity and wound healing. A chemical system using sodium nitrite and ascorbic acid has been developed which generates significant amounts of NO. The originally described system was messy and impractical, and the high acidity may cause pain and further tissue damage in ulcerated skin. To overcome this, a selectively permeable, hydrophilic polyester co-polymer membrane system (Sympatex ) has been identified that can be placed between the NO-generating chemicals and the skin. The aim of the present study was to determine whether NO derived from this chemical system was able to diffuse through the membrane and have a measurable vasodilatory effect on forearm skin in healthy volunteers. The Sympatex 10 microm membrane was found to be highly permeable to NO, while preventing passage of the constituents of the NO-generation gel to the skin. The transmembrane NO-generation system had a vasodilatory effect comparable with that resulting from direct topical application. Additionally, the NO generated was effective in killing Staphylococcus aureus and Escherichia coli at doses lower than those required to increase skin blood flow. The vasodilatory and anti-microbial effects of this system may be useful as a patch-based topical therapy for skin ulceration, particularly when there is concomitant ischaemia and infection.

Administration, Cutaneous↗
PubMed2000

Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX.

Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.

Amino Acid Sequence↗
PubMed1999

Living wills.

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Ethics, Medical↗
PubMed1997

Detection of enterotoxigenic Bacteroides fragilis by PCR.

Strains of enterotoxigenic Bacteroides fragilis (ETBF) are associated with diarrhea in young farm animals and, at least in particular settings, in children. Enterotoxin production by ETBF is currently detected by a tissue culture assay with HT-29 cells. We have developed a PCR assay based on the detection of the enterotoxin gene to identify ETBF in culture and in stool samples. Overall, 113 bacterial strains were examined, including 3 B. fragilis reference strains, 75 B. fragilis isolates (comprising 40 ETBF isolates), 20 Bacteroides spp. other than B. fragilis, and 15 strains belonging to other genera. Complete agreement was found between the results of the tissue culture assay and those of the PCR for our strains. PCR was also used to detect ETBF directly in fecal samples. Stools from two healthy volunteers were spiked with known numbers of ETBF and were processed by three different methods. A culture method, which required inoculation of the stools on selective plates and the collection of the whole bacterial growth ("sweeps"), was found to be the most sensitive. PCR performed with the plate sweeps yielded amplification products with a detection limit of 10(5) to 10(4) CFU/g of feces. By this method 18 samples of diarrheic stools (10 positive and 8 negative for ETBF) were examined. The results of the PCR were in accordance with the culture results in all cases. The proposed PCR assay represents a diagnostic tool for the rapid identification of ETBF in culture as well as in fecal samples.

Bacterial Toxins↗
PubMed1996

A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile.

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.

Cloning, Molecular↗
PubMed1995

Transfer of macrolide-lincosamide-streptogramin B (MLS) resistance in Clostridium difficile is linked to a gene homologous with toxin A and is mediated by a conjugative transposon, Tn5398.

An MLS resistance gene designated ermBZ, from a toxigenic Clostridium difficile strain (630) could be transferred between C. difficile strains, and to and from Bacillus subtilis. The intergeneric transfer occurred in the absence of any detectable plasmid DNA and the element responsible for gene transfer entered the recipient's chromosome, behaviour which is characteristic of a conjugative transposon. The element was designated Tn5398 and was found in six C. difficile strains. Tn5398 could be transferred to the non-toxigenic strain C. difficile CD37 which lacks the genes for toxins A and B. Transconjugants from both C. difficile and B. subtilis that had received the ermBZ gene also acquired a sequence of DNA that was homologous to the part of the toxin A gene that coded for the C-terminal repeat region.

Anti-Bacterial Agents↗
PubMed1995

Detection by PCR and analysis of the distribution of a fibronectin-binding protein gene (fbn) among staphylococcal isolates.

The fibronectin-binding proteins of Staphylococcus aureus are considered to be important virulence factors for colonisation and infection. The polymerase chain reaction (PCR) was used to detect part of a gene equivalent to the fbnA gene of S. aureus in 120 isolates of staphylococci (S. aureus, S. epidermidis, S. haemolyticus, S. simulans, S. hominis, S. warneri, S. cohnii and S. lugdunensis). Primers specific for the binding domain region of the fbnA gene of S. aureus produced PCR products of the predicted sizes (93 and 207 bp). The identity of the PCR products was confirmed by digestion with DdeI and nucleic acid hybridisation. The fibronectin-binding activity of the staphylococci was determined with a particle agglutination assay (PAA). The fbn gene was found to be present by PCR in 107 of the 120 staphylococci tested, irrespective of their site of isolation, and expression of the gene was detected by PAA in 101 of the 120 strains.

Adhesins, Bacterial↗
PubMed1994

Gene cloning in Clostridium difficile using Tn916 as a shuttle conjugative transposon.

A pBR322-based vector, pCI195, containing a 4.2-kb region of the conjugative transposon Tn919 was used as a vector for gene cloning in Clostridium difficile. The plasmid was found to integrate into the chromosome of a Bacillus subtilis strain that contained Tn916 delta E. Southern blot analysis of the recombinant demonstrated that pCI195 had inserted into Tn916 delta E by a recombination event. The transposon::plasmid structure could be transferred, by filter mating, from B. subtilis to C. difficile (at a frequency of 10(-8) per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transposon markers was observed on transfer, although the Tn916 delta E::pCI195 was stably maintained in C. difficile. To demonstrate that pCI195 could be used for gene cloning in C. difficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM100. Tn916 delta E::pPPM100 was transferred into a nontoxigenic C. difficile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. difficile, as the transposon::plasmid structure could be transferred to different C. difficile strains. This is the first report of gene cloning in C. difficile.

Bacillus subtilis↗
PubMed1994

Serial electroencephalograms in a patient with D-lactic acidosis.

A patient with previous bowel surgery was followed through the course of her recurrent encephalopathy with biochemically demonstrated D-lactic acidosis by detailed serial electroencephalography (EEG). Although the EEG changes were marked and parallelled the clinical and biochemical abnormalities closely, they were essentially non-specific. This diagnosis should be considered in encephalopathic patients with a history of bowel disease particularly after extensive small bowel resection.

Acidosis, Lactic↗
PubMed1994

Typing of Clostridium difficile by polymerase chain reaction with an arbitrary primer.

We assessed the use of the polymerase chain reactions (PCR) with an arbitrary primer (AP-PCR) to investigate a major hospital outbreak of diarrhoea due to Clostridium difficile. A single pattern consisting of three bands of 240, 580 and 1100 bp was obtained from all isolates studied. AP-PCR is a simple, rapid technique which should find increased application in the rapid investigation of suspected outbreaks of many different bacterial species, particularly new pathogens or those for which no accepted typing scheme exists.

Bacterial Typing Techniques↗
PubMed1993

Inhibition of human neutrophil migration in vitro by low-molecular-mass products of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae commonly causes infections in the lower and upper respiratory tract, although the mechanisms of its colonization and persistence in the airways are unclear. Culture filtrates from six clinical isolates of this bacterium were assessed for their abilities to influence neutrophil function in vitro. Each culture filtrate was assessed on six separate occasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth (0 to 18 h), culture filtrates contained primarily neutrophil chemokinetic activity but no activity affecting neutrophil migration toward the chemotactic factors N-formyl-L-methionyl-L-leucyl-L-phenylalanine and leukotriene B4. In contrast, filtrates obtained after 24 h of culture contained factors which inhibited neutrophil migration toward both of these chemotactic factors. This chemotaxis-inhibitory activity persisted between 24 and 72 h of bacterial culture, and it was not associated with the presence of either chemotactic or chemokinetic activity as assessed by checkerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of chemotaxis-inhibitory activity. Endotoxin was present together with two other low-molecular-mass hydrophobic factors of approximately 8 and 2 kDa. These low-molecular-mass factors are chloroform insoluble and heat stable, and they are inactivated by protease, periodate, and diborane reduction. Activity was completely retained on a wheat germ agglutinin column, and it could be eluted with N-acetyl-D-glucosamine. These data suggest that inhibitory activity is associated with N-acetyl-D-glucosamine-containing glycopeptides, possibly derived from the bacterial cell wall. The production of these compounds may contribute to the persistence of this bacterium in vivo by inhibiting neutrophil chemotaxis in the microenvironment of the respiratory mucosa.

Biological Factors↗
PubMed1992

Epidemiological aspects of infections caused by Bacteroides fragilis and Clostridium difficile.

Bacteroides fragilis and Clostridium difficile are two of the most common anaerobes associated with human disease. Studies on the epidemiology of Bacteroides fragilis are limited and are based predominantly on serogrouping, which suggests intraspecies differences. Further studies using newer techniques for typing are required to elucidate the epidemiological characteristics of this important pathogen. By contrast, numerous phenotypic, immunological and molecular methods have been developed for typing and fingerprinting of Clostridium difficile and applied in epidemiological studies to show conclusively that Clostridium difficile is nosocomially acquired and that there is transmission and cross-infection between hospital patients.

Animals↗
PubMed1991

Transfer of Tn916 and Tn916 delta E into Clostridium difficile: demonstration of a hot-spot for these elements in the C. difficile genome.

The conjugative transposon Tn916 and a derivative Tn916 delta E was transferred from Bacillus subtilis into Clostridium difficile CD37 by filter mating. All the C. difficile transconjugants appeared to contain one copy of the transposon integrated into the same position in the genome. Transposition from the original site of integration was not observed. Like Tn916 the transferable tetracycline resistance determinant (Tc-CD) of C. difficile has a preferred site of integration in C. difficile and is homologous with Tn916 along the whole length of Tn916. However comparisons of the distribution of TaqI and Sau3AI sites in the homologous regions of the two elements did not demonstrate any hybridizing fragments in common.

Bacillus subtilis↗