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Biomedical subjects

M Wilks

Publications and source records attributed to M Wilks.

At least 37 records · Page 2Linked to original sources

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

A double-blind comparison of the effects of cefaclor and amoxycillin on respiratory tract and oropharyngeal flora and clinical response in acute exacerbations of bronchitis.

Fifty-one patients admitted to hospital with severe exacerbations of chronic bronchitis entered a double-blind trial of treatment with cefaclor (500 mg tds) compared with amoxycillin (500 mg tds) for 7 days. Twenty-six patients received cefaclor and 25 amoxycillin. Sputum and throat swabs were collected on admission, after 7 days of therapy and at outpatient follow-up, 3 weeks after treatment had finished. Clinical status and spirometry were assessed on admission and at the third, seventh and 28th day. There was no significant difference between the two regimes for clinical outcome, spirometry or numbers of infecting pathogens. Opportunistic colonization with resistant Gram-negative organisms and Candida species was highly prevalent on admission (56%) in both groups, perhaps because of previous antibiotic administration and general debility of the majority of patients. The high prevalence of opportunistic colonizing organisms persisted at follow-up (48%) with a significant excess of new organisms (Enterobacter cloacae, Klebsiella species and Candida species) present in sputum in the amoxycillin-treated patients. Cefaclor may be less damaging to normal flora than amoxycillin with a consequently reduced risk of colonization and superinfection of the respiratory tract with resistant Gram-negative organisms and yeasts.

Acute Disease↗

Histidine decarboxylases from bacteria that colonise the human respiratory tract.

We investigated whether production of histamine by bacteria isolated from sputum of patients with infective lung diseases could be attributed to the presence of histidine decarboxylase (HD). Twenty gram-positive and 20 gram-negative organisms were studied for their ability to decarboxylate 14C-histidine in vitro over the pH range 4.5-7.5. Of the bacteria investigated, lysates from the gram-negative species Haemophilus influenzae, H. parainfluenzae, Moraxella (Branhamella) catarrhalis and Pseudomonas aeruginosa liberated 14CO2 and histamine from 14C-histidine in the presence of the cofactor pyridoxal phosphate. In contrast, results obtained in the absence of cofactor were similar to those of negative (lysate-free) controls suggesting that the HD enzymes of these species resembled those previously described in other gram-negative bacteria. No HD activity was detected over this pH range in lysates from gram-positive species. This finding correlated with earlier observations that these gram-positive organisms did not produce histamine in vitro.

Carbon Dioxide↗

Mycobacterium paratuberculosis and Crohn's disease.

The possible aetiological role of Mycobacterium paratuberculosis in Crohn's disease was investigated. The immunological response was studied using an enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunocytochemistry. The antibody response to two protoplasmic antigen preparations of M paratuberculosis in the sera of patients with inflammatory bowel disease was measured by ELISA. IgG and IgM antibodies to these antigens were measured in serum samples from 52 patients with Crohn's disease, 15 patients with ulcerative colitis, and 41 control patients without inflammatory bowel disease. Although there was wide variation in the concentrations of antibody detected, patients with Crohn's disease had concentrations that were not significantly different from those of the other two groups. In addition, mycobacterial antigens were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the immune response to each antigen was then examined separately and assayed for IgG and IgM in 10 patients from each of the three groups. An indirect peroxidase test was also used to detect M paratuberculosis in sections of tissue from 18 patients with Crohn's disease and 10 with ulcerative colitis. The results were negative in all cases. This study does not support a role for M paratuberculosis in Crohn's disease.

Adolescent↗

Lack of effect of ACTH in porcine Escherichia coli septic shock.

The cardiovascular and metabolic responses to treatment with ACTH(1-24) were investigated in a porcine model of septicaemia. Sixteen anaesthetised, instrumented animals were infused with live Escherichia coli over 2 hr. During the first hour of the infusion, significant reductions in cardiac index, mean arterial pressure, and pH were observed together with a significant increase in mixed venous blood lactate concentrations and packed cell volumes. ACTH(1-24) 160 micrograms kg-1, given 1 hr after starting the E. coli infusion (n = 8), had no significant effect on these haemodynamic or metabolic measurements when compared with the control group (n = 8). These results do not support the suggestion that intravenous ACTH(1-24) reverses cardiovascular depression in septicaemic shock.

Adrenocorticotropic Hormone↗

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis.

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.

Bacillus subtilis↗

Beneficial effects of buprenorphine (a partial opiate agonist) in porcine Escherichia coli septicaemia: a comparison with naloxone.

The cardiovascular and metabolic responses to treatment with naloxone or buprenorphine (a partial opiate agonist) were investigated in a porcine model of septicaemia. Animals anaesthetised with alpha-chloralose were infused with live E. coli over two hours. They were then divided into three groups and received either naloxone (2 mg kg-1 + 1.5 mg kg-1 hr-1) or buprenorphine (0.3 mg kg-1) or an equivalent volume of normal saline. Treatment was started one hour after commencing the infusion, by which time a significant fall in cardiac index (CI), stroke index (SI), mean arterial pressure (MAP), and pH had occurred in all groups, together with a significant rise in mixed venous blood lactate and packed cell volume. Treatment with both naloxone and buprenorphine resulted in significant improvements in CI, pH, and base excess and in a fall in mixed venous lactate and packed cell volume. Although no significant effect on survival was seen at three hours after the start of treatment, buprenorphine may prove to be a suitable alternative to naloxone in the management of septic shock.

Animals↗

Beta 2-microglobulin kinetics during haemofiltration.

To study the kinetics of beta 2-microglobulin during haemofiltration, seven patients with end-stage renal failure were treated with the AN 69 (acrylonitrile), Duo-Flux (cellulose acetate) and F 60 (polysulphone) haemofilter. Low beta 2-microglobulin sieving coefficients and a highly negative filter mass balance error were observed during the initial phase of treatment with AN 69 but not with Duo-Flux or F 60, indicating a high degree of beta 2-microglobulin adsorption by AN 69. Total removal of beta 2-microglobulin was calculated by addition of the total amount adsorbed by the membrane and the total amount recovered in the collected ultrafiltrate. With AN 69 and F 60, total removal of beta 2-microglobulin amounted to 393 +/- 135 (SD) and 316 +/- 35 mg per treatment, while total removal with Duo-Flux was 242 +/- 79 mg per treatment. Thus, highly permeable membranes such as AN 69 or F 60 used in a haemofiltration mode may nearly balance the presumed generation of beta 2-microglobulin in uraemic patients. During treatment, an increase of the calculated beta 2-microglobulin distribution volume occurred with all three membranes, probably representing extra-to-intracellular water shifts. The water shifts occurring during haemofiltration reduce the value of precision of beta 2-microglobulin kinetics and limit the value of the plasma level decrease as an index of beta 2-microglobulin removal.

Acrylonitrile↗

Quantitative bacteriology of the vaginal flora during the menstrual cycle.

The vaginal bacteriology of 10 healthy asymptomatic women was assessed during the menstrual cycle. Samples were taken from the posterior vaginal fornix for quantitative analysis. There were no significant alterations in the total vaginal flora at different stages of the menstrual cycle. The mean number of species isolated per specimen declined from 4.6 in week 1 to 2.9 in week 4. This decline was not caused by a decrease in the occurrence or concentration of any one organism or group of organisms. The vaginal pH decreased from a mean of 6.6 in week 1 to 4.3 in week 4, this increased acidity could not be attributed to the action of lactobacilli as their total incidence or concentration did not change during the menstrual cycle.

Bacteria, Aerobic↗