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ABSTRACT: 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.
Vaccine 08/2001; 19(28-29):3927-35. · 3.77 Impact Factor
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ABSTRACT: Antiserum raised against whole Helicobacter pylori cells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress, indicating that the clpB gene product functions as a stress response protein in H. pylori.
Journal of Bacteriology 02/1998; 180(2):426-9. · 3.83 Impact Factor
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ABSTRACT: Clostridium difficile is now established as the most common nosocomial enteric pathogen causing pseudomembranous colitis, antibiotic-associated colitis and antibiotic-associated diarrhoea. Antibiotic therapy is the most important risk factor in colonization and infection with C. difficile. However, other factors are involved such as age and underlying illness. The introduction of reliable typing and fingerprinting methods has demonstrated hospital acquisition and cross-infection with C. difficile and has been important in improving our understanding of the epidemiology and pathogenicity of C. difficile.
European Journal of Gastroenterology & Hepatology 12/1996; 8(11):1035-40. · 1.76 Impact Factor
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ABSTRACT: 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.
Gene 10/1996; 174(1):145-50. · 2.34 Impact Factor
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ABSTRACT: Pleural infection with Clostridium difficile is extremely rare. A case of nosocomial empyema following chest drain insertion in a 46 year old man is described. The potential of C difficile to cause extra-intestinal infections should be recognised and its isolation from other sites should not be ignored.
Journal of Clinical Pathology 03/1996; 49(2):172-3. · 2.31 Impact Factor
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ABSTRACT: Samples of pus aspirated from 53 peritonsillar abscesses were examined in detail for aerobic and anaerobic bacteria, and the microbiological results correlated with clinical data in 44 cases. In 45 samples (85%) cultures were positive: 7 yielded organisms consistent with an aerobic infection, mainly Lancefield group A beta-haemolytic streptococci (5/7), and 38 yielded organisms consistent with an anaerobic infection. The anaerobic infections were usually mixed, but in two cases Fusobacterium necrophorum was isolated in pure culture. Peptostreptococcus micros and Streptococcus milleri were the predominant isolates in this group. Direct Gram stain smear and gas-liquid chromatography were useful indicators of the type of infection present. Samples from ten patients (18.9%) grew one or more beta-lactamase-producing isolates. Of the 25 patients prescribed antibiotics by their general practitioners prior to admission, 18 received one or more beta-lactam antibiotics. Most cases of peritonsillar abscess were due to mixed anaerobic infections, Lancefield group A beta-haemolytic streptococci playing a central role in only a minority of cases. In light of these findings and the possibility of infection with beta-lactamase-producing isolates, it is suggested that the first-line antibiotic therapy in this group of patients should include a chemotherapeutic agent directed against anaerobic bacteria.
European Journal of Clinical Microbiology 11/1995; 14(10):870-7. · 2.86 Impact Factor
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BMJ 06/1995; 310(6991):1375-80. · 14.09 Impact Factor
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ABSTRACT: 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.
Journal of Medical Microbiology 03/1995; 42(2):96-101. · 2.50 Impact Factor
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ABSTRACT: 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.
Journal of Antimicrobial Chemotherapy 03/1995; 35(2):305-15. · 5.07 Impact Factor
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ABSTRACT: 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.
Electroencephalography and Clinical Neurophysiology 12/1994; 91(5):403-5.
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ABSTRACT: Screening of a Clostridium difficile lambda EMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb HindIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, beta-hydroxybutyryl coenzyme A dehydrogenase and thiolase).
FEMS Microbiology Letters 12/1994; 124(1):61-7. · 2.04 Impact Factor
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ABSTRACT: 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.
Journal of Hospital Infection 12/1994; 28(3):231-4. · 3.39 Impact Factor
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ABSTRACT: The bacterial flora of the tonsil surface and core was compared in patients suffering from recurrent tonsillitis. Surface swabs and tonsil core tissues were received as paired samples from 50 patients admitted for elective tonsillectomy. Analysis of paired samples from individual patients revealed differences in the bacterial flora of the tonsil core and the tonsil surface. Of 366 aerobic isolates, 30% grew from the surface alone, 26% from the core only and 44% from both sites. Of 290 anaerobic isolates, 35% grew from the surface alone, 33% from the core only and 31% from both sites. The total number of isolates from surface and core samples was similar (average 9.2 and 8.8, respectively). The range of species isolated was also similar for both surface and core samples, as was the proportion of organisms producing beta-lactamase from each site (10.7% and 9.5%, respectively). Eighty-two percent of patients carried beta-lactamase-producing organisms on either the tonsil surface or in the core tissue. A surface swab does not reliably reflect the types of organisms present in the tonsil core in individual patients. Anaerobes are a major component of tonsil surface and core bacterial flora in patients with recurrent tonsillitis. The high carriage rate of beta-lactamase-producing organisms in the tonsils should be considered when selecting antimicrobial therapy for persistent or recurrent tonsillitis.
European Journal of Clinical Microbiology 08/1994; 13(7):542-8. · 2.86 Impact Factor
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ABSTRACT: The clinical importance of the gram-positive anaerobic cocci (GPAC) isolated in 1987 at St Bartholomew's Hospital, London, is assessed. Of about 800 anaerobic isolates, 209 (27%) were GPAC, of which 67 (32%) were from abscesses and 22 (11%) were in pure growth. Four species comprised 77% of the 168 isolates available for study: Peptostreptococcus magnus (55 isolates, 33%), P. micros (23, 14%), P. asaccharolyticus (24, 14%) P. asaccharolyticus (24, 14%) and P. anaerobius (27, 16%). Different species were associated with different sites, from P. magnus (usually skin-associated sites; normally cultured with aerobes, infrequently with other anaerobes), P. asaccharolyticus (distributed widely) and P. anaerobius (usually genitourinary and gastrointestinal; always below the diaphragm) to P. micros (always deep sites with other anaerobes). P. magnus was isolated from 15 abscesses and was obtained in pure culture from 11 specimens, six of them abscesses developing from infected sebaceous cysts. P. micros was usually isolated from soft tissue abscesses, never from the skin, and with a characteristic mixed flora consisting of "Streptococcus milleri" and anaerobic gram-negative rods. P. heliotrinreducens was a rare isolate from similar specimens. P. asaccharolyticus was cultured from a wide variety of sites, typically mixed with both aerobes and anaerobes, and frequently from abscesses. Most isolates of P. anaerobius came from gastrointestinal or female genitourinary specimens, never from above the diaphragm and rarely from the skin; cultures were usually heavily mixed. Isolates of P. vaginalis and the "bGAL" group made up 11% of str ains and were usally cultured from superficial sites, P. vaginalis often from post-operative wound infections with Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Medical Microbiology 08/1994; 41(1):36-44. · 2.50 Impact Factor
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ABSTRACT: 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.
Plasmid 06/1994; 31(3):320-3. · 1.52 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 07/1993; 61(6):2419-24. · 4.16 Impact Factor
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ABSTRACT: A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.
Journal of Clinical Microbiology 07/1993; 31(6):1420-5. · 4.15 Impact Factor
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ABSTRACT: A total of 218 Clostridium difficile strains was examined for production of toxin A by ELISA, production of toxin B by a cytotoxin assay and the presence of toxin A and B gene-associated sequences by the polymerase chain reaction (PCR). After saturation amplification with toxin B-specific primers, the characteristic amplification product (591 bp) was detected in all 184 toxigenic strains examined. PCR with toxin A-specific primers gave positive results with all but one of the toxigenic strains. By contrast, PCR with toxin A- and toxin B-specific primers yielded negative results with all 34 non-toxigenic strains tested. This suggests that PCR detection of either the toxin A or B gene is a good indication of toxin production. PCR did not require DNA extraction or hybridisation and was convenient, sensitive and rapid. Toxigenic C. difficile could be detected in mixed cultures, suggesting a role for PCR in the identification of toxigenic C. difficile in primary culture.
Journal of Medical Microbiology 03/1993; 38(2):109-13. · 2.50 Impact Factor
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ABSTRACT: 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.
European Journal of Clinical Microbiology 12/1992; 11(11):1049-57. · 2.86 Impact Factor
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ABSTRACT: A cytotoxigenic Clostridium difficile strain that fails to produce toxin A but causes hemorrhage and bloody fluid accumulation in ligated ileal loops of rabbits and hemorrhage and diarrhea in hamsters is described. The lack of reaction of DNA from this strain in hybridization studies with a toxin A gene-specific 4.5-kb probe and polymerase chain reaction studies with six toxin A-specific primers indicate the absence of the toxin A gene. The cytotoxin produced by this strain was not responsible for the enterotoxic or hemorrhagic activity and shared characteristics with toxin B, i.e., its cytotoxicity was neutralized by antibodies to toxigenic strains of C. difficile and Clostridium sordellii. Polymerase chain reaction studies with toxin B-specific primers showed that the DNA from this strain produced a 690-bp product in addition to the expected 591-bp product.
Infection and Immunity 11/1992; 60(10):4192-9. · 4.16 Impact Factor
