- Volume 46, Issue 2, 1997

Research in periodontal disease has shown the presence of oral spirochaetes repeatedly in subgingival plaque. There is uncertainty as to whether these spirochaetes are involved in the actual disease process; however, it has been shown that their presence is a definite marker for disease occurrence. An understanding of their role in periodontal disease requires further characterisation of these organisms. Diagnostic tests would be useful for the clinician and enable treatment for the patient to be planned. Studies on characterising the different treponemal species have been limited by difficulties in culturing these organisms. Moreover, there is a need to obtain pure cultures of these organisms and to identify them in order to associate particular species with disease and, ultimately, to make probes for their easy detection directly from dental plaque. This review examines the methods used, and reports our own experience, in obtaining pure cultures of oral spirochaetes. The techniques available and the problems that occur when identifying these organisms are also considered.

Methicillin-resistant Staphylococcus aureus (MRSA) are important nosocomial pathogens. Diseases caused by these resistant bacteria frequently are serious and there is a need to control the spread of epidemic MRSA clones in hospitals. However, detection is complicated by the fact that expression of the resistance is variable and, commonly, heterogeneous within strains. The reliability of several tests recommended to discriminate heterogeneous MRSA isolates from borderline-resistant and susceptible strains was evaluated. Screening for growth on agar with methicillin 25 mg/L was the only method that detected all MRSA strains tested, but screening on agar with methicillin 10 mg/L or oxacillin 6 mg/L detected all but one of 10 heterogeneously resistant strains tested. None of the borderline-resistant nor any truly susceptible staphylococci tested grew on any of these screening plates.

Forty-seven Penner heat-stable (HS) serotype reference strains for Campylobacter jejuni and 47 serologically non-typable strains were examined by pulsed field gel electrophoresis (PFGE) DNA restriction analysis. The SmaI and KpnI digest profiles were compared by numerical analysis. Most strains grouped differently in the two analyses but strain lineages were inferred where the two agreed. Genetic relationships between reference strains in the cross-reacting HS4 complex were examined. Three clonal lines were evident and comprised: (i) HS4, HS13 and HS16; (ii) HS50 and HS65; (iii) HS43. The majority of those C. jejuni expressing HS antigens not recognised by currently available antisera had >50% PFGE DNA digest similarity to one or more Penner scheme reference strain(s) and so did not necessarily represent distinct genetic lineages. PFGE analysis provided a high level of discrimination amongst strains of C. jejuni but overall similarity estimates for defining types must be based on the analysis of more than one restriction pattern.

The adhesion of Helicobacter pylori to gastric carcinoma cells (MKN45, KatoIII and MKN28) and Intestine-407 cells was tested by flow cytometric analysis. The mean adhesion rates of H. pylori strains to MKN45, KatoIII and Intestine-407 cells were 90.5, 42.7 and 15.1%, respectively. There was no statistical correlation between the adhesion rates to MKN45 cells and haemagglutination (HA) activity of H. pylori strains, although H. pylori strains with high HA activity with human type O erythrocytes tended to adhere effectively to MKN45 cells. No correlation between adhesion and production of vacuolating toxin was observed.

Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201–224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed.

The role of the polymerase chain reaction (PCR) in the diagnosis of tuberculosis in clinical practice remains to be defined: most results have been based on sputum samples. This study systematically compared the relative sensitivity and specificity of a single simplified method for different clinical samples. A wide range of clinical samples, including sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, pleural fluid, gastric aspirate, pus and tissues (both fresh and paraffin-embedded) was tested. This method did not require routine DNA extraction before PCR, and consisted of an optimised single tube PCR amplification designed with different sets of time and temperature profiles. A total of 398 samples from 293 patients was studied. The sensitivity was 100% for all types of specimens, while the specificity ranged from 95% for sputum to 88% for bronchoalveolar lavage fluid and pleural fluid and to 85% for non-pulmonary specimens. This study showed that it was possible to employ a single simplified method with minor modifications for a wide range of specimens in clinical practice without loss of sensitivity and specificity.