- Volume 47, Issue 8, 1998

The heat-shock response of Streptococcus pyogenes following exposure to elevated growth temperatures, and the immunological reactivity of heat-shock proteins (HSPs) in streptococcal infections were studied. Two major proteins of 65 and 75 kDa were expressed when a S. pyogenes strain was shifted from 37°C to heat-shock temperatures of 40, 42 and 45°C. Such proteins are members of the GroEL and DnaK families recognised in a Western blot assay with polyclonal antibodies against Escherichia coli GroEL and E. coli DnaK, respectively. Two-dimensional autoradiograms of polypeptides labelled at 37 or 42°C showed an increased intensity of three spots at 42°C. A monoclonal antibody (MAb) against HSP 63 of Bordetella pertussis also recognised the 65-kDa inducible protein, although MAbs against Mycobacterium tuberculosis HSP 65 failed to recognise this protein. Immunoblot analysis of sera from individuals with rheumatic fever or uncomplicated streptococcal diseases revealed seven major immunogenic protein bands, two of which also reacted with anti-E. coli GroEL and DnaK polyclonal antibodies. Furthermore, antibodies to the GroEL and DnaK proteins were also detected in sera from patients with either rheumatoid arthritis or systemic lupus erythematosus. These results demonstrated a heat-shock response of S. pyogenes, and indicated the presence of an immune response against HSPs in streptococcal diseases.

The ability of different serotypes of group B streptococci (GBS) to induce septic arthritis in mice was compared. Types II, III, IV, V, VI and VII GBS were investigated. A highly capsulate strain of type III GBS, COH1, and its mutants, COH1–11 (lacking capsular sialic acid) and COH1–13 (non-capsulate), obtained by transposon insertional mutagenesis, were used to assess the role of type-specific polysaccharide on the induction of arthritis. At an intravenous dose of 107 cfu/mouse, reference strains of types II, III, IV, VI and VII and type III strain COH1 induced arthritis with an incidence ranging from 70 to 90%. For type V and strain COH1-11, 108 cfu/mouse was required to obtain a 50% incidence of arthritis; lesions were not evident with strain COH1–13. The presence of the capsule played a major role in the induction of GBS septic arthritis. The presence and amount of sialic acid in capsular polysaccharide influenced the incidence of articular lesions. The bacterial dose affected the manifestations of arthritis; the less virulent strains of GBS also induced articular lesions when an adequate number of micro-organisms reached the joints.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS). Patients with AIDS appear to acquire M. avium mainly through the gastrointestinal tract. Previous studies have shown that healthy mice given M. avium orally develop disseminated infection after 2-4 weeks. The chief site of M. avium invasion of the intestinal mucosa is the terminal ileum. To learn more about the pathophysiology of M. avium infection of the intestinal mucosa, C57BL/6 bg+ bg+ mice were infected orally with M. avium strain 101 and groups of six mice were killed each week for 8 weeks. The terminal ileum was then prepared for histopathological studies and electron microscopy. A delayed inflammatory response was observed and influx of neutrophils in the Peyer's patches was the only abnormality seen at 1 week. A severe inflammatory response was seen from week 2 to week 5 and necrosis of intestinal villi was observed 6 weeks after infection. These results indicate that invasion and infection of the normal intestine by M. avium results in a severe inflammatory response with segmental necrosis of the intestinal mucosa.

There are currently more than 40 species of Legionella and the identification of most of these by standard methods is technically difficult. The aim of this study was to assess the suitability of a previously published PCR-based method of identifying Legionella spp. Intergenic 16S–23S rDNA spacer regions were amplified with primers complementary to conserved regions of the rRNA genes. Following electrophoretic separation of the products, data analyses were performed with the Taxotron® software package. Computer-assisted analysis (with an empirically derived error tolerance of 3%) could differentiate only 26 of the 43 strains (representing 43 species), with the remaining 17 species clustering into four groups (group I, comprising 10 species; group II, three species; group III, two species and group IV, two species). Analysis of well-characterised ‘non-type’ strains of some Legionella spp. (e.g., from type culture collections) resulted in patterns distinct from the corresponding type strain in most cases. Furthermore, recent isolates (identified by conventional methods) were identified by this PCR method to the presumed correct species (or species group) in only a minority of cases. Well characterised strains and recent isolates of Legionella showed heterogeneity within many species. This intra-species variation severely limits the usefulness of the method for the identification of isolates. However, this property may be useful for epidemiological typing within such species.

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

The flagellin gene sequence from a clinical isolate of Burkholderia pseudomallei was used to design oligonucleotide primers for PCR/RFLP analysis of flagellin gene variation among clinical and environmental isolates of B. pseudomallei. Genes from four clinical and six environmental isolates were amplified and compared by RFLP. The clinical isolates were indistinguishable, but variation was detected among some of the environmental isolates. Sequence analysis of flagellin gene amplified products demonstrated high levels of conservation amongst the flagellin genes of clinical isolates (>99% similarity), compared to the variation observed between the clinical isolates and one of the environmental isolates (<90% similarity). Genomic comparisons with pulsed-field gel electrophoresis (PFGE) revealed differences between the relationships inferred by flagellin genotyping and PFGE, suggesting that a combination of molecular methods may be useful for the subtyping of B. pseudomallei strains.

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.