- Volume 48, Issue 8, 1999

Knowledge of the genetic structure of populations of potentially pathogenic bacteria is important in understanding the epidemiology of diseases. Porphyromonas gingivalis is thought to be an important aetiological agent in periodontal diseases and several methods have been used for typing strains of this species. Here, PCR with primers to repetitive extragenic palindromic sequences (REP-PCR) was compared with three other widely used molecular fingerprinting techniques - restriction endonuclease analysis (REA), ribotyping and PCR with arbitrary primers (AP-PCR) - to type P. gingivalis isolates from healthy and diseased periodontal sites. The data obtained with all four methods were in broad agreement and, with one exception, each subject harboured a single unique genotype of P. gingivalis. REP-PCR of P. gingivalis resulted in the production of 5-10 amplicons, which gave unique electrophoretic patterns in each individual (10 REP-PCR types in 10 patients) and similar results were obtained with AP-PCR. Two isolates from one subject appeared identical by REP-PCR and AP-PCR, but could be differentiated by ribotyping, although there was only minor polymorphism. Thus, ribotyping and REA were the most discriminating methods; however, these are time-consuming and expensive relative to the PCR-based techniques. REP-PCR has the advantage that the same pair of primers is used for all species, whereas AP-PCR needs to be optimised by screening a range of primers. These results show that REP-PCR is a useful and rapid technique for typing P. gingivalis.

Pulsed-field gel electrophoresis (PFGE) has been used successfully to discriminate between strains of many different bacterial species. In this study, digestion of bacterial DNA with the restriction endonuclease NotI and PFGE were evaluated for the typing of isolates of Shigella dysenteriae type 1, an important cause of epidemic dysentery. There were 27 isolates from four outbreaks of dysentery, and 44 isolates from endemic dysentery cases and a laboratory culture collection. The epidemic isolates yielded two types each with two subtypes, whereas the endemic isolates and culture collection yielded eight types with numerous subtypes. These findings suggest that S. dysenteriae 1 can be typed by PFGE.

To gain an understanding of the role of fimbriae and flagella in the adherence and colonisation of Salmonella enterica serotype Enteritidis in chickens, an in-vitro gut adherence assay was developed and used to assess the adherence of a wild-type Enteritidis strain and isogenic non-fimbriate and non-flagellate mutant strains. Enteritidis strain S1400/94, a clinical isolate virulent in chickens, was shown to possess genes which encoded type 1, SEF14, SEF17, plasmid-encoded and long polar fimbriae. Mutant strains unable to elaborate these fimbriae were created by allelic exchange. Each fimbrial operon was inactivated by the insertion of an antibiotic resistance gene cassette. In addition, fliC, motAB and cheA loci, which encode the major subunit of the flagellum, the energy-translation system for motility and one of the chemotaxis signalling proteins, respectively, were similarly inactivated. Non-flagellate mutant strains were significantly less adherent than the wild-type strain, whereas mutant strains defective for the elaboration of any of the types of fimbriae adhered as well as the wild-type strain. A flagellate but non-motile (paralysed) mutant strain and a smooth-swimming chemotaxis-deficient mutant strain were shown to be less adherent than the wild-type strain, but that observation depended on the assay conditions used.

Rabbit anti-idiotypic antibodies (anti-Id-ab) against Yersinia pestis lipopolysaccharide (LPS) were obtained with monoclonal immunoglobulins. Their complementary character to the original antigen was confirmed by immunohistochemical analysis and ELISA and gel precipitation tests. The anti-Id-ab were shown to possess all essential properties of Ab2β subtype. Both in-vitro and in-vivo experiments demonstrated a pronounced adjuvant activity of anti-Id-ab without the toxic effect characteristic of Y. pestis LPS. Combined immunisation with anti-Id-ab plus the FI capsular antigen led to a significant increase in the protective immune response against experimental acute challenge with virulent Y. pestis.

The immunoprotective behaviour of the 30-kDa secretory glycoprotein of Mycobacterium tuberculosis H37Rv has been investigated in different adjuvant systems in two mouse strains, NMR1 and C57BL/6J. In comparison with Freund's incomplete adjuvant (FIA) and dimethyldioctadecyl ammonium bromide (DDA), the 30-kDa glycoprotein complexed with polylactide-co-glycolide microparticles (PLG-MPs) induced maximum immuno-reactivity in the two mouse strains. As compared with controls, immunisation with 30-kDa-PLG-MPs resulted in significantly greater protection in animals challenged with 1 × LD50 of M. tuberculosis H37Rv on the basis of survival rates and number of cfu in the infected organs 30 days after challenge. The degree of protection provided by 30-kDa-PLG-MPs was similar to that obtained with 30-kDa-FIA and higher than BCG immunisation. These findings suggest that biodegradable PLG microparticles can be used as an efficient carrier system for the key immunoprotective 30-kDa secretory protein antigen of M. tuberculosis H37Rv.

The effect of passive immunotherapy with antisera against heat-killed Pseudomonas aeruginosa and three of its exo-enzymes (elastase, alkaline protease and exotoxin A) in gut-derived P. aeruginosa sepsis was evaluated. Mice were given a suspension of P. aeruginosa strain D4 in their drinking water, together with ampicillin (200 mg/kg) to disrupt the normal bacterial flora. Cyclophosphamide was then administered to induce translocation of P. aeruginosa that had colonised the gastrointestinal tract so that gut-derived septicaemia was produced. In this model, intraperitoneal administration of antiserum against heat-killed bacteria, 100 μl/mouse, twice a day for 3 consecutive days significantly increased the survival rate over that of mice treated with normal rabbit serum. Antiserum against elastase, alkaline protease, or a combination of these two antisera, failed to provide significant protection. In contrast, antiserum against exotoxin A significantly increased the survival rate over that of mice treated with normal rabbit serum. These results indicate that passive immunisation with antiserum against heat-killed bacteria and exotoxin A, but not with antiserum against either elastase or alkaline protease, protects mice against gut-derived sepsis caused by P. aeruginosa.

The expression of secreted aspartyl proteinases (Saps) by clinical isolates of Candida albicans, C. tropicalis and C. parapsilosis in human saliva supplemented with glucose and in a proteinase-inducing medium (YCB-BSA), was investigated. Also, yeast growth, pH fluctuation and total protein concentration of the saliva cultures during incubation were measured. Sap expression was assessed by evaluating the enzyme activity as well as the antigen concentration. Saps were expressed well in human saliva supplemented with glucose by all three Candida species, although greater expressions was found in YCB-BSA medium. C. albicans isolates were significantly more proteolytic than the non-albicans isolates. In general, for all three species, the rate of yeast growth, pH fluctuation and percentage reduction of total salivary protein concentration concurred with the degree of expression of Saps. These data strongly suggest that Saps of C. albicans, C. tropicalis and C. parapsilosis may play an active role in the progression of oral candidoses, particularly with regard to the abundance of low pH micro-environments in the oral cavity, which are regularly replenished with dietary carbohydrates.

Lysozyme (muramidase) is a non-specific, antimicrobial protein ubiquitous in human mucosal secretions such as saliva. Although its antibacterial and antifungal activities are well recognised, there are no data on the specific concentrations necessary to affect the growth of Candida albicans or about the effect of lysozyme on the production of secreted aspartyl proteinase (Sap), a putative virulence factor of C. albicans. Five Sap-producing isolates of C. albicans were cultured in YCB-BSA medium with various concentrations of lysozyme to examine its effect on yeast cell growth, ultrastructural cellular topography and extracellular and intracellular Sap concentration and activity. Lysozyme was candidacidal at high concentrations and decreased significantly the extracellular Sap concentration at sublethal doses, accompanied by intracellular accumulation of the enzyme. At low concentrations of lysozyme (c. 10 μg/ml), Sap activity decreased more than two-fold and Sap concentration decreased five-fold without any appreciable effect on cell growth or viability. Ultrastructural investigations showed ballooned cells and cells with invaginations (expecially present near bud scars), indicating that cell-wall components may be possible targets for this enzyme. All concentrations of lysozyme tested were well within physiologically attainable levels. These data suggest that lysozyme has, at least, a bimodal action on C. albicans, killing the organism at higher concentrations and modulating Sap metabolism at lower concentrations.

A recent study demonstrated that phospholipase B (PLB), lysophospholipase (LPL) and lysophopholipase transacylase (LPTA) are secreted by Cryptococcus neoformans var. neoformans and showed that the amount of enzyme production correlated with virulence in mice. The present study characterised the extracellular enzyme activities further by radiometric assays and 31P nuclear magnetic resonance spectroscopy (NMR). All three enzymes were most active between 25 and 40°C. Bovine lung surfactant and its major lipid components, disaturated phosphatidylcholine and phosphatidylglycerol, were the optimal substrates for PLB. Lysophosphatidylcholine was the favoured substrate for LPL and LPTA. PLB and LPL/LPTA were differentially affected by Triton X-100, and palmitoyl carnitine was a potent inhibitor of the three phospholipases. LPL and PLB activities were inhibited by dithiothreitol; N-ethylmaleimide inhibited LPL and LPTA activities. None of the enzymes was inhibited by N-bromosuccinimide or p-bromo-phenacyl bromide. Cellular disruption experiments indicated that >85% of the phospholipase activities were cell-associated, with LPL and LPTA being more easily released than PLB. At pH 5.5 and 7.0, the heat-inactivated secreted enzyme preparations decreased the viability of human neutrophils. This effect was attenuated by active supernates. The relative activities of the PLB, LPL and LPTA in the environment of neutrophils are likely to determine the fate of these cells in vivo. Both phospholipases and heat-stable substances secreted by C. neoformans at 37°C could contribute to membrane degradation and virulence.