- Volume 46, Issue 8, 1997

On the assumption that specific host defences are lower in newborn and infant animals, the susceptibility of CD1 suckling mice to Legionella pneumophila was studied with the hypothesis that this model could detect consistent differences in virulence among Legionella isolates from various clinical and environmental sources. Mice 3−14 days old were indeed markedly susceptible to intraperitoneal challenge with fresh clinical isolates, but not to serially subcultured or type collection strains of L. pneumophila. For example, intraperitoneal inoculation of 107cells of a fresh clinical isolate of L. pneumophila (strain Monza 3) caused 60% mortality of suckling mice in 1 day whereas the same number of cells of a culture-attenuated derivative (strain Monza 3p50) caused <10% mortality in >15 days. Lethal infection by the ‘virulent’ Monza 3 strain was strictly dependent on mouse age (no death was observed in mice >26 days old), required the inoculation of viable cells and was not related to endotoxin production. The ‘virulent’ L. pneumophila strain was cleared from mouse lungs less rapidly, while adhering to, and being internalised into the peritoneal exudate cells (PEC) of suckling mice to a greater extent, than the avirulent derivative, as shown by immunofluorescence and confocal microscopy. The Monza 3 strain also induced the production by PEC in vivo of 5-to-10 times more tumour necrosis factor-alpha (TNF-α) mRNA than the Monza 3p50 strain. Overall, suckling CD1 mice appear to provide a promising, easily handled, highly reproducible and relatively inexpensive animal model for studies of the virulence of L. pneumophila, and possibly, of the role of pro-inflammatory cytokine production in this phenomenon.

Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolation in cultured mammalian cells. Screening for such strains makes use of HeLa cells which may not be sensitive enough to detect minimal changes. The aim of this study was to develop a more sensitive cell line. Vacuole formation was examined in HeLa cells, as well as four other cell lines established in this laboratory by ammonium chloride induction. Among five cell lines tested, LYM-1 cells were most sensitive for the detection of intracellular vacuolation with this agent. Loss of cell viability of LYM-1 and HeLa cells induced by H. pylori culture supernates was also examined: LYM-1 were more sensitive than HeLa cells. Cell death was not always accompanied by vacuole formation. This suggests that the mechanism whereby cell death occurs must be different from that for vacuole formation. LYM-1 cells may be useful when measuring vacuole formation and cell death of the cultured cells induced by culture supernates of clinical isolates of H. pylori.

Although V. parahaemolyticus does not generally produce urease, several studies have reported urease-positive V. parahaemolyticus isolates from clinical sources. Recently, studies have shown a complete coincidence between the urease-producing phenotype of V. parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh). TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V. parahaemolyticus gastroenteritis. The present study attempted to identify the gene ure encoding urease in V. parahaemolyticus to clarify the relationship between urease production and possession of trh. The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V. parahaemolyticus ure gene. Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease. Considering the coincidence between production of urease and possession of trh in V. parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V. parahaemolyticus strains. Furthermore, the relative location of ure and trh on V. parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis. The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA. These results suggest that the ure and trh genes are genetically linked in V. parahaemolyticus strains.

Bacterial entry into intestinal host cells is the result of a fairly sophisticated manipulation of host cell machinery by the pathogens. To study further the potential cell target of Listeria spp., the in-vitro entry of L. monocytogenes strains into intestinal cells was examined in relation to the metabolism, proliferation and differentiation of the cells by the alamarBlue assay, [3H] thymidine incorporation, and brush border-associated enzyme activities, respectively. The study showed that cell metabolism was not involved in the entry of L. monocytogenes in three cell models (two human and one porcine). On the other hand, entry was closely related to the proliferation process and poorly related to the differentiation state of the cells. The use of L. monocytogenes mutants lacking invasion proteins showed that InIA and InIB acted in synergy to mediate the entry of L. monocytogenes into proliferative cells, whereas InIA alone seemed to be involved in the entry into non-proliferative cells. These two entry pathways could correspond to the two cellular processes used by L. monocytogenes to enter proliferative and non-proliferative cells, as suggested by the use of cytochalasin D, nocodazole, chloroquine and monodansylcadaverine. Taken together, we propose a hypothesis in which the entry of L. monocytogenes is mediated by interaction between randomly distributed E-cadherin on the surface of proliferative cells. In contrast, the entry into non-proliferative cells may involve pp60c-src, a proto-oncogenic tyrosine kinase signal that modifies E-cadherin localisation. In conclusion, these results suggest that L. monocytogenes may preferentially enter crypt cells in vivo by a microfilament-dependent process, whereas the few bacteria that infect villus cells enter by an E-cadherin-internalin interaction that mediates microtubule-dependent endocytosis.

The role of lysozyme in the immune immobilisation of Treponema pallidum is not yet fully understood. The T. pallidum immobilisation assay was used to demonstrate that the immobilisation and lysis of T. pallidum in vitro by antibodies (serum, IgG fraction or IgM fraction) and complement proceed in a lysozyme-independent mode. In the presence of lysozyme the rate of immobilisation increased. In contrast with its effect on Escherichia coli, the effect of lysozyme on T. pallidum was governed exclusively by its enzymic activity rather than by the cationic protein nature of the molecule. Lysozyme, released from stimulated phagocytes, induced formation of lysozyme antibodies in 59.6% of syphilis patients as determined by lysozyme antibody ELISA. The highest frequency was found in patients with untreated secondary syphilis, whereas untreated primary syphilis was only rarely accompanied by the presence of lysozyme antibodies. Cross-reactivities between lysozyme and treponemal antigens were excluded by immunoblot-ting. The autoantibodies did not influence the lysozyme activity. It was concluded that the formation of lysozyme antibodies is only an epiphenomenon in the host defence against treponemal infection.

A 44-year-old immunocompromised man presented with multiple tissue abscesses, covering the entire left limb. A dematiaceous fungus compatible with Pleurophomopsis lignicola Petrak was isolated from the diseased tissue in pure culture. This is the second reported isolation of this fungus from man and the first report of fatal soft tissue infection. A detailed morphological description of the isolate is provided.

Pneumocystosis is usually a disease of the lungs, but the number of cases of extrapulmonary pneumocystosis has greatly increased during the AIDS epidemic. Much remains unknown about the frequency and mechanisms of dissemination. In the present study, a systematic search for Pneumocystis carinii by PCR with primers specific for mitochondrial rRNA was performed in the lung, liver, spleen and kidney of 12 immunosuppressed rats and two immunocompetent rats. The amplified products were analysed by Southern hybridisation with a digoxigenin-11-dUTP labeled probe. P. carinii DNA was found in lungs in all 14 rats and in at least one organ other than lung in 11 immunosuppressed rats and the two control rats. We suggest that extrapulmonary dissemination may not be an exceptional phenomenon in the course of pneumocystosis, but rather part of the natural evolution of the disease.