RESUMO
AIMS: To use fluorescent antibody (FA) and PCR studies on fixed lung tissue to investigate whether Legionella pneumophila was the cause of pneumonia in a cluster of three haematology patients. METHODS: Cut sections of paraffin blocks of lung tissue were examined by direct FA (DFA) using fluorescently labelled antibody to serogroup 1 and Pontiac strains of L. pneumophila. In addition, a single tube 'hanging drop' nested PCR targeting the mip gene of Legionella was performed on DNA extracted from the lung sections. Products were sequenced using dye terminator chemistry. RESULTS: Numerous fluorescing bacteria were seen on staining with both antibodies in lung tissue from two of the patients. Identical L. pneumophila mip gene sequences were amplified from both DFA-positive lung sections. Two differing L. pneumophila mip sequences were obtained on three separate occasions from the tissue sections from the third patient negative by DFA. These sequences differed slightly from those obtained from the two DFA positive lung tissues. CONCLUSIONS: There is good epidemiological evidence to link the first two cases who had been treated in the same ward prior to development of fever within two days of each other. The significance of results is controversial for the third patient.
Assuntos
Proteínas de Bactérias/genética , Imunofilinas/genética , Legionella pneumophila/isolamento & purificação , Doença dos Legionários/diagnóstico , Proteínas de Membrana/genética , Peptidilprolil Isomerase , Pneumonia/diagnóstico , Sequência de Bases , Biópsia , Imunofluorescência , Genes Bacterianos/genética , Humanos , Imunofilinas/metabolismo , Legionella pneumophila/classificação , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , Doença dos Legionários/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Pneumonia/epidemiologia , Pneumonia/etiologia , Reação em Cadeia da PolimeraseRESUMO
Examination of a panel of Legionella longbeachae serogroup 1 strains using a guinea pig model of virulence determined that this clonal species of Legionella shows a remarkable variation in symptoms and disease outcome (Doyle et al., Infect. Immun. 69, 5335-5344, 2001). The presence of plasmids was investigated, as plasmid encoded functions may contribute to the virulence of genetically similar strains. Partial sequence analysis of a large native plasmid (approximately 120 kb), designated pA5H5, from a highly virulent Australian isolate revealed a putative two-component regulatory system with inferred identity to the OmpR family of two-component transcriptional regulatory proteins and EnvZ sensor kinases. An isogenic mutant was constructed in the transcriptional regulatory gene, designated lrpR (L. longbeachae sg 1 regulatory protein) and this strain was tested in Acanthamoeba, U937 cells and in a guinea pig animal model. The mutant was reduced in intracellular multiplication within Acanthamoeba but not U937 macrophage-like cells. However, the lrpR mutant did appear reduced in invasion at the early stages of infection of U937 cells. The lrpR mutant was also attenuated for virulence in a guinea pig animal model of infection. The importance of plasmid-encoded functions for the pathogenicity of Legionella longbeachae serogroup 1 strains is discussed.