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Growth-supporting activity for Legionella pneumophila in tap water cultures and implication of hartmannellid amoebae as growth factors.

Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures.

Animals↗

Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia.

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.

Animals↗

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin.

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.

Amino Acid Sequence↗

Isolation of Acanthamoeba culbertsoni from a patient with meningitis.

A case of amoebic meningitis, presumably primary, was encountered in the Christian Medical College Hospital, Vellore, South India, in November 1983. The patient, a 40-year-old man, had cerebrospinal fluid rhinorrhea before the meningitis developed. Acanthamoeba culbertsoni was repeatedly demonstrated in and cultured from the cerebrospinal fluid. The patient responded dramatically to a combination therapy of penicillin and chloramphenicol.

Adult↗

Retrospective identification of Acanthamoeba culbertsoni in a case of amoebic meningoencephalitis.

Acanthamoeba culbertsoni was identified retrospectively in a case of amoebic meningoencephalitis, previously reported by Jager and Stamm (Lancet, 2, 1343, 1972). This is the second report of this species causing secondary infection in man. Positive results were obtained only with anti-A. culbertsoni sera when the brain sections were stained by the indirect immunofluorescence antibody test with various antisera produced against different Acanthamoeba species. Antiserum raised against purified plasma membranes of A. culbertsoni showed once more its highly specific diagnostic value.

Adult↗

Fulminant amebic meningoencephalitis due to Acanthamoeba.

A 57-year-old chronically immunosuppressed woman with systemic lupus erythematosus developed fulminant meningoencephalitis due to Acanthamoeba castellanii. Amebic trophozoites were also found in the lungs, suggesting a primary pulmonary focus of infection. This case illustrates that Acanthamoeba can cause a fulminant, rapidly fatal meningoencephalitis, as well as the previously reported chronic granulomatous meningoencephalitis. X

Amebiasis↗

Primary amoebic meningoencephalitis.

As a serendipitous by-product of polio virus research, a highly fatal amoebic meningoencephalitis was recognized in animals. The causative microorganisms, contaminants of the viral cultures, were identified as small soil amoebae. These organisms, previously considered non-pathogenic, are prevalent throughout the world. Based on animal studies, the original investigators suggested the possibility of a similar disease in humans. Seven years later, human cases of amoebic meningoencephalitis were reported from widely separated areas of the world. Since 1965, a total of 79 cases have been reported. The literature of primary amoebic meningoencephalitis is presented. The history of the discovery and elucidation of this disease is reviewed. The 79 cases reported in the world literature are divided into two groups, those diagnosed retrospectively after reviewing previous deaths from meningoencephalitis, and those diagnosed at the time of the illness. The classification, morphology, pathogenicity, virulence and distribution of pathogenic soil amoebae are reviewed. The presenting clinical findings, diagnostic procedures, pathology, and management of this recently recognized, highly fatal, human disease is presented along with a report of a new case. Otolaryngologists should become familiar with this serious disorder with a transnasal portal of entry.

Amebiasis↗

Interactions of "Limax amoebae" and gram-negative bacteria: experimental studies and review of current problems.

Free-living amoebae can harbour bacteria inside their cysts giving them a microhabitat and protecting them from disinfectants. The aim of this study was to evaluate the potential importance of "Limax amoebae" as vectors for environmental and nosocomial bacteria in a hospital. It was shown that free-living amoebae are ubiquitous in the investigated hospital, occur syntopically with facultative human pathogens (Comamonas acidovorans and Pseudomonas aeruginosa) and may serve as hosts not only for these but also for bacteria isolated from clinical specimens (Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa); temperature is apparently of crucial importance for the interactions between these microorganisms. Recent studies have shown that "Limax amoebae" apart from acting as protective hosts, may also play a role for the thermotolerance, invasiveness and antibiotic-resistance of bacteria. Considering also the reduced immune-status of many patients, this "symbiosis" of free-living amoebae and bacteria might still be of underestimated hospital-hygienic importance.

Acanthamoeba↗