Five sporadic cases of nosocomial Legionnaires’ disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes. Legionella pneumophila serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of L. pneumophila serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of rrnoperon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of Legionellainfection. The long-term persistence in the hot-water system of different clones of L. pneumophila serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.
Summary. The polyether antibiotic monensin exhibited bacteriostatic activity against a clinical isolate of Legionellapneumophila in vitro. Experiments designed to test the effect of the compound on the invasiveness and multiplication of L. pneumophila in HeLa cells showed that, in the presence of the antibiotic, legionellas that penetrated the cells did not multiply. However, monensin did not alter the characteristics of phagosomes that contained ingested legionellas. In the presence of monensin, infected cells exhibited extensive vacuolation and a noticeable reduction in the number of intracellular micro-organisms was evident a few hours after infection.
Fatty acid profile analysis, monoclonal antibody (MAb) subtyping, pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (AP-PCR), and ribotyping were used to compare clinical and environmental Legionella pneumophila serogroup 1 isolates from an outbreak of Legionnaires' disease presumptively associated with cooling towers. According to the Oxford subtyping scheme, the MAb subtype of patients' isolates and of two strains originating from a cooling tower was Pontiac, whereas the other isolates were subtype Olda. The strains showed no intrinsic strain-to-strain difference in fatty acid profiles, and ribotyping and length polymorphism of the 16S-23S rDNA intervening regions failed to reveal any differences between the isolates. Conversely, PFGE and AP-PCR appeared to be more discriminatory, as the same genomic profile was found for the clinical and some environmental strains. Meteorologic and epidemiological data and the results of molecular analysis of the Legionella pneumophila serogroup 1 isolates support the hypothesis that the infection was transmitted from one of the cooling towers to the indoor environment of the same building, to homes in proximity that had open windows, and to the streets. In fact, the outbreak diminished and later ended after a part in the tower was replaced. This investigation demonstrates the utility of combined molecular methods (i.e., phenotypic and genomic typing) in comparing epidemiologically linked clinical and environmental isolates. Finally, the outbreak confirms the risk of Legionnaires' disease posed by cooling towers, mainly when atmospheric thermal and humidity inversions occur. This finding emphasizes the need to determine whether the source of infection is in the living or working environment or somewhere else.
Multiplication of Legionella pneumophila in HeLa cells was found to be inhibited by noncytotoxic concentrations of bafilomycin A 1 , with blockage of bacterial growth at a concentration 15.6 nM. The inhibiting action was evident only when the antibiotic was present during the initial phase of intracellular multiplication, i.e., during the formation of the phagosome, whereas the addition of the drug did not affect microorganisms already actively multiplying within the phagosome.
A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-D-glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mm glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.
The effect of sub-inhibitory concentrations of various antibiotics on the hemolytic activity of different strains of Legionella has been tested. By means of a gradient plate technique it was possible to demonstrate that in a limited range of sub-inhibitory concentrations, antibiotics did not affect bacterial growth but inhibited the hemolytic activity of the strains examined.
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