Environmental regulation of the growth, physiology and virulence of Legionella pneumophila

Mauchline, William Stuart (1996). Environmental regulation of the growth, physiology and virulence of Legionella pneumophila. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000e0d1


Members of the Legionellaceae cause respiratory infections in man; the most severe, pneumonic form is known as Legionnaires' disease. Of the 39 species described to date 16 have been associated with human disease, however the majority of reported cases of legionellosis are caused by Legionella pneumophila serogroup 1. A number of pathogenic bacteria regulate their virulence gene expression in response to environmental stimuli. Temperature and the availability of iron are considered to be stimuli which signal entry to a host environment.

The first part of this study utilised chemostat culture to investigate the influence of growth temperature and the availability of iron on the physiology, morphology and virulence of L. pneumophila serogroup 1. This study demonstrated, for the first time, that the virulence of L. pneumophila was significantly reduced (P < 0.05) when the culture temperature was lowered from 37 to 24°C and this modulation was reversed by returning the temperature to 37°C which resulted in a statistically significant (P < 0.05) increase in virulence. Further experiments demonstrated that the concentration of iron in the growth medium also had an effect on virulence. Contrary to expectations iron-limited cultures were less virulent than those grown iron-replete. This modulation was also reversible with a return to virulence when iron-replete conditions were restored.

The physiology and morphology of L. pneumophila were also influenced by both growth temperature and iron-limitation. At 24°C cultures consisted of flagellated short rods, whereas cultures grown at 37°C were pleomorphic and flagella were not evident. It was demonstrated that L. pneumophila accumulates the intracellular carbon storage compound, polyhydroxybutyrate, and that the proportion of the cell dry weight which it comprised varied with growth temperature, being maximal at 24°C. The ratio of saturated to unsaturated fatty acids in L. pneumophila decreased as the temperature was reduced to 24QC; this is a common strategy designed to maintain membrane fluidity. Siderophore production was detected in iron-limited cultures but not in iron replete cultures. Protease production was also affected by both growth temperature and iron-limitation.

The BIOLOG bacterial identification system was modified for use with legionellae and this was used to investigate the metabolic versatility of these bacteria. A database containing substrate utilisation profiles of Legionella species was constructed using the modified system; this was then used to identify legionella isolates to species level.

Evaporative cooling towers are a significant source of Legionnaires' disease accounting for the majority of outbreak cases in the United Kingdom. In the second part of this study a microbiologically-contained, fully-functional evaporative cooling tower was constructed and used to investigate factors that could influence the growth of L. pneumophila in such systems. The mode of operation of the cooling tower was found to influence the multiplication of legionellae in the system. Low-usage situations resulted in enhanced growth of L. pneumophila. Growth of L. pneumophila demonstrated a significant positive correlation with water temperature but its concentration decreased with increased conductivity. The concentrations of calcium, magnesium, potassium and zinc and the total hardness of the water all exhibited inverse relationships with legionella population size. The protocol for the emergency disinfection of cooling systems recommended in the Report of the Department of Health Expert Advisory Committee on Biocides did not eradicate L. pneumophila from the experimental cooling tower.

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