The integration of an avirulent Legionella pneumophila into aquatic biofilms
A continuous culture model system was set up in the laboratory and inoculated with a diverse range of microorganisms, including several bacteria and protozoa, obtained from the local mains water tap supply. This inoculum and was added to the system without any prior culture or other selection process. Biofilms readily developed on glass tiles suspended in the planktonic phase of the system. An avirulent Legionella pneumophila was inoculated into the system and was subsequently isolated from both biofilms and also from the aqueous(planktonic) phase of the chemostat. The attenuation of this strain, determined by its inability to cause disease and death in guinea pigs, remained unaltered despite the long term survival of this strain within the system. Investigations to determine whether the avirulent L. pneumophila was able to infect and proliferate within protozoa were carried out. The results of the present study show that this avirulent L. pneumophila did not proliferate intracellularly and suggest that the association of L. pneumophila with protozoa although probably important in the long term survival of this bacterium especially during periods where adverse conditions prevail, is not essential but opportunistic. In chapter 3 the importance of the presence of these non-legionellae bacteria, which included Flavobacterium sp., Acinetobacter spp. and several species of Pseudomonas, was investigated. The results suggest that the presence of the non-legionellae are relevant to the survival of Legionella especially in environments which favour it's growth, for example water distribution systems. In order that we may gain a further insight into the ecology of microorganisms in their natural environment, it is necessary to visualise them in conditions which allow them to interact in a way which mimics as closely as possible the natural environment. Biofilms were developed on surfaces which could be removed from the model system in their entirety. Direct visualisation techniques, including atomic force microscopy and Hoffman modulation microscopy could then be used which allowed the in vivo examination of biofilms in situ on the surface upon which they had developed. More traditional microscopy methods were also used. Atomic force microscope images of biofilms and individual biofilm bacteria including Legionella were obtained, which clearly showed the presence of exopolymeric substances (EPS). Hoffman modulation contrast microscopy and scanning electron microscopy showed the diverse nature of the biofilms being studied. The results of these investigations suggest that a more complete understanding of the complex nature of biofiims is achieved by the use of a combination of several microscopy techniques. The response of a L. pneumophila serogroup 6 and the avirulent L. pneumophila serogroup 1 to a commercially available biocide, Vantocil IB, was investigated. Both the serogroup 6 and the avirulent serogroup 1 could not be detected following biocide teatment in either the planktonic phase or biofilms. These results suggest that this avirulent L. pneumophila is a suitable model substitute for the virulent L. pneumophila.