Legionella pneumophila, the causative agent of Legionnaires' disease, is an aquatic intracellular organism capable of replicating within both amoebae and human phagocytic cells. A comprehensive study has been undertaken to examine the hypothesis that intracellular replication of L. pneumophila induces enhanced resistance to external stress stimuli relative to that of in vitro grown legionellae. Microscopical studies have shown that L. pneumophila grown in YE broth consistently develop a rod-shaped morphology and are non-motile. In contrast, L. pneumophila grown within Acanthamoeba polyphaga or U937 monocytes exhibit a smaller, rounded morphology and are highly motile. After ca. 72 h post lysis, the intracellular bacteria adopted a morphology similar to that of broth grown legionellae in stationary phase. These bacteria were termed 'aged'. Time-kill assays have shown that L. pneumophila grown within A. polyphaga or U937 monocytes are more resistant than broth grown legionellae to a number of different stress conditions including exposure to the antibiotics traditionally used in the treatment of Legionnaires' disease, elevated temperatures associated with water treatment processes, mechanical stress and starvation. 'Ageing' of intracellular L. pneumophila before exposure to the stress stimuli, resulted in a marked loss of stress resistance. To study the physiological basis of the increased stress resistance of intracellular grown L. pneumophila, a preliminary study of the surface properties of the variously grown legionellae was undertaken. The outer membrane protein (OMP) profiles of the L. pneumophila were prepared using sarkosyl and analysed by SDS-PAGE. Proteinase K digestion of the outer membrane (OM) was employed to prepare the LPS layer. The preliminary comparative study of the L. pneumophila OM has established that the mode of growth influences both the OMP profile and the LPS layer. In particular, intra-amoebic grown L. pneumophila possesses a novel protein of 15 kDa and intra-monocytic grown legionellae one of 24 kDa, both of which are lost upon 'ageing'.The results of the project have shown a link between the presence of novel sarkosyl insoluble OMPs, bacterial morphology and enhanced resistance of the intracellular grown L. pneumophila to external stress stimuli. It could, therefore, be suggested that L. pneumophila undergo a prior adaptation to stress conditions during intracellular replication. Upon 'ageing' of intracellular grown legionellae the novel insoluble OMPs were lost with a concomitant change in morphology and loss in resistance to external stress stimuli. The findings of this work have practical implications with respect to the clinical treatment of Legionnaires' disease and the eradication of the causative agent from water systems.

The attachment and/or penetration of animal cells by two strains of Legionella pneumophila was studied in three vertebrae cell lines in vitro . The study focused on (1) differences in attachment and penetration between the two bacterial strains (an environmental isolate, Johannesburg-2, and a clinical isolate, Chicago-8) and between the cell lines (Hep-2, WI-38 and a murine line); (2) effects of L. pneumophila on cell morphology and growth; and (3) the effects of pyruvate and six sugars or sugar derivatives (D-mannose, D-Galactose, D-Glucose, L-glucose, D-fructose, and 2-deoxy-D-glucose).

Outer membrane proteins (OMPs) were recovered from eleven strains (eight serogroups) of Legionella pneumophila by sequential treatment with Tris buffer (pH 8), citrate buffer(pH 2.75) and Tris buffer (pH 8). Transmission electron microscopy revealed clearly the separation of the outer membrane from the bacteria. The development of delayed hypersensitivity was also noted by measuring the area of arythema and induration produced by intradermal injections of the MPSs from Chicago 8 strain. The adjuvants enhanced greatly both active and cell-meditated immunity (CMI). Transient lymphocytopenia with a slight rise in neutrophils was noted in each of the immunized groups. Intraperitoneal challenge, seven days after the OMP booster, of one LD (1.5 x10^6) of legionellae resulted in lymphocytopenia with elevated neutrophils. All immunized rats survived the challenge, although those in the saline-OMP group were clearly the sickest. Post-challenge, legionella antibody titers rose greatly and CMI was heightened. Passive immunization (homologous and heterologous) was found to protect the rats from a challenge of on LD. Actively-immunized rats retained their immunity for at least six months as determined by their resistance to a second challenge.

Legionella pneumophila is a gram-negative bacterium responsible for Legionnaire's disease, and is commonly transmitted via aerosolized water. Legionella colonization of emergency eyewash and shower stations may pose an exposure hazard to users of these stations. There is little information about the role of these stations as significant reservoirs for Legionella. Samples were collected from 67 stations in an industrial facility. At the time of this study, the stations within this facility were under a routine maintenance program that included at least monthly flushing. This study also included the analysis for other bacterial organisms to determine an association between the presence and concentration of other bacteria and Legionella. All samples resulted in no detection of Legionella, yet 12 of the samples contained large counts of other bacteria. Thus, this study supports that properly maintained emergency eyewash and shower stations do not appear to be a significant source for aerosol transmission of Legionella.