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

Abstract

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°C. The growth-supporting factor(s) was separated from each culture by filtration through 1-jim-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°C but unaffected at 0, 25, and 45°C after 30-min incubations. Filtration experiments indicated that the growthsupporting factor(s) in WS1 was 2 to 5 jum 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 (103/ml) and the 1-jim 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.

The role of biofilms and protozoa inLegionellapathogenesis: implications for drinking water

“…Rowbotham (1980) was the first to report the growth of L. pneumophila within Acanthamoeba and Naegleria as a method to enrich for environmental strains of L. pneumophila. Interestingly, others have shown that growth of L. pneumophila in potable water occurs only in the presence of amoebae (Wadowsky et al 1988) and L. pneumophila may remain culturable for up to 6 months in a medium containing Acanthamoeba castellanii (Bouyer et al 2007), whereas free-living Legionella within biofilms may be inactivated within a few weeks (Murga et al 2001;Declerck et al 2007b). Furthermore, active but nonculturable Legionella species have only been propagated or 'resuscitated' culture via co-culture with A. polyphaga and A. castellanii (Hay et al 1995;Steinert et al 1997;Ohno et al 2003;Seno et al 2006;Garcia et al 2007).…”

Legionella pneumophilaEnhC is required for efficient replication in tumour necrosis factor α-stimulated macrophages

“…Legionella pneumophila is a Gram-negative intracellular pathogen that is the causative agent of Legionnaires' pneumonia (McDade et al, 1977;Burnsed et al, 2007). The major natural reservoir for L. pneumophila is freshwater amoebae (Fields et al, 1984;Wadowsky et al, 1988). The disease in humans is initiated by inhalation of contaminated water sources, and progresses as a result of intracellular replication of L. pneumophila in alveolar macrophages (McDade et al, 1977;Horwitz and Silverstein, 1980).…”

Legionella pneumophila Is Internalized by a Macropinocytotic Uptake Pathway Controlled by the Dot/Icm System and the Mouse Lgn1 Locus✪

“…A central aspect of Legionnaires' disease is the growth of Legionella pneumophila within alveolar macrophages after inhalation of aerosolized fresh water supplies containing L. pneumophila . In culture, growth of L. pneumophila can be observed within monocytes and macrophages (1), as well as within amoebae, the presumed natural reservoir (2)(3)(4). Intracellular replication has been demonstrated to be an essential virulence determinant in animal models (5).…”