Characterising the relationship between Legionella longbeachae and their amoebal hosts: Implications for Legionnaires' disease prevention

Abstract

In New Zealand, Legionella longbeachae is the leading cause of Legionnaires’ disease, a severe form of pneumonia that often results in hospitalisation and an intensive course of treatment. Every year, during spring and summer, cases of Legionnaires’ disease caused by L. longbeachae infection are seen to increase. These cases are often associated with the exposure to composted plant materials, such as those found in commercial potting mix. In both natural and man-made environments, Legionella can parasitize free-living amoeba and subvert host cell signalling to support their replication. Whilst this interaction has been well characterised for several species of Legionella, very little is known about the relationship between L. longbeachae and its amoebal hosts and as a result, our ability to plan strategies to reduce the potential risk of human infection is limited. Using qPCR, we tested DNA from a variety of previously collected and stored environmental samples for the presence of Acanthamoeba spp. and Naegleria fowleri. Low levels of Acanthamoeba DNA were detected in several samples where L. longbeachae was present, and while there did not appear to be a relationship between Acanthamoeba and L. longbeachae in nature, Acanthamoeba may still be able to support the replication of L. longbeachae in vitro. To investigate this, a Legionella co-culture system was established with Acanthamoeba polyphaga (ATCC® 50372™). Amoebal uptake of L. longbeachae was observed using fluorescent microscopy, and a comparison was made using a type-strain of Legionella pneumophila (ATCC® 33152™). After two hours, both L. pneumophila and L. longbeachae were seen to co-localise with A. polyphaga. However, these findings alone were not enough to conclude whether L. longbeachae was being internalised or replicating within A. polyphaga. In an effort to help identify other potential amoebal hosts for L. longbeachae, culture-based techniques were used to screen a variety of environmental samples for protozoa. Viable amoeba were successfully recovered from a potting mix sample in which L. longbeachae had been previously isolated, and ongoing experiments have been planned to identify these species using 18S-ITS2 universal primers. In summary, this study included a significant methodological development that will be utilised to enhance our ability to better define the relationship that L. longbeachae has with its amoebal hosts. Although we were unable to identify a suitable host for L. longbeachae in the given time frame, we have established a strong foundation for future research.