Amoebae as vehicles for bacteria

Free-living amoebae are highly abundant in the environment, but have also repeatedly been isolated from typical man made habitats, such as air conditioning and hot water systems. Owing to their extremely resistant cysts these amoebae, particularly the genera Acanthamoeba and Hartmannella, can withstand antimicrobials, heat, ultraviolet radiation and gamma irradiation - and there is ample evidence that they play an important role as vehicles of dispersal for bacteria, including known and new emerging pathogens. In the past years such associations have among others been reported for legionellae and mycobacteria, two groups of pathogens that have been causing smaller outbreaks also in Austria, e.g. in hospitals and hotels. This is not only of ecological interest, but it is also a public health issue, because within the amoebae the bacteria survive disinfection enabling them to recolonize water systems after disinfection measures. Yet, information on the occurrence of free-living amoebae and amoeba- associated bacteria in water facilities and large air-conditioning units in Austria is largely missing. In addition, we are lacking suitable rapid and large scale detection assays for the amoebae and rapid and reliable typing assays for the bacteria. The aim of this project is twofold, firstly, to develop new screening modules, which can then individually be integrated into the obligatory routine screening of water facilities in Austria, depending on demand and feasibility. And secondly, the generation of inventories of amoebae and amoeba-associated bacteria in Austria allowing the improvement of disinfection strategies. The modules will include a high throughput detection system for the various amoebae relevant as hosts and fast and reliable new genotyping methods for Legionella spp. and Mycobacterium spp. In Austria, community-relevant water facilities are screened for legionellae on a routine basis, however, the presence of free-living amoebae and their role for persistence and transmission of Legionella and other opportunists such as mycobacteria are currently not considered. A two years` pilot study performed during this project will evaluate the respective modules and will be the basis for planning the implementation into the routine screening. The accomplishment of this project becomes only possible by translational research, as it requires the access to large water and air-conditioning facilities and the expertise with water sampling and analysis, as well as specific experience in microbiology/ diagnostics of free-living amoebae and endosymbiotic/ endoparasitic bacteria. In order to achieve this, the current project brings together the Austrian agency for health and food safety (AGES), the largest public health organisation in Austria, the Department of Parasitology of the Medical University of Vienna with one of Europe`s largest free-living amoeba research group, and the Department of Microbial Ecology of the University of Vienna with one of the leading groups in the field of research on amoeba-associated bacteria.

Free-living amoebae are widely spread in the environment and also known to cause rare but often serious infections. Besides this, amoebae have indirect public health significance as they may serve as vehicles of dispersal and replication for bacterial pathogens, including also Legionella pneumophila, the causative agent of Legionnaires disease. This intracellular replication in amoebae seems to trigger the ability of the legionellae to infect human cells, besides intracellular bacteria are also protected against disinfection. The aim of this project was to evaluate the diversity of amoebae in water samples that are routinely screened for legionellae. To achieve this, a new highly sensitive but also specific molecular biological screening system for synchronous detection all amoebae relevant as bacterial hosts was developed. Moreover, we investigated all amoebal isolates for intracellular bacteria. Two hospital cooling towers and a cooling tower from a large office building were sampled periodically over the period of one year and investigated by culture and molecular methods for amoebae and bacteria in parallel. Altogether, 83.3% of all cooling tower samples were positive for amoebae. Acanthamoebae, being themselves potential pathogens and being the most relevant bacterial host amoebae, were the most prevalent amoebae, detected in 71.2% of the cooling tower samples. Moreover, several amoebal isolates revealed endosymbionts, including totally new genera of bacteria. Interestingly, 68.8% of the cooling tower samples were not suitable for standard screening methods for Legionella due to their high organic burden. In the remaining samples positivity for Legionella spp. was 25%, but positivity was 50% in a random set of samples screened with molecular methods. In a so-called amplicon study, each hospital cooling tower showed a unique set of Legionella strains. L. pneumophila, although present, was at no time point dominating. Cooling towers, being semi-open water systems, provide an environment for more complex microbial communities compared to drinking water systems and they each seem to have unique microbial communities. In the current study, the community composition varied in the systems over the year despite of the generally constant temperature. Network analysis of co-occurrence revealed a number of striking patterns and also identified a few keystone species, likely important for shaping the community by providing vital metabolic compounds to others. In conclusion, it was shown that there is a high prevalence of amoebae suited as bacterial vehicles in cooling towers and regular disinfection does not affect amoebal survival. Moreover, it was shown that results obtained from current Legionella routine screenings are not reliable for water samples with high organic burden, such as cooling tower waters.