The cooling tower water microbiota: Seasonal dynamics and co-occurrence of bacterial and protist phylotypes.

Cooling towers for heating, ventilation and air conditioning are ubiquitous in the built environment. Often located on rooftops, their semi-open water basins provide a suitable environment for microbial growth. They are recognized as a potential source of bacterial pathogens and have been associated with disease outbreaks such as Legionnaires' disease. While measures to minimize public health risks are in place, the general microbial and protist community structure and dynamics in these systems remain largely elusive. In this study, we analysed the microbiome of the bulk water from the basins of three cooling towers by 16S and 18S rRNA gene amplicon sequencing over the course of one year. Bacterial diversity in all three towers was broadly comparable to other freshwater systems, yet less diverse than natural environments; the most abundant taxa are also frequently found in freshwater or drinking water. While each cooling tower had a pronounced site-specific microbial community, taxa shared among all locations mainly included groups generally associated with biofilm formation. We also detected several groups related to known opportunistic pathogens, such as Legionella, Mycobacterium, and Pseudomonas species, albeit at generally low abundance. Although cooling towers represent a rather stable environment, microbial community composition was highly dynamic and subject to seasonal change. Protists are important members of the cooling tower water microbiome and known reservoirs for bacterial pathogens. Co-occurrence analysis of bacteria and protist taxa successfully captured known interactions between amoeba-associated bacteria and their hosts, and predicted a large number of additional relationships involving ciliates and other protists. Together, this study provides an unbiased and comprehensive overview of microbial diversity of cooling tower water basins, establishing a framework for investigating and assessing public health risks associated with these man-made freshwater environments.

In situ architecture, function, and evolution of a contractile injection system.

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

'Candidatus Cochliophilus cryoturris' (Coxiellaceae), a symbiont of the testate amoeba Cochliopodium minus.

Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as 'Candidatus Cochliophilus cryoturris'. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

Free-living amoebae and their associated bacteria in Austrian cooling towers: a 1-year routine screening.

Free-living amoebae (FLA) are widely spread in the environment and known to cause rare but often serious infections. Besides this, FLA may serve as vehicles for bacterial pathogens. In particular, Legionella pneumophila is known to replicate within FLA thereby also gaining enhanced infectivity. Cooling towers have been the source of outbreaks of Legionnaires' disease in the past and are thus usually screened for legionellae on a routine basis, not considering, however, FLA and their vehicle function. The aim of this study was to incorporate a screening system for host amoebae into a Legionella routine screening. A new real-time PCR-based screening system for various groups of FLA was established. Three cooling towers were screened every 2 weeks over the period of 1 year for FLA and Legionella spp., by culture and molecular methods in parallel. Altogether, 83.3 % of the cooling tower samples were positive for FLA, Acanthamoeba being the dominating genus. Interestingly, 69.7 % of the cooling tower samples were not suitable for the standard Legionella screening due to their high organic burden. In the remaining samples, positivity for Legionella spp. was 25 % by culture, but overall positivity was 50 % by molecular methods. Several amoebal isolates revealed intracellular bacteria.