Hot water recirculation systems (HWRS) in hotels and nursing homes, which are common in countries such as Spain, have been related to outbreaks of legionellosis. To establish the relationships of microbial and physicochemical parameters, especially protozoa, with the occurrence of Legionella in HWRS, 231 samples from hotels and nursing homes were analysed for Legionella, protozoa, heterotrophic plate counts (HPC) at 22 and 37 °C, Pseudomonas, metals, temperature and others. Legionella pneumophila was the dominant species isolated, and 22 % were sg. 1. The sampling method became particularly important in order to define which factors were involved on the occurrence of Legionella. Results showed that the bacteria and the accompanying microbiota were more abundant in the first flush water whose temperature was lower. The bacteria occurred in those samples with high HPC and were inversely correlated with high temperatures. Multivariate regression showed that a concentration above 1 × 10(5) CFU/100 mL of HPC at 37 °C, Fe above 0.095 ppm and the presence of protozoa increased significantly the risk of Legionella colonization, while univariant regression showed that the presence of Cu above 0.76 ppm and temperature above 55 °C diminished it. Therefore, to reduce the risk associated with Legionella occurrence in HWRS these parameters should be taken into consideration.

The deep sea provides global vital functions such as sequestration of carbon from the atmosphere. The increased anthropogenic pressures and interest in expanding deep-sea fisheries make this pristine ecosystem particularly vulnerable, whose conservation largely depends on rapid knowledge acquisition. In view of the limitations of traditional methods to explore the biodiversity of this vast ecosystem, the analysis of traces of macroorganismal DNA released into the water column arises as a cost-effective, noninvasive alternative. Yet, the success of this approach requires understanding of the stratification of DNA traces in the ocean. This study provides evidence that fish DNA traces can be used to establish depth-specific fish diversity and abundance throughout the water column, opening a promising avenue for gathering knowledge about the deep-sea ecosystem.Establishing the foundations for a sustainable use of deep-sea resources relies on increasing knowledge on this inaccessible ecosystem, which is challenging with traditional methods. The analysis of environmental DNA (eDNA) emerges as an alternative, but it has been scarcely applied to deep-sea fish. Here, we have analyzed the fish eDNA contained in oceanic vertical profile samples (up to 2000 m depth) collected throughout the continental slope of the Bay of Biscay. We detected 52 different fish species, of which 25 were classified as deep-sea fish. We found an increase of deep-sea fish richness and abundance with depth, and that eDNA reflects daynight community patterns and species-specific vertical distributions that are consistent with the known diel migratory behavior of many mesopelagic fishes. These findings highlight the potential of eDNA to improve knowledge on the fish species inhabiting the dark ocean before this still pristine ecosystem is further exploited.

Ciliates are globally distributed eukaryotic organisms inhabiting virtually all environments on Earth. Although ciliates range from 10 µm to few mm in cell size, they are repeatedly reported in the pico-sized fraction (smaller than 2-3 µm) of molecular surveys. Here, we used existing datasets (BioMarKs and Tara Oceans) with different size fractions to demonstrate that the ciliate pico-sized signal, likely derived from cell breakage during filtration, is informative and reliable to study marine ciliate biodiversity and biogeography. Then, we used sequences from the picoeukaryotic fraction of two circumnavigation expeditions, Malaspina-2010 and Tara Oceans, to give insights into the taxonomic composition and horizontal and vertical distribution of ciliates in the global ocean. Results suggested a high homogeneity of ciliate communities along the ocean surface from temperate to tropical waters, with ciliate assemblages dominated by a few abundant and widely distributed taxa. Very few taxa were found in a single oceanic region, therefore suggesting a high level of ciliate cosmopolitanism in the global ocean. In vertical profiles, ciliates were detected up to 4000 m depth, and a clear vertical community structuring was observed. Our results provided evidence supporting ciliates as deeply integrated organisms in the deep-sea trophic web, where they may play a relevant role as symbionts of metazoans and grazers of prokaryotes and small eukaryotes in the water column and in aggregates.

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Aims: To evaluate the effect of temperature on two amoeba strains of the genera Acanthamoeba and two amoeba strains of the genera Hartmannella separately treated depending on their life stage, trophozoite or cyst, when cells are directly exposed under controlled conditions. Methods and Results: For thermal treatments, three temperatures were selected 50, 60 and 70°C, and a microcosm was designed using dialysis bags. The inactivation of each strain was determined using a method based on the most probable number quantification on agar plates. The results showed that for all amoeba strains, thermal treatment was more effective against trophozoites compared with cyst stages. The inactivation patterns showed statistical differences between the two genera analysed at temperatures above 50°C. The effectiveness of the thermal treatments at 60 and 70°C was higher for both life stages of Hartmannella vermiformis strains compared with Acanthamoeba strains, being the most resistant Acanthamoeba cysts. Conclusions: Free-living amoebae have been isolated in a wide range of environments worldwide due to their capacity to survive under harsh conditions. This capacity is mainly based on the formation of resistant forms, such as double-walled cysts, which confers a high level of resistance as shown here for thermal treatments. Significance and Impact of Study: Free-living amoebae survival can promote a rapid recolonization of drinking water systems and is a likely source of emerging opportunistic pathogens such as Legionella. Because of that a better understanding of the factors that affect micro-organism inactivation in water systems would allow more efficient application of disinfection treatments.

The deep-sea remains among the most unknown ecosystems on Earth despite its relevant role in carbon sequestration and increasing threat due to interest by fishing and mining industries. This, together with the recent discovery that the upper layer of this ecosystem (mesopelagic zone) harbors about 90% of the fish biomass on Earth, claims for a deeper understanding of the deep-sea so that the foundations for a sustainable use of its resources can be established. The analysis of environmental DNA (eDNA) collected from the water column emerges as an alternative to traditional methods to acquire this elusive information, but its application to the deep ocean is still incipient. Here, we have amplified and sequenced the fish eDNA contained in vertical profile samples (from surface to 2000 m depth) collected during day and night-time throughout the Bay of Biscay. We found that eDNA-derived deep-sea fish richness and abundance follow a day-night pattern that is consistent with the diel migratory behavior of many mesopelagic species, and that eDNA can reveal species-specific distribution and movement through the water column. These results highlight the potential of eDNA-based studies to improve our knowledge on the species inhabiting the dark ocean before this still pristine ecosystem is exploited.

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In recent decades, free-living protozoa (FLP) have gained prominence as the focus of research studies due to their pathogenicity to humans and their close relationship with the survival and growth of pathogenic amoeba-resisting bacteria. In the present work, we studied the presence of FLP in operational man-made water systems, i.e. cooling towers (CT) and hot sanitary water systems (HSWS), related to a high risk of Legionella spp. outbreaks, as well as the effect of the biocides used, i.e. chlorine in CT and high temperature in HSWS, on FLP. In CT samples, high-chlorine concentrations (7.5 ± 1.5 mg chlorine L(-1)) reduced the presence of FLP by 63.8 % compared to samples with low-chlorine concentrations (0.04 ± 0.08 mg chlorine L(-1)). Flagellates and amoebae were observed in samples collected with a level of 8 mg chlorine L(-1), which would indicate that some FLP, including the free-living amoeba (FLA) Acanthamoeba spp., are resistant to the discontinuous chlorine disinfection method used in the CT studied. Regarding HSWS samples, the amount of FLP detected in high-temperatures samples (53.1 ± 5.7 °C) was 38 % lower than in low-temperature samples (27.8 ± 5.8 °C). The effect of high temperature on FLP was chiefly observed in the results obtained by the culture method, in which there was a clear reduction in the presence of FLP at temperatures higher than 50 °C, but not in those obtained by PCR. The findings presented here show that the presence of FLP in operational man-made water systems should be taken into account in future regulations.