A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Presence of Non-Tuberculous Mycobacteria Including Mycobacterium avium subsp. paratuberculosis Associated with Environmental Amoebae.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis (Map) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis (M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map.

Identifying Eukaryotes and Factors Influencing Their Biogeography in Drinking Water Metagenomes.

The biogeography of eukaryotes in drinking water systems is poorly understood relative to that of prokaryotes or viruses, limiting the understanding of their role and management. A challenge with studying complex eukaryotic communities is that metagenomic analysis workflows are currently not as mature as those that focus on prokaryotes or viruses. In this study, we benchmarked different strategies to recover eukaryotic sequences and genomes from metagenomic data and applied the best-performing workflow to explore the factors affecting the relative abundance and diversity of eukaryotic communities in drinking water distribution systems (DWDSs). We developed an ensemble approach exploiting k-mer- and reference-based strategies to improve eukaryotic sequence identification and identified MetaBAT2 as the best-performing binning approach for their clustering. Applying this workflow to the DWDS metagenomes showed that eukaryotic sequences typically constituted small proportions (i.e., <1%) of the overall metagenomic data with higher relative abundances in surface water-fed or chlorinated systems with high residuals. The α and ß diversities of eukaryotes were correlated with those of prokaryotic and viral communities, highlighting the common role of environmental/management factors. Finally, a co-occurrence analysis highlighted clusters of eukaryotes whose members' presence and abundance in DWDSs were affected by disinfection strategies, climate conditions, and source water types.

Here, there and everywhere: Ecology and biology of the Dependentiae phylum.

Our view of bacterial diversity has been dramatically impacted by cultivation-independent approaches such as metagenomics and 16S rRNA gene sequencing. Consequently, most bacterial phyla known to date are only documented by the presence of DNA sequences in databases and lack cultivated representatives. This bacterial majority that is yet-to-be cultivated, is forming the 'Microbial Dark Matter', (MDM) a consortium, whose ecology and biology remain largely unexplored. The Candidatus Dependentiae stands as one of many phyla within this MDM, found worldwide in various environments. Genomic evidence suggests ancestral, unusual adaptations of all Ca. Dependentiae to a host dependent lifestyle. In line with this, protists appear to be important for Ca. Dependentiae biology, as revealed by few recent studies, which enabled their growth in laboratory through host cultivation. However, the Ca. Dependentiae still remain to this day a poorly documented phylum. The present review aims to summarize the current knowledge accumulated on this often found, but rarely highlighted, bacterial phylum.

Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis.

Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. While a partial 18S rRNA gene has allowed us to suggest that this amoeba was Rosculus-like, at that time we were not able to sub-cultivate it. In the present study, we succeeded in cultivating this strain at 20-25°C. This amoeba is among the smallest (5-7 µm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the Rosculus genus, i.e., R. vilicus. Of note, the mitochondrial genome is particularly large (184,954 bp). Finally, we showed that this amoeba was able to phagocyte Mycobacterium avium subsp. paratuberculosis and that the bacterium was still observed within amoebae after at least 3 days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. As a result, R. vilicus is a potential candidate as environmental reservoir for Mycobacterium avium subsp. paratuberculosis but further experiments are needed to test this hypothesis.

Comparison of DNA purification methods for high-throughput sequencing of fungal communities from wine fermentation.

High-throughput sequencing approaches, which target a taxonomically discriminant locus, allow for in-depth insight into microbial communities' compositions. Although microorganisms are historically investigated by cultivation on artificial culture media, this method presents strong limitations, since only a limited proportion of microorganisms can be grown in vitro. This pitfall appears even more limiting in enological and winemaking processes, during which a wide range of molds, yeasts, and bacteria are observed at the different stages of the fermentation course. Such an understanding of those dynamic communities and how they impact wine quality therefore stands as a major challenge for the future of enology. As of now, although high-throughput sequencing has already allowed for the investigation of fungal communities, there is no available comparative study focusing on the performance of microbial deoxyribonucleic acid (DNA) extraction in enological matrixes. This study aims to provide a comparison of five selected extraction methods, assayed on both must and fermenting must, as well as on finished wine. These procedures were evaluated according to their extraction yields, the purity of their extracted DNA, and the robustness of downstream molecular analyses, including polymerase chain reaction and high-throughput sequencing of fungal communities. Altogether, two out of the five assessed microbial DNA extraction methods (DNeasy PowerSoil Pro Kit and E.Z.N.A.® Food DNA Kit) appeared suitable for robust evaluations of the microbial communities in wine samples. Consequently, this study provides robust tools for facilitated upcoming studies to further investigate microbial communities during winemaking using high-throughput sequencing.

Defensive symbiosis against giant viruses in amoebae.

Protists are important regulators of microbial communities and key components in food webs with impact on nutrient cycling and ecosystem functioning. In turn, their activity is shaped by diverse intracellular parasites, including bacterial symbionts and viruses. Yet, bacteria-virus interactions within protists are poorly understood. Here, we studied the role of bacterial symbionts of free-living amoebae in the establishment of infections with nucleocytoplasmic large DNA viruses (Nucleocytoviricota). To investigate these interactions in a system that would also be relevant in nature, we first isolated and characterized a giant virus (Viennavirus, family Marseilleviridae) and a sympatric potential Acanthamoeba host infected with bacterial symbionts. Subsequently, coinfection experiments were carried out, using the fresh environmental isolates as well as additional amoeba laboratory strains. Employing fluorescence in situ hybridization and qPCR, we show that the bacterial symbiont, identified as Parachlamydia acanthamoebae, represses the replication of the sympatric Viennavirus in both recent environmental isolates as well as Acanthamoeba laboratory strains. In the presence of the symbiont, virions are still taken up, but viral factory maturation is inhibited, leading to survival of the amoeba host. The symbiont also suppressed the replication of the more complex Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean (Mimiviridae). Our work provides an example of an intracellular bacterial symbiont protecting a protist host against virus infections. The impact of virus-symbiont interactions on microbial population dynamics and eventually ecosystem processes requires further attention.

Identifying group-specific primers for environmental Heterolobosa by high-throughput sequencing.

Diversity of Heterolobosea (Excavata) in environments is poorly understood despite their ecological occurrence and health-associated risk, partly because this group tends to be under-covered by most universal eukaryotic primers used for sequencing. To overcome the limits of the traditional morpho-taxonomy-based biomonitoring, we constructed a primer database listing existing and newly designed specific primer pairs that have been evaluated for Heterolobosea 18S rRNA sequencing. In silico taxonomy performance against the current SILVA SSU database allowed the selection of primer pairs that were next evaluated on reference culture amoebal strains. Two primer pairs were retained for monitoring the diversity of Heterolobosea in freshwater environments, using high-throughput sequencing. Results showed that one of the newly designed primer pairs allowed species-level identification of most heterolobosean sequences. Such primer pair could enable informative, cultivation-free assays for characterizing heterolobosean populations in various environments.

Isolation and Genome Analysis of an Amoeba-Associated Bacterium Dyella terrae Strain Ely Copper Mine From Acid Rock Drainage in Vermont, United States.

Protozoa play important roles in microbial communities, regulating populations via predation and contributing to nutrient cycling. While amoebae have been identified in acid rock drainage (ARD) systems, our understanding of their symbioses in these extreme environments is limited. Here, we report the first isolation of the amoeba Stemonitis from an ARD environment as well as the genome sequence and annotation of an associated bacterium, Dyella terrae strain Ely Copper Mine, from Ely Brook at the Ely Copper Mine Superfund site in Vershire, Vermont, United States. Fluorescent in situ hybridization analysis showed this bacterium colonizing cells of Stemonitis sp. in addition to being outside of amoebal cells. This amoeba-resistant bacterium is Gram-negative with a genome size of 5.36 Mbp and GC content of 62.5%. The genome of the D. terrae strain Ely Copper Mine encodes de novo biosynthetic pathways for amino acids, carbohydrates, nucleic acids, and lipids. Genes involved in nitrate (1) and sulfate (7) reduction, metal (229) and antibiotic resistance (37), and secondary metabolite production (6) were identified. Notably, 26 hydrolases were identified by RAST as well as other biomass degradation genes, suggesting roles in carbon and energy cycling within the microbial community. The genome also contains type IV secretion system genes involved in amoebae resistance, revealing how this bacterium likely survives predation from Stemonitis sp. This genome analysis and the association of D. terrae strain Ely Copper Mine with Stemonitis sp. provide insight into the functional roles of amoebae and bacteria within ARD environments.

Exploring the Individual Bacterial Microbiota of Questing Ixodes ricinus Nymphs.

Ixodes ricinus is the most common hard tick species in Europe and an important vector of pathogens of human and animal health concerns. The rise of high-throughput sequencing has facilitated the identification of many tick-borne pathogens and, more globally, of various microbiota members depending on the scale of concern. In this study, we aimed to assess the bacterial diversity of individual I. ricinus questing nymphs collected in France using high-throughput 16S gene metabarcoding. From 180 dragging-collected nymphs, we identified more than 700 bacterial genera, of which about 20 are abundantly represented (>1% of total reads). Together with 136 other genera assigned, they constitute a core internal microbiota in this study. We also identified 20 individuals carrying Borreliella. The most abundant species is B. afzelii, known to be one of the bacteria responsible for Lyme disease in Europe. Co-detection of up to four Borreliella genospecies within the same individual has also been retrieved. The detection and co-detection rate of Borreliella in I. ricinus nymphs is high and raises the question of interactions between these bacteria and the communities constituting the internal microbiota.

Morphology and Ecology of Two New Amoebae, Isolated From a Thalassohaline Lake, Dziani Dzaha.

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus.

Vermamoeba vermiformis in hospital network: a benefit for Aeromonas hydrophila.

Aeromonas hydrophila, considered as an emerging pathogen, is increasingly involved in opportunistic human infections. This bacterium, mainly present in aquatic environments, can therefore develop relationships with the free-living amoeba Vermamoeba vermiformis in hospital water networks. We showed in this study that the joint presence of V. vermiformis and A. hydrophila led to an increased bacterial growth in the first 48 h of contact and moreover to the protection of the bacteria in adverse conditions even after 28 days. These results highlight the fact that strategies should be implemented to control the development of FLA in hospital water systems.

Free-living amoebae and squatters in the wild: ecological and molecular features.

Free-living amoebae are protists frequently found in water and soils. They feed on other microorganisms, mainly bacteria, and digest them through phagocytosis. It is accepted that these amoebae play an important role in the microbial ecology of these environments. There is a renewed interest for the free-living amoebae since the discovery of pathogenic bacteria that can resist phagocytosis and of giant viruses, underlying that amoebae might play a role in the evolution of other microorganisms, including several human pathogens. Recent advances, using molecular methods, allow to bring together new information about free-living amoebae. This review aims to provide a comprehensive overview of the newly gathered insights into (1) the free-living amoeba diversity, assessed with molecular tools, (2) the gene functions described to decipher the biology of the amoebae and (3) their interactions with other microorganisms in the environment.

Targeted Metagenomics of Microbial Diversity in Free-Living Amoebae and Water Samples.

The presence of Legionella spp. in natural and man-made water systems is a great public health concern and heavily depends on the presence of free-living amoebae. Taking advantage of the development and affordability of next-generation sequencing technology, we present here a method to characterize the whole bacterial community directly from water samples, as well as from isolated free-living amoebae.

Microbiome of drinking water: A full-scale spatio-temporal study to monitor water quality in the Paris distribution system.

The microbiological water quality of drinking water distribution system (DWDS) is of primary importance for human health. High-throughput sequencing has gained more and more attention in the last decade to describe this microbial diversity in water networks. However, there are few studies describing this approach on large drinking water distribution systems and for extended periods of time. To fill this gap and observe the potential subtle variation in microbiota of a water network through time and space, we aimed to apply high-throughput sequencing of the 16S rRNA gene approach to characterize bacterial communities of the Paris' DWDS over a one-year period. In this study, the Paris network, composed of four different DWDSs, was sampled at 31 sites, each month for one year. The sampling campaign was one of the largest described so far (n = 368) and the importance of key spatio-temporal and physico-chemical parameters was investigated. Overall, 1321 taxa were identified within the Paris network, although only fifteen of them were found in high relative abundance (>1%) in all samples. Two genera, Phreatobacter and Hyphomicrobium were dominant. The whole bacterial diversity was not significantly affected between the four DWDSs (spatial parameter) and by physico-chemical parameters. However, the bacterial diversity was slightly modified over the one-year period (temporal parameter) as we were able to observe DWDS microbiome perturbations, presumably linked to a preceding flood event. Comparison of high-throughput sequencing of the 16S rRNA gene amplicons vs. cultivation-based techniques showed that only 1.8% of bacterial diversity was recovered through cultivation. High throughput sequencing has made it possible to monitor DWDS more accurately than conventional methods by describing the whole diversity and detecting slight fluctuations in bacterial communities. This method would be further used to supervise drinking water networks, to follow any perturbations due to internals events (such as treatments) or external events (such as flooding).

Vermamoeba vermiformis: a Free-Living Amoeba of Interest.

Free-living amoebae are protists that are widely distributed in the environment including water, soil, and air. Although the amoebae of the genus Acanthamoeba are still the most studied, other species, such as Vermamoeba vermiformis (formerly Hartmannella vermiformis), are the subject of increased interest. Found in natural or man-made aquatic environments, V. vermiformis can support the multiplication of other microorganisms and is able to harbor and potentially protect pathogenic bacteria or viruses. This feature is to be noted because of the presence of this thermotolerant amoeba in hospital water networks. As a consequence, this protist could be implicated in health concerns and be indirectly responsible for healthcare-related infections. This review highlights, among others, the consequences of V. vermiformis relationships with other microorganisms and shows that this free-living amoeba species is therefore of interest for public health.

Environmental Mycobacterium avium subsp. paratuberculosis Hosted by Free-Living Amoebae.

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and non-pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis.

Exploiting the Richness of Environmental Waterborne Bacterial Species to Find Natural Legionella pneumophila Competitors.

Legionella pneumophila is one of the most tracked waterborne pathogens and remains an important threat to human health. Despite the use of biocides, L. pneumophila is able to persist in engineered water systems with the help of multispecies biofilms and phagocytic protists. For few years now, high-throughput sequencing methods have enabled a better understanding of microbial communities in freshwater environments. Those unexplored and complex communities compete for nutrients using antagonistic molecules as war weapons. Up to now, few of these molecules were characterized in regards of L. pneumophila sensitivity. In this context, we established, from five freshwater environments, a vast collection of culturable bacteria and investigated their ability to inhibit the growth of L. pneumophila. All bacterial isolates were classified within 4 phyla, namely Proteobacteria (179/273), Bacteroidetes (48/273), Firmicutes (43/273), and Actinobacteria (3/273) according to 16S rRNA coding sequences. Aeromonas, Bacillus, Flavobacterium, and Pseudomonas were the most abundant genera (154/273). Among the 273 isolates, 178 (65.2%) were shown to be active against L. pneumophila including 137 isolates of the four previously cited main genera. Additionally, other less represented genera depicted anti-Legionella activity such as Acinetobacter, Kluyvera, Rahnella, or Sphingobacterium. Furthermore, various inhibition diameters were observed among active isolates, ranging from 0.4 to 9 cm. Such variability suggests the presence of numerous and diverse natural compounds in the microenvironment of L. pneumophila. These molecules include both diffusible secreted compounds and volatile organic compounds, the latter being mainly produced by Pseudomonas strains. Altogether, this work sheds light on unexplored freshwater bacterial communities that could be relevant for the biological control of L. pneumophila in manmade water systems.

Mycobacterium llatzerense, a waterborne Mycobacterium, that resists phagocytosis by Acanthamoeba castellanii.

Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.

Environmental factors shaping cultured free-living amoebae and their associated bacterial community within drinking water network.

Free-living amoebae (FLA) constitute an important part of eukaryotic populations colonising drinking water networks. However, little is known about the factors influencing their ecology in such environments. Because of their status as reservoir of potentially pathogenic bacteria, understanding environmental factors impacting FLA populations and their associated bacterial community is crucial. Through sampling of a large drinking water network, the diversity of cultivable FLA and their bacterial community were investigated by an amplicon sequencing approach, and their correlation with physicochemical parameters was studied. While FLA ubiquitously colonised the water network all year long, significant changes in population composition were observed. These changes were partially explained by several environmental parameters, namely water origin, temperature, pH and chlorine concentration. The characterisation of FLA associated bacterial community reflected a diverse but rather stable consortium composed of nearly 1400 OTUs. The definition of a core community highlighted the predominance of only few genera, majorly dominated by Pseudomonas and Stenotrophomonas. Co-occurrence analysis also showed significant patterns of FLA-bacteria association, and allowed uncovering potentially new FLA - bacteria interactions. From our knowledge, this study is the first that combines a large sampling scheme with high-throughput identification of FLA together with associated bacteria, along with their influencing environmental parameters. Our results demonstrate the importance of physicochemical parameters in the ecology of FLA and their bacterial community in water networks.