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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.

Opportunistic premise plumbing pathogens present in drinking water are linked to a significant number of infections for health compromised patients. However, their monitoring is not required in current water potability standards and they have been poorly studied in a full-scale network. In this study, we quantified, by qPCR, three opportunistic pathogens, Mycobacterium spp., Legionella pneumophila, Pseudomonas aeruginosa throughout the Paris drinking water network over a one-year sampling campaign. While Mycobacteria spp. seemed ubiquitous whatever the distribution system and the time of the year, the occurrence of L. pneumophila and P. aeruginosa showed seasonal variations. Unlike L. pneumophila and P. aeruginosa, the concentration (copies number/L) of Mycobacterium spp. varied between sampling sites. The variation in microbial numbers did not demonstrate any correlations with temperature, pH, chlorine, conductivity, orthophosphate or nitrate levels. In conclusion, Mycobacterium spp. are common inhabitants of the Paris network while L. pneumophila and P. aeruginosa presence fluctuate over space and time. Such qPCR approach would help to better understand the behaviour of opportunistic premise plumbing pathogens.

Acanthamoeba castellanii is an amphizoïc free-living amoeba as it can be found in humans and in the environment. This amoeba represents an important reservoir of pathogenic microorganisms. Persistence of A. castellanii in the environment or in humans is allowed by the ability of the vegetative form to differentiate under cysts when surrounding conditions are unfavorable. In this study, we investigate the role of the ACA1_383450 gene during encystment of A. castellanii. This gene encodes a putative G-protein coupled receptor, which shares homology with human GPR107 and murine GPR108. Expression of the ACA1_383450 gene is transiently repressed at the early phase of encystment and its overexpression affects encystment of A. castellanii. This study reveals a new Acanthamoeba gene which could affect the encystment process.HighlightsThe ACA1_383450 gene encodes for a putative G-protein coupled receptor (GPCR).The ACA1_383450 mRNA levels are down-regulated during the early phase of encystment.Overexpression of the ACA1_383450 gene affects formation of cysts.

International audience; 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.

Multinuclearity is a widespread phenomenon across the living world, yet how it is achieved, and the potential related advantages, are not systematically understood. In this study, we investigate multinuclearity in amoebae. We observe that non-adherent amoebae are giant multinucleate cells compared to adherent ones. The cells solve their multinuclearity by a stretchy cytokinesis process with cytosolic bridge formation when adherence resumes. After initial adhesion to a new substrate, the progeny of the multinucleate cells is more numerous than the sibling cells generated from uninucleate amoebae. Hence, multinucleate amoebae show an advantage for population growth when the number of cells is quantified over time. Multiple nuclei per cell are observed in different amoeba species, and the lack of adhesion induces multinuclearity in diverse protists such as Acanthamoebacastellanii, Vermamoebavermiformis, Naegleriagruberi and Hartmannellarhysodes. In this study, we observe that agitation induces a cytokinesis delay, which promotes multinuclearity. Hence, we propose the hypothesis that multinuclearity represents a physiological adaptation under non-adherent conditions that can lead to biologically relevant advantages.

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment.

International audience; The present study provides an insight on physiological parameters and root-associated microbiome of two complementary Arabidopsis thaliana culture systems suitable for physiological studies. Methods: A. thaliana plants were grown in rhizobox and in classic pot culture. An analysis of plant growth parameters along with the characterization of the bacterial communities of the compost and the rhizosphere, rhizoplane and endosphere compartments were performed. Results: A. thaliana plants grown in rhizobox exhibited a plant habitus similar to those grown in pots during the first month, but with a delayed leaf initiation and slower rate of growth over the experiment. No nutrient deficiency symptoms were observed in these plants. The rhizobox design permits the migration of bacteria from compost to plant roots and allowed root-bacteria interactions to be established as in traditional pot culture. Some differences into the composition of the rhizobiome were highlighted between the two culture systems. Nevertheless, key bacterial taxa that help to ensure plant growth and health colonized the rhizo- and endosphere compartments in both systems. Conclusions: This study gives some clues for future research programs and selection of efficient root systems with the use of such specific culture devices.

International audience; Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii.

Les amibes libres sont des micro-organismes eucaryotes omnipresents dans les reseaux d’eau naturels et anthropiques. Bien que les amibes libres soient rarement decrites comme directement pathogenes pour l’homme, elles sont considerees comme un reservoir permettant la persistance et la propagation de bacteries pathogenes et indesirables dans les reseaux d’eau. Ce constat justifie ainsi la necessite de mieux comprendre la dynamique des populations amibiennes dans le contexte de l’approvisionnement en eau potable. Alors que leur presence dans les reseaux d’eau potable est largement documentee, l’ecologie de ces micro-organismes dans ce type d’environnement reste a ce jour tres peu decrite. Sur la base d’une campagne de prelevement d’eau couvrant le reseau d’eau potable de la ville de Paris, sur une periode d’un an, la presence et la composition des communautes amibiennes, ainsi que l’ensemble des bacteries en association avec ces dernieres (designees comme leur microbiome) ont ete caracterises par pyrosequencage. La collection de divers parametres physico-chimiques tels que l’origine de l’eau, la temperature, le pH et la concentration en chlore, a permis de mettre en evidence le fort impact de ces facteurs abiotiques dans la composition et la dynamique des communautes amibiennes omnipresentes au sein du reseau d’eau potable etudie.

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.

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.

International audience; Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events and behave as facultative intracellular pathogen of oyster haemocytes. Herein, we wondered whether V. tasmaniensis LGP32 resistance to phagocytosis is specific to oyster immune cells or contributes to resistance to other phagocytes, like marine amoebae. To address this question, we developed an integrative study, from the first description of amoeba diversity in oyster farms to the characterization of LGP32 interactions with amoebae. An isolate of the Vannella genus, Vannella sp. AP1411, which was collected from oyster farms, is ubiquitous, and belongs to one clade of Vannella that could be found associated with Vibrionaceae. LGP32 was shown to be resistant to grazing by Vannella sp. AP1411 and this phenotype depends on some previously identified virulence factors: secreted metalloprotease Vsm and copper efflux p-ATPase CopA, which act at different steps during amoeba-vibrio interactions, whereas some other virulence factors were not involved. Altogether, our work indicates that some virulence factors can be involved in multi-host interactions of V. tasmaniensis ranging from protozoans to metazoans, potentially favouring their opportunistic behaviour.

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).

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Vibrios are ubiquitous in marine environments and opportunistically colonize a broad range of hosts. Strains of Vibrio tasmaniensis present in oyster farms can thrive in oysters during juvenile mortality events. Among them, V. tasmaniensis LGP32 behaves as a facultative intracellular pathogen of oyster hemocytes, a property rather unusual in vibrios. Herein, we asked whether LGP32 resistance to phagocytosis could result from coincidental selection of virulence factors during interactions with heterotrophic protists, such as amoeba, in the environment. To answer that question, we developed an integrative study, from the first description of amoeba diversity in oyster-farming areas to the characterization of LGP32 interactions with amoebae of the Vannella genus that were found abundant in the oyster environment. LGP32 was shown to be resistant to grazing by amoebae and this phenotype was dependent on previously identified virulence factors: the secreted metalloprotease Vsm and the copper efflux p-ATPase CopA. Using dedicated in vitro assays, our results showed that these virulence factors act at different steps during amoeba-vibrio interactions than they do in oysters-vibrio interactions. Hence, the virulence factors of LGP32 are key determinants of biotic interactions with multiple hosts ranging from protozoans to metazoans, suggesting that the selective pressure exerted by amoebae in marine coastal environments favor coincidental selection of virulence factors.

The TM6 phylum belongs to the so-called microbial dark matter that gathers uncultivated bacteria detected only via DNA sequencing. Recently, the genome sequence of a TM6 bacterium (TM6SC1) has led to suggest that this bacterium would adopt an endosymbiotic life. In the present paper, free-living amoebae bearing a TM6 strain were isolated from a water network. The amoebae were identified as Vermamoeba vermiformis and the presence of a TM6 strain was detected by polymerase chain reaction and microscopy. The partial sequence of its 16S rRNA gene showed this strain to be closely related to the sequenced TM6SC1 strain. These bacteria displayed a pyriform shape and were found within V. vermiformis. Therefore, these bacteria were named Vermiphilus pyriformis. Interactions studies showed that V. pyriformis was highly infectious and that its relation with V. vermiformis was specific and highly stable. Finally, it was found that V. pyriformis inhibited the encystment of V. vermiformis. Overall, this study describes for the first time an endosymbiotic relationship between a TM6 bacterium and a free-living amoeba in the environment. It suggests that other bacteria of the TM6 phylum might also be endosymbiotic bacteria and may be found in other free-living amoebae or other organisms.

International audience; Autophagy is a eukaryotic process responsible for the degradation of intracellular content such as damaged organelles. Several putative autophagy-related genes have been identified within the annotated genome of the free-living amoeba Acanthamoeba castellanii. However, the involvement of the corresponding proteins in the autophagy pathway had not been formerly established. Here, we report that AcAtg8 cDNA can complement ATG8-deficient Saccharomyces cerevisiae.

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.

Free-living amoebae of the genus Acanthamoeba are the causal agents of a sight-threatening ulceration of the cornea called Acanthamoeba keratitis, as well as the rare but usually fatal disease granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba infections, they are generally lengthy and/or have limited efficacy. For the best clinical outcome, treatments should target both the trophozoite and the cyst stages, as cysts are known to confer resistance to treatment. In this study, we document the activities of caffeine and maslinic acid against both the trophozoite and the cyst stages of three clinical strains of Acanthamoeba . These drugs were chosen because they are reported to inhibit glycogen phosphorylase, which is required for encystation. Maslinic acid is also reported to be an inhibitor of extracellular proteases, which may be relevant since the protease activities of Acanthamoeba species are correlated with their pathogenicity. We also provide evidence for the first time that both drugs exert their anti-amoebal effects through programmed cell death.

Legionella pneumophila is a pathogenic bacterium commonly found in water. Eventually, it could be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts. Many studies were performed to understand iron utilization by L. pneumophila but no global approaches were conducted. In this study, transcriptomic analyses were performed, comparing gene expression in L. pneumophila in standard vs. iron restricted conditions. Among the regulated genes, a newly described one, lpp_2867, was highly induced in iron restricted conditions. Mutants lacking this gene in L. pneumophila were not affected in siderophore synthesis or utilization. On the contrary, they were defective for growth on iron depleted solid media and for ferrous iron uptake. A sequence analysis predicts that Lpp_2867 is a membrane protein, suggesting that it is involved in ferrous iron transport. We thus named it IroT, for iron transporter. Infection assays showed that the mutants are highly impaired in intracellular growth within their environmental host Acanthamoeba castellanii and human macrophages. Taken together, our results show that IroT is involved, directly or indirectly, in ferrous iron transport and is a key virulence factor for L. pneumophila. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

International audience; : Free-living amoebae are naturally present in water. These protozoa could be pathogenic and could also shelter pathogenic bacteria. Thus, they are described as a potential hazard for health. Also, free-living amoebae have been described to be resistant to biocides, especially under their cyst resistant form. There are several studies on amoeba treatments but none of them compare sensitivity of trophozoites and cysts from different genus to various water disinfectants. In our study, we tested chlorine, monochloramine and chlorine dioxide on both cysts and trophozoites from three strains, belonging to the three main genera of free-living amoebae. The results show that, comparing cysts to trophozoites inactivation, only the Acanthamoeba cysts were highly more resistant to treatment than trophozoites. Comparison of the disinfectant efficiency led to conclude that chlorine dioxide was the most efficient treatment in our conditions and was particularly efficient against cysts. In conclusion, our results would help to adapt water treatments in order to target free-living amoebae in water networks.

Resume Les amibes libres sont des protozoaires cosmopolites particulierement retrouves dans les environnements hydriques. Elles peuvent etre responsables de pathologies graves mais exceptionnelles, comme la meningoencephalite amibienne primitive due a Naegleria fowleri ou l’encephalite granulomateuse amibienne due a Acanthamoeba sp. ou a Balamuthia mandrillaris. En revanche, les keratites causees par les amibes du genre Acanthamoeba sont plus frequemment rapportees, le plus souvent liees a un defaut d’entretien de lentilles de contact. Les amibes libres ont de plus la particularite de pouvoir heberger d’autres micro-organismes (bacteries, champignons, virus…), leur accordant ainsi une certaine forme de protection et permettant un entrainement a leur survie ulterieure dans les macrophages. Les amibes retrouvees dans les reseaux d’eau peuvent ainsi endosser le role de reservoir de pathogenes potentiels et etre indirectement en lien avec differentes infections.

International audience; Free-living amoebae (FLA) are protozoa that can be found in water networks where they prey on bacteria within biofilms. Most bacteria are digested rapidly by phagocytosis, however some are able to survive within amoebae and some are even able to multiply, as it is the case for Legionella pneumophila. These resisting bacteria are a potential health problem as they could also resist to macrophage phagocytosis. Several publications already reported intra-amoebal bacteria but the methods of identification did not allow metagenomic analysis and are partly based on co-culture with one selected amoebal strain. The aim of our study was to conduct a rRNA-targeted metagenomic analysis on amoebae and intra-amoebal bacteria found in drinking water network, to provide the first FLA microbiome in environmental strains. Three sites of a water network were sampled during four months. Culturable FLA were isolated and total DNA was prepared, allowing purification of both amoebal and bacterial DNA. Metagenomic studies were then conducted through 18S or 16S amplicons sequencing. Hartmannella was by far the most represented genus of FLA. Regarding intra-amoebal bacteria, 54 genera were identified, among which 21 were newly described intra-amoebal bacteria, underlying the power of our approach. There were high differences in bacterial diversity between the three sites. Several genera were highly represented and/or found at least in two sites, underlying that these bacteria could be able to multiply within FLA. Our method is therefore useful to identify FLA microbiome and could be applied to other networks to have a more comprehensive view of intra-amoebal diversity.

Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.

International audience; Free-living amoebae (FLA) constitute an important part of eukaryotic populations colonising drinkingwater 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 environmentalfactors impacting FLA populations and their associated bacterial community is crucial. Throughsampling of a large drinking water network, the diversity of cultivable FLA and their bacterial communitywere investigated by an amplicon sequencing approach, and their correlation with physicochemicalparameters was studied. While FLA ubiquitously colonised the water network all year long, significantchanges in population composition were observed. These changes were partially explained by severalenvironmental parameters, namely water origin, temperature, pH and chlorine concentration. Thecharacterisation of FLA associated bacterial community reflected a diverse but rather stable consortiumcomposed of nearly 1400 OTUs. The definition of a core community highlighted the predominance ofonly few genera, majorly dominated by Pseudomonas and Stenotrophomonas. Co-occurrence analysis alsoshowed 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 schemewith high-throughput identification of FLA together with associated bacteria, along with their influencingenvironmental parameters. Our results demonstrate the importance of physicochemical parametersin the ecology of FLA and their bacterial community in water networks.

International audience; Electromagnetic field (EMF) treatment has proven to be effective against mineral scaling in water systems. Therefore, it should be assessed for the treatment of other deposits such as biofilms. In this study, a commercial device producing low-frequency EMF (1-10 kHz) was applied to a reactor fed with natural water for 45 days. The treatment promoted the concentration of microorganisms in suspension and limited the amount of sessile microorganisms in the biofilm, as determined by the measurement of total DNA, qPCR and microscopy. The structure of the bacterial community was assessed by t-RFLP and pyrosequencing analysis. The results showed that EMF treatment affected both planktonic and sessile community composition. EMFs were responsible for a shift in classes of Proteobacteria during development of the biofilm. It may be speculated that the EMF treatment affected particle solubility and/or microorganism hydration. This study indicated that EMFs modulated biofilm formation in natural water.

International audience; Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera.

Acinetobacter baumanii, which may be found in water, is an important emerging hospital-acquired pathogen. Free-living amoebae can be recovered from the same water networks, and it has been shown that these protozoa may support the growth of other bacteria. In this paper, we have studied potential relationships between A. baumanii and Acanthamoeba species. Two strains of A. baumanii isolated from hospital water were co-cultivated with the trophozoites or supernatants of two free-living amoebae strains: Acanthamoeba castellanii or Acanthamoeba culbertsoni. Firstly, the presence of the amoebae or their supernatants induced a major increase in A. baumanii growth, compared with controls. Secondly, A. baumanii affected only the viability of A. culbertsonii, with no effect on A. castellanii. Electron microscopy observations of the cultures investigating the bacterial location in the protozoa showed persistence of the bacteria within cyst wall even after 60 days of incubation. In our study, the survival and growth of A. baumanii could be favored by Acanthamoeba strains. Special attention should consequently be paid to the presence of free-living amoebae in hospital water systems, which can promote A. baumanii persistence.

Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chlorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.

Chlorination is a well-known disinfection method, used in water treatment to inactivate various microorganisms, it induces numerous cellular changes. Even though Acanthamoebae are frequently found in water, the cellular changes induced in Acanthamoebae have not been described in the literature. Acanthamoebae are pathogenic amoebae and may provide a reservoir for pathogenic bacteria such as Legionella pneumophila; it is consequently important to understand the response of this amoeba to chlorination, and our study was indeed aimed at examining cellular changes in Acanthamoebae following chlorination. Acanthamoeba trophozoites were treated at various chlorine concentrations (1–5 mg/L). A 3-log reduction in Acanthamoebae population was achieved with 5 mg/L of free chlorine. Confocal microscopy and flow cytometry experiments indicated that chlorination induced cell permeabilization, size reduction and likely intracellular thiol concentration. Our data show that among the non-cultivable cells some remained impermeabilized (negative staining with propidium iodide), thereby suggesting that these cells might remained viable. A similar state is described in other microorganisms as a VBNC (viable but not cultivable) state. Electron microscopy observations illustrate drastic morphological changes: the pseudopods disappeared and subcellular components, such as mitochondrion, were pronouncedly affected. In conclusion, depending on the concentration used, chlorination leads to many cellular effects on Acanthamoeba that could well arise in cell inactivation.

Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis.

delta-hemolysin is a hemolytic peptide produced by Staphylococcus, and it has been studied for nearly 50 years. Therefore, it has become a model in the study of peptides interacting with membranes. In this review, we report some recent findings and compare them with previous works. delta-hemolysin is a 26 amino acid peptide, somewhat hydrophobic and presenting a zero net charge. Study of its structure has shown that delta-hemolysin is alpha-helical and amphipathic, such as many antimicrobial peptides (e.g. magainin and melittin). However, delta-hemolysin had not displayed any reported antimicrobial activity until a recent publication showed its high potency against Legionella. Its mode of action is based on direct interaction with target membranes. In accordance with its concentration, delta-hemolysin may slightly perturb a membrane or lead to cell lysis. Peptide charge plays an important role in its interaction with membranes, as is shown in the study of peptide variants. Some positively charged variants become highly hemolytic and even active against Escherichia coli and Staphylococcus aureus. Finally, it has recently been demonstrated that peptide preferentially binds to lipid-disordered domains. It has been postulated that as a result, enrichment in lipid-ordered domains might increase peptide concentration in lipid-disordered domains and thereby improve its activity.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a waterborne bacteria. It can multiply in man-made water systems and infect people who inhale contaminated droplets. We have previously reported a Staphylococcus warneri strain that display an anti-Legionella activity. In this work, we characterized three anti-Legionella peptides that are produced by S. warneri. One peptide, warnericin RK, is original, while the two others are delta-lysin I and delta-lysin II, whose genes were previously described. Due to high sequence similarity of the two delta-lysins, further characterization was performed only on delta-lysin I. Warnericin RK and delta-lysin I displayed the same antibacterial spectrum, which is almost restricted to the Legionella genus. Also, both peptides have a hemolytic activity. These results led to the hypothesis that warnericin RK and delta-lysin I share a similar mode of action, and that Legionella should have a specific feature that may explain the high specificity of these antibacterial peptides.

Les amibes libres sont des protozoaires (unicellulaires) tres presents dans l’environnement et notamment dans les milieux hydriques et ont la capacite a s’y multiplier sans l’aide d’un hote. Certaines especes sont directement pathogenes pour l’homme mais d’autres peuvent egalement etre responsables, de facon indirecte, d’infections en jouant le role de reservoir de bacteries pathogenes comme Legionella pneumophila. Afin d’evaluer l’occurrence des amibes libres en eau chaude sanitaire, une campagne d’analyse a ete realisee sur 36 reseaux interieurs reels (eau froide et eau chaude). La campagne d’analyse sur sites a montre que les amibes libres thermophiles sont retrouvees dans 19 % des prelevements effectues et les amibes mesophiles dans 46 %. Le genre amibien le plus retrouve est Hartmanella. A priori, ce genre d’amibe n’est pas intrinsequement pathogene pour l’homme. Sur quelques echantillons, des amibes du genre Acanthamoeba ont ete retrouvees. Les points les plus frequemment contamines par les amibes libres sont l’eau froide et les points d’usage. Les caracteristiques des sites ont ete mis au regard de la contamination par les amibes totales. Aucune correlation n’a pu etre trouvee entre la presence d’amibes et le type d’etablissement, le type de production d’eau chaude, la qualite du bouclage, le type de materiaux. Tres peu d’amibes sont retrouvees lorsque la temperature de l’eau est superieure a 60 °C.

Summary Legionella pneumophila, the causative agent of Legionnaires' disease, is ubiquitously found in aquatic environments, associated with free living amoebae. Trophozoite forms of the genus Acanthamoeba have been shown to support the intracellular growth of Legionella while it has been proposed that cyst forms are related to survival in harsh environments. This underlines that amoebae are of primary importance in Legionella spreading. In this study, we followed the survival of L. pneumophila Lens over 6 months in a poor medium, with or without Acanthamoeba castellanii. The results demonstrated that L. pneumophila Lens could survive for at least 6 months in association with A. castellanii and that cultivable bacteria are to be found within expelled vesicles rather than within cysts. Our findings suggest that vesicles might be further studied in order to elucidate their production and their role in the environmental spreading of Legionella.

Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts.

International audience; Free-living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.

Strains of the food-borne pathogenListeria monocytogenes, showing either intermediate or high-level resistance to class IIa bacteriocins, were investigated to determine characteristics that correlated with their sensitivity levels. Two intermediate and one highly resistant spontaneous mutant ofL. monocytogenesB73, a highly resistant mutant ofL. monocytogenes412, and a highly resistant, defined (mptA) mutant ofL. monocytogenesEGDe were compared with their respective wild-type strains in order to investigate the contribution of different factors to resistance. Decreased mannose-specific phosphotransferase system gene expression (mptA, EIIABMancomponent) was implicated in all levels of resistance, confirming previous studies by the authors' group. However, a clear correlation betweend-alanine content in teichoic acid (TA), in particular the alanine : phosphorus ratio, and a more positive cell surface, as determined by cytochromecbinding, were found for the highly resistant strains. Furthermore, two of the three highly resistant strains showed a significant increase in sensitivity towardsd-cycloserine (DCS). However, real-time PCR of thedltA(d-alanine esterification), anddalandddlAgenes (peptidoglycan biosynthesis) showed no change in transcriptional levels. The link between DCS sensitivity and increasedd-alanine esterification of TA may be that DCS competes with alanine for transport via the alanine transporter. A possible tendency towards increased lysinylation of membrane phospholipid in the highly resistant strains was also found. A previous study reported that cell membranes of all the resistant strains, including the intermediate resistant strains, contained more unsaturated phosphatidylglycerol, which is an indication of a more fluid cell membrane. The results of that study correlate with the possible lysinylation, decreasedmptAexpression,d-alanine esterification of TA and more positive cell surface charge found in this study for resistant strains. The authors' findings strongly indicate that all these factors could contribute to class IIa bacteriocin resistance and that the combination and contribution of each of these factors determine the level of bacteriocin resistance.

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EIItMan mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on σ54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.

The role of the alternativeσ54factor, encoded by therpoNgene, was investigated inListeria monocytogenesby comparing the global gene expression of the wild-type EGDe strain and anrpoNmutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in therpoNmutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only themptACDoperon was shown to be directly controlled byσ54. Therefore, the remaining modifications seem to be due to indirect effects. In parallel, anin silicoanalysis suggests thatσ54may directly control the expression of four different phosphotransferase system (PTS) operons, includingmptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest thatσ54is mainly involved in the control of carbohydrate metabolism inL. monocytogenesvia direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.

The σ 54 subunit of the RNA polymerase directs the expression of specific operons in association with cognate activators. Three different activators have been detected in the Listeria monocytogenes genome on the basis of the high conservation of a specific domain. Among them, the LacR activator, of the LevR family, was found just upstream from a newly described σ 54 –dependent operon, lpo , which presents a classical –24/–12 consensus promoter. The lpo operon encodes proteins similar to subunits of a PTS permease (EII) of the lactose family, namely LpoA (IIA) and LpoB (IIB). It also encodes a third putative protein, LpoO, with an unknown function but sharing high similarity with proteins also encoded within PTS operons from other bacteria and bearing a RGD motif. The expression of lpo was clearly dependent on LacR and σ 54 , and was induced by cellobiose, chitobiose and lactose. It underlies that the lpo operon likely encodes proteins involved in the utilization of these sugars by L. monocytogenes .