Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms.

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae - Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum - and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis. The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

Amoeboid swimming in a compliant channel.

Several prokaryotes and eukaryotic cells swim in the presence of deformable and rigid surfaces that form confinement. The most commonly observed examples from biological systems are motility of leukocytes and pathogens present within the blood suspension through a microvascular network, and locomotion of eukaryotic cells such as immune system cells and cancerous cells through interstices between soft interstitial cells and the extracellular matrix within the interstitial tissue. This motivated us to investigate numerically the flow dynamics of amoeboid swimming in a flexible channel. The effects of wall stiffness and channel confinement on the flow dynamics and swimmer motion are studied. The swimmer motion through the flexible channel is substantially decelerated compared to the rigid channel. The strong confinement in the amply flexible channel imprisons the swimmer by severely restricting its forward motion. The swimmer velocity in a stiff channel displays nonmonotonic variation with the confinement while it shows monotonic reduction in a highly flexible channel. The physical rationale behind such distinct velocity behaviour in flexible and rigid channels is illustrated using an instantaneous flow field and flow history displayed by the swimmer. This behavior follows from a subtle interplay between the shape changes exhibited by the swimmer and the wall compliance. This study may aid in understanding the influence of elasticity of the surrounding environment on cell motility in immunological surveillance and invasiveness of cancer cells.

Prevalence of contamination by intestinal parasites in vegetables (Lactuca sativa L. and Coriandrum sativum L.) sold in markets in Belém, northern Brazil.

BACKGROUND: Previous studies have recorded a high prevalence of intestinal parasites in lettuce (Lactuca sativa L.) and coriander (Coriandrum sativum L.) destined for human consumption. This study determined the prevalence of contamination by intestinal parasites in these two plants sold in two street markets and two supermarkets in the city of Belém, northern Brazil. RESULTS: A total of 200 plant samples were analyzed (100 of each species). The samples were collected randomly between August and October 2018, examined by the spontaneous sedimentation method with two washes, and stored for 24 h. The analysis found that 89% (89/100) of the lettuce samples and 86% (86/100) of the coriander samples were contaminated. Polyparasitism was more frequent in lettuce, but monoparasitism predominated in the coriander. A total of 226 intestinal parasites were found in the lettuce, with a predominance of non-pathogenic parasites in the supermarket samples and more pathogenic parasites in the samples from street markets. In the coriander samples, 172 intestinal parasites were identified, with a predominance of pathogenic parasites in samples from both types of market. In the case of the protozoans, the most prevalent species was Endolimax nana, followed by Blastocystis hominis, in both vegetables. In the helminths, hookworms predominated in the lettuce, and Trichuris trichiura in the coriander. CONCLUSIONS: These results highlight the need for the monitoring of parasite contamination in vegetables destined for human consumption. Public health initiatives should include educational campaigns on the importance of disinfecting vegetables prior to consumption. © 2020 Society of Chemical Industry.

Eukaryotic organisms of continental hydrothermal systems.

Continental hydrothermal systems are a dynamic component of global thermal and geochemical cycles, exerting a pronounced impact on water chemistry and heat storage. As such, these environments are commonly classified by temperature, thermal fluid ionic concentration, and pH. Terrestrial hydrothermal systems are a refuge for extremophilic organisms, as extremes in temperature, metal concentration, and pH profoundly impact microorganism assemblage composition. While numerous studies focus on Bacteria and Archaea in these environments, few focus on Eukarya-likely due to lower temperature tolerances and because they are not model organisms for understanding the evolution of early life. However, where present, eukaryotic organisms are significant members of continental hydrothermal microorganism communities. Thus, this manuscript focuses on the eukaryotic occupants of terrestrial hydrothermal systems and provides a review of the current status of research, including microbe-eukaryote interactions and suggestions for future directions.

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella.

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.

Unraveling past impacts of climate change and land management on historic peatland development using proxy-based reconstruction, monitoring data and process modeling.

Peatlands represent globally significant soil carbon stores that have been accumulating for millennia under water-logged conditions. However, deepening water-table depths (WTD) from climate change or human-induced drainage could stimulate decomposition resulting in peatlands turning from carbon sinks to carbon sources. Contemporary WTD ranges of testate amoebae (TA) are commonly used to predict past WTD in peatlands using quantitative transfer function models. Here we present, for the first time, a study comparing TA-based WTD reconstructions to instrumentally monitored WTD and hydrological model predictions using the MILLENNIA peatland model to examine past peatland responses to climate change and land management. Although there was very good agreement between monitored and modeled WTD, TA-reconstructed water table was consistently deeper. Predictions from a larger European TA transfer function data set were wetter, but the overall directional fit to observed WTD was better for a TA transfer function based on data from northern England. We applied a regression-based offset correction to the reconstructed WTD for the validation period (1931-2010). We then predicted WTD using available climate records as MILLENNIA model input and compared the offset-corrected TA reconstruction to MILLENNIA WTD predictions over an extended period (1750-1931) with available climate reconstructions. Although the comparison revealed striking similarities in predicted overall WTD patterns, particularly for a recent drier period (1965-1995), there were clear periods when TA-based WTD predictions underestimated (i.e. drier during 1830-1930) and overestimated (i.e. wetter during 1760-1830) past WTD compared to MILLENNIA model predictions. Importantly, simulated grouse moor management scenarios may explain the drier TA WTD predictions, resulting in considerable model predicted carbon losses and reduced methane emissions, mainly due to drainage. This study demonstrates the value of a site-specific and combined data-model validation step toward using TA-derived moisture conditions to understand past climate-driven peatland development and carbon budgets alongside modeling likely management impacts.

Gastrointestinal protists and helminths of habituated agile mangabeys (Cercocebus agilis) at Bai Hokou, Central African Republic.

Infectious diseases including those caused by parasites can be a major threat to the conservation of endangered species. There is thus a great need for studies describing parasite infections of these species in the wild. Here we present data on parasite diversity in an agile mangabey (Cercocebus agilis) group in Bai Hokou, Dzanga-Sangha Protected Areas (DSPA), Central African Republic. We coproscopically analyzed 140 mangabey fecal samples by concentration techniques (flotation and sedimentation). Agile mangabeys hosted a broad diversity of protistan parasites/commensals, namely amoebas (Entamoeba spp., Iodamoeba buetschlli), a Buxtonella-like ciliate and several parasitic helminths: strongylid and spirurid nematodes, Primasubulura sp., Enterobius sp., and Trichuris sp. Importantly, some of the detected parasite taxa might be of potential zoonotic importance, such as Entamoeba spp. and the helminths Enterobius sp., Trichuris sp., and strongylid nematodes. Detailed morphological examination of ciliate cysts found in mangabeys and comparison with cysts of Balantioides coli from domestic pigs showed no distinguishing structures, although significant differences in cyst size were recorded. Scanning or transmission electron microscopy combined with molecular taxonomy methods are needed to properly identify these ciliates. Further studies using molecular epidemiology are warranted to better understand cross-species transmission and the zoonotic potential of parasites in sympatric non-human primates and humans cohabiting DSPA.

Repertoire of free-living protozoa in contact lens solutions.

BACKGROUND: The repertoire of free-living protozoa in contact lens solutions is poorly known despite the fact that such protozoa may act as direct pathogens and may harbor intra-cellular pathogens. METHODS: Between 2009 and 2014, the contact lens solutions collected from patients presenting at our Ophthalmology Department for clinically suspected keratitis, were cultured on non-nutrient agar examined by microscope for the presence of free-living protozoa. All protozoa were identified by 18S rRNA gene sequencing. RESULTS: A total of 20 of 233 (8.6 %) contact lens solution specimens collected from 16 patients were cultured. Acanthamoeba amoeba in 16 solutions (80 %) collected from 12 patients and Colpoda steini, Cercozoa sp., Protostelium sp. and a eukaryotic more closely related to Vermamoeba sp., were each isolated in one solution. Cercozoa sp., Colpoda sp., Protostelium sp. and Vermamoeba sp. are reported for the first time as contaminating contact lens solutions. CONCLUSION: The repertoire of protozoa in contact lens solutions is larger than previously known.

Diversity and distribution of freshwater testate amoebae (protozoa) along latitudinal and trophic gradients in China.

Freshwater microbial diversity is subject to multiple stressors in the Anthropocene epoch. However, the effects of climate changes and human activities on freshwater protozoa remain poorly understood. In this study, the diversity and distribution of testate amoebae from the surface sediments were investigated in 51 Chinese lakes and reservoirs along two gradients, latitude and trophic status. A total of 169 taxa belonging to 24 genera were identified, and the most diverse and dominant genera were Difflugia (78 taxa), Centropyxis (26 taxa) and Arcella (12 taxa). Our analysis revealed that biomass of testate amoebae decreased significantly along the latitudinal gradient, while Shannon-Wiener indices and species richness presented an opposite trend (P < 0.05). The relationship of diversity and latitude is, we suspect, an artifact of the altitudinal distribution of our sites. Furthermore, biomass-based Shannon-Wiener index and species richness of testate amoebae were significantly unimodally related to trophic status (P < 0.05). This is the first large-scale study showing the effects of latitude and trophic status on diversity and distribution of testate amoebae in China. Therefore, our results provide valuable baseline data on testate amoebae and contribute to lake management and our understanding of the large-scale global patterns in microorganism diversity.

Apparent mortality as a result of an elevated parasite infection in Presbytis rubicunda.

We provide the first parasite assessment of wild red langurs (Presbytis rubicunda). Seven helminth taxa and 4 protozoa were recovered from faecal samples. We report the details of the death of an adult female red langur with potential association of an elevated parasite infection. There is a paucity of published literature on red langurs and nothing is known of the factors affecting interspecific parasite transmission and disease spread between other primate species. Parasites may be substantial determinants of host health and may present a significant influence on the survival and reproduction of individuals and the subsequent fecundity of populations. Further research into the connection of parasite burden with individual deaths and potential population declines is recommended for monitoring population health.

[Spatial distribution of intestinal parasites in the City of La Plata, Argentina]. / Distribución espacial de las parasitosis intestinales en la Ciudad de La Plata, Argentina.

Parasitosis analysis at regional levels is simplified by the use of the Geographic Information System (GIS), which enables the identification of areas with different degrees of vulnerability. We analyzed the spatial distribution of intestinal parasites in La Plata district and their relationship with socio-environmental conditions in order to identify areas with different degrees of epidemiological risk. An epidemiological survey was completed; stool samples were collected and analyzed by Ritchie's method. Levels of precariousness and vulnerability (Iv rate) were calculated and compared to the parasitological results. Parasitological and environmental analysis were carried out on a total of 653 individuals, of whom 585 (89.6%) were children and 68 (10.4%) adults. The analysis indicated that the most vulnerable (Iv = 3-4) were those located in areas with less access to urban infrastructure services. The Iv was strongly associated with the overall prevalence of intestinal parasitosis and the 4 pathogenic species related to the fecal contamination of the environment: Trichuris trichiura, Ascaris lumbricoides, Hymenolepis nana and Giardia lamblia (p < 0.01). On the other hand, Enterobius vermicularis, a parasite not related to the sanitary condition of its host, was not associated with the Iv. Statistical association between poverty and parasitosis was noted; the greater the Iv, higher the incidence of parasites (p < 0.01). GIS allowed zoning socio-environmental variables in an increasing gradient of unfavorable conditions and their relationship to the presence of pathogenic species. The continuity of these studies in different regions of Argentina contributes to the determination of health risk areas.

Arcellacea (testate lobose amoebae) as pH indicators in a pyrite mine-acidified lake, Northeastern Ontario, Canada.

Arcellacea (testate lobose amoebae) were examined in 24 sediment-water interface samples collected over two late August field seasons in 2010 and 2011, from James and Granite lakes, Temagami Region, Northeastern Ontario. The work was carried out to quantitatively test species-environment relationships in a lake system known to be characterized by a significant pH gradient, partially the result of contamination from the early twentieth century Northland Pyrite Mine Co., located on the shoreline in the southern basin of James Lake. Redundancy analysis confirmed that arcellacean assemblage structure was most strongly controlled by pH, explaining 14.06 % (p < 0.002) of the total variance. Q- and R-mode cluster analysis supported by detrended correspondence analysis yielded two major faunal assemblages. The Oligotrophic Assemblage (1) had a Shannon Diversity Index (SDI) ranging up to 2.45, typical of healthy boreal lakes. This assemblage characterized samples collected from higher pH stations within James and Granite lakes away from the immediate area of the mine site, while the Low pH Assemblage 2010 (2a) and Low pH Assemblage 2011 (2b) groupings were from the very low pH environments of James Lake adjacent to the former mine site. Both low diversity assemblages (SDI ranging from 0.62 to 1.22) were characterized by Arcella vulgaris, a species known to thrive in hostile lacustrine environments. Differing depositional conditions during August 2010, a probable result of different prevailing wind patterns that summer, led to allochthonous specimens of the seasonally planktic Cucurbitella tricuspis dominating the Low pH Assemblage 2010 (2a) fauna.

'Run-and-tumble' or 'look-and-run'? A mechanical model to explore the behavior of a migrating amoeboid cell.

Single cell migration constitutes a fundamental phenomenon involved in many biological events. Amoeboid cells are single cell organisms that migrate in a cyclic manner like worms. In this paper, we propose a 3D finite element model of an amoeboid cell migrating over a 2D surface. In particular, we focus on the mechanical aspect of the problem. The cell is able to generate cyclic active deformations, such as protrusion and contraction, in any direction. The progression of the cell is governed by a tight synchronization between the adhesion forces, which are alternatively applied at the front and at the rear edges of the cell, and the protrusion-contraction phases of the cell body. Finally, two important aspects have been taken into account: (1) the external stimuli in response to which the cell migrates (e.g. need to feed, morphogenetic events, normal or abnormal environment cues), (2) the heterogeneity of the 2D substrate (e.g. obstacles, rugosity, slippy regions) for which two distinct approaches have been evaluated: the 'run-and-tumble' strategy and the 'look-and-run' strategy. Overall, the results show a good agreement with respect to the experimental observations and the data from the literature (e.g. velocity and strains). Therefore, the present model helps, on one hand, to better understand the intimate relationship between the deformation modes of a cell and the adhesion strength that is required by the cell to crawl over a substrate, and, on the other hand, to put in evidence the crucial role played by mechanics during the migration process.

Could the canopy structure of bryophytes serve as an indicator of microbial biodiversity? A test for testate amoebae and microcrustaceans from a subtropical cloud forest in Dominican Republic.

The mechanisms that ultimately regulate the diversity of microbial eukaryotic communities in bryophyte ecosystems remain a contentious topic in microbial ecology. Although there is robust consensus that abiotic factors, such as water chemistry of the bryophyte and pH, explain a significant proportion of protist and microcrustacean diversity, there is no systematic assessment of the role of bryophyte habitat complexity on such prominent microbial groups. Water-holding capacity is correlated with bryophyte morphology and canopy structure. Similarly, canopy structure explains biodiversity dynamics of the macrobiota suggesting that canopy structure may also be a potential parameter for understanding microbial diversity. Canopy roughness of the dominant bryophyte species within the Bahoruco Cloud Forest, Cachote, Dominican Republic, concomitant with their associated diversity of testate amoebae and microcrustaceans was estimated to determine whether canopy structure could be added to the list of factors explaining microbial biodiversity in bryophytes. We hypothesized that smooth (with high moisture content) canopies will have higher species richness, density, and biomass of testate amoebae and higher richness and density of microcrustaceans than rough (desiccation-prone) canopies. For testate amoebae, we found 83 morphospecies with relative low abundances. Species richness and density differed among bryophytes with different bryophyte canopy structures and based on non-metric multidimensional scaling, canopy roughness explained 25% of the variation in species composition although not as predicted. Acroporium pungens (low roughness, LR) had the lowest species richness (2 ± 0.61 SD per gram dry weight bryophyte), and density (2.1 ± 0.61 SD individual per gram of dry weight bryophyte); whereas Thuidium urceolatum (high roughness) had the highest richness (24 ± 10.82 SD) and density (94 ± 64.30 SD). The fact that the bryophyte with the highest roughness had the highest levels of diversity for testate amoebae suggests that moisture levels at the level of the bryophyte canopy may not represent a biodiversity driver in a cloud forest with high relative humidity; however, high roughness could generate a dynamic and fluctuating moisture environment with concomitant alternating microbial communities. A total of 26 microcrustacean morphospecies were found across 11 bryophytes; however, no bryophyte canopy effect was detected on their richness and density. Microcrustacean mean density was low ranging from less than one individual per 50 cm2 of bryophyte in Leucobryum (LR) to a maximum of 6 ± 3.37 SD individuals/50 cm2 in Monoclea (LR). This lack of pattern suggests that possible explanatory variables may be related to larger scale processes than those examined in this study.

Effect of a temperature gradient on Sphagnum fallax and its associated living microbial communities: a study under controlled conditions.

Microbial communities living in Sphagnum are known to constitute early indicators of ecosystem disturbances, but little is known about their response (including their trophic relationships) to climate change. A microcosm experiment was designed to test the effects of a temperature gradient (15, 20, and 25°C) on microbial communities including different trophic groups (primary producers, decomposers, and unicellular predators) in Sphagnum segments (0-3 cm and 3-6 cm of the capitulum). Relationships between microbial communities and abiotic factors (pH, conductivity, temperature, and polyphenols) were also studied. The density and the biomass of testate amoebae in Sphagnum upper segments increased and their community structure changed in heated treatments. The biomass of testate amoebae was linked to the biomass of bacteria and to the total biomass of other groups added and, thus, suggests that indirect effects on the food web structure occurred. Redundancy analysis revealed that microbial assemblages differed strongly in Sphagnum upper segments along a temperature gradient in relation to abiotic factors. The sensitivity of these assemblages made them interesting indicators of climate change. Phenolic compounds represented an important explicative factor in microbial assemblages and outlined the potential direct and (or) indirect effects of phenolics on microbial communities.

Amoebae and Legionella pneumophila in saline environments.

Amoeboid protists that harbor bacterial pathogens are of significant interest as potential reservoirs of disease-causing organisms in the environment, but little is known about them in marine and other saline environments. We enriched amoeba cultures from sediments from four sites in the New England estuarine system of Mt. Hope Bay, Massachusetts and from sediments from six sites in the Great Salt Lake, Utah. Cultures of amoebae were enriched using both minimal- and non-nutrient agar plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested polymerase chain reactions (PCR) and primers specific for the genus. Positive samples were then screened with nested amplification using primers specific for the macrophage infectivity potentiator surface protein (mip) gene from L. pneumophila. Forty-eight percent (185 out of 388) of isolated amoeba cultures were positive for the presence of Legionella species. Legionella pneumophila was detected by PCR in 4% of the amoeba cultures (17 out of 388), and most of these amoebae were growing on marine media. Our results show that amoebae capable of growing in saline environments may harbor not only a diverse collection of Legionella species, but also species potentially pathogenic to humans.

Novel Endosymbionts in Rhizarian Amoebae Imply Universal Infection of Unrelated Free-Living Amoebae by Legionellales.

Legionellales-infected water is a frequent cause of local outbreaks of Legionnaires' disease and Pontiac fever. Decontaminations are difficult because Legionellales reproduce in eukaryotic microorganisms (protists). Most often, Legionellales have been isolated from amoebae; however, the culture-based sampling methods are taxonomically biased. Sequencing studies show that amoebae in the cercozoan class Thecofilosea are dominant in soils and wastewater treatment plants, prompting us to screen their capability to serve as potential hosts of endosymbiotic bacteria. Environmental isolates of Thecofilosea contained a surprising richness of endosymbiotic Legionellales, including Legionella. Considering the widespread dispersal of Legionellales in apparently unrelated amoeboid protist taxa, it appears that the morphotype and not the evolutionary origin of amoebae determines their suitability as hosts for Legionellales. We further provide a protocol for gnotobiotic cultivation of Legionellales and their respective hosts, facilitating future genomic and transcriptomic research of host-symbiont relationships.

The morphogenesis of different giant viruses as additional evidence for a common origin of Nucleocytoviricota.

Large and giant DNA viruses are a monophyletic group constituting the recently established phylum Nucleocytoviricota. The virus particle morphogenesis of these viruses exhibit striking similarities. Viral factories are established in the host cells where new virions are assembled by recruiting host membranes, forming an inner lipid layer. An outer protein layer starts as a lamellar structure, commonly referred to as viral crescents, coded by the major capsid protein gene. Also, these viruses have a conserved ATPase-coding gene related to genome encapsidation. Similar properties are described for tectiviruses, putative small ancestors of giant viruses. Here we review the morphogenesis of giant viruses and discuss how the process similarities constitute additional evidence to the common origin of Nucleocytoviricota.

Investigation of the transcriptomic response in Atlantic salmon (Salmo salar) gill exposed to Paramoeba perurans during early onset of disease.

Amoebic Gill Disease (AGD), caused by the protozoan extracellular parasite Paramoeba perurans (P. perurans) is a disease affecting Atlantic salmon (Salmo salar). This study investigated the gill transcriptomic profile of pre-clinical AGD using RNA-sequencing (RNA-seq) technology. RNA-seq libraries generated at 0, 4, 7, 14 and 16 days post infection (dpi) identified 19,251 differentially expressed genes (DEGs) of which 56.2% were up-regulated. DEGs mapped to 224 Gene Ontology (GO) terms including 140 biological processes (BP), 45 cellular components (CC), and 39 molecular functions (MF). A total of 27 reference pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and 15 Reactome gene sets were identified. The RNA-seq data was validated using real-time, quantitative PCR (qPCR). A host immune response though the activation of complement and the acute phase genes was evident at 7 dpi, with a concurrent immune suppression involving cytokine signalling, notably in interleukins, interferon regulatory factors and tumour necrosis factor-alpha (tnf-α) genes. Down-regulated gene expression with involvement in receptor signalling pathways (NOD-like, Toll-like and RIG-1) were also identified. The results of this study support the theory that P. perurans can evade immune surveillance during the initial stages of gill colonisation through interference of signal transduction pathways.