Environmental amoebae do not support the long‐term survival of virulent mycobacteria

Aims

To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa.

Methods and Results

In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid‐fast micro‐organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M. bovis detected by IS6110 PCR. Co‐incubation of Acanthamoeba castellanii with virulent M. bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M. bovis.

Conclusions

The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M. bovis rather than representing a potential environmental reservoir.

Significance and Impact of the Study

Protozoa have been suggested to act as an environmental reservoir for M. bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M. bovis and that protozoa might inhibit persistence of M. bovis in the environment.     

Spatial distribution of gymnamoebae (Rhizopoda, Lobosea) in brackish-water sediments at the scale of centimeters and millimeters

In order to study micro-spatial distribution of amoebae, an intact slice of sandy sediment from the brackish-water Nivå Bay (Baltic Sea, The Sound), 40 × 24 mm in size and 2 mm in thickness was gently sectioned into cubes, 2 × 2 × 2 mm in size. Each cube was inoculated into enrichment media to reveal the biodiversity of amoebae. Seventeen species of amoebae were recovered. The 2-D map of amoebae species distribution in the slice, consisting of 240 2 × 2 mm cells was drawn and analyzed. Results show heterogeneous distribution of amoebae at the scale of centimeters and millimeters and confirm the idea of the presence of microhabitats, selectively occupied by amoebae species. Three types of distribution patterns were found: random, aggregated and equally spaced. Microelectrode studies indicated that amoebae distribution was not related to the dissolved oxygen content in the sediment. The studied slice of sediment contained several pronounced “hotspots” of amoebae biodiversity, where up to four species co-occur in the same area. Seven species of amoebae numbered 1–4 specimens in the studied slice (i.e. there was 0.5–2 cell ml^–1). Analysis of the amoebae distribution map shows the high probability of undersampling rare amoebae species during faunistic studies.     

Vertical distribution of gymnamoebae (rhizopoda, lobosea) in the top layer of brackish-water sediments

The paper reports on the distribution of amoebae in a vertical slice of sandy sediment from the brackish-water Nivå Bay (Baltic Sea, The Sound). The 24 × 20 mm slice 2 mm in thickness was gently cut from the top of sediment to a depth of 20 mm using coverslips, and further sectioned into 2 × 2 × 2 mm cubes. Each cube was inoculated into enrichment media to reveal the biodiversity of amoebae. The map of amoebae species distribution in the slice was drawn and analyzed. The results show patchy distributions of many amoebae species. Amoebae tend to form a band of maximum diversity within the depth interval 2–16 mm. Two mini-cores of sediment located close to the butt-ends of the slice were collected using cut 1 ml syringes. They were sliced and inoculated to reveal the biodiversity of amoebae at different depths. The distribution patterns of amoebae in the mini-cores were similar to the ones obtained in the slice, and show evident patchiness of some species. It seems that abundant species mostly form irregular patches 1–2 cm across, whereas many species are rare and appear in few number of specimens or in patches smaller than 2 mm.     

A seasonal study of the carbon content of planktonic naked amoebae in the Hudson Estuary and in a productive freshwater pond with comparative data for ciliates

The total carbon contents of gymnamoebae and ciliates, dwelling in the water column of the Hudson Estuary and a highly productive freshwater pond, were monitored during a 7-month period from April through October 2006. The carbon contents of the gymnamoebae and the ciliates were greater in the pond compared with the estuary, and carbon contents of gymnamoebae were greater in the spring and autumn in both locations than those of ciliates. Given the global distribution of gymnamoebae, these results suggest that greater attention should be given to the potential role of gymnamoebae in microbial food webs.     

Testate amoebae analysis in ecological and paleoecological studies of wetlands: past, present and future

Testate amoebae are an abundant and diverse polyphyletic group of shelled protozoa living in aquatic to moist habitats ranging from estuaries to lakes, rivers, wetlands, soils, litter, and moss habitats. Owing to the preservation of shells in sediments, testate amoebae are useful proxy indicators complementary to long-established indicators such as pollen and spores or macrofossils. Their primary use to date has been for inferring past moisture conditions and climate in ombrotrophic peatlands and, to a lesser extent, to infer pH in peatlands and the trophic or nutrient status of lakes. Recent research on these organisms suggests other possible uses in paleoecology and ecology such as sea-level reconstruction in estuarine environments, as indicators of soil or air pollution, and monitoring recovery of peatland. We review the past and present use of testate amoebae, the challenges in current research, and provide some ideas on future research directions.     

The new mycobacteria: an update

The continuous evolution of mycobacterial taxonomy may represent a source of confusion for laboratories and clinicians. Apart from the obvious pathogenic strains of the Mycobacterium tuberculosis complex, Mycobacterium leprae and Mycobacterium ulcerans, the role of other mycobacteria may be associated with varying conditions ranging from contamination to specific disease processes. Of the more than 120 mycobacterial species recognized currently, very few have not been reported as pathogenic in humans or animals. Although the attempt to keep pace with the steadily increasing number of mycobacterial species seems hopeless, a careful review of the recent literature relevant to the newly described species may be advantageous. The aim of this present update is to provide epidemiological and clinical information along with major phenotypic and genotypic characteristics of the species described in the last 3 years.     

The rapidly growing mycobacteria: saprophytes and parasites

Rapidly growing mycobacteria are widespread saprophytes, but approximately one-third of identified species are also opportunistic pathogens in humans and animals, associated with skin, soft tissue, bone, and pulmonary infections as well as disseminated disease. Clinical and experimental evidence indicates a major role for the cell-mediated immune response in the pathogenesis of infection.     

Vertical micro-distribution and response to nitrogen deposition of testate amoebae in Sphagnum

Previous studies have shown the existence of a vertical micro-distribution of testate amoebae in the first centimeters of Sphagnum and their response to nutrient enrichment. In order to test the response of testate amoebae to depth and N addition in dry moss carpets recolonizing cutover peatlands, we sampled Sphagnum that had received 0, 1, 3, or 10g N m(-2) yr(-1) for three years. The mosses were cut into three segments: 0-1cm, 1-3cm and 3-5cm and analyzed for testate amoebae. The overall diversity (22 taxa) was high considering the dryness of the site, but the species richness of individual samples was low (mean 6.6). The presence of several species characteristic of wetter conditions suggests that they have a broader tolerance than usually believed and/or have a high colonization potential. Species richness increased with depth. Assulina muscorum was most abundant in the top segment, while Phryganella acropodia, Heleopera rosea and Nebela militaris were most abundant in the deepest segment. Neither the metabolism type nor the shell characteristics significantly explained the vertical distribution of species. There was no overall response of testate amoebae to N, although one species, Bullinularia indica, was significantly more abundant in the fertilized than in the control plots.     

Surrounded by mycobacteria: nontuberculous mycobacteria in the human environment

A majority of the Mycobacterium species, called the nontuberculous mycobacteria (NTM), are natural inhabitants of natural waters, engineered water systems, and soils. As a consequence of their ubiquitous distribution, humans are surrounded by these opportunistic pathogens. A cardinal feature of mycobacterial cells is the presence of a hydrophobic, lipid-rich outer membrane. The hydrophobicity of NTM is a major determinant of aerosolization, surface adherence, biofilm-formation, and disinfectant- and antibiotic resistance. The NTM are oligotrophs, able to grow at low carbon levels [>50 μg assimilable organic carbon (AOC) l⁻¹], making them effective competitors in low nutrient, and disinfected environments (drinking water). Biofilm formation and oligotrophy lead to survival, persistence, and growth in drinking water distribution systems. In addition to their role as human and animal pathogens, the widespread distribution of NTM in the environment, coupled with their ability to degrade and metabolize a variety of complex hydrocarbons including pollutants, suggests that NTM may be agents of nutrient cycling.     

Free-living amoebae promote Candida auris survival and proliferation in water

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics

Infectious disease ecology has recently raised its public profile beyond the scientific community due to the major threats that wildlife infections pose to biological conservation, animal welfare, human health and food security. As we start unravelling the full extent of emerging infectious diseases, there is an urgent need to facilitate multidisciplinary research in this area. Even though research in ecology has always had a strong theoretical component, cultural and technical hurdles often hamper direct collaboration between theoreticians and empiricists. Building upon our collective experience of multidisciplinary research and teaching in this area, we propose practical guidelines to help with effective integration among mathematical modelling, fieldwork and laboratory work. Modelling tools can be used at all steps of a field-based research programme, from the formulation of working hypotheses to field study design and data analysis. We illustrate our model-guided fieldwork framework with two case studies we have been conducting on wildlife infectious diseases: plague transmission in prairie dogs and lyssavirus dynamics in American and African bats. These demonstrate that mechanistic models, if properly integrated in research programmes, can provide a framework for holistic approaches to complex biological systems.     

COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.     

Panglobalism and pandemics: ecological and ethical concerns

A pandemic is a human medical problem but must be understood at multiple levels. Analysis of social and commercial forces is vital, and, more comprehensively, an ecological framework is necessary for an inclusive picture. Ecological health webworked with political and social determinants surrounds issues of human health. In this constellation of both natural and social factors, ethical concerns will arise at these multiple levels, from human health to the conservation and health of wild nature.     

Molecular ecology of parasites: elucidating ecological and microevolutionary processes

We review studies that have used molecular markers to address ecological and microevolutionary processes in parasites. Our goal is to highlight areas of research that may be of particular interest in relation to the parasitic lifestyle, and to draw attention to areas that require additional study. Topics include species identification, phylogeography, host specificity and speciation, population genetic structure, modes of reproduction and transmission patterns, and searching for loci under selection.     

Effect of growth media and phase on Raman spectra and discrimination of mycobacteria

When developing a Raman spectral library to identify bacteria, differences between laboratory and real world conditions must be considered. For example, culturing bacteria in laboratory settings is performed under conditions for ideal bacteria growth. In contrast, culture conditions in the human body may differ and may not support optimized bacterial growth. To address these differences, researchers have studied the effect of conditions such as growth media and phase on Raman spectra. However, the majority of these studies focused on Gram‐positive or Gram‐negative bacteria. This article focuses on the influence of growth media and phase on Raman spectra and discrimination of mycobacteria, an acid‐fast genus. Results showed that spectral differences from growth phase and media can be distinguished by spectral observation and multivariate analysis. Results were comparable to those found for other types of bacteria, such as Gram‐positive and Gram‐negative. In addition, the influence of growth phase and media had a significant impact on machine learning models and their resulting classification accuracy. This study highlights the need for machine learning models and their associated spectral libraries to account for various growth parameters and stages to further the transition of Raman spectral analysis of bacteria from laboratory to clinical settings.      

A method for estimating cell volume of amoebae based on measurements of cell length of motile forms: physiological and ecological applications

A novel method of estimating the cell volume of non-testate, free-living amoebae is presented. Measurements of the length (L) of motile forms of some amoebae, obtained by applying some basic rules, can be used to calculate diameter (D) using a relatively simple formula, derived from regression analysis (N=39): D=0.6L. Important physiological and ecological variables, including carbon content and respiratory rate, can then be easily calculated using this estimated cell volume.