Isolation and Culture of Leptospira from Clinical and Environmental Samples.

Leptospires, the etiological agents of leptospirosis, are fastidious slow-growing organisms. Here we describe the isolation and routine maintenance of leptospires from clinical (blood, urine, or tissue) and environmental (water or soil) samples. Using combinations of filtration, agar plating, and selective agents, leptospires can be isolated in pure cultures even from complex contaminated sources in standard EMJH culture medium.

Use of MALDI-ToF Mass Spectrometry for Identification of Leptospira.

Medical microbiology has used phenotypical and metabolic criteria to identify bacterial pathogens for decades. However, no such criteria have been applied to identify leptospires at the species level. In the recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) has emerged as new tool for the identification of bacterial species in the medical microbiology laboratory. This technology has rapidly gained more and more popularity. Actually, this technique is sensitive and economic, saving both labor and bench costs, but also rapid, significantly reducing turnaround time from isolation to identification. MALDI-ToF MS provides an unprecedented tool for the rapid identification of Leptospira at the species level.

Biofilm Formation and Quantification Using the 96-Microtiter Plate.

Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flat-bottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile structure.

Leptospira interrogans Retains Direct Virulence After Long Starvation in Water.

Mostly studied as a zoonosis, leptospirosis is also an environment-borne infection and most human cases originate from soil or water contaminations. Yet, only few studies have been interested in the survival of pathogenic Leptospira in freshwater. In this study, water microcosms were designed to evaluate the survival and virulence of Leptospira spp. for 2 years. Four commercial bottled drinking waters and a non-ionized water, all previously filter-sterilized, were studied. Either one of two Leptospira interrogans strains, one Leptospira borgpetersenii strain, or a saprophytic Leptospira biflexa was inoculated in these waters under nutrient-deprived conditions. Molecular, microscopic and cultural approaches were used to study Leptospira survival. Direct virulence of the pathogens was assessed using animal challenge without re-culturing. Our results confirmed the capacity of pathogenic Leptospira to survive for more than a year in water. In addition, we showed the ability of L. interrogans in nutrient-deprived conditions to directly cause systemic infection in susceptible animals. To our knowledge, this is the first report of direct infection of a susceptible host with Leptospira following a long starvation and survival period in nutrient-deprived water. Our results also suggest that Leptospira turned into a physiological "survival" state in harsh freshwater conditions. These data are of prime importance considering that freshwater is a major source of Leptospira infections. Environmental survival and virulence of pathogenic Leptospira spp. are becoming a crucial challenge to determine the environmental risk and adopt relevant prevention and control strategies.

The zoonotic pathogen Leptospira interrogans mitigates environmental stress through cyclic-di-GMP-controlled biofilm production.

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm's cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium's ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.

A systematic review of Leptospira in water and soil environments.

BACKGROUND: Leptospirosis, caused by pathogenic Leptospira, is a zoonosis of global distribution. This infectious disease is mainly transmitted by indirect exposure to urine of asymptomatic animals via the environment. As human cases generally occur after heavy rain, an emerging hypothesis suggests that rainfall re-suspend leptospires together with soil particles. Bacteria are then carried to surface water, where humans get exposed. It is currently assumed that pathogenic leptospires can survive in the environment but do not multiply. However, little is known on their capacity to survive in a soil and freshwater environment. METHODS: We conducted a systematic review on Leptospira and leptospirosis in the environment in order to collect current knowledge on the lifestyle of Leptospira in soil and water. In total, 86 scientific articles retrieved from online databases or institutional libraries were included in this study. PRINCIPALS FINDINGS/SIGNIFICANCE: This work identified evidence of survival of Leptospira in the environment but major gaps remain about the survival of virulent species associated with human and animal diseases. Studies providing quantitative data on Leptospira in soil and water are a very recent trend, but must be interpreted with caution because of the uncertainty in the species identification. Several studies mentioned the presence of Leptospira in soils more frequently than in waters, supporting the hypothesis of the soil habitat and dispersion of Leptospira with re-suspended soil particles during heavy rain. In a near future, the growing use of high throughput sequencing will offer new opportunities to improve our understanding of the habitat of Leptospira in the environment. This better insight into the risk of leptospirosis will allow implementing efficient control measures and prevention for the human and animal populations exposed.

Assessment of the interplay between scaffold geometry, induced shear stresses, and cell proliferation within a packed bed perfusion bioreactor.

By favoring cell proliferation and differentiation, perfusion bioreactors proved efficient at optimizing cell culture. The aim of this study was to quantify cell proliferation within a perfusion bioreactor and correlate it to the wall shear stress (WSS) distribution by combining 3-D imaging and computational fluid dynamics simulations.NIH-3T3 fibroblasts were cultured onto a scaffold model made of impermeable polyacetal spheres or Polydimethylsiloxane cubes. After 1, 2, and 3 weeks of culture, constructs were analyzed by micro-computed tomography (µCT) and quantification of cell proliferation was assessed. After 3 weeks, the volume of cells was found four times higher in the stacking of spheres than in the stacking of cube.3D-µCT reconstruction of bioreactors was used as input for the numerical simulations. Using a lattice-Boltzmann method, we simulated the fluid flow within the bioreactors. We retrieved the WSS distribution (PDF) on the scaffolds surface at the beginning of cultivation and correlated this distribution to the local presence of cells after 3 weeks of cultivation. We found that the WSS distributions strongly differ between spheres and cubes even if the porosity and the specific wetted area of the stackings were very similar. The PDF is narrower and the mean WSS is lower for cubes (11 mPa) than for spheres (20 mPa). For the stacking of spheres, the relative occupancy of the surface sites by cells is maximal when WSS is greater than 20 mPa. For cubes, the relative occupancy is maximal when the WSS is lower than 10 mPa. The discrepancies between spheres and cubes are attributed to the more numerous sites in stacking of spheres that may induce 3-D (multi-layered) proliferation.

Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species.

Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis.

The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis - an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.

Biodiversity of Environmental Leptospira: Improving Identification and Revisiting the Diagnosis.

Leptospirosis is an important environmental disease and a major threat to human health causing at least 1 million clinical infections annually. There has recently been a growing interest in understanding the environmental lifestyle of Leptospira. However, Leptospira isolation from complex environmental samples is difficult and time-consuming and few tools are available to identify Leptospira isolates at the species level. Here, we propose a polyphasic isolation and identification scheme, which might prove useful to recover and identify environmental isolates and select those to be submitted to whole-genome sequencing. Using this approach, we recently described 12 novel Leptospira species for which we propose names. We also show that MALDI-ToF MS allows rapid and reliable identification and provide an extensive database of Leptospira MALDI-ToF mass spectra, which will be valuable to researchers in the leptospirosis community for species identification. Lastly, we also re-evaluate some of the current techniques for the molecular diagnosis of leptospirosis taking into account the extensive and recently revealed biodiversity of Leptospira in the environment. In conclusion, we describe our method for isolating Leptospira from the environment, confirm the usefulness of mass spectrometry for species identification and propose names for 12 novel species. This also offers the opportunity to refine current molecular diagnostic tools.

Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral 'pathogens' cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.

BioFlow: a non-invasive, image-based method to measure speed, pressure and forces inside living cells.

Cell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software.

Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils.

BACKGROUND: Leptospirosis is an important re-emerging infectious disease that affects humans worldwide. Infection occurs from indirect environment-mediated exposure to pathogenic leptospires through contaminated watered environments. The ability of pathogenic leptospires to persist in the aqueous environment is a key factor in transmission to new hosts. Hence, an effort was made to detect pathogenic leptospires in complex environmental samples, to genotype positive samples and to assess leptospiral viability over time. METHODOLOGY/PRINCIPAL FINDINGS: We focused our study on human leptospirosis cases infected with the New Caledonian Leptospira interrogans serovar Pyrogenes. Epidemiologically related to freshwater contaminations, this strain is responsible for ca. 25% of human cases in New Caledonia. We screened soil and water samples retrieved from suspected environmental infection sites for the pathogen-specific leptospiral gene lipL-32. Soil samples from all suspected infection sites tested showed detectable levels of pathogenic leptospiral DNA. More importantly, we demonstrated by viability qPCR that those pathogenic leptospires were viable and persisted in infection sites for several weeks after the index contamination event. Further, molecular phylogenetic analyses of the leptospiral lfb-1 gene successfully linked the identity of environmental Leptospira to the corresponding human-infecting strain. CONCLUSIONS/SIGNIFICANCE: Altogether, this study illustrates the potential of quantitative viability-PCR assay for the rapid detection of viable leptospires in environmental samples, which might open avenues to strategies aimed at assessing environmental risk.

In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor.

BACKGROUND: Entamoeba histolytica cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction). The tissue inflammation associated with tumour necrosis factor (TNF) secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. METHODS: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. RESULTS: an antibody against human TNF receptor 1 (TNFR1) stained the E. histolytica trophozoite surface and (on immunoblots) binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as "cell surface protein", CSP, in view of its cellular location). Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. CONCLUSIONS: there is a clear link between TNF chemotaxis, CSP and pathogenesis.

The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue.

Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.

Identification of the virulence landscape essential for Entamoeba histolytica invasion of the human colon.

Entamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (ß-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica.

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain.

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.

Newly visualized fibrillar collagen scaffolds dictate Entamoeba histolytica invasion route in the human colon.

The extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5.

3-D active meshes: fast discrete deformable models for cell tracking in 3-D time-lapse microscopy.

Variational deformable models have proven over the past decades a high efficiency for segmentation and tracking in 2-D sequences. Yet, their application to 3-D time-lapse images has been hampered by discretization issues, heavy computational loads and lack of proper user visualization and interaction, limiting their use for routine analysis of large data-sets. We propose here to address these limitations by reformulating the problem entirely in the discrete domain using 3-D active meshes, which express a surface as a discrete triangular mesh, and minimize the energy functional accordingly. By performing computations in the discrete domain, computational costs are drastically reduced, whilst the mesh formalism allows to benefit from real-time 3-D rendering and other GPU-based optimizations. Performance evaluations on both simulated and real biological data sets show that this novel framework outperforms current state-of-the-art methods, constituting a light and fast alternative to traditional variational models for segmentation and tracking applications.