Leptospira biodiversity

An unexpected Leptospira biodiversity in New Caledonian soils
Principal investigator Cyrille Goarant
Focal point IPNC Roman Thibeaux, Cyrille Goarant
Collaborators at IPNC Marie-Estelle Soupé-Gilbert, Dominique Girault, Emilie Bierque
Other Collaborators Mathieu Picardeau, IPP, Gregorio Iraola and Ignacio Ferres, IP Montevideo
Total budget of project 3000 € Budget devoted to IPNC : NA
Funding IPNC
Timeline Start date: 2016 End date: 2018
Context

Following the demonstration of the environmental survival of pathogenic leptospires at sites of human contamination in New Caledonia, isolation of Leptospira were undertaken.

Infectious strains were not re-isolated from the environment, but a large number of isolates were obtained. Difficulties of identification (MALDI-TOF, 16S rRNA sequencing) led us to suspect novel species and to undertake the sequencing of their complete genome.

Objectives
Perform a genomic study of leptospires isolated from Caledonian soils.
Methods

Isolations were carried out from field soil or water samples. Liquid cultures in EMJH medium were supplemented with a combination of antibiotics described to select growth of leptospires (Chakraborty et al, 2011). The cultures were then spread on EMJH agar medium for isolation.

The isolates were identified with MALDI-TOF and by sequencing their complete genome. Strains of the pathogenic and intermediate clusters are tested in animal models.

Results
More than 100 environmental isolates have been obtained. The first 26 strains for which the complete genome was sequenced showed the presence of 12 novel species: 3 in the pathogen cluster, 5 intermediates and 4 saprophytes.

Strains of the pathogen cluster were unable to cause acute infection in hamsters or renal carriage in mice.

A detailed comparison of core genomes and accessory genomes supports the hypothesis of a polyphyletic group of leptospires with low virulence within the pathogen cluster. This result suggests, in terms of evolution, that virulence was acquired independently in the pathogen and intermediate clusters.

Preliminary results from other isolates suggest other new species.

 

 

Prospects
These 12 novel species should be validly described and named. The new isolates will also be studied. Specific PCR will be designed and tested.
Valorisation 

Thibeaux R, Iraola G, Ferrés I, Bierque E, Girault D, Soupé-Gilbert ME, Picardeau M, Goarant C. Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence. Under review Microbial Genomics.

Thibeaux R, Iraola G, Ferrés I, Bierque E, Girault D, Soupé-Gilbert ME, Picardeau M, Goarant C. Of soils, Leptospira and humans. Présentation orale à la conférence de l’ILS, Palmerston North, NZ, November 2017.

Biofilm and Leptospira

Project title Leptospira Biofilm: an unexplored reservoir for environmental survival and persistence of infectious bacteria.
Principal investigator Roman Thibeaux
Focal point IPNC Roman Thibeaux, Cyrille Goarant
Collaborators at IPNC Marie-Estelle Soupé-Gilbert, Dominique Girault, Emilie Bierque
Other Collaborators Mathieu Picardeau, IPP, Jean-Marc Ghigo, IPP, Michaël Meyer, UNC
Total budget of project 8000 euros Budget devoted  to IPNCNA
Funding IPNC
Timeline Start: 2018 End :2019
Objectives
The specific objectives of this project are to investigate the bacterial protection provided by the biofilm against both harsh ecological conditions and upon kidney chronic colonization.
Methods

We developed a 96-well method to grow Leptospira biofilm in vitro. After 4 weeks, biofilm formation was quantified using an in-house image analysis workflow based on phase-contrast optical images. To identify leptospiral genes regulating biofilm formation, we quantified impaired or enhanced ability of L. interrogans transposon insertion mutants to produce a biofilm. We then investigated mutants’ infectivity in hamsters, while renal colonization ability was challenged in mice. Protection against environmental stresses (T°, UV light, NaCl) was also assessed using a cell viability Alamar blue assay.

Finally, fluorescently-labelled lectins were used to visualized in situ exo-polysaccharide (EPS) presence and distribution within Leptospira biofilm.

Results

By screening defective mutants, we identified the c-di-GMP signaling pathway as a major regulator of biofilm formation. Indeed, “loss of function” mutants impaired for c-di-GMP synthesis enzymes (GGDEF containing motif) display a decreased ability to produce a biofilm; conversely, “Loss of function” mutants for c-di-GMP recycling enzymes (HD-GYP containing motif) exhibit an enhanced capacity to produce a biofilm. In addition, we show that a defective mutant for an aldo-keto-reductase involved in carbohydrate metabolism exhibits a decreased capacity to produce a biofilm.

We next investigated the ecological relevance of leptospires’ biofilm. Challenge of mutants’ infectivity in hamsters shows that biofilm-deficient mutants are more virulent than mutants displaying an enhanced biofilm production. In a mice model of chronic leptospirosis, urinary excretion load of leptospires was higher for biofilm-deficient mutants as compared to their counterparts. Finally, a protective effect on cell viability was observed for mutants with increased biofilm formation when exposed to high doses of NaCl and UV-c. Further, the increase in viability was strongly correlated to biofilm production in a dose-dependent manner.

Finally, fluorescently-labelled lectins allowed a preliminary characterization of biofilm composition, demonstrating the the presence of α-linked d-glucopyranosyl repeated units as well as x-mannopyranosyl and x-glucopyranosyl residues.

Prospects

This work reveals that, although apparently not required for host infection, biofilm formation improves leptospires’ survival in detrimental environmental-like conditions, thus probably contributing to their persistence in natural habitats.

Ultimately, this work could suggest strategies to impair bacterial biofilm production and organization, thus rendering bacteria accessible to the immune system or harsh ecological conditions. These approaches would hence open new avenues to prevention and treatment of leptospirosis.

Valorisation 
Thibeaux R, Soupé-Gilbert ME, Picardeau M, Goarant C. 2017. Biofilm of pathogenic Leptospira: A compromise between virulence and environmental survival? Présentation orale à la conférence de l’ILS, Palmerston North, NZ, November 2017.

Maldi-tof Leptospire identification

Maldi-Tof identification of Leptospira spp.
Principal investigator Cyrille Goarant
Focal point IPNC Dominique Girault, Cyrille Goarant
Collaborators at IPNC Roman Thibeaux, Julien Colot
Other Collaborators Anna Rettinger, Faculty of Veterinary Medicine, LMU Munich, Germany
Total budget of project 1 000 euros Budget devoted to IPNC : NA
Funding IPNC
Timeline Start date: 2015 End date: 2018
Context
IPNC has implemented MALDI-ToF mass spectrometry for routine identification of bacterial isolates. We aimed to use this system to identify bacteria of the genus Leptospira.
Objectives
The objectives were to create a database of mass spectra of reference strains of the genus Leptospira and then to use it to control the identification of strains used for MAT serology as well as unknown isolates.
Methods

A bibliographic review identified 3 teams that had a mass spectra database of Leptospira spp. The corresponding authors of the articles were asked to share their database. Dr. Anna Rettinger (University of Munich) agreed to transmit her reference spectra.

We have also created reference spectra of known collection strains belonging to different species. Unknown (patient isolates) or known strains (from the MAT serology panel) were then identified blindly.

 

Results

The preliminary tests made it possible to optimize the technique of preparation of the leptospires for reading their mass spectrum on the Maldi-Tof Bruker. The reference spectra of 57 strains representing all 22 known Leptospira species were created and grouped into a dedicated “research” database.

Blind analysis of strains shows very good reliability for species-level identification, but insufficient precision to identify individual strains used as antigens for MAT serology. The strains isolated from patients were properly identified at the species-level with high scores considered as diagnostic.

Perspectives
The reference spectrum of Leptospira venezuelensis (currently being described) will also be created. The transfer of the MALDI-TOF and computer breakdowns slowed down the work. This database will be incremented with novel species and used to identify unknown isolates. Threshold scores for species identification will be determined.
Valorisation 
These data should be included in a draft publication on the identification of leptospires.

Leptospira survival in water

 

The survival of pathogenic leptospires in nutrient-poor water: towards a better understanding of the environmental reservoir of leptospirosis
Principal investigator Emilie Bierque
Focal point IPNC Emilie Bierque, Cyrille Goarant
Collaborators at IPNC Marie-Estelle Soupé-Gilbert, Sophie Geroult
Total budget of project 8 300 euros Budget devoted to IPNC: NA
Funding IPNC
Timeline Start date: 2016 End date:2018
Context
The transmission of leptospirosis to humans occurs via exposure to virulent leptospires through contact with urine or tissues of infected animals, most often indirectly via a hydrotelluric reservoir. However, the factors determining the survival of pathogenic leptospires in the environment remain very poorly known.
Objectives
The objective is to describe the survival of leptospires in waters of different mineral compositions.
Methods
We used 0.1µm filter-sterilized water to study the effect of the ionic composition of the waters without the complex interactions that could occur in a complex microbiota and prevent precipitation of salts, which would occur during autoclaving. Waters were dispensed into 50-mL plastic culture flasks with 2 sterile glass slides to assess the adhesion of leptospires and observe possible morphological changes. Leptospires were seeded in each flask at a final concentration of 106 Leptospires / mL. The culture flasks were incubated at 30 ° C in the dark. After various incubation times, individual flasks are used to assess leptospiral counts, viability, cultivability and virulence, for a maximum of 2 years.
Results
After 12 months of experimentation, results reveal different survival rates depending on water, observed from 30 days post-incubation on. We noticed different outcomes between Leptospira strains only for the most mineralized water. Two waters appear to allow a better survival for 3 of the 4 Leptospira strains studied. Furthermore, the most mineralized water seems to become supportive for the survival of one of the strains after one year of experimentation. The 3 strains remain alive and virulent (for pathogenic ones) during at least 9 months in water while the forth strain died after only 2 days. Microscopic techniques allow observing cell aggregates formation as of 60 days post-incubation for pathogens and as early as 2 days for the saprophytic stain.
Prospects
Our study will help identify the ionic composition of the water that favours or compromise the survival of leptospires and thus acquire important data on the survival and maintenance of virulence of leptospires in nutrient-poor conditions.
Valorisation 

Mélanie Faure, L2 student at University of New Caledonia made a 2-month training period on this subject.

Bierque E, Soupé-Gilbert ME, Geroult S, Thibeaux R and Goarant C. Leptospira survival in freshwater microcosms. Poster at the ILS conference, Palmerston North, NZ, November 2017.