Author: Cyrille Goarant

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.

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.

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.

Thibeaux R, Iraola G, Ferrés I, Bierque E, Girault D, Soupé-Gilbert ME, Picardeau M, Goarant C, 2018. Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence. Microbial Genomics 4: 000144.

Thibeaux R, Girault D, Bierque E, Soupé-Gilbert ME, Rettinger A, Douyere A, Meyer M, Iraola G, Picardeau M, Goarant C, 2018. Biodiversity of environmental Leptospira: improving identification and revisiting the diagnosis. Front. Microbiol. 9: 816.

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.

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.

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.

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.

Thibeaux R, Soupé-Gilbert ME, Kainiu M, Girault D, Bierque E, Fernandes J, Bähre H, Douyère A, Eskenazi N, Vinh J, Picardeau M, Goarant C. The Zoonotic Pathogen Leptospira interrogans Mitigates Environmental Stress Through cyclic-di-GMP-controlled Biofilm Production. npj Biofilms and Microbiomes 6(1):24 Texte intégral / Full text

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.

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.

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.