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Recherches

Development and validation of a MALDI-TOF MS microbial database for rapid identification of public-, animal-, and plant-health relevant environmental bacteria

Background

This is a project of three years (2010-2013) in collaboration with Dr. Brion Duffy (Agroscope, Wädenswil) and MABRITEC AG, Basel (Dr. Guido Vogel) which received a grant from the CTI for the production of a marketable database. MALDI-TOF MS is an emerging tool for routine identification of microorganisms. Our group introduced this technique for the first time in Switzerland during the year 2007 for the identification of pathogenic bacteria in the bacteriology lab at the Institute of microbiology of Canton Tessin. Reliable identification using MALDI-TOF MS depends upon a validated and comprehensive database of peptide mass fingerprints. Such databases are currently only available for selected human pathogenic bacteria. Potential applications in wider areas of microbiology present an interesting challenge. This project started in July 2010. In this first phase we are dealing, with the genetic and mass spectrometric analysis of selected microorganisms to serve as a reference for the database. During this first six months, 136 strains were analyzed. These bacteria are generally difficult to identify using conventional methods therefore the genetic analysis based on the sequencing of the 16S rRNA genes were performed for all isolates.

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Fig. 1: MALDI-TOF mass spectrometry

Ongoing Projects

  • Commission for Technology and Innovation project (Cinzia Benagli and Sophie De Respinis) in collaboration with Dr. Guido Vogel (MABRITEC, Basel) and Swiss Confederation (CTI).
  • In the frame of this CTI-project and in collaboration with Dr. Xavier Perret (Microbiology unit, UNI GE) and (MABRITEC, Basel) a PhD student (Dominik Ziegler) and a Master student (Anna Mariotti-Nesurini) both at the University of Geneva started a project dealing with the characterization of symbiotic bacteria. The chosen model organism is Sinorhizobium sp. NGR234. The idea is to characterize the components of the MALDI-TOF MS spectrum. 
  • Determination of the clinical and pathological significance of the species of the bacterial genus Yersinia through the use MALDI-TOF mass spectrometry. (Damiana Ravasi)

Fate of Bacillus thuringensis var. israelensis (BTI) applied as a bioagent against mosquito larvae in the wetland reserve "Bolle di Magadini"

Background

Bacillus thuringiensis var. israelensis (Bti) is a spore forming bacterium belonging to the Bacillus cereus group. During sporulation, Bti forms intracellular insecticidal protein crystal aggregates composed of different delta-endotoxins (encoded by the genes cry4Aa, cry4Ba, cry10Aa, and cry11Aa) and hemolytic factors (encoded by cyt1Aa and cyt2Ba). The genes encoding these proteins are located on a 127.9 kb plasmid. The activation of the toxins in the alkaline midgut environment of mosquito larvae leads to the disruption of the membrane integrity and eventual cytolysis of the epithelial cells, with the consequent blocking of the intestinal functions and the death of the larvae.

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Fig. 2: Mechanism of toxicity of Bacillus thuringiensis var. israelensis

Biopesticides based on Bti are used worldwide to control mosquito and black fly larvae, belonging to the families Culicidae and Simulidae. In Switzerland, at the natural wetland reserve of the “Bolle di Magadino”, regular treatments with VectoBac-G® (Valent Biosciences, USA), a preparation containing crystals and living spores of Bti, have been carried out regularly since 1988 to control flood water mosquitoes, in particular Aedes vexans and Ochlerotatus sticticus, that proliferate during the frequent flooding of this area. Every year, the frequency of Bti applications depends on the degree of flooding and on the number of mosquito larvae developing in the area. The persistence and accumulation of allochthonous Bti in the environment as a consequence of these regular treatments, although not proven to have a negative impact on the ecosystem, may result in the acquisition of resistance by mosquito larvae after prolonged exposure to this biological control agent. The long term treatments with VectoBac-G® makes “Bolle di Magadino” an optimal location for an ecological survey and a follow-up of both fate and impact of the Bti released in the environment. During a previous PhD thesis (Chappuis, 2002), an autochthonous strain of Bti (called A19) was isolated from a sediment sample. This autochthonous strain could be theoretically used against mosquito larvae of this reserve, avoiding therefore to introduce allochthonous microorganisms in this valuable natural area.

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Fig. 3: Natural wetland reserve of the “Bolle di Magadino” (photo: Fondazione Bolle di Magadino)

Ongoing Projects

  • The development of a specific molecular technique enabling a rapid and cost effective detection and quantification of Bti spores in the environment is necessary for an accurate long term monitoring of areas treated with this biological control agent. The first aim of the project is to develop a real-time quantitative PCR for the rapid detection and quantification of Bti spores present in soil and sediment samples. A large scale analysis of the “Bolle di Magadino” area using a fast quantitative method could help to determine the overall distribution of Bti spores in the soil and to analyze factors influencing their distribution. Major aim of this project is to analyze the spatial distribution of Bti resting spores in the soil of the “Bolle di Magadino” reserve after more than 20 years of treatments. The project encompass the influence of the topography, the water flooding dynamics and the frequency of the Vectobac-G® applications on the Bti spores distribution. Limited information is available on the potential proliferation of the biopesticide in the treated area. The ability of Bti spores to recycles in mosquito larvae is also subject of investigation. (Valeria Guidi)
  • Characterization of strain A19 belonging to the species Bacillus thuringiensis var. israelensis (Bti) isolated from soil of a protected wetland reserve in Switzerland. (Ines Cabral)

Role of the purple sulfur bacterium Thiodictyon sp. Cad16 in the primary production from the chemocline of the Lake Cadagno

Background

The Lake Cadagno located in the Piora valley in the southern Alps of Switzerland (46°33' N, 8°43'E) is a crenogenic meromictic lake. This lake is characterized by a compact chemocline with high concentrations of sulphate; steep gradients of oxygen, sulfide, and light; and a turbidity maximum that correlates to large numbers of bacteria (up to 107 cells ml-1). The chemocline of the meromictic Lake Cadagno, situated at 11-13 m of depths, is characterized by the presence of a dense community of phototrophic sulfur bacteria, as well as purple sulfur bacteria (PSB) belonging to the genera Lamprocystis, Thiodyction, Thiocystis and Chromatium and green sulfur bacteria (GSB) of the genus Chlorobium. PSB and GSB are able to carry out anoxygenic photosynthesis on the basis of bacteriochlorophyll mediated processes. In general GSB are regarded as obligate photoautotrophs, while PBS are capable of both photoautotrophy and photoheterotrophy, with some strains being able to grow as chemotrophs in microaerophilic conditions. GSB are often found below PSB in stratified microbial communities, essentially because they have more efficient light-harvesting antennas (chlorosomes) and are adapted to lower light intensities compared to PSB. The photosynthetic process depends on oxygen-deficient condition, because synthesis of photosynthetic pigments is repressed by oxygen, so they use sulfide or, other reduced sulfur compounds, as electrons donors. Since up to 40% of the total photoassimilation of inorganic carbon (CO2) in the lake takes place in this transition zone. Moreover in the chemocline the CO2 dark assimilation reached values comparable to the photo-assimilation.

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Fig. 4: Lake Cadagno

Ongoing Projects

  • Study of the fonctional ecology of the PSB Thiodictyon sp. strain Cad16, especially in the primary production (CO2 assimilation process). (Nicola Storelli)
  • Proteomics analysis of the purple sulfur bacterium (PSB) Thiodictyon sp. Strain Cad16. 2D gels and MALDI-TOF mass spectrometry are used to detect chenages in proteins expression for different growning conditions. (Nicola Storelli)
  • Structure analysis of mutualistic aggregats isolated from the chmocline of the Lake Cadagno. This aggregate is composed of the purple sulfur bacterium Thiodictyon sp. Strain Cad16 and the sulfate-reducing bacterium Desulfocapsa thiozymogenes Cad 626. (Nicholas Vacchietti)
  • Paleomicrobiological studies of the phototrophic sulfur bacteria in the anoxic sediment of Lake Cadagno. (Damiana Ravasi)