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Work Package 2: Listeria Protozoa interaction

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part of Food Microbiology:

In short:

The key objectives of this module are (I) to explore environmental reservoirs of Listeria and (II) to characterize strategies of transfer from the environment to foods and the human host.

Project:

Objectives:

In this study the interaction of Listeria and environmental Protozoa is characterized to determine if Protozoa represent a vector for Listeria transmission.

Methods:

Environmental protozoa were isolated and investigated for possible interactions with Listeria.

Intracellular survival of L. monocytogenes was determined by gentamicin protection assays and co-incubation experiments. In addition fluorescence microscopy and electron microscopy were applied (Fig. 1 - 3).

Fig. 1: Isolated Protozoa used to investigate interactions with Listeria.

Fig. 2: Agglomeration ("backpacks") of gfp-tagged L. monocytogenes at the uroid of Acanthamoeba castellanii.

Fig. 3: Electron microscopy picture of L. monocytogenes agglomerations at Acanthamoeba castellanii.



Results:

After phagocytosis Listeria does not survive intracellularily in the tested protozoa. However, they specifically attach to the exterior cell pole (uroid) of Acanthamoba castellanii.

Listeria monocytogenes also benefit from this interaction with the amoeba resulting in an increase of cell numbers compared to cultures without the presence of protozoa.

The examination of the factors that are required for the observed interaction of the pathogen Listeria monocytogenes and the environmental protozoa demonstrated that virulence factors of the pathogen play no role, whereas bacterial motility is required to form "backpacks".

A small soluble substance (produced by the Acanthamoeba) leads to the benefit for Listeria monocytogenes. This explains the observation that direct contact between the bacteria with Acanthamoeba castellanii is not necessary for the growth effect.



References:

Doyscher D., Haggenmüller M., Fieseler L., Schuppler M., Loessner M.J. „Interaction of Listeria spp. with environmental protozoa“ Switzerland, Zurich, Latsis Symposium 2007 „Research Frontiers in Environment und Sustainability“, 17-19.09.07 (poster and presentation)

Doyscher D., Fieseler L., Schuppler M., Loessner M.J. „Interactions of Listeria and Acanthamoeba“ Switzerland, Stels, D-Biol PhD Summer School “Evolution - From Molecules to Civilization”, 20.-22.10.08 (poster and presentation)

Doyscher D., Fieseler L., Schuppler M., Dons L., Loessner M.J „Interactions of Listeria monocytogenes with Acanthamoeba spp.“ Sweden, Gothenburg, 3rd FEMS Congress of European Microbiologists, 28.06-02.07.09 (poster and presentation)

Doyscher D., Fieseler L., Dons L., Loessner M.J., Schuppler M. “The Interaction of Listeria monocytogenes with Acanthamoeba spp.“, Switzerland, ETH Zurich, CCES Latsis Symposium, 15.-17.11.2010 (poster)

Briers Y., Staubli T., Schnell B., Schuppler M., Loessner M.J. “Multiplication of Listeria monocytogenes L-forms by intracellular budding”, Switzerland, Geneva, FEMS 2011, 4th Congress of European microbiologists, 26.-30.06.2011 (poster)

Doyscher D., Fieseler L., Dons L., Loessner M.J., Schuppler M. “From hitchhikers and backpackers: co-cultures of Listeria monocytogenes and Acanthamoeba spp. feature a peculiar type of interaction”, Switzerland, Geneva, FEMS 2011, 4th Congress of European microbiologists, 26.-30.06.2011 (oral presentation)

Schnell B., Briers Y., Schuppler M., Loessner M.J. “Growth properties and pathogenicity of Listeria monocytogenes L-forms”, Switzerland, Geneva, FEMS 2011, 4th Congress of European Microbiologists 26.-30.06.2011 (poster)

Schuppler M., Doyscher D., Fieseler L., Loessner M.J. “Per Anhalter durch die Mikrowelt: Welche Rolle spielen Acanthamöben als potentielles Umweltreservoir für Listeria monocytogenes?”, Germany, Damp, 11. Fachsymposium Lebensmittelmikrobiologie, 22.-24.07.2011 (oral presentation)



part of Food Biotechnology:

T2.4: Susceptibility to antibiotics (AB) within amoeba

It could be disproven that Listeria spp. survive or even multiply within protozoa. However it could be shown that certain motile bacteria (including Listeria monocytogenes) are attracted by amoebae and form agglomerations (backpacks) on the surface of these protozoa. Although the human pathogen Listeria monocytogenes cannot use the amoebae tested as environmental hosts to prime for virulence, there clearly is an interaction between these organisms.

At the moment it is under investigation, if backpacks are places for horizontal gene transfer (HGT) between motile bacteria. Therefore, it was tested whether different bacteria form backpacks together. So-called "mixed backpacks" (Fig. 3 b)could be shown for Listeria spp., Bacillus subtilis and Salmonella typhimurium. For the visualization, bacteria were stained with different dyes (DAPI and gfp). 

The transfer of the resistance genes tet(M) and the resistance to clindamycin, which were transferable in vitro by filter matings, was tested in a setup with amoebae and the putative donor and the putative recipient of the ABR. First results show that gene transfer of tet(M) occurred between Listeria monocytogenes strains in this setup at transfer frequencies comparable to filter matings. Parameters of the experimental setup will be varied to investigate their influence on transfer frequencies.

A second approach is to determine if HGT happens within amoebae from Listeria spp. to Legionella spp. It is known and could be shown by confocal microscopy that legionella survive within Acanthamoeba castellanii Neff and can be detected after four days (Fig. 3a). As listeria are slowly phagocytosed by amoebae after accumulation on their surface, DNA with resistance genes of digested Listeria spp. might be present within amoebae and could easily be taken up by the persisting Legionella spp.

Like this, amoebae could provide a platform for motile bacteria in the environment, where bacterial counts are very low. If different bacteria are attracted by the protozoa, they could get in contact and gene transfer could be favored.

legionella
legionella
mixed backpacks
mixed backpacks

Fig. 3: Interaction of a) Legionella pneumophila and b) Listeria monocytogenes & Bacillus subtilis with amoebae

References:

Bertsch D., Muelli M., Lacroix C., Meile L. “Is the antibiotic resistance trait a problem in Listeria isolated from Swiss foods?“, Switzerland, Schlieren, 1st Swiss FoodTec Day 2009, 15.05.09 (poster)

Bertsch D., Muelli M., Lacroix C., Meile L. “Susceptibility of listeria isolates from Swiss food to antibiotics“, Sweden, Gothenburg, 3rd FEMS Congress of European Microbiologists, 28.06-02.07.09 (poster)

Bertsch D., Muelli M., Weller M., Lacroix C., Meile L. “Antibiotic resistance genes in foodborne and human listeria isolates from Switzerland: prevalence and transfer potential“, Switzerland, Gwattzentrum Thunersee, SWIMM2010, 5th Swiss Molecular Microbiology Meeting, 24.-26.03.2010 (poster and oral presentation)

Bertsch D., Muelli M., Weller M., Lacroix C., Meile L. “Antimicrobial resistance situation and horizontal gene transfer of foodborne, clinical and environmental Listeria isolates from Switzerland“, Canada, Toronto, 2nd ASM Conference on Antimicrobial Resistance in Zoonootic Bacteria and Foodborne Pathogens, 08.-11.06.2010 (poster)

Bertsch D., Muelli M., Weller M., Lacroix C., Meile L. “Prevalence and transfer potential of antibiotic resistance genes in swiss foodborne and human listeria isolates“, Switzerland, ETH Zurich, 69th annual assembly of the SSM, 24.-25.06.2010 (poster)

Bertsch D., Muelli M., Weller M., Lacroix C., Meile L. “Antibiotic resistance in Listeria isolates from Switzerland“, Switzerland, ETH Zurich, CCES Latsis Symposium, 15.-17.11.2010 (poster)

Bertsch D., Haug M., Tanner S., Mülli M., Weller M., Stevens M., Lacroix C. and Meile L. “Antibiotic resistance and horizontal gene transfer potential of foodborne, clinical and environmental Listeria isolates from Switzerland“, Switzerland, Engelberg, Swiss Microbial Ecology Meeting (SME), 02.-04.02.2011 (oral presentation)

Bertsch D., Muelli M., Weller M., Lacroix C., Meile L. “Antibiotic resistance genes and their horizontal gene transfer potential in clinical, environmental and foodborne Listeria isolates from Switzerland“ Switzerland, Geneva, FEMS 2011, 4th Congress of European microbiologists, 26.-30.06.2011 (poster)

 
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