Date Published: February 5, 2018
Publisher: Public Library of Science
Author(s): Elodie Denet, Valentin Vasselon, Béatrice Burdin, Sylvie Nazaret, Sabine Favre-Bonté, Riccardo Manganelli.
Stenotrophomonas maltophilia is found ubiquitously in the environment and is an important emerging nosocomial pathogen. S. maltophilia has been recently described as an Amoebae-Resistant Bacteria (ARB) that exists as part of the microbiome of various free-living amoebae (FLA) from waters. Co-culture approaches with Vermamoeba vermiformis demonstrated the ability of this bacterium to resist amoebal digestion. In the present study, we assessed the survival and growth of six environmental and one clinical S. maltophilia strains within two amoebal species: Acanthamoeba castellanii and Willaertia magna. We also evaluated bacterial virulence properties using the social amoeba Dictyostelium discoideum. A co-culture approach was carried out over 96 hours and the abundance of S. maltophilia cells was measured using quantitative PCR and culture approach. The presence of bacteria inside the amoeba was confirmed using confocal microscopy. Our results showed that some S. maltophilia strains were able to multiply within both amoebae and exhibited multiplication rates up to 17.5 and 1166 for A. castellanii and W. magna, respectively. In contrast, some strains were unable to multiply in either amoeba. Out of the six environmental S. maltophilia strains tested, one was found to be virulent. Surprisingly, this strain previously isolated from a soil amoeba, Micriamoeba, was unable to infect both amoebal species tested. We further performed an assay with a mutant strain of S. maltophilia BurA1 lacking the efflux pump ebyCAB gene and found the mutant to be more virulent and more efficient for intra-amoebal multiplication. Overall, the results obtained strongly indicated that free-living amoebae could be an important ecological niche for S. maltophilia.
Stenotrophomonas maltophilia is a non-fermentative Gram-negative bacterium occurring ubiquitously in various natural and anthropogenic environments . The presence of S. maltophilia has been reported in various water sources such as rivers , petroleum reservoir waste water in Iran , high altitude lakes, as well as in sediment  and deep-sea invertebrates . This species also occurs in various soil types all around the world [6,7] where it is a frequent colonizer of the rhizosphere [8,9]. This bacterium shows plant-growth promoting activity as well as antagonistic properties against bacterial and fungal plant pathogens due to its production of phytohormones  and chitinolytic activities . It can also degrade a variety of xenobiotics [12,13] and hydrocarbons  with a significant role in bioremediation of polluted sites . This bacterium was also found associated with the gut of a bark beetle where it could be implicated in the oxidation, fermentation, and hydrolysis of cellulose and lignin derived aromatic products . Recently, we showed that S. maltophilia is also part of the microbiome of several free-living amoebal genera from soils collected in Burkina Faso and Vietnam . However, its role in the context of amoebal interactions is poorly known.
Free-living amoebae may constitute a host for some bacterial species . Currently, most studies have focused on species known to be endosymbionts of Acanthamoeba, such as the members of the bacterial genera Legionella, Chlamydia or Mycobacterium avium . Other analyses characterizing amoebal microbiomes have shown the presence of various associated bacteria including several human opportunistic pathogens, such as P. aeruginosa and S. maltophilia [17,35,36]. The present study demonstrated that various strains of S. maltophilia regardless of their origin i.e. environmental or clinical, were capable of intra-cellular survival and/or growth inside two different FLA, A. castellanii and W. magna. Recently, Cateau et al. (2014)  also reported that both clinical and environmental isolates of S. maltophilia were able to survive and multiply inside V. vermiformis. Our results are the first to compare the behavior of several environmental isolates of S. maltophilia with two amoebal genera and provide insight on whether selectivity towards specific amoebal genera exists or not. We showed that at an early step of the interaction i.e. bacterial internalization (time 0) by the amoeba, differences can be seen in the number of cells internalized from one strain to another. Indeed, with A. castellanii L6a, the numbers of S. maltophilia PierC1 and R551.3 cells internalized is lower than for the other strains. Regarding W. magna C2c, S. maltophilia PierC1 and K29a were the least internalized strains. These differences were confirmed using culture approach.
In conclusion, our results showed for the first time that S. maltophilia isolates with contrasting phenotypes of virulence are able to grow inside two amoebae, A. castellanii and W. magna. These results suggest that in the environment, S. maltophilia could have the potential to infect and proliferate within a large panel of FLA. The fact that this emerging opportunistic pathogen is often found in the amoebal microbiome  and that it can multiply in the amoeba support the hypothesis that in the environment, FLA could be a reservoir and vector for the transmission of S. maltophilia. Thus, S. maltophilia could use the amoebae as a “training ground” in order to better resist human macrophages, as demonstrated for L. pneumophila , or to increase their virulence and antibiotic resistance properties . In conclusion, FLA constitute an ecological niche for opportunistic bacterial pathogens in which important genetic exchanges between species could occur and contribute to the propagation of antibiotic resistance and virulence genes in the environment .