Survival and growth of Stenotrophomonas maltophilia in free-living amoebae (FLA) and bacterial virulence properties

Abstract

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.

Fig 1. Growth of S. maltophilia strains expressed in number of copies of bacterial smeD gene per mL, in co-culture with amoeba.

a) co-culture with Acanthamoeba castellanii L6a; b) co-culture with Willaertia magna C2c. Means +/- standard deviations from three independent experiments in duplicate are presented.

Fig 2. Fluorescent confocal microscopy images of Acanthamoeba castellanii L6a in co-culture with S. maltophilia strains.

(a) after 48 hours with BurA1, (b) after 24 hours with BurE1, (c) after 24 hours with PierC1 and (d) after 48 hours with MEEB-Am6.2.

Fig 3. Fluorescent confocal microscopy images of Willaertia magna C2c in co-culture with S. maltophilia strains.

(a) after 48 hours with BurA1, (b) after 72 hours with BurE1, (c) after 24 hours with PierC1 and after (d) 48 hours with K279a.

Fig 4. Growth of S. maltophilia strains in co-culture with amoeba, expressed in number of colony forming unit of S. maltophilia per mL.

a) co-culture with Acanthamoeba castellanii L6a; b) co-culture with Willaertia magna C2c. Means +/- standard deviations from two independent experiments in triplicate are presented.

Fig 5. D. discoideum plate killing assay with seven S. maltophilia strains.

Bars representing the number of amoebae necessary to form a lysis plaque on the bacterial lawn. P. aeruginosa PT5 and K. pneumoniae KpGe were used as negative and positive controls, respectively. Means +/- standard deviations from 3 independent experiments in triplicate are presented.

Fig 6. Growth of S. maltophilia BurA1 and BurA1ΔebyCAB expressed in number of copies of bacterial smeD gene per mL, in co-culture with amoeba and confocal microscopy.

(a) co-culture with A. castellanii L6a; (b) co-culture with W. magna C2c. Means +/- standard deviations from three independent experiments in duplicate are presented; (c) Fluorescent confocal microscopy images of A. castellanii in co-culture with S. maltophilia BurA1ΔebyCAB after 48 hours; (d) Fluorescent confocal microscopy images of W. magna in co-culture with S. maltophilia BurA1ΔebyCAB after 48 hours.

Fig 7. D. discoideum plate killing assay with two S. maltophilia strains, BurA1 and BurA1ΔebyCAB.

Bars representing the number of amoebae necessary to form a lysis plaque on the bacterial lawn. Pseudomonas aeruginosa PT5 and Klebsiella pneumoniae KpGe are used as negative and positive controls, respectively. Means +/- standard deviations from three independent experiments in triplicate are presented.