S. maltophilia virulence was determined as previously described [31 (link)] using the social amoeba, D. discoideum. Strains of P. aeruginosa PT5 and K. pneumoniae KpGe were used as negative and positive controls, respectively, for each assay. From the overnight bacterial culture, the optical density (OD) at 600 nm was adjusted to 1.5 by dilution in LB. For co-cultures between bacteria and D. discoideum, Sm Agar (FORMEDIUM, Hustanton, United Kingdom) medium was used. One mL of each bacterial suspension was spread on Sm Agar and plates were allowed to dry for one hour to obtain a dry bacterial layer.
Meanwhile, cells of D. discoideum were washed twice in PAS buffer by centrifugation at 1000 g for 10 minutes. The amoebal suspension was adjusted to 2 x 106 cells.mL-1 and diluted in series to reach a final concentration of 7812 cells.mL-1. Five μL of each serial dilution was spotted on the bacterial lawn. Plates were incubated at 22.5°C for five days and appearance of phagocytic plaques was checked at the end of the incubation time. This assay was performed in triplicate.
In order to interpret the results, we used the categories defined by Adamek et al. (2011) [23 (link)]: non virulent (less than 400 amoebae for lysis plaque formation), low-virulent (400–2500 amoebae for lysis plaque formation) and virulent (more than 2500 amoebae).
Meanwhile, cells of D. discoideum were washed twice in PAS buffer by centrifugation at 1000 g for 10 minutes. The amoebal suspension was adjusted to 2 x 106 cells.mL-1 and diluted in series to reach a final concentration of 7812 cells.mL-1. Five μL of each serial dilution was spotted on the bacterial lawn. Plates were incubated at 22.5°C for five days and appearance of phagocytic plaques was checked at the end of the incubation time. This assay was performed in triplicate.
In order to interpret the results, we used the categories defined by Adamek et al. (2011) [23 (link)]: non virulent (less than 400 amoebae for lysis plaque formation), low-virulent (400–2500 amoebae for lysis plaque formation) and virulent (more than 2500 amoebae).
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