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Legionella pneumophila are aquatic bacteria that rely on parasitizing amoebae for survival and replication. L. pneumophila have evolved a wide range of effector proteins to ensure their own survival, but these same effectors enable Legionella pnemophila to infect human alveolar macrophages. To gain knowledge regarding the mechanisms in legionella that make them able to evolve and adapt to hosts a long term evolution experiment was performed. Legionella pneumophila was passaged in two different hosts- Acanthamoeba Castellani and human macrophage- like cells U937- separately and in alternation twice a week throughout a year. One notable result was the development of streptomycin resistance after five passages, even without streptomycin use. This was linked to two separate mutations in rpsL, K43 and K88. Compensatory mutations were also observed: K43 was linked to mutations in rpsD, and K88 was connected to mutations in groES. Fluctuation assays using different fluorophore-marked Legionella strains were performed to determine the mutation rate. The wild-type strain had a mutation rate of 6.3∙10−10, while strains marked with dTomato and SYFP had rates of were 7.5∙10−10 and 7.7∙10−10, respectively, indicating that the fluorophores did not affect the mutation rate. To assess amoebas ability to facilitate an environment more prone to resistance development a short evolution experiment was conducted for five passages. A. Castellani pre- grown with and without penicillin-streptomycin (Pen-strep) was used to investigate its ability to accumulate antibiotic intracellularly. Resistance was developed in the condition where A. Castellani had been pre- grown with Pen- strep indicating that amoeba may have the ability to accumulate pen- strep intracellularly and protect them.

Legionella pneumophila are aquatic bacteria that rely on parasitizing amoebae for survival and replication. L. pneumophila have evolved a wide range of effector proteins to ensure their own survival, but these same effectors enable Legionella pnemophila to infect human alveolar macrophages. To gain knowledge regarding the mechanisms in legionella that make them able to evolve and adapt to hosts a long term evolution experiment was performed. Legionella pneumophila was passaged in two different hosts- Acanthamoeba Castellani and human macrophage- like cells U937- separately and in alternation twice a week throughout a year. One notable result was the development of streptomycin resistance after five passages, even without streptomycin use. This was linked to two separate mutations in rpsL, K43 and K88. Compensatory mutations were also observed: K43 was linked to mutations in rpsD, and K88 was connected to mutations in groES. Fluctuation assays using different fluorophore-marked Legionella strains were performed to determine the mutation rate. The wild-type strain had a mutation rate of 6.3∙10−10, while strains marked with dTomato and SYFP had rates of were 7.5∙10−10 and 7.7∙10−10, respectively, indicating that the fluorophores did not affect the mutation rate. To assess amoebas ability to facilitate an environment more prone to resistance development a short evolution experiment was conducted for five passages. A. Castellani pre- grown with and without penicillin-streptomycin (Pen-strep) was used to investigate its ability to accumulate antibiotic intracellularly. Resistance was developed in the condition where A. Castellani had been pre- grown with Pen- strep indicating that amoeba may have the ability to accumulate pen- strep intracellularly and protect them.