Improved axenization method reveals complexity of symbiotic associations between bacteria and acanthamoebae

Bacteria associated with free-living amoebae have attracted considerable attention because of their role in human disease and as models for studying endosymbiosis. However, the identification and analysis of such novel associations are hindered by the limitations of methods for isolation and axenization of amoebae. Here, we replaced the heat-inactivated Escherichia coli, which is typically used as food source during axenization, with a live E. coli tolC knockout mutant strain hypersensitive to antibiotics. Together with the addition of otherwise sublethal amounts of ampicillin, this approach tripled the success rate and reduced the time required for axenization by at least 3 days. Using this method for two environmental samples, 10 Acanthamoeba strains were isolated, seven of which contained bacterial symbionts. In three cases, amoebae harbouring two phylogenetically distinct symbionts were recovered, supporting a more widespread occurrence of multi-partner symbiotic associations among free-living amoebae.