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Despite its prominent role as an intracellular human pathogen,Listeria monocytogenesnormally features a saprophytic lifestyle, and shares many environmental habitats with predatory protozoa. Earlier studies claimed thatAcanthamoebamay act as environmental reservoirs forL. monocytogenes,whereas others failed to confirm this hypothesis. Our findings support the latter and provide clear evidence thatL. monocytogenesis unable to persist inAcanthamoeba castellaniiandA. polyphaga. Instead, externalListeriacells are rapidly immobilized on the surface ofAcanthamoebatrophozoites, forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulentL. monocytogenesmutants, non-pathogenicListeria, and other motile bacteria, indicating that formation of backpacks is not specific forL. monocytogenes,and independent of bacterial pathogenicity or virulence.Hence, backpacking appears to represent a unique and highly effective strategy ofAcanthamoebato trap and feed on motile bacteria.