A Sporulation-Independent Way of Life for Bacillus thuringiensis in the Late Stages of an Infection

The formation of endospores has been considered the unique survival and transmission mode of sporulating Firmicutes due to the exceptional resistance and persistence of this bacterial form. However, nonsporulated bacteria (Spo(–)) were reported at the early stages following the death of a host infected with Bacillus thuringiensis, an entomopathogenic sporulating bacterium. Here, we investigated the characteristics of the bacterial population in the late stages of an infection in the B. thuringiensis/Galleria mellonella infection model. Using fluorescent reporters and molecular markers coupled to flow cytometry, we demonstrated that the Spo(–) cells persist and constitute about half of the population 2 weeks post-infection (p.i.). Protein synthesis and growth recovery assays indicated that they are in a metabolically slowed-down state. These bacteria were extremely resistant to the insect cadaver environment, which did not support growth of in vitro-grown vegetative cells and spores. A transcriptomic analysis of this subpopulation at 7 days p.i. revealed a signature profile of this state, and the expression analysis of individual genes at the cell level showed that more bacteria mount an oxidative stress response as their survival time increases, in agreement with the increase of the free radical level in the host cadaver and in the number of reactive oxygen species (ROS)-producing bacteria. Altogether, these data show for the first time that nonsporulated bacteria are able to survive for a prolonged period of time in the context of an infection and indicate that they engage in a profound adaptation process that leads to their persistence in the host cadaver.