Evaluation of the plasmid copy number in B. cereus spores, during germination, bacterial growth and sporulation using real-time PCR.

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.