Genome adaptive evolution of Lactobacillus casei under long-term antibiotic selection pressures

Jicheng Wang, Xiao Dong, Yuyu Shao, Huiling Guo, Lin Pan, Wenyan Hui, Lai-Yu Kwok, Heping Zhang, Wenyi Zhang

The extensive use of antibiotics in medicine has raised serious concerns about biosafety. However, the effect of antibiotic application on the adaptive evolution of microorganisms, especially to probiotic bacteria, has not been well characterized. Thus, the objective of the current work was to investigate how antibiotic selection forces might drive genome adaptation using Lactobacillus (L.) casei Zhang as a model. Two antibiotics, amoxicillin and gentamicin, were consistently applied to the laboratory culture of L. casei Zhang. We then monitored the mutations in the bacterial genome and changes in the minimum inhibitory concentrations (MICs) of these two antibiotics along a 2000-generation-cultivation lasted over 10 months. We found an approximately 4-fold increase in the genome mutation frequency of L. casei Zhang, i.e. 3.5 × 10(-9) per base pair per generation under either amoxicillin or gentamicin stress, when compared with the parallel controls grown without application of any antibiotics. The increase in mutation frequency is significantly lower than that previously reported in Escherichia (E.) coli. The rate of de novo mutations, i.e. 20 per genome, remained low and stable throughout the long-term cultivation. Moreover, the accumulation of new mutations stopped shortly after the maximum bacterial fitness (i.e. the antibiotic MICs) was reached. Our study has shown that the probiotic species, L. casei Zhang, has high genome stability even in the presence of long-term antibiotic stresses. However, whether this is a species-specific or universal characteristic for all probiotic bacteria remains to be explored.