Whole-genome sequencing reveals the mechanisms for evolution of streptomycin resistance in Lactobacillus plantarum

摘要

ABSTRACTIn this research, we investigated the evolution of streptomycin resistance inLactobacillus plantarumATCC14917, which was passaged in medium containing a gradually increasing concentration of streptomycin. After 25 d, the minimum inhibitory concentration (MIC) ofL. plantarumATCC14917 had reached 131,072 µg/mL, which was 8,192-fold higher than the MIC of the original parent isolate. The highly resistantL. plantarumATCC14917 isolate was then passaged in antibiotic-free medium to determine the stability of resistance. The MIC value of theL. plantarumATCC14917 isolate decreased to 2,048 µg/mL after 35 d but remained constant thereafter, indicating that resistance was irreversible even in the absence of selection pressure. Whole-genome sequencing of parent isolates, control isolates, and isolates following passage was used to study the resistance mechanism ofL. plantarumATCC14917 to streptomycin and adaptation in the presence and absence of selection pressure. Five mutated genes (single nucleotide polymorphisms and structural variants) were verified in highly resistantL. plantarumATCC14917 isolates, which were related to ribosomal protein S12, LPXTG-motif cell wall anchor domain protein, LrgA family protein, Ser/Thr phosphatase family protein, and a hypothetical protein that may correlate with resistance to streptomycin. After passage in streptomycin-free medium, only the mutant gene encoding ribosomal protein S12 remained; the other 4 mutant genes had reverted to the wild type as found in the parent isolate. Although the MIC value ofL. plantarumATCC14917 was reduced in the absence of selection pressure, it remained 128-fold higher than the MIC value of the parent isolate, indicating that ribosomal protein S12 may play an important role in streptomycin resistance. Using the mobile elements database, we demonstrated that streptomycin resistance–related genes inL. plantarumATCC14917 were not located on mobile elements. This research offers a way of combining laboratory evolution techniques and whole-genome sequencing for evaluating antibiotic resistance in probiotics.