The aim of this study was to obtain the Lactobacillus plantarum ATCC14917 with high-level resistance to penicillin and evaluate their probiotic traits using laboratory evolution assay and whole-genome sequencing. In penicillin environment, the minimum inhibitory concentration (MIC) of L. plantarum to penicillin increased from 1 μg/mL to 16 μg/mL and remained stable after the removal of antibiotic pressure, suggesting that the resistance acquisition to penicillin was an irreversible process. Subsequently, change of probiotic characteristics was further evaluated, and the results showed that the acid tolerance, bile tolerance and adhesion ability were significantly declined in the highly resistant strains. Whole-genome sequencing indicated that genes encoding hypothetical protein, LPXTG-motif cell wall anchor domain protein and acetyltransferase were detected in highly resistant L. plantarum, and these genes were still present after the following subculture in the absence of penicillin, suggesting that these three mutants might be the main reason for the development of penicillin resistance. The homology-based analysis of surrounding DNA regions of mutant genes was further performed and indicated that no resistant genes were located on mobile elements in evolved L. plantarum strains, signifying that the spread of antibiotic resistance genes in the gut would not occur for these mutant genes. This study provided a basis for the combined use of highly resistant L. plantarum and penicillin in the treatment of pathogen induced gut diseases.
Keywords: Antibiotic resistance gene; Mutant gene; Probiotic trait; Whole-genome sequencing.
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