Gain and Loss of Acquired Doxycycline Resistance in Lactiplantibacillus plantarum: An Adaptive Laboratory Evolution Study

Probiotics interact in a mutualistic way with humans. Exposure of probiotics to sublethal concentrations of commonly prescribed antibiotics can cause resistance to arise. Over 1000 generations, we performed an adaptive laboratory evolution (ALE) experiment to determine if the fitness of the probiotic Lactobacillus plantarum is altered by exposure to doxycycline (DOX) at 1/10 MIC. Compared to the original culture, L. plantarum exposed to sublethal DOX acquired modest resistance (~4-fold) over time. When the selection pressure was removed, resistance was lost rapidly, in ~50 generations. This suggests that resistance, once acquired, is not fixed. The mechanism by which resistance is acquired and subsequently lost was investigated by whole genome sequencing (WGS). Analysis of the single nucleotide variants (SNVs) identified in the WGS of the generation 1000 DOX-treated cultures reveal 16 distinct variants across 15 genes. Two of these variants are in the rpsJ gene, which encodes ribosomal protein s10, a component of the 30S ribosomal subunit, and result in synonymous mutations (H56Y and S94N). This gene has been previously reported to harbor mutations associated with tetracycline class resistance, including DOX and tigecycline. WGS of archived cells from generations 350 and 750 reveals that one of these rpsJ variants (H56Y) arises early in the experiment. Additional rpsJ variants at position 57 (K57M, K57I) could be identified within intermediate generations. Most rpsJ SNVs identified from the WGS could be verified by colony PCR and Sanger sequencing. Importantly, no rpsJ variants are observed in the original culture sequences or in same-generation controls.