Gain and loss of acquired doxycycline resistance in <i>Lactiplantibacillus plantarum</i> : an adaptive laboratory evolution study

ABSTRACT Probiotics interact in a mutualistic way with humans. Exposure of probiotics to sublethal concentrations of commonly prescribed antibiotics can cause resistance to arise. Over 1,000 generations, we performed an adaptive laboratory evolution experiment to determine if the fitness of the probiotic Lactiplantibacillus plantarum is altered by exposure to doxycycline (DOX) at one-tenth of the minimal inhibitory concentration. 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 1,000 DOX-treated cultures reveals 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 non-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 and 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. IMPORTANCE Probiotics are beneficial bacteria often found in fermented foods and beverages. When consumed, they can colonize the gut and provide several advantages. Antibiotics, even at levels too low to kill bacteria, can exert pressure that leads to antibiotic resistance. Though well studied in harmful bacteria, the effects this may have on probiotics are less well understood. Using experimental evolution, we grew a popular probiotic, Lactiplantibacillus plantarum , in the presence of a low concentration of doxycycline, one of the most highly prescribed antibiotics in the US. Over the course of 5 months, roughly a thousand generations, the probiotic became ~4-fold more resistant; once we removed the antibiotic and continued the experiment, this resistance disappeared rapidly. DNA sequencing identified variants in the rpsJ gene, which encodes a protein important for translation. As doxycycline interferes with this process, the rpsJ variants may underlie the emergence of the modest resistance we observe.