Selective labeling and eradication of antibiotic-tolerant subpopulations in bacterial biofilms

Microorganisms are able to aggregate and form surface-attached biofilm communities in nature to counter potentially harmful environmental conditions. The physiological differentiation of microbial cells within biofilms enables them to develop into subpopulations with distinct antibiotic-tolerance profiles and social behaviors. Thus, it remains a great challenge to investigate the mechanisms of antibiotic-tolerance in biofilm cells. Here, we developed a pulsed-stable isotope labeling with amino acids (pulsed-SILAC) proteomic strategy to investigate the antibiotic-tolerant subpopulation of biofilms formed by Pseudomonas aeruginosa. P. aeruginosa biofilms were initially cultivated in minimal medium supplemented with C12-lysine and then subjected to colistin treatment. The colistin-susceptible subpopulations in the biofilms were rapidly killed, with a small fraction of colistin-tolerant P. aeruginosa cells left. After which, the biofilm cultivation medium was replaced by a minimal medium containing colistin and heavy C13-lysine so that we can label and quantify the newly expressed proteins in the colistin-tolerant subpopulations of biofilms with heavy C13-lysine. This analysis together with real-time imaging revealed that group migration is essential for the formation of colistin-tolerant biofilm subpopulations, as colistin-tolerant cell-aggregates migrate by using type IV pili, onto the top of the colistin-killed biofilms. We also found that the colistin-tolerant cells employ quorum sensing to initiate the formation of new colistin-tolerant microcolonies, highlighting the importance of social behavior in antibiotic tolerance development. In summary, our pulsed-SILAC strategy is an ideal approach to study antibiotic-tolerant bacterial subpopulations in complex microbial biofilm communities, which can provide novel insights for understanding biofilm physiology.