Recent laboratory evolution studies have shown that upon repetitive antibiotic treatments, bacterial populations will adapt and eventually became tolerant and resistant to the drug. Drug tolerance rapidly evolves upon frequent, intermittent antibiotic treatments, and such emerging drug tolerance seems to be specific to the treatment conditions, complicating clinical practice. Moreover, it has been shown that tolerance often promotes the development of resistance, which further reinforces the need of clinical diagnostics for antibiotic tolerance to reduce the occurrence of acquired resistance. Here, we discuss the laboratory evolution studies that were performed to track the development of tolerance in bacterial populations, and highlight the urgency of developing a comprehensive knowledge base of various tolerance phenotypes and their detection in clinics. Finally, we propose future directions for basic research in this growing field.

Bacterial persisters, a dormant and multidrug tolerant subpopulation that are able to resuscitate after antibiotic treatment, have recently received considerable attention as a major cause of relapse of various infectious diseases in the clinic. However, because of their low abundance and inherent transience, it is extremely difficult to study them by proteomics. Here we developed a magnetic-beads-based separation approach to enrich Escherichia coli persisters and then subjected them to shotgun proteomics. Rifampin pretreatment was employed to increase persister formation, and the resulting cells were exposed to a high concentration of ampicillin (10× MIC) to remove nonpersisters. The survivors were analyzed by spectral counting-based quantitative proteomics. On average, 710 proteins were identified at a false discovery rate of 0.01 for enriched E. coli persisters. By spectral counting-based quantification, 105 proteins (70 down-regulated, 35 up-regulated) were shown to be differentially expressed compared with normal cells. A comparison of the differentially expressed proteins between the magnetic beads-enriched persisters and nonenriched persisters (a mixture of persisters and intact dead cells) shows only around half (∼58%) overlap and different protein–protein interaction networks. This suggest that persister enrichment is important to eliminate the cumulative effect of dead cells that will obscure the proteome of persisters. As expected, proteins involved in carbohydrate metabolism, fatty acid and amino acid biosynthesis, and bacterial chemotaxis were found to be down-regulated in the persisters. Interestingly, membrane proteins including some transport proteins were up-regulated, indicating that they might be important for the drug tolerance of persisters. Knockout of the pal gene expressing peptidoglycan-associated lipoprotein, one of the most up-regulated proteins detected in persisters, led to 10-fold reduced persister formation under ampicillin treatment.

Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.